Agents and methods for targeted delivery of immune effector cells

Abstract

The present invention relates to reagents and methods for targeted delivery of immune effector cells to target cells. In some embodiments, the invention relates to providing a subject with an immune effector cell genetically modified to express a chimeric antigen receptor (CAR) and a compound (docking compound) comprising a binding moiety against an antigen on a target cell and a binding moiety against the CAR. The CAR and the binding moiety for the CAR are bound to each other by an ALFA tag and a single domain antibody that binds to the ALFA tag.

Description

Technical Field

[0001] This invention relates to reagents and methods for targeted delivery of immune effector cells to target cells. In some embodiments, the invention relates to providing a subject with genetically modified immune effector cells expressing a chimeric antigen receptor (CAR) and a compound (docking compound) comprising a binding moiety against an antigen on a target cell and a binding moiety against the CAR. The CAR and the binding moiety against the CAR bind to each other via an ALFA tag and a single-domain antibody bound to the ALFA tag. Background Technology

[0002] Chimeric antigen receptors (CARs)—also known as chimeric immune receptors, chimeric T-cell receptors, or artificial T-cell receptors—are receptor proteins that have been engineered to give T cells a new ability to target specific antigens. These receptors are chimeric because they combine antigen-binding and T-cell activation functions into a single receptor. For example, CAR-T cell therapy uses CAR-modified T cells to treat cancer. CAR-T cell therapy aims to modify T cells to recognize target cells, such as cancer cells, thereby attacking and destroying them more effectively. However, for this purpose, it may be necessary to generate T cells modified to express different CARs to recognize different targets on the target cells, which can involve a great deal of work and difficulty. This workload can be significantly reduced if the CAR-modified T cells no longer target the target cells directly, but indirectly through compounds that bind to the target cells. This allows the use of universal CAR-T cells, but requires the use of one or more compounds that bind to the target cells and interact with the CAR-T cells. Therefore, a system with two interacting members, one on the CAR and the other on the compound that binds to the target cells, is needed for this purpose.

[0003] In 2019, Götzke et al. published the article "The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications" (Götzke et al., Nat Commun. 2019 Sep 27;10(1):4403. doi: 10.1038 / s41467-019-12301-7). ALFA ®The tag is a rationally designed peptide with a unique sequence that forms a stable α-helix in solution, is electrically neutral, and is highly soluble under physiological conditions. It can fuse with its binding partner in different orientations (N- or C-terminus or between two domains of the fusion protein) without loss of structure or function, and can be detected using a high-affinity (two-digit pM Kd) single-domain antibody (VHH), which the authors identified from immunized alpacas using a target-specific B-cell separation selection method (CELLINE). ALFA ® A detailed description of the / NbALFA epitope tagging system can be found in PCT patent publication WO 2020 / 053239A1. Therefore, ALFA ® Tags and NbALFA single-domain antibodies specific to those tags have great potential in bioscience applications.

[0004] NbALFA is derived from immunized alpacas, and alpaca-derived single-domain antibodies (VHHs) typically exhibit low homology to human sequences. However, for ALFA... ® The in vivo application of the / NbALFA tool in the aforementioned CAR-T cell therapy in humans is expected to yield higher sequence homology with the corresponding human sequences. Summary of the Invention

[0005] This invention relates to reagents and methods for targeted delivery of immune effector cells to target cells (e.g., diseased cells). Targeted delivery of immune effector cells to target cells can be achieved using docking compounds that bind to immune effector cells via an antigen receptor, said docking compound comprising a targeting portion for targeting the target cell. The docking compound binds to the antigen receptor via a tag / conjugate system comprising an ALFA tag and a single-domain antibody as described herein.

[0006] In some embodiments, the present invention relates to genetically modified immune effector cells expressing a chimeric antigen receptor (CAR) and a compound comprising a binding portion targeting an antigen on a target cell and another binding portion targeting the CAR (a docking compound). Providing the immune effector cells and the docking compound to a subject can deliver the immune effector cells to the target cells. The CAR and the binding portion targeting the CAR bind to each other via an ALFA tag and a single-domain antibody bound to the ALFA tag. In some embodiments, the portion of the CAR that binds to the binding portion targeting the CAR comprises an ALFA tag, and the binding portion targeting the CAR comprises a single-domain antibody. In some embodiments, the portion of the CAR that binds to the binding portion targeting the CAR comprises a single-domain antibody, and the binding portion targeting the CAR comprises an ALFA tag.

[0007] Immune effector cells can be genetically modified to express antigen receptors in vitro / in vitro or in vivo. Genetic modification can be accomplished by transfecting immune effector cells with nucleic acids encoding antigen receptors.

[0008] In some embodiments, genetically modified immune effector cells expressing CAR are provided to the subject by administration of the immune effector cells. In some embodiments, genetically modified immune effector cells expressing CAR are provided to the subject by generating the immune effector cells in the subject, for example, by transfecting the immune effector cells with a nucleic acid (e.g., DNA or RNA) encoding the CAR.

[0009] Docking compounds can be provided by adding them to target cells (e.g., applying the docking compound to a subject) or by adding nucleic acids (e.g., RNA) that encode the docking compound (e.g., applying RNA that encodes the docking compound to a subject to express the docking compound).

[0010] In some embodiments, the docking compound is delivered to the subject by administering a nucleic acid (e.g., RNA) encoding the docking compound. In some embodiments, the docking compound is delivered to the subject by administration. In some embodiments, a pre-formed complex is delivered to the subject by administration, wherein the docking compound binds to immune effector cells that are genetically modified to express a CAR.

[0011] The binding site for antigens on target cells can bind to target antigens (such as cancer antigens on cancer cells), and immune effector cells can target the binding site for CARs, thereby precisely delivering immune effector cells to target cells, such as cancer cells.

[0012] Therefore, this disclosure relates to a method in which a docking compound is used to label target cells, for example, by binding to a cell surface antigen. The docking compound comprises a portion that will be targeted by immune effector cells having an antigen receptor that targets that portion.

[0013] The methods described herein can deliver immune effector cells to target cells in vitro / in vitro and in vivo. The genetically modified immune effector cells described herein to express antigen receptors can be used to treat diseases in which cells (e.g., diseased cells) targeting antigens (e.g., tumor antigens) are beneficial. Target cells may express antigens on their cell surface for recognition by the docking compound. The treatments described herein can provide selective eradication of such antigen-expressing cells, thereby minimizing adverse effects on normal cells that do not express antigens. The methods and reagents described herein are particularly useful for treating diseases characterized by diseased cells expressing antigens targeted by the docking compound.

[0014] The concepts described in this article allow for the use of a single type of immune effector cell to target a broad range of target cells, i.e., by using a combination of a single type of immune effector cell with different docking compounds that target different antigen targets and contain the same CAR-binding moiety.

[0015] In one aspect, the present invention provides a system comprising:

[0016] (i) genetically modified immune effector cells to express chimeric antigen receptors (CARs), or nucleic acids encoding CARs, said nucleic acids being used to genetically modify immune effector cells to express said CARs; and

[0017] (ii) A compound comprising a binding moiety targeting a CAR and a binding moiety targeting an antigen on a target cell, or a nucleic acid encoding said compound,

[0018] The CAR and the binding portion targeting the CAR comprise a binding system comprising an ALFA tag and a single-domain antibody that binds to the ALFA tag.

[0019] The antibody comprises a CDR1 sequence as shown in SEQ ID NO: 94 or a sequence having one or two mutations relative to the sequence, a CDR2 sequence as shown in SEQ ID NO: 97 or a sequence having one or two mutations relative to the sequence, and a CDR3 sequence as shown in SEQ ID NO: 98 or a sequence having one or two mutations relative to the sequence;

[0020] The antibody further comprises the following amino acid residues at positions 47, 48, and 50 of the sequence corresponding to SEQ ID NO: 1: Arg at position 47, Arg at position 48, and Met at position 50;

[0021] And the antibody described herein contains a sequence having at least 90% sequence identity with any of SEQ ID NO: 118-125.

[0022] In some embodiments, the antibody comprises the CDR1 sequence as shown in SEQ ID NO: 94, the CDR2 sequence as shown in SEQ ID NO: 97, and the CDR3 sequence as shown in SEQ ID NO: 98.

[0023] In some implementations, the immune effector cells comprise T cells.

[0024] In some implementations, the immune effector cells express CAR on their cell surface.

[0025] In some implementations, the CAR includes a portion that is coupled to a coupling portion for the CAR.

[0026] In some implementations, the portion of the CAR that binds to the binding portion of the CAR is contained in the extracellular portion of the CAR.

[0027] In some embodiments, the portion of the CAR that binds to the CAR-targeting binding site includes an ALFA tag, and the CAR-targeting binding site includes an antibody.

[0028] In some embodiments, the portion of the CAR that binds to the CAR-targeting binding site comprises an antibody, and the CAR-targeting binding site comprises an ALFA tag.

[0029] In some embodiments, the antibody contains a sequence Epitope tag specific binding.

[0030] In some embodiments, the antibody is humanized and / or the antibody contains a sequence having at least about 70% V segment identity with the closest human lineage sequence.

[0031] In some embodiments, the antibody has a higher Tm compared to the antibody of SEQ ID NO: 1.

[0032] In some embodiments, the antibody contains a His residue at position 99 of the sequence corresponding to SEQ ID NO: 1, a Val residue at position 100 of the sequence corresponding to SEQ ID NO: 1, and / or a Tyr residue at position 40 of the sequence corresponding to SEQ ID NO: 1.

[0033] In some embodiments, the antibody comprises FR1, FR2, FR3, and FR4 sequences that have at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with, or are identical to, the sequences shown in SEQ ID NO:99, 102, 108, and 109, respectively.

[0034] In some implementations, the antibody includes a protein A binding site.

[0035] In some embodiments, the antibody comprises a sequence having at least 95% sequence identity with any of SEQ ID NO: 118-125, or the antibody comprises a sequence shown in any of SEQ ID NO: 118-125.

[0036] In some embodiments, the ALFA tag contains the amino acid sequence X1-X2-LE-X5-E-X7-RRRL-X12-X13 (SEQ ID NO: 3), wherein X1 is G or S or P or T, X2 is R or G or P, X5 is E or Q, X7 is L or I, X12 is S or T or P or A or D or E, and X13 is P or A or S or K or D or E or has no amino acid.

[0037] In some implementations, the binding portion targeting the CAR is connected to the binding portion targeting the antigen on the target cell via a connecting portion.

[0038] In some implementations, the connecting portion comprises a polymer.

[0039] In some implementations, the connecting portion includes a non-peptide portion.

[0040] In some embodiments, the connecting portion comprises a polymer selected from: polyethylene glycol (PEG), polysarcosine (pSar) (poly(N-methylglycine)), poly(N-methylglycine)... POX, polyoxo-zoline Azine (POZ), and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA), its derivatives and combinations thereof.

[0041] In some embodiments, the linker portion comprises a peptide portion.

[0042] In some implementations, the binding portion of an antigen on a target cell comprises a portion selected from: an antibody that binds to the antigen, an antibody derivative that binds to the antigen, and a ligand for the antigen.

[0043] In some implementations, the antibody derivative that binds to the antigen contains an antibody fragment.

[0044] In some implementations, the binding portion of the antigen on the target cell includes a peptide portion.

[0045] In some embodiments, the compound comprises a fusion polypeptide that includes a binding portion for CAR and a binding portion for antigens on target cells.

[0046] In some implementations, the ligand for the antigen contains DUPA or FAPI.

[0047] In some implementations, the target cells are diseased cells.

[0048] In some implementations, the target cells are cancer cells.

[0049] In some implementations, the antigen on the target cell includes cell surface antigens.

[0050] In some implementations, the antigens on the target cells include tumor antigens.

[0051] In some implementations, the nucleic acid encoding the CAR contains DNA.

[0052] In some implementations, the nucleic acid encoding the compound comprises RNA.

[0053] In some implementations, the binding of a complex comprising the following components to cells expressing the antigen results in the killing of cells expressing the antigen:

[0054] (i) the genetically modified immune effector cells expressing chimeric antigen receptors (CARs); and

[0055] (ii) The compound comprising a binding portion for CAR and a binding portion for antigens on target cells.

[0056] In another aspect, the present invention provides a method for treating a subject suffering from a disease, disorder, or symptom characterized by cells expressing antigens, comprising:

[0057] (i) Providing the subject with genetically modified immune effector cells that express chimeric antigen receptors (CARs); and

[0058] (ii) Providing the object with a compound comprising a binding moiety against a CAR and a binding moiety against an antigen,

[0059] The CAR and the binding portion targeting the CAR comprise a binding system comprising an ALFA tag and a single-domain antibody that binds to the ALFA tag.

[0060] The antibody comprises a CDR1 sequence as shown in SEQ ID NO: 94 or a sequence having one or two mutations relative to the sequence, a CDR2 sequence as shown in SEQ ID NO: 97 or a sequence having one or two mutations relative to the sequence, and a CDR3 sequence as shown in SEQ ID NO: 98 or a sequence having one or two mutations relative to the sequence;

[0061] The antibody further comprises the following amino acid residues at positions 47, 48, and 50 of the sequence corresponding to SEQ ID NO: 1: Arg at position 47, Arg at position 48, and Met at position 50;

[0062] And the antibody described herein contains a sequence having at least 90% sequence identity with any of SEQ ID NO: 118-125.

[0063] In some embodiments, the antibody comprises the CDR1 sequence as shown in SEQ ID NO: 94, the CDR2 sequence as shown in SEQ ID NO: 97, and the CDR3 sequence as shown in SEQ ID NO: 98.

[0064] In some implementations, the immune effector cells comprise T cells.

[0065] In some implementations, the immune effector cells express CAR on their cell surface.

[0066] In some implementations, the CAR includes a portion that is coupled to a coupling portion for the CAR.

[0067] In some implementations, the portion of the CAR that binds to the binding portion of the CAR is contained in the extracellular portion of the CAR.

[0068] In some embodiments, the portion of the CAR that binds to the CAR-binding portion contains an ALFA tag, and the CAR-binding portion contains an antibody.

[0069] In some embodiments, the portion of the CAR that binds to the CAR-targeting binding site comprises an antibody, and the CAR-targeting binding site comprises an ALFA tag.

[0070] In some embodiments, the antibody contains a sequence Epitope tag specific binding.

[0071] In some embodiments, the antibody is humanized and / or the antibody contains a sequence having at least about 70% V segment identity with the closest human lineage sequence.

[0072] In some embodiments, the antibody has a higher Tm compared to the antibody of SEQ ID NO: 1.

[0073] In some embodiments, the antibody contains a His residue at position 99 of the sequence corresponding to SEQ ID NO: 1, a Val residue at position 100 of the sequence corresponding to SEQ ID NO: 1, and / or a Tyr residue at position 40 of the sequence corresponding to SEQ ID NO: 1.

[0074] In some embodiments, the antibody comprises FR1, FR2, FR3, and FR4 sequences that have at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with, or are identical to, the sequences shown in SEQ ID NO:99, 102, 108, and 109, respectively.

[0075] In some implementations, the antibody includes a protein A binding site.

[0076] In some embodiments, the antibody comprises a sequence having at least 95% sequence identity with any of SEQ ID NO: 118-125, or the antibody comprises a sequence shown in any of SEQ ID NO: 118-125.

[0077] In some embodiments, the ALFA tag contains the amino acid sequence X1-X2-LE-X5-E-X7-RRRL-X12-X13 (SEQ ID NO: 3), wherein X1 is G or S or P or T, X2 is R or G or P, X5 is E or Q, X7 is L or I, X12 is S or T or P or A or D or E, and X13 is P or A or S or K or D or E or has no amino acid.

[0078] In some implementations, the binding portion targeting the CAR is connected to the binding portion targeting the antigen on the target cell via a connecting portion.

[0079] In some implementations, the connecting portion comprises a polymer.

[0080] In some implementations, the connecting portion includes a non-peptide portion.

[0081] In some embodiments, the connecting portion comprises a polymer selected from: polyethylene glycol (PEG), polysarcosine (pSar) (poly(N-methylglycine)), poly(N-methylglycine)... POX, polyoxo-zoline Azine (POZ), and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA), its derivatives and combinations thereof.

[0082] In some embodiments, the linker portion comprises a peptide portion.

[0083] In some implementations, the binding portion against the antigen comprises a portion selected from: an antibody that binds to the antigen, an antibody derivative that binds to the antigen, and a ligand of the antigen.

[0084] In some implementations, the antibody derivative that binds to the antigen contains an antibody fragment.

[0085] In some implementations, the binding portion against the antigen includes a peptide portion.

[0086] In some embodiments, the compound comprises a fusion polypeptide that includes a binding portion for CAR and a binding portion for antigen.

[0087] In some implementations, the ligand for the antigen contains DUPA or FAPI.

[0088] In some implementations, the cells expressing the antigen are diseased cells.

[0089] In some implementations, the cells expressing the antigen are cancer cells.

[0090] In some implementations, the antigen comprises a cell surface antigen.

[0091] In some implementations, the antigen includes a tumor antigen.

[0092] In some implementations, the binding of a complex comprising the following components to cells expressing the antigen results in the killing of cells expressing the antigen:

[0093] (i) the genetically modified immune effector cells expressing chimeric antigen receptors (CARs); and

[0094] (ii) The compound comprising a binding portion for CAR and a binding portion for antigen.

[0095] In some implementations, the method includes administering genetically modified immune effector cells to a subject to express CAR.

[0096] In some implementations, the method includes generating genetically modified immune effector cells in the subject to express CAR.

[0097] In some implementations, the method includes administering a nucleic acid encoding a CAR to the subject.

[0098] In some implementations, the nucleic acid encoding the CAR contains DNA.

[0099] In some implementations, the method includes applying a compound to a target.

[0100] In some implementations, the method includes generating a compound in the object.

[0101] In some implementations, the method includes administering a nucleic acid encoding a compound to the subject.

[0102] In some implementations, the nucleic acid encoding the compound comprises RNA.

[0103] In some embodiments, the method includes causing cells of the object to express a compound such that the compound is associated with cells expressing an antigen and that the binding portion of the CAR can be bound by immune effector cells.

[0104] In some implementations, cells expressing the compound are transfected with nucleic acids encoding the compound.

[0105] In some implementations, the cells expressing the compound secrete the compound.

[0106] In some implementations, cells expressing the compound express the compound so that it can be released into the bloodstream.

[0107] In some implementations, the nucleic acid encoding the CAR and / or the nucleic acid encoding the compound are administered as a particulate formulation, for example, formulated as lipid nanoparticles.

[0108] In some implementations, the disease, obstacle, or ailment is cancer.

[0109] In another aspect, the present invention provides a chimeric antigen receptor (CAR) comprising a single-domain antibody that binds to an ALFA tag.

[0110] The antibody comprises a CDR1 sequence as shown in SEQ ID NO: 94 or a sequence having one or two mutations relative to the sequence, a CDR2 sequence as shown in SEQ ID NO: 97 or a sequence having one or two mutations relative to the sequence, and a CDR3 sequence as shown in SEQ ID NO: 98 or a sequence having one or two mutations relative to the sequence;

[0111] The antibody further comprises the following amino acid residues at positions 47, 48, and 50 of the sequence corresponding to SEQ ID NO: 1: Arg at position 47, Arg at position 48, and Met at position 50;

[0112] And the antibody described herein contains a sequence having at least 90% sequence identity with any of SEQ ID NO: 118-125.

[0113] In some embodiments, the antibody comprises the CDR1 sequence as shown in SEQ ID NO: 94, the CDR2 sequence as shown in SEQ ID NO: 97, and the CDR3 sequence as shown in SEQ ID NO: 98.

[0114] In some embodiments, the antibody contains a sequence Epitope tag specific binding.

[0115] In some embodiments, the antibody is humanized and / or the antibody contains a sequence having at least about 70% V segment identity with the closest human lineage sequence.

[0116] In some embodiments, the antibody has a higher Tm compared to the antibody of SEQ ID NO: 1.

[0117] In some embodiments, the antibody contains a His residue at position 99 of the sequence corresponding to SEQ ID NO: 1, a Val residue at position 100 of the sequence corresponding to SEQ ID NO: 1, and / or a Tyr residue at position 40 of the sequence corresponding to SEQ ID NO: 1.

[0118] In some embodiments, the antibody comprises FR1, FR2, FR3, and FR4 sequences that have at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with, or are identical to, the sequences shown in SEQ ID NO:99, 102, 108, and 109, respectively.

[0119] In some implementations, the antibody includes a protein A binding site.

[0120] In some embodiments, the antibody comprises a sequence having at least 95% sequence identity with any of SEQ ID NO: 118-125, or the antibody comprises a sequence shown in any of SEQ ID NO: 118-125.

[0121] In some implementations, the antibody is contained within the extracellular domain of the CAR.

[0122] In another aspect, the present invention provides nucleic acids encoding the CAR described herein.

[0123] In some implementations, the nucleic acid is DNA.

[0124] In another aspect, the present invention provides a host cell comprising nucleic acid encoding the CAR described herein and / or expressing the CAR described herein.

[0125] In some implementations, the host cell comprises immune effector cells.

[0126] In some implementations, the immune effector cells comprise T cells.

[0127] In some implementations, the host cell is genetically modified to contain the nucleic acid and / or express the CAR.

[0128] In some implementations, the host cell expresses the CAR on its cell surface.

[0129] In another aspect, the present invention provides compounds comprising a single-domain antibody that binds to an ALFA tag and a binding moiety against an antigen on a target cell.

[0130] The antibody comprises a CDR1 sequence as shown in SEQ ID NO: 94 or a sequence having one or two mutations relative to the sequence, a CDR2 sequence as shown in SEQ ID NO: 97 or a sequence having one or two mutations relative to the sequence, and a CDR3 sequence as shown in SEQ ID NO: 98 or a sequence having one or two mutations relative to the sequence;

[0131] The antibody further comprises the following amino acid residues at positions 47, 48, and 50 of the sequence corresponding to SEQ ID NO: 1: Arg at position 47, Arg at position 48, and Met at position 50;

[0132] And the antibody described herein contains a sequence having at least 90% sequence identity with any of SEQ ID NO: 118-125.

[0133] In some embodiments, the antibody comprises the CDR1 sequence as shown in SEQ ID NO: 94, the CDR2 sequence as shown in SEQ ID NO: 97, and the CDR3 sequence as shown in SEQ ID NO: 98.

[0134] In some embodiments, the antibody contains a sequence Epitope tag specific binding.

[0135] In some embodiments, the antibody is humanized and / or the antibody contains a sequence having at least about 70% V segment identity with the closest human lineage sequence.

[0136] In some embodiments, the antibody has a higher Tm compared to the antibody of SEQ ID NO: 1.

[0137] In some embodiments, the antibody contains a His residue at position 99 of the sequence corresponding to SEQ ID NO: 1, a Val residue at position 100 of the sequence corresponding to SEQ ID NO: 1, and / or a Tyr residue at position 40 of the sequence corresponding to SEQ ID NO: 1.

[0138] In some embodiments, the antibody comprises FR1, FR2, FR3, and FR4 sequences that have at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with, or are identical to, the sequences shown in SEQ ID NO:99, 102, 108, and 109, respectively.

[0139] In some implementations, the antibody includes a protein A binding site.

[0140] In some embodiments, the antibody comprises a sequence having at least 95% sequence identity with any of SEQ ID NO: 118-125, or the antibody comprises a sequence shown in any of SEQ ID NO: 118-125.

[0141] In some implementations, the antibody is connected to the binding portion of the antigen on the target cell via a linker portion.

[0142] In some implementations, the connecting portion comprises a polymer.

[0143] In some implementations, the connecting portion includes a non-peptide portion.

[0144] In some embodiments, the connecting portion comprises a polymer selected from: polyethylene glycol (PEG), polysarcosine (pSar) (poly(N-methylglycine)), poly(N-methylglycine)... POX, polyoxo-zoline Azine (POZ), and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA), its derivatives and combinations thereof.

[0145] In some embodiments, the linker portion comprises a peptide portion.

[0146] In some implementations, the binding portion of an antigen on a target cell comprises a portion selected from: an antibody that binds to the antigen, an antibody derivative that binds to the antigen, and a ligand for the antigen.

[0147] In some implementations, the antibody derivative that binds to the antigen contains an antibody fragment.

[0148] In some implementations, the binding portion of the antigen on the target cell includes a peptide portion.

[0149] In some embodiments, the compound comprises a fusion polypeptide containing an antibody and a binding portion against an antigen on a target cell.

[0150] In some implementations, the ligand for the antigen contains DUPA or FAPI.

[0151] In some implementations, the target cells are diseased cells.

[0152] In some implementations, the target cells are cancer cells.

[0153] In some implementations, the antigen on the target cell includes cell surface antigens.

[0154] In some implementations, the antigens on the target cells include tumor antigens.

[0155] In another aspect, the present invention provides a nucleic acid encoding a compound comprising a single-domain antibody that binds to an ALFA tag as described herein and a binding moiety against an antigen on a target cell.

[0156] In some implementations, the nucleic acid is RNA.

[0157] In another aspect, the present invention provides a host cell comprising a nucleic acid encoding a compound described herein, the compound comprising a single-domain antibody that binds to an ALFA tag and a binding moiety against an antigen on a target cell, and / or expressing the compound described herein, the compound comprising a single-domain antibody that binds to an ALFA tag and a binding moiety against an antigen on a target cell.

[0158] In another aspect, the present invention provides reagents, compositions, and systems described herein for use in the methods described herein. Attached Figure Description

[0159] Figure 1 Affinity of humanized NbALFA variants generated by CDR grafting

[0160] A, Affinity measurement results of NbALFA (wtNbALFA) of virgin alpaca origin against the peptide of SEQ ID NO: 2. B, Affinity measurement results of variant 3 against the peptide of SEQ ID NO: 2. C, Affinity measurement results of variant 4 against the peptide of SEQ ID NO: 2. D, Affinity measurement results of variant 5 against the peptide of SEQ ID NO: 2. E, Affinity measurement results of variant 6 against the peptide of SEQ ID NO: 2. F, Affinity measurement results of variant 7 against the peptide of SEQ ID NO: 2. Affinity was determined using biolayer interferometry as described in Method 1 of Example.

[0161] Figure 2 Affinity of other modified humanized NbALFA variants

[0162] A, Affinity measurement results for the peptide of SEQ ID NO: 2, Variant 8. B, Affinity measurement results for the peptide of SEQ ID NO: 2, Variant 9. C, Affinity measurement results for the peptide of SEQ ID NO: 2, Variant 10. D, Affinity measurement results for the peptide of SEQ ID NO: 2, Variant 11. E, Affinity measurement results for the peptide of SEQ ID NO: 2, Variant 12. F, Affinity measurement results for the peptide of SEQ ID NO: 2, Variant 13. G, Affinity measurement results for the peptide of SEQ ID NO: 2, Variant 14. H, Affinity measurement results for the peptide of SEQ ID NO: 2, Variant 15. Affinity was determined using biolayer interferometry as described in Method 1 of Example.

[0163] Figure 3 SpA-assisted purification of humanized NbALFA variants

[0164] SpA purification using optimized variants. Spiking experiments were performed on the variants in cell culture supernatant, followed by SpA chromatography using a rotating column.

[0165] Figure 4 Modular CAR-T cell approach

[0166] This diagram illustrates a general CAR-T approach based on a modular interaction pair with an ALFA tag / NbALFA. The CAR-T carries a tag-binding unit, NbALFA VHH, on its surface. A second binding unit, consisting of an ALFA tag fused with a tumor antigen-specific ligand (here: CLDN6-specific scFv), can bind to tumor cells due to its specificity for the tumor antigen and is also bound by NbALFA-CAR-T cells, leading to specific lysis of the tumor cells.

[0167] Figure 5 T cells were genetically modified to stably express wild-type ModCAR (NbALFA-CAR) or humanized ModCAR (variant 11 / variant 13). On day 7, the CAR positivity rate was adjusted to 50%, and then CLDN6 was used. + Tumor cells at a 5:1 CAR ratio +Effector cells were co-cultured at an effector-to-target cell ratio (E:T). At the 0-hour time point, either 0% supernatant containing the adaptor was not added to the co-culture, or different dilutions (0.125%, 0.03%, 0.5%, 2%) of supernatant containing the adaptor were added. After 24 hours of co-culture, the surface expression (% of parent or mean fluorescence intensity, MFI) of activation markers (CD25, CD69) and degranulation marker (CD107a) was analyzed by flow cytometry. The mean and standard deviation of two technical replicates are shown.

[0168] Figure 6 T cells were genetically modified to stably express wild-type ModCAR (NbALFA-CAR) or humanized ModCAR (variant 11 / variant 13). On day 7, the CAR positivity rate was adjusted to 50%, and the cells were co-cultured with CLDN6+ tumor cells at a 5:1 CAR+ effector cell to target cell ratio (E:T). At time point 0 h, either no (0%) supernatant containing the adaptor was added to the co-culture, or different dilutions (0.125%, 0.03%, 0.5%, 2%) of supernatant containing the adaptor were added. After 24 h of co-culture, cell culture supernatant was collected and analyzed for GM-CSF, IFN-γ, IL-2, and TNF-α. The mean and standard deviation of two technical replicates are shown.

[0169] Figure 7 T cells were genetically modified to stably express wild-type ModCAR (NbALFA-CAR) or humanized ModCAR (variant 11 / variant 13). On day 7, the CAR positivity rate was adjusted to 50%, and then CLDN6 was used. + Tumor cells were co-cultured at a 5:1 CAR+ effector cell to target cell ratio (E:T). At time point 0 h, either no (0%) supernatant containing the adaptor was added to the co-culture, or different dilutions (0.125%, 0.5%, 2%) of supernatant containing the adaptor were added. Tumor cell lysis over 96 hours was measured using xCELLigence. Data were normalized relative to a Mock (unmodified) T cell control and relative to maximum lysis via Triton-X-100. The mean and standard deviation of three technical replicates are shown.

[0170] Figure 8 Genetically modified wild-type ModCAR (NbALFA-CAR) T cells and humanized ModCAR (variant 11 / variant 13) T cells were regulated to a CAR positivity rate of 70%, and the CAR ratio was 7:1. +The effector cell to target cell ratio was increased by adding GFP generated 24 hours prior to co-culture. + CLDN6 + Tumor spheroids. At time point 0, no (0%) supernatant containing the adaptor was added to the co-culture, or supernatant containing the adaptor at different dilutions (0.125%, 0.5%, 2%) was added. Second and third CLDN6 were added at 166 and 334 hours, respectively. + Tumor spheroids (using) (Target representation). Tumor spheroid lysis over 480 hours was measured using Incucyte. Data were normalized relative to a simulated (unmodified) T-cell control. The mean and standard deviation of three technical replicates are shown.

[0171] Figure 9 Genetically modified wild-type ModCAR (NbALFA-CAR) T cells and humanized ModCAR (variant 11 / variant 13) T cells were regulated to a CAR positivity rate of 70%, and the CAR ratio was 7:1. + The effector cell to target cell ratio was increased by adding GFP generated 24 hours prior to co-culture. + CLDN6 - Tumor spheroids. No supernatant containing the adaptor was added to the co-culture. Tumor spheroid lysis and T cell expansion over 168 hours were measured using Incucyte. Data were normalized relative to a simulated (ungenetically modified) T cell control. The mean and standard deviation of three technical replicates are shown.

[0172] Figure 10 Lysis of CD19+, CLDN6+, and CLDN18.2+ tumor spheroids by adaptor-functionalized ModCAR T cells. Lysis of CD19+ (A), CLDN6+ (B), or CLDN18.2+ (C) spheroids co-cultured with mimic (unmodified) or ModCAR T cells (NbALFA-CAR / variant 13) at a 5:1 effector-to-target cell ratio in 0.5% supernatant containing the matched adaptor. Adaptor was administered at 0 h. ModCAR-T cells (---- spheroids) were repeatedly challenged with antigen + spheroids at specified time points. Data were normalized relative to a mimic T cell control and represent the mean ± standard deviation of three technical replicates from two donors.

[0173] Figure 11 Schematic diagram of the in vivo study workflow for functional comparison of NbALFA-CAR variants. CLDN6+ OV90-SC12 tumors were established in NSG mice via subcutaneous tumor cell injection. On day 38 after tumor transplantation, 1×10⁻⁶ tumor cells were injected into the tumor cells. 6T cells expressing NbALFA-CAR or variant 13, or a corresponding number of T cells derived from human PBMCs, were adopted intravenously into mice. Antitumor activity was assessed by administering 5 µg, 30 µg, or no Riboadaptor LNP at designated time points weekly. n = 10 mice / group.

[0174] Figure 12 Antitumor response of NbALFA-CAR variants after in vivo adaptor functionalization. Tumor volumes are shown in animals treated with NbALFA-CAR, variant 13, and control T cells after adoptive cell transfer (ACT) and subsequent treatment with no (A), 5 µg (B), or 30 µg (C) Riboadaptor. Vertical lines represent ACT (solid lines) and Riboadaptor LNP (dashed lines) treatment. Data for n≥4 are shown and expressed as mean ± SEM for all mice / groups.

[0175] Figure 13 Peripheral CAR T cell expansion of NbALFA-CAR variants during antitumor response. The kinetics of CAR T cell numbers in the blood of mice treated with NbALFA-CAR or variant 13 during tumor studies are shown. CAR T cell expansion in each individual mouse is shown, including those not receiving (A), receiving 5 µg (B), or receiving 30 µg (C) Riboadaptor. Mean expansion for each treatment group is shown (D, E, F). Vertical dashed lines indicate Riboadaptor LNP treatment. Detailed Implementation

[0176] Although this disclosure is described in more detail below, it should be understood that this disclosure is not limited to the specific methods, schemes, and reagents described herein, as these are subject to variation. It should also be understood that the terminology used herein is for the purpose of describing some specific embodiments only and is not intended to limit the scope of this disclosure, which will be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0177] The elements of this disclosure will be described in more detail below. These elements are listed along with some specific embodiments; however, it should be understood that they can be combined in any way and in any number to produce other embodiments. The various described examples and preferred embodiments should not be construed as limiting this disclosure to the explicitly described embodiments. This description should be understood to support and cover embodiments that combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, unless the context otherwise requires, any permutation and combination of all elements described in this application should be considered as disclosed in the specification of this application.

[0178] Unless otherwise stated, the practice of this disclosure will be carried out using conventional chemical, biochemical, pharmaceutical, cell biological, immunological, and recombinant DNA techniques as explained in the literature in this field.

[0179] Throughout this specification and the appended claims, unless the context otherwise requires, the term "comprising / including" and its variations shall be understood to mean including the stated features, elements, members, integers, or steps, or groups of features, elements, members, integers, or steps, but not excluding any other features, elements, members, integers, or steps, or groups of features, elements, members, integers, or steps. The term "consistently composed of" limits the scope of the claims or disclosure to the specified features, elements, members, integers, or steps, as well as those that do not substantially affect the essential features and novel features of the claims or disclosure. The term "consistently composed of" limits the scope of the claims or disclosure to the specified features, elements, members, integers, or steps. The term "comprising / including" encompasses the term "consistently composed of," and "consistently composed of" in turn encompasses the term "consistently composed of." Therefore, whenever the term "comprising / including" appears in this application, it may be replaced by the terms "consistently composed of" or "composed of." Similarly, whenever the term "consistently composed of" appears in this application, it may be replaced by the term "composed of."

[0180] Unless otherwise specified herein or clearly contradicted by the context, nouns without quantifiers and similar references used in the context of describing this disclosure (especially in the context of the claims) mean one / agent or more / agents.

[0181] Unless otherwise specified in this document or clearly contradicted by the context, all methods described herein may be performed in any suitable order.

[0182] The use of any and all instances or exemplary language (e.g., "for example") provided herein is intended only to better illustrate the scope of this disclosure and does not limit the scope of any additional claims made herein. The language used in this specification should not be construed as indicating that any unclaimed element is necessary for the practice of this disclosure.

[0183] As used herein, the terms “optional” or “optionally” mean that the event, condition, or situation subsequently described may or may not occur, and the description includes instances where the event, condition, or situation occurs and instances where the event, condition, or situation does not occur.

[0184] As used herein, “and / or” is considered to be a specific disclosure of each of two specified features or components, with or without the other. For example, “X and / or Y” is considered to be a specific disclosure of (i) X, (ii) Y, and (iii) X and Y, as if each were listed separately herein.

[0185] In the context of this disclosure, the term "about" represents an accuracy range that a person skilled in the art would understand, while still ensuring the technical effectiveness of the features under discussion. This term typically indicates a deviation from an indicated value of ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, and, for example, ±0.01%. In some embodiments, "about" indicates a deviation from an indicated value of ±10%. In some embodiments, "about" indicates a deviation from an indicated value of ±5%. In some embodiments, "about" indicates a deviation from an indicated value of ±4%. In some embodiments, "about" indicates a deviation from an indicated value of ±3%. In some embodiments, "about" indicates a deviation from an indicated value of ±2%. In some embodiments, "about" indicates a deviation from an indicated value of ±1%. In some embodiments, "about" indicates a deviation from an indicated value of ±0.9%. In some embodiments, "about" indicates a deviation from an indicated value of ±0.8%. In some embodiments, "about" indicates a deviation from the indicated value of ±0.7%. In some embodiments, "about" indicates a deviation from the indicated value of ±0.6%. In some embodiments, "about" indicates a deviation from the indicated value of ±0.5%. In some embodiments, "about" indicates a deviation from the indicated value of ±0.4%. In some embodiments, "about" indicates a deviation from the indicated value of ±0.3%. In some embodiments, "about" indicates a deviation from the indicated value of ±0.2%. In some embodiments, "about" indicates a deviation from the indicated value of ±0.1%. In some embodiments, "about" indicates a deviation from the indicated value of ±0.05%. In some embodiments, "about" indicates a deviation from the indicated value of ±0.01%. As will be understood by those skilled in the art, the specific deviation of such a value for a given technical effect will depend on the nature of the technical effect. For example, natural or biotechnological effects may generally have a larger such deviation than artificial or engineered effects.

[0186] The description of ranges of values ​​in this document is intended only as a way of abbreviating the individual values ​​falling within the range. Unless otherwise stated herein, each individual value is incorporated into the specification as if described separately herein.

[0187] Numerous references are cited throughout the main text of this specification. Each reference cited herein (including all patents, patent applications, scientific publications, manufacturers' specifications, guidelines, etc.) is incorporated herein by reference in its entirety. Nothing herein should be construed as an admission that the invention is not entitled to claim prior invention rights over such disclosure.

[0188] The following provides definitions and implementation schemes applicable to all aspects of this disclosure. Unless otherwise stated, the terms defined below have their defined meanings. Any undefined term has its generally accepted meaning in its field.

[0189] As used herein, terms such as “reduce” or “inhibit” mean the ability to cause an overall reduction in level, for example, a reduction of about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, or about 75% or more. The term “inhibit” or similar phrases include complete or substantially complete inhibition, i.e., reduction to zero or substantially reduction to zero.

[0190] The term “enhancement” as used in this document means the ability to cause an overall increase or enhancement in level, such as by at least about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, about 75% or more, or about 100% or more.

[0191] As used herein, “physiological pH” refers to a pH of approximately 7.4. In some embodiments, the physiological pH is 7.3 to 7.5. In some embodiments, the physiological pH is 7.35 to 7.45. In some embodiments, the physiological pH is 7.3, 7.35, 7.4, 7.45, or 7.5.

[0192] As used in this disclosure, "% w / v" refers to weight-to-volume percentage, which is a unit of concentration of the amount of solute (in grams (g)) expressed as a percentage of the total volume of the solution (in milliliters (mL)).

[0193] As used in this disclosure, “weight%” means weight percentage, which is a unit of concentration of substance (in grams (g)) expressed as a percentage of the total weight (in grams (g) of the total composition.

[0194] As used in this disclosure, “mol %” is defined as the ratio of the number of moles of one component to the total number of moles of all components multiplied by 100.

[0195] As used in this disclosure, "total lipids in mol%" is defined as the ratio of the number of moles of one lipid component to the total number of moles of all lipids multiplied by 100. In some embodiments herein, the term "total lipids" includes both lipids and lipid-like substances.

[0196] The term "ionic strength" refers to the mathematical relationship between the number of different types of ions in a given solution and their respective charges. Therefore, ionic strength I is mathematically expressed by the following formula:

[0197]

[0198] Where c is the molar concentration of a specific ionic substance, and z is the absolute value of its charge. The sum Σ is the sum of all different types of ions (i) in the solution.

[0199] According to this disclosure, in some embodiments, the term "ionic strength" refers to the presence of monovalent ions. Regarding the presence of divalent ions, particularly divalent cations, in some embodiments, their concentration or effective concentration (the presence of free ions) is sufficiently low to prevent nucleic acid degradation due to the presence of a chelating agent. In some embodiments, the concentration or effective concentration of divalent ions is below the catalytic level required for hydrolyzing phosphodiester bonds between nucleotides (e.g., RNA nucleotides). In some embodiments, the concentration of free divalent ions is 20 μM or less. In some embodiments, free divalent ions are absent or substantially absent.

[0200] "Osmotic pressure" refers to the concentration of a specific solute, expressed as the number of osmole of solute per kilogram of solvent.

[0201] The term "lyophilization" and its variations refer to the freezing of a substance by freezing the substance and subsequently reducing the ambient pressure (e.g., below 15 Pa, such as below 10 Pa, below 5 Pa, or 1 Pa or lower) to cause the freezing medium in the substance to sublimate directly from the solid phase to the gas phase. Therefore, the terms "lyophilization" and "freeze-drying" are used interchangeably herein.

[0202] The term "spray drying" refers to the process of drying a substance by mixing (heated) gas with a fluid that is atomized (sprayed) inside a container (spray dryer), in which the solvent from the formed droplets evaporates to obtain a dried powder.

[0203] The term “reconstruction” refers to adding a solvent (such as water) to a dried product to return it to a liquid state, such as its original liquid state.

[0204] In the context of this disclosure, the term "recombinant" means "prepared by genetic engineering." In some embodiments, the "recombinant object" in the context of this disclosure is not naturally occurring.

[0205] As used herein, the term "naturally occurring" refers to the fact that an object can be found in nature. For example, peptides or nucleic acids that exist in organisms (including viruses) and can be isolated from natural sources without being intentionally modified by humans in a laboratory are naturally occurring. The term "found in nature" means "existing in nature" and includes both known objects and objects that have not yet been discovered and / or isolated from nature but may be discovered and / or isolated from natural sources in the future.

[0206] The terms “room temperature” and “ambient temperature” as used herein are used interchangeably and refer to a temperature of at least about 15°C, for example, about 15°C to about 35°C, about 15°C to about 30°C, about 15°C to about 25°C, or about 17°C to about 22°C. Such temperatures will include 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, and 22°C.

[0207] The term "EDTA" refers to disodium ethylenediaminetetraacetate. All concentrations are expressed as disodium EDTA.

[0208] The term "cryoprotectant" refers to substances added to formulations to protect the active ingredient during the freezing phase.

[0209] The term "lyophilization protectant" refers to substances added to formulations to protect the active ingredient during the drying phase.

[0210] According to this disclosure, the term "peptide" refers to a substance comprising about two or more, about three or more, about four or more, about six or more, about eight or more, about ten or more, about thirteen or more, about sixteen or more, about 20 or more, and more than about 50, about 100, or about 150 consecutive amino acids linked together by peptide bonds. The term "polypeptide" refers to a large peptide, particularly a peptide having at least about 151 amino acids. Both "peptide" and "polypeptide" are protein molecules. Therefore, the terms "peptide," "protein," and "polypeptide" are generally used as synonyms herein.

[0211] The peptides and polypeptides disclosed herein may contain linear or cyclic peptide sequences.

[0212] In some embodiments, the peptides disclosed herein comprise at least one cyclic portion, i.e., a polypeptide chain containing a cyclic sequence of bonds, referred to herein as a "cyclic peptide". The cyclic sequence can be generated by: a link between the amino terminus and the carboxyl terminus of the peptide; a link between the amino terminus and a side chain; a link between the carboxyl terminus and a side chain; or by forming disulfide bonds between two side chains containing sulfur groups of two cysteine ​​amino acids, or more complex arrangements.

[0213] In some embodiments, the peptides and polypeptides disclosed herein consist of naturally occurring amino acids, non-naturally occurring amino acids, amino acid derivatives and non-amino acid components, or mixtures thereof. In some embodiments, the peptides and polypeptides disclosed herein comprise amino acid mimics and amino acid analogs. In some embodiments, the peptides and polypeptides disclosed herein comprise non-naturally occurring amino acid sequences resistant to enzyme cleavage.

[0214] In some embodiments, one or more positions of the peptide or polypeptide disclosed herein are replaced by amino acids that are not naturally occurring. In some embodiments, the replaced amino acids are chemically related to the original residues (e.g., aliphatic, charged, basic, acidic, aromatic, hydrophilic), or are isosteres of the original residues.

[0215] As used herein in its broadest sense, the term "amino acid" or "amino acid residue" refers to compounds and / or substances that can be incorporated into, are incorporated into, or have been incorporated into peptides, for example, by forming one or more peptide bonds. In some embodiments, the term "amino acid" refers to α-aminocarboxylic acid. In some embodiments, amino acids have the general structure H₂N-C(H)(R)-COOH. In some embodiments, amino acids are naturally occurring amino acids. In some embodiments, amino acids are non-natural amino acids. In some embodiments, amino acids are D-amino acids. In some embodiments, amino acids are L-amino acids.

[0216] "Standard amino acid" refers to any one of the twenty standard L-amino acids commonly found in naturally occurring peptides and polypeptides. "Non-standard amino acid" refers to any amino acid other than the standard amino acids, whether it is synthetically prepared or obtained from a natural source. In some embodiments, the amino acids (including carboxyl and / or amino-terminal amino acids) in a peptide or polypeptide may contain structural modifications compared to the general structure described above. For example, in some embodiments, amino acids may be modified by methylation, amidation, acetylation, PEGylation, glycosylation, phosphorylation, and / or substitution (e.g., amino, carboxylic acid, one or more protons, and / or hydroxyl groups) compared to the general structure. In some embodiments, such modifications may, for example, alter the cyclic half-life of a peptide or polypeptide containing modified amino acids compared to a peptide or polypeptide containing otherwise identical unmodified amino acids. In some embodiments, such modifications do not significantly alter the relevant activity of a peptide or polypeptide containing modified amino acids compared to a peptide or polypeptide containing otherwise identical unmodified amino acids. As will be clear from the context, in some embodiments, the term "amino acid" may be used to refer to a free amino acid. In some embodiments, it may be used to refer to the amino acid residues of a peptide or polypeptide. In some embodiments, the amino acid is selected from the following: L-alanine (Ala or A); L-arginine (Arg or R); L-asparagine (Asn or N); L-aspartic acid (Asp or D); L-cysteine ​​(Cys or C); L-glutamine (Gln or Q); L-glutamic acid (Glu or E); glycine (Gly or G); L-histidine (His or H); L-isoleucine (Ile or I); L-leucine (Leu or L); L-lysine (Lys or K); L-methionine (Met or M); L-phenylalanine (Phe or F); L-proline (Pro or P); L-serine (Ser or S); L-threonine (Thr or T); L-tryptophan (Trp or W); L-tyrosine (Tyr or Y); and L-valine (Val or V), although modified, synthetic, or rare amino acids such as taurine, ornithine, selenocysteine, homocysteine, hydroxyproline, thioproline, iodotyrosine, 3-nitrotyrosine, ornithine, citrulline, canavalialine, 5-hydroxytryptophan, carnosine, cycloleucine, 3,4-dihydroxyphenylalanine, N-acetylcysteine, proline, allylglycine, or acetidine-2-carboxylic acid may be used as needed.

[0217] Generally speaking, amino acids are L-amino acids, while D-amino acids are indicated by the prefix "D". The prefix "high" or "h" indicates an α-amino acid that is otherwise similar to one of the common α-amino acids but contains an additional methylene group in its carbon chain.

[0218] In this article, “Orn” refers to ornithine or 2,5-diaminovaleric acid, “Dab” refers to 2,4-diaminobutyric acid, “Dap” refers to 2,3-diaminopropionic acid, “hLys” refers to 2,7-diaminoheptanoic acid, “hCys” refers to 2-amino-4-mercaptobutyric acid, and “Pen” refers to penicillamine or 2-amino-3-methyl-3-thioalkylbutyric acid.

[0219] It is also possible to include non-peptide bonds and other chemical modifications. For example, a portion or all of a peptide or polypeptide can be synthesized as a peptide mimic, such as peptide-like compounds (see, for example, Simon et al. (1992) Proc. Natl. Acad. Sci. USA 89:9367-71 and Horwell (1995) Trends Biotechnol. 13:132-4). A peptide or polypeptide may contain one or more (e.g., all) non-hydrolyzable bonds. Many non-hydrolyzable peptide bonds and synthetic procedures for peptides containing such bonds are known in the art. Exemplary non-hydrolyzable bonds include -[CH2NH]-reducing amide peptide bond, -[COCH2]-ketomethylene peptide bond, -[CH(CN)NH]-(cyanomethylene)amino peptide bond, -[CH2CH(OH)]-hydroxyethylidene peptide bond, -[CH2O]-oxymethylene peptide bond, and -[CH2S]-thiomethylene peptide bond (see, for example, U.S. Patent No. 6,172,043).

[0220] The term “amide” used in this article stands for the “-NHC(O)-” group.

[0221] The term "thioamide" represents the "-NHC(S)-" group.

[0222] As used herein, the terms “disulfide bond,” “disulfide bridge,” or “disulfide” refer to a covalent bond formed between two sulfur atoms. The amino acid cysteine ​​contains a thiol group, which can form a disulfide bond or bridge with a second thiol group.

[0223] The term "ether" refers to a group or compound with oxygen between two carbon atoms.

[0224] The term "thioether" refers to a group or compound containing sulfur between two carbon atoms.

[0225] The term "ester" refers to a compound derived from a carboxylic acid and an alcohol, formed by the formal dehydration of the hydroxyl group of the former's -C(=O)OH group and the latter's hydroxyl group. Therefore, the term refers to the -C(O)O- group.

[0226] The term "thioester" refers to the group -C(O)S- or -C(S)O-.

[0227] The term "triazole" refers to a compound whose structure contains any heterocyclic structure having a five-membered ring consisting of two carbon atoms and three nitrogen atoms (e.g., 1,2,3-triazole).

[0228] The term "portion" refers to a fraction. For example, an amino acid sequence or a specific structure of a protein, the term "portion" can refer to a continuous or discontinuous fraction of that structure.

[0229] The terms “part” and “fraction” are used interchangeably herein and refer to a continuous element. For example, a part of a structure (e.g., an amino acid sequence or a protein) refers to a continuous element of said structure. When used in the context of a composition, the term “part” means a portion of the composition. For example, a part of the composition can be any portion from 0.1% to 99.9% (e.g., 0.1%, 0.5%, 1%, 5%, 10%, 50%, 90%, or 99%) of said composition.

[0230] Regarding amino acid sequences (peptides or polypeptides), a "fragment" refers to a portion of the amino acid sequence, specifically a sequence representing a shortened amino acid sequence at the N-terminus and / or C-terminus. A C-terminal shortened fragment (N-terminal fragment) can be obtained, for example, by translating a truncated open reading frame (OPF) lacking the 3' end. An N-terminal shortened fragment (C-terminal fragment) can be obtained, for example, by translating a truncated OPF lacking the 5' end, provided that the truncated OPF contains a start codon for initiating translation. A fragment of an amino acid sequence contains, for example, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the amino acid residues from the amino acid sequence. A fragment of an amino acid sequence contains, for example, at least 6, particularly at least 8, at least 10, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids from the amino acid sequence. The amino acid sequence fragment contains, for example, a sequence of up to 8, particularly up to 10, 12, 15, 20, 30, or 55 consecutive amino acids.

[0231] As used herein, the term "variant" of an amino acid sequence (peptide or polypeptide) means an amino acid sequence that differs from a parent amino acid sequence by at least one amino acid (e.g., a different amino acid, or a modification of the same amino acid). The parent amino acid sequence may be a naturally occurring or wild-type (WT) amino acid sequence, or it may be a modified form of a wild-type amino acid sequence. In some embodiments, the variant amino acid sequence has at least one amino acid difference compared to the parent amino acid sequence, for example, a difference of 1 to about 20 amino acids, or, for example, a difference of 1 to about 10 or 1 to about 5 amino acids compared to the parent.

[0232] As used in this article, “wild-type”, “WT”, or “natural” refers to an amino acid sequence (peptide or polypeptide) that exists in nature, including allelic variations. Wild-type amino acid sequences, peptides, or polypeptides have amino acid sequences that have not been intentionally modified.

[0233] For the purposes of this disclosure, a “variant” of an amino acid sequence (peptide or polypeptide) may include amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and / or amino acid substitution variants. The term “variant” includes all mutants, splicing variants, post-translational modification variants, conformations, isotypes, allele variants, species variants, and species homologs, particularly those that are naturally occurring. The term “variant” specifically includes fragments of amino acid sequences.

[0234] Amino acid insertion variants include the insertion of one, two, or more amino acids into a specific amino acid sequence. In the case of amino acid sequence variants with insertions, one or more amino acid residues are inserted into a specific site in the amino acid sequence, although random insertion and appropriate screening of the resulting product are also possible. Amino acid addition variants contain amino and / or carboxyl-terminal fusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, for example, the removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Deletions can occur at any position in the protein. Amino acid deletion variants containing deletions at the N-terminus and / or C-terminus of a protein are also called N-terminal and / or C-terminal truncation variants. Amino acid substitution variants are characterized by the removal of at least one residue from the sequence and the insertion of another residue at its position. Preferred modifications are made at non-conserved positions in the amino acid sequence between homologous peptides or polypeptides and / or substitutions of amino acids with other amino acids having similar properties. In some embodiments, amino acid changes in peptide and polypeptide variants are conserved amino acid changes, i.e., substitutions of similar charged or uncharged amino acids. Conserved amino acid changes involve the substitution of one of the families of amino acids associated with their side chains. Naturally occurring amino acids are generally classified into four families: acidic amino acids (aspartic acid, glutamic acid); basic amino acids (lysine, arginine, histidine); nonpolar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); and uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes collectively classified as aromatic amino acids. In some embodiments, conserved amino acid substitutions include substitutions within the following groups:

[0235] Glycine, alanine;

[0236] Valine, isoleucine, leucine;

[0237] Aspartic acid, glutamic acid;

[0238] Asparagine, glutamine;

[0239] Serine, threonine;

[0240] Lysine, arginine; and

[0241] Phenylalanine, tyrosine.

[0242] In some embodiments, the degree of similarity (e.g., identity) between a given amino acid sequence and an amino acid sequence that is a variant of said given amino acid sequence is at least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the degree of similarity or identity is given for an amino acid region that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the full length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is given, for example, for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids (continuous amino acids in some embodiments). In some embodiments, the degree of similarity or identity is given for the full length of the reference amino acid sequence. Alignments used to determine sequence similarity, such as sequence identity, can be performed using tools known in the art, such as optimal sequence alignment, for example, using Align, using standard settings, preferably EMBOSS::needle, matrix: Blosum62, gapopen 10.0, gap extend 0.5.

[0243] "Sequence similarity" indicates the percentage of identical or conserved amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of identical amino acids between the sequences. "Sequence identity" between two nucleic acid sequences indicates the percentage of identical nucleotides between the sequences.

[0244] The terms “identical (%)” and “identity (%)” or similar terms are specifically intended to refer to the percentage of identical nucleotides or amino acids between sequences to be compared in an optimal alignment. These percentages are purely statistical, and the differences between the two sequences may (but are not necessarily) be randomly distributed across the full length of the sequences to be compared. Comparison of two sequences is typically performed by comparing the sequences after an optimal alignment with respect to a segment or “comparison window” to identify local regions of the respective sequences. The best alignment for comparison can be performed manually or by means of algorithms, such as the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482; the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443; and the similarity retrieval algorithm of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or by means of computer programs using said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 ScienceDrive, Madison, Wisconsin). In some implementations, it is performed using the website of the National Center for Biotechnology Information (NCBI), for example, on [website address missing]. The BLASTN or BLASTP algorithm obtained from the NCBI website is used to determine the percentage identity of two sequences. In some implementations, the algorithm parameters used for the BLASTN algorithm on the NCBI website include: (i) an expected threshold set to 10; (ii) a word length set to 28; (iii) a maximum match within the query range set to 0; (iv) match / non-match scores set to 1, -2; (v) a gap cost set to linear; and (vi) a filter using low-complexity regions. In some implementations, the algorithm parameters used for the BLASTP algorithm on the NCBI website include: (i) an expected threshold set to 10; (ii) a word length set to 3; (iii) a maximum match within the query range set to 0; (iv) a matrix set to BLOSUM62; (v) a gap cost set to exist:11, extend:1; and (vi) conditional composition score matrix adjustment.

[0245] Percentage identity is obtained by determining the number of identical positions corresponding to the sequences to be compared, dividing that number by the number of positions being compared (e.g., the number of positions in the reference sequence), and multiplying the result by 100.

[0246] In some embodiments, a degree of similarity or identity is given for regions that comprise at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the full length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, a degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides (continuous nucleotides in some embodiments). In some embodiments, a degree of similarity or identity is given for the full length of the reference sequence.

[0247] According to this disclosure, the homologous amino acid sequences show at least 40%, particularly at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and, for example, at least 95%, at least 98%, or at least 99% identity of the amino acid residues.

[0248] The amino acid sequence variants described herein can be readily prepared by a technician, for example, through recombinant DNA manipulation. DNA sequence manipulations for preparing peptides or polypeptides with substitutions, additions, insertions, or deletions are described in detail, for example, *Molecular Cloning: A Laboratory Manual, 4th Edition, MR Green and J. Sambrook et al. (1989), eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 2012. Furthermore, the peptides, polypeptides, and amino acid variants described herein can be readily prepared with the aid of known peptide synthesis techniques, such as solid-phase synthesis and similar methods.

[0249] In some embodiments, fragments or variants of an amino acid sequence (peptide or polypeptide) are "functional fragments" or "functional variants." The term "functional fragment" or "functional variant" of an amino acid sequence refers to any fragment or variant that exhibits one or more functional properties that are the same as or similar to the functional properties of the amino acid sequence from which the fragment or variant originates (i.e., it is functionally equivalent). Regarding the sequence of a binder (e.g., an antibody), a specific function is one or more binding activities exhibited by the amino acid sequence from which the fragment or variant originates. As used herein, the term "functional fragment" or "functional variant" specifically refers to a variant molecule or sequence that comprises an amino acid sequence altered by one or more amino acids compared to the amino acid sequence of the parent molecule or sequence and still capable of achieving one or more functions of the parent molecule or sequence (e.g., binding to a target molecule). In some embodiments, modifications to the amino acid sequence of the parent molecule or sequence do not significantly affect or alter the properties of the molecule or sequence. In different embodiments, the function of the functional fragment or functional variant may be reduced but still significantly present; for example, the function of the functional fragment or functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of that of the parent molecule or sequence. However, in other embodiments, the function of the functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.

[0250] The amino acid sequence (peptide or polypeptide) “derived from” the specified amino acid sequence (peptide or polypeptide) refers to the source of the first amino acid sequence. In some embodiments, the amino acid sequence derived from a particular amino acid sequence has the same, substantially the same, or homologous amino acid sequence as that particular sequence or a fragment thereof. The amino acid sequence derived from a particular amino acid sequence can be a variant of that particular sequence or a fragment thereof. For example, those skilled in the art will understand that sequences applicable herein can be modified such that their sequence differs from that of the naturally occurring or native sequence from which they are derived, while retaining the desired activity of the native sequence.

[0251] In some implementations, "isolated" means removed (e.g., purified) from a natural state or from an artificial composition (e.g., a composition derived from a production process). For example, nucleic acids, peptides, or polypeptides naturally present in living organisms are not "isolated," but the same nucleic acids, peptides, or polypeptides isolated, either partially or completely, from their natural coexisting forms are "isolated." Isolated nucleic acids, peptides, or polypeptides may exist in a substantially purified form or may exist in a non-natural environment, such as in host cells.

[0252] The term "binding" or variations thereof refers to non-covalent interactions with a target. In some implementations, the term "binding" or variations thereof refers to specific binding.

[0253] The term "k" used in this article d "(Second -1 ) refers to the dissociation rate constant of a specific interaction (e.g., antibody-antigen interaction). This value is also called k. off value.

[0254] The term "K" used in this article D "(M) refers to the dissociation equilibrium constant of a specific interaction (e.g., antibody-antigen interaction).

[0255] Generally speaking, the terms “combination” or its variations and “targeting” or its variations are used interchangeably in this article.

[0256] The term “genetic modification” or simply “modification” includes transfecting cells with nucleic acids. The term “transfection” involves introducing nucleic acids (e.g., DNA and / or RNA) into cells. For the purposes of this disclosure, the term “transfection” also includes introducing nucleic acids into cells or taking up nucleic acids by such cells, wherein said cells may be present in a subject (e.g., a patient) or may be in vitro (e.g., outside the patient). Thus, according to this disclosure, cells used for transfecting the nucleic acids described herein may be present in vitro or in vivo, for example, said cells may form an organ, tissue, and / or part of a patient’s body. According to this disclosure, transfection can be transient or stable. For some applications of transfection, transient expression of the transfected genetic material alone is sufficient. RNA can be transfected into cells to transiently express the protein it encodes. Since the nucleic acids introduced during transfection typically do not integrate into the nuclear genome, the exogenous nucleic acids will be diluted or degraded by mitosis. Cells that allow for free amplification of nucleic acids greatly reduce the dilution rate. Stable transfection must occur if it is desired that the transfected nucleic acids actually remain in the genome of the cell and its daughter cells. Such stable transfection can be achieved using, for example, virus-based or transposon-based systems. Typically, cells genetically modified to express an antigen receptor are stably transfected with a nucleic acid encoding that receptor. Typically, cells transfected with a nucleic acid encoding a docking compound are transiently transfected with a nucleic acid encoding that docking compound. RNA can be transfected into cells to transiently express its encoded protein.

[0257] The terms “connected,” “fused,” or “fused / fused body” used in this document are used interchangeably. These terms refer to two or more elements, components, or domains connected together.

[0258] As used herein, the terms "fusion polypeptide" or "fusion protein" refer to a polypeptide or protein comprising two or more subunits. Preferably, a fusion protein is a translational fusion between two or more subunits. Translational fusions can be generated through genetic modification so that the coding nucleotide sequence of one subunit is in the same reading frame as the coding nucleotide sequence of another subunit. The subunits may be distributed with linkers.

[0259] As used in this article, “endogenous” means any substance that originates from or is produced within an organism, cell, tissue, or system.

[0260] As used in this article, the term "exogenous" means any substance introduced or produced from outside an organism, cell, tissue, or system.

[0261] The term "autologous" is used to describe anything derived from the same object. For example, "autologous transplantation" refers to the transplantation of tissue or organ derived from the same object. Such procedures are advantageous because they overcome the immune barrier, which would otherwise lead to rejection.

[0262] The term "alien" is used to describe anything that originates from different individuals of the same species. Two or more individuals are considered alliens when the genes at one or more loci are different.

[0263] The term "homogeneous" is used to describe anything that originates from an individual or tissue with the same genotype (i.e., identical twins or animals of the same inbred strain, or their tissues).

[0264] The term "heterogeneous" is used to describe something composed of multiple different elements. As an example, transferring bone marrow from one individual to another constitutes a heterologous transplant. Heterogeneous genes are genes derived from sources other than the recipient.

[0265] According to various embodiments of this disclosure, nucleic acids encoding peptides or polypeptides are absorbed or introduced into cells, i.e., transfected or transduced into cells that may be present in vitro or in a subject, resulting in the expression of said peptides or polypeptides. Cells may express the encoded peptides or polypeptides, for example, intracellularly (e.g., in the cytoplasm and / or nucleus), secrete the encoded peptides or polypeptides, and / or express the encoded peptides or polypeptides on their surface. In some embodiments, if the encoded peptide or polypeptide is an antigen receptor, the cell expresses the antigen receptor on its cell surface. In some embodiments, if the encoded peptide or polypeptide is a docking compound, the cell secretes the encoded peptide or polypeptide.

[0266] According to this disclosure, terms such as “nucleic acid expressing…” and “nucleic acid encoding…” or similar terms are used interchangeably herein, and with respect to a particular peptide or polypeptide, it means that the nucleic acid, if present in a suitable environment, such as within a cell, can be expressed to produce the peptide or polypeptide.

[0267] The term “expression” as used in this article includes transcription and / or translation of a specific nucleotide sequence.

[0268] In the context of this disclosure, the term "transcription" refers to the process in which the genetic code in a DNA sequence is transcribed into RNA (specifically mRNA). The RNA can then be translated into peptides or polypeptides.

[0269] Regarding RNA, the terms "expression" or "translation" refer to the process in the ribosomes of the cell through which the strand of mRNA directs the assembly of an amino acid sequence to produce a peptide or polypeptide.

[0270] The term "host cell" includes prokaryotic or eukaryotic cells. Prokaryotic host cells include bacterial cells, such as *Escherichia coli* (E. coli) or *Bacillus subtilis*. Eukaryotic host cells can be microbial cells, such as yeast cells, such as *Saccharomyces cerevisiae* or *Pichia pastoris*; plant cells; or animal cells, such as insect cells, such as SF9 or High5 insect cells, avian cells, or mammalian cells. Mammalian cells can be derived from any species, such as mice, rats, rabbits, monkeys, apes, or humans. In some embodiments, the host cell is a non-human host cell. The host cell can be an immortalized mammalian cell (e.g., HeLa cells or CHO cells) or a primary mammalian cell. The host cell is preferably an isolated host cell.

[0271] In some embodiments, in systems containing multiple components, these components are present in close spatial relationships, for example, in formulations containing components, particularly in kits. Pharmaceutical formulations described herein, particularly kits, may contain instruction materials or instructions. As used herein, “instruction materials” or “instructions” include publications, recordings, diagrams, or any other medium of expression that can be used to convey the usefulness of the compositions and methods of this disclosure. For example, instruction materials for a kit of this disclosure may be attached to or shipped with a container containing the composition / formulation of this disclosure. Alternatively, the instruction materials may be shipped separately from the container, with the intention that the instruction materials and the composition be used synergistically by the recipient.

[0272] The term "mean diameter" refers to the average hydrodynamic diameter of a particle, such as that measured by dynamic light scattering (DLS) and analyzed using the so-called cumulant algorithm, which provides a so-called Z-axis with a length dimension. 平均值 The results are related to the dimensionless polydispersity index (PDI) (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321). Here, the particle's "mean diameter," "diameter," or "size" is related to Z. 平均值 This value is synonymous with.

[0273] In some implementations, the "polydispersity index" is calculated based on dynamic light scattering measurements through so-called cumulative analysis, as mentioned in the definition of "mean diameter." Under certain conditions, it can serve as a measure of the size distribution of a population of nanoparticles.

[0274] The term "alkyl" refers to a monovalent group in a saturated straight-chain or branched hydrocarbon. Preferably, the alkyl group comprises 1 to 12 (e.g., 1 to 10) carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms, abbreviated as C. 1-12 Alkyl groups (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, abbreviated as C) 1-10 Alkyl groups, more preferably 1 to 8 carbon atoms, such as 1 to 6 or 1 to 4 carbon atoms. Exemplary alkyl groups include methyl, ethyl, propyl, isopropyl (also known as 2-propyl or 1-methylethyl), butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, neopentyl, 1,2-dimethyl-propyl, isopentyl, n-hexyl, isohexyl, sec-hexyl, n-heptyl, isoheptyl, n-octyl, 2-ethylhexyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, etc. "Substituted alkyl" means that one or more hydrogen atoms of the alkyl group (e.g., from 1 to the maximum number of hydrogen atoms bonded to the alkyl group, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, such as 1 to 5, 1 to 4, or 1 to 3 or 1 or 2) are replaced by substituents other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). Preferably, the substituents other than hydrogen are first-order substituents as specified herein.

[0275] The term "alkylene" refers to a divalent group of a saturated straight-chain or branched hydrocarbon. Preferably, the alkylene comprises 1 to 12 (e.g., 1 to 10) carbon atoms, that is, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms), more preferably 1 to 8 carbon atoms, such as 1 to 6 or 1 to 4 carbon atoms. Exemplary alkylenes include methylene, ethylene (i.e., 1,1-ethylene, 1,2-ethylene), propylene (i.e., 1,1-propylene, 1,2-propylene (-CH(CH3)CH2-), 2,2-propylene (-C(CH3)2-), and 1,3-propylene), butylene isomers (e.g., 1,1-butylene, 1,2-butylene, 2,2-butylene, 1,3-butylene, 2,3-butylene (cis or trans or mixtures thereof), 1,4-butylene, 1,1-isobutylene, 1,2-isobutylene, and 1,3-isobutylene), and pentylene isomers (e.g., 1,1-pentylene, 1,2-pentylene, 1,3-pentylene, 1,4-pentylene, 1,5-pentylene, 1,1- Isopentyl, 1,1-sec-pentyl, 1,1-neopentyl), hexane isomers (e.g., 1,1-hexane, 1,2-hexane, 1,3-hexane, 1,4-hexane, 1,5-hexane, 1,6-hexane and 1,1-isohexane), heptamethrin isomers (e.g., 1,1-heptamethrin, 1,2-heptamethrin, 1,3-heptamethrin, 1,4-hexane, 1,5-hexane, 1,6-hexane and 1,1-isohexane), and heptamethrin isomers (e.g., 1,1-heptamethrin, 1,2-heptamethrin, 1,3-heptamethrin, 1,4-hexane, 1,5-hexane, 1,6 Examples of alkylene derivatives include 4-heptenyl, 1,5-heptenyl, 1,6-heptenyl, 1,7-heptenyl, and 1,1-isoheptenyl, as well as octyl isomers (e.g., 1,1-octyl, 1,2-octyl, 1,3-octyl, 1,4-octyl, 1,5-octyl, 1,6-octyl, 1,7-octyl, 1,8-octyl, and 1,1-isooctyl). A straight-chain alkylene moiety having at least three carbon atoms and a free valence at each end can also be referred to as multiple methylene groups (e.g., 1,4-butylene can also be referred to as tetramethylene). Generally, as an alternative to using the suffix "ylene" for the aforementioned alkylene moiety, the suffix "diyl" can also be used (e.g., 1,2-butylene can also be referred to as butyl-1,2-diyl). "Substituted alkylene" means that one or more hydrogen atoms of an alkylene group (e.g., from 1 to the maximum number of hydrogen atoms bonded to the alkylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) are replaced by substituents other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). Preferably, the substituents other than hydrogen are first-order substituents as specified herein.

[0276] The term "cycloalkylene" refers to the cyclic non-aromatic form of "alkylene" and can be saturated or unsaturated. Cycloalkylene is a divalent group at the fused, ortho, or isolated position. In some embodiments, the cycloalkylene (i) is monocyclic or polycyclic (e.g., bicyclic or tricyclic) and / or (ii) is 3 to 14-membered (i.e., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14-membered, e.g., 3 to 12-membered or 3 to 10-membered). In one embodiment, the cycloalkylene is a monocyclic, bicyclic, or tricyclic 3 to 14-membered (i.e., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14-membered, e.g., 3 to 12-membered or 3 to 10-membered) cycloalkylene. Generally, for the above-described cycloalkylene portion, as an alternative to using the term "alkylene group," the term "diyl" (e.g., 1,2-cyclopropylene may also be referred to as cyclopropane-1,2-diyl) may also be used. Exemplary cycloalkylene compounds include cyclohexylene, cycloheptylene, cyclopropylene, cyclobutylene, cyclobutenylene, cyclopentylene, cyclooctylene, bicyclo[3.2.1]octylene, bicyclo[3.2.2]nonylene, and adamantaneylene (e.g., tricyclo[3.3.1.1]octylene, bicyclo[3.2.2]nonylene, and adamantaneylene). 3,7 [Decane-2,2-diyl]. "Substituted cycloalkylene" means that one or more hydrogen atoms of the cycloalkylene group (e.g., from 1 to the maximum number of hydrogen atoms bonded to the cycloalkylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) are replaced by substituents other than hydrogen (when more than one hydrogen atom is replaced, the substituents can be the same or different). Preferably, the substituents other than hydrogen are primary substituents as specified herein.

[0277] The term "aryl" refers to a monovalent group in an aromatic cyclic hydrocarbon. Preferably, the aryl group comprises 3 to 14 (e.g., 5, 6, 7, 8, 9, or 10, e.g., 5, 6, or 10) carbon atoms, which may be arranged in a single ring (e.g., phenyl) or two or more fused rings (e.g., naphthyl). Exemplary aryl groups include cyclopropene. The aryl group includes cyclopropenyl, cyclopentadienyl, phenyl, indene, naphthyl, azulel, fluorenyl, anthracene, and phenanthryl. Preferably, "aryl" refers to a monocyclic aromatic ring containing 6 carbon atoms or an aromatic bicyclic system containing 10 carbon atoms. Some preferred examples are phenyl and naphthyl. Aryl does not cover fullerenes. "Substituted aryl" means that one or more hydrogen atoms of the aryl group (e.g., from 1 to the maximum number of hydrogen atoms bonded to the aryl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) are replaced by substituents other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). Preferably, the substituents other than hydrogen are first-order substituents as specified herein.

[0278] The term "arylene" refers to the divalent group of the aforementioned aryl group. Preferably, the arylene comprises 3 to 14 carbon atoms, which may be arranged in a single ring (e.g., phenylene) or two or more fused rings (e.g., naphthylene). An exemplary arylene is derived from cyclopropylene by removing two hydrogen atoms. The compounds are alkyl, cyclopentadienyl, benzene, indene, naphthalene, azulene, fluorene, anthracene, or phenanthrene. Preferably, "arylene" refers to a monocyclic aromatic ring containing 6 carbon atoms or an aromatic bicyclic system containing 10 carbon atoms. Some preferred examples are phenylene and naphthylene. "Substituted arylene" means that one or more hydrogen atoms of the arylene (e.g., from 1 to the maximum number of hydrogen atoms bonded to the arylene, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) are replaced by substituents other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). Preferably, the substituents other than hydrogen are first-order substituents as specified herein.

[0279] The term "heteroaryl" or "heteroary ring" refers to an aryl group as defined above, wherein one or more carbon atoms of the aryl group are replaced by O, S, or N heteroatoms. Preferably, a heteroaryl refers to a five- or six-membered aromatic monocyclic ring, wherein one, two, or three carbon atoms are replaced by the same or different O, N, or S heteroatoms. Alternatively, it refers to an aromatic bicyclic or tricyclic system, wherein one, two, three, four, or five carbon atoms are replaced by the same or different O, N, or S heteroatoms. Preferably, the maximum number of O atoms in each ring of the heteroaryl is one, the maximum number of S atoms is one, and the maximum total number of O and S atoms is two. Exemplary heteroaryl groups include furanyl, thiophene, etc. azole group, iso azole group, Diazolyl, pyrrolyl, imidazoleyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyrimidinyl, pyrazinyl, triazinyl, benzofuranyl, indoleyl, isoindoleyl, benzothiophenyl, 1H-indazoleyl, benzimidazoleyl, benzo[…] azole group, indole Indoxazinyl, benzo[a]azinyl Azolyl, benzothiazolyl, benzoisothiazolyl, benzotriazolyl, quinolinyl, isoquinolinyl, benzodiazinyl, quinoxalinyl, quinazolinyl, benzotriazinyl, pyridazinyl, phen Zinyl, thiazopyridyl, pyrrolothiazolyl, phenthiazyl, isobenzofuranyl, chromenyl, xanthyl, pyrrolizinyl, indoleazinyl, indazoleyl, purinyl, quinolizinyl, phthalazinyl, naphthinyl, cinnamyl, pteridinyl, carbazoleyl, phenanthinyl, acridineyl, perimidinyl, phenanthrolinyl, and phenazinyl. Exemplary 5- or 6-membered heteroaryl groups include furanyl, thiophenyl, azole group, iso azole group, Diazolyl, pyrroloyl, imidazolyl (e.g., 2-imidazolyl), pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridinyl (e.g., 4-pyridinyl), pyrimidinyl, pyrazinyl, triazinyl, and pyridazinyl. "Substituted heteroaryl" means that one or more hydrogen atoms of the heteroaryl group (e.g., from 1 to a maximum number of hydrogen atoms bonded to the heteroaryl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1 to 5, 1 to 4, 1 to 3, or 1 or 2) are replaced by substituents other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). Preferably, the substituents other than hydrogen are first-order substituents as specified herein.

[0280] The term "heterocyclic group" or "heterocycle" means a cycloalkyl group as defined above, wherein 1, 2, 3, or 4 carbon atoms in the cycloalkyl group are replaced by heteroatoms of oxygen, nitrogen, silicon, selenium, phosphorus, or sulfur, preferably O, S, or N. The heterocyclic group preferably has 1 or 2 rings, which contain 3 to 10, for example 3, 4, 5, 6, or 7 ring atoms. Preferably, the maximum number of O atoms in each ring of the heterocyclic group is 1, the maximum number of S atoms is 1, and the maximum total number of O and S atoms is 2. The term "heterocyclic group" also means the partially or fully hydrogenated form (e.g., dihydro, tetrahydro, or all-hydrogen form) encompassing the above-described heteroaryl groups. Exemplary heterocyclic groups include morpholino, pyrrolyl, imidazoalkyl, pyrazolyl, piperidinyl (also known as piperidyl), piperazinyl, dihydrofuranyl and tetrahydrofuranyl, dihydrothiophenyl and tetrahydrothiophenyl, dihydropyranyl and tetrahydropyranyl, hexamethylenetetramine (urotropinyl), lactone, lactam, cyclic imide, and cyclic anhydride. "Substituted heterocyclic group" means that one or more hydrogen atoms of the heterocyclic group (e.g., from 1 to the maximum number of hydrogen atoms bonded to the heterocyclic group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) are replaced by substituents other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). Preferably, the substituents other than hydrogen are first-order substituents as specified herein.

[0281] As used herein, the term "heterocyclic alkylene" means a heterocyclic group as defined above, comprising at least one cyclic heteroatom (e.g., a cyclic heteroatom selected from O, S, N, B, Si, and P) and one of its hydrogen atoms has been removed to produce a homocyclic, ortho, or isolated divalent group. In some embodiments, the heteroatom of the heterocyclic alkylene is selected from O, S, and N. For example, the heterocyclic alkylene may be O / S-heterocyclic alkylene, such as O-heterocyclic alkylene. In some embodiments, the maximum number of O atoms and the maximum number of S atoms in each ring of the heterocyclic alkylene is 1, and the maximum total number of O and S atoms is 2. The heterocyclic alkylene may be monocyclic or polycyclic (e.g., bicyclic or tricyclic). In some embodiments, the heterocyclic alkylene is a monocyclic, bicyclic, or tricyclic 4- to 14-membered (i.e., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14-membered, e.g., 4 to 12-membered or 4 to 10-membered) heterocyclic alkylene. The term "heterocyclic alkylene" also refers to the partially or fully hydrogenated form (e.g., dihydro, tetrahydro, or all-hydrogen form) of the aforementioned heteroaryl groups (preferably the partially or fully hydrogenated forms of the aforementioned mono-, di-, or tricyclic heteroaryl groups), in which one hydrogen atom has been removed from the same carbon atom, resulting in a homodivalent group. Thus, in some embodiments, the heterocyclic alkylene is saturated or unsaturated (i.e., containing one or more double bonds in the ring), but cannot be aromatic. "Substituted heterocyclic alkylene" means that one or more hydrogen atoms of the heterocyclic alkylene (e.g., from 1 to the maximum number of hydrogen atoms bonded to the heterocyclic alkylene, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) are replaced by substituents other than hydrogen (when more than one hydrogen atom is replaced, the substituents can be the same or different). Preferably, the substituents other than hydrogen are first-order substituents as specified herein.

[0282] As used herein, the term "partially hydrogenated form" of an unsaturated compound or group means that a portion of the unsaturation is removed by formally adding hydrogen to the original unsaturated compound or group without removing all of the unsaturation. The phrase "fully hydrogenated form" of an unsaturated compound or group is used interchangeably with the term "fully hydrogenated" herein and means that all of the unsaturation is removed by formally adding hydrogen to the original unsaturated compound or group. For example, a partially hydrogenated form of a 5-membered heteroaryl group (containing two double bonds in the ring, such as furan) includes the dihydrogen form of the 5-membered heteroaryl group (e.g., 2,3-dihydrofuran or 2,5-dihydrofuran), while the tetrahydrogen form of the 5-membered heteroaryl group (e.g., tetrahydrofuran, or THF) is the fully hydrogenated (or fully hydrogenated) form of the 5-membered heteroaryl group. Similarly, for a 6-membered heteroaryl group (e.g., pyridyl) having three double bonds in the ring, the partially hydrogenated forms include dihydro and tetrahydro forms (e.g., dihydropyridyl and tetrahydropyridyl), while the hexahydro forms (in the case of heteroarylpyridyl, e.g., piperidyl) are fully hydrogenated (or all-hydrogenated) derivatives of the 6-membered heteroaryl group. Therefore, if an aryl or heteroaryl group contains at least four unsaturated moieties consisting of double and triple bonds between ring atoms, the hexahydro forms of the aryl or heteroaryl group can be considered only as partially hydrogenated forms according to this disclosure.

[0283] In the context of hydrocarbons, the term "aromatic" means that the entire molecule must be aromatic. For example, if a monocyclic aryl group is hydrogenated (partially or completely), the resulting hydrogenated cyclic structure is classified as cycloalkyl for the purposes of this disclosure. Similarly, if a bicyclic or polycyclic aryl group (e.g., naphthyl) is hydrogenated, the resulting hydrogenated bicyclic or polycyclic structure (e.g., 1,2-dihydronaphthyl) is classified as cycloalkyl for the purposes of this disclosure (even if one ring (e.g., in 1,2-dihydronaphthyl) is still aromatic). In this application, a similar distinction is made between heteroaryl and heterocyclic groups. For example, dihydroindolyl, i.e., a dihydro variant of indolyl, is classified as a heterocyclic group for the purposes of this disclosure because only one ring of the bicyclic structure is aromatic and one of the ring atoms is a heteroatom.

[0284] Typical primary substituents are preferably selected from C. 1-3 Alkyl, phenyl, halogen, -CF3, -OH, -OCH3, -SCH3, -NH 2-z (CH3) z -C(=O)OH, oxo, and -C(=O)OCH3, where z is 0, 1, or 2 and C 1-3 The alkyl group is methyl, ethyl, propyl, or isopropyl. Particularly preferred primary substituents are selected from methyl, ethyl, propyl, isopropyl, halogens (e.g., F, Cl, or Br) and -CF3, such as halogens (e.g., F, Cl, or Br) and -CF3.

[0285] docking compounds

[0286] According to this disclosure, immune effector cells are specifically delivered to target cells by providing a docking compound having a portion that binds to a target on the target cell (e.g., an antigen on the target cell) and a portion that binds to an antigen receptor, which is an essential part of the immune effector cell.

[0287] A "docking compound" is used to form a connection, such as a non-covalent connection, between an antigen and a target cell or on a target cell. The docking compound can form a connection, such as a non-covalent connection, with an immune effector cell to be delivered to the target cell via an antigen receptor on the immune effector cell. The antigen receptor contains a binding portion for binding to the docking compound and forms part of the immune effector cell.

[0288] In some embodiments, the docking compound comprises a binding portion targeting an antigen on a target cell, such as a portion targeting a cell surface antigen on a target cell, which is capable of binding to a target (e.g., a cell surface antigen on a target cell). As used herein, "binding portion targeting an antigen on a target cell" refers to a portion of the docking compound that binds to an antigen on a target cell. Such a targeting portion is a portion that typically has an affinity for a cell surface target. These portions can be any peptide or protein (e.g., an antibody or antibody fragment) that binds to an antigen on a target cell. Some specific embodiments of suitable binding portions targeting antigens on target cells used herein include cell surface antigen binding portions, such as antibodies, antibody fragments, and DARPin. Other examples of binding portions targeting antigens on target cells are peptides or proteins that bind to receptors.

[0289] The binding site to the antigen on the target cell preferably binds with high specificity and / or high affinity, and the binding to the antigen on the target cell is preferably stable in vivo.

[0290] To allow for specific targeting of antigens on target cells, the binding portion of the docking compound to the antigen on the target cell may contain the compound, including but not limited to antibodies, antibody fragments (e.g., Fab2, Fab, scFV, VHH domains), and other proteins or peptides.

[0291] According to some implementation schemes, the antigen on the target cell is a cell surface antigen (e.g., a cancer antigen), and suitable binding portions against the antigen on the target cell include, but are not limited to, peptides and polypeptides that target cell surface antigens, such as antibodies, antibody fragments, and DARPin.

[0292] According to some implementation schemes, the antigen on the target cell is a receptor, and suitable binding portions against the antigen on the target cell include, but are not limited to, ligands of such receptors or portions thereof that are still bound to the receptor, such as receptor-binding peptides in the case of receptor-binding protein ligands.

[0293] In some embodiments, the binding portion of the antigen on the target cell comprises a non-protein or non-peptide compound. In some embodiments, the binding portion of the antigen on the target cell comprises a small compound.

[0294] In some embodiments, the binding moiety against the antigen on the target cell is a tumor receptor ligand. In some embodiments, the binding moiety against the antigen on the target cell is folic acid, 2-[3-(1,3-dicarboxypropyl)ureo]glutaric acid (DUPA), or cholecystokinin 2 receptor (CCK2R) ligand.

[0295] For example, DUPA, which belongs to the glutamate class, can be used as a target for selectively delivering immune effector cells to prostate cancer cells that express prostate-specific membrane antigen (PSMA).

[0296] As used in this article, the term "DUPA" refers to 2-[3-(1,3-dicarboxypropyl)ureo]glutaric acid with the following structure:

[0297]

[0298] As used herein, the term "DUPA derivative" refers to a compound containing a DUPA substructure that is still bound to prostate-specific membrane antigen (PSMA). Preferably, the DUPA substructure comprises the following formula:

[0299]

[0300] in This represents the bond that connects the DUPA substructure to the rest of the DUPA derivative. Preferably, it is with... The connected C atoms have an S configuration.

[0301] In some embodiments, the binding portion of the antigen on the target cell comprises a DUPA derivative. In some embodiments, the DUPA derivative comprises the following formula:

[0302]

[0303] Where R 1 Yes - (L 1 )-(L 2 ) s -(L 3 ) t -H; where L 1 L 2 and L 3Each of them is a connecting part; s is 0 or 1; and t is 0 or 1, where t is 0 when s is 0.

[0304] In some implementations, s is 0 and t is 0, i.e., R 1 Yes - (L 1 )-H.

[0305] In some alternative implementations, s is 1 and t is 1, i.e., R 1 Yes - (L 1 )-(L 2 )-(L 3 )-H.

[0306] In some implementations, L 1 Selected from -alkylene-N(R) 10 -, -alkylene-O-, -alkylene-C(O)-, -alkylene-C(O)O-, -alkylene-OC(O)-, -alkylene-C(O)N(R) 10 )- and -alkylene-N(R 10 )C(O)-, where each R 10 Independently H or alkyl; and each alkylene moiety is optionally surrounded by 1, 2 or 3 R. 11 Replace, where each R 11 Independently selected from OH, halogen, C 1-6 Alkyl and =O. In some embodiments, L 1 Selected from -alkylene-N(R) 10 -, -alkylene-O- and -alkylene-C(O)-, wherein each R 10 Independently H or alkyl; and each alkylene moiety is optionally surrounded by 1, 2 or 3 R. 11 Replace, where each R 11 Independently selected from OH, halogen, C 1-6 Alkyl and =O.

[0307] In some implementations, L 1 Selected from -alkylene-N(R) 10 )-、 -alkylene-O-, -alkylene-C(O)-, -alkylene-C(O)O-, -alkylene-OC(O)-, -alkylene-C(O)N(R) 10 )-and -alkylene-N(R) 10 )C(O)-, where Represents the connection point with the DUPA substructure; each R10 Independently H or alkyl; and each alkylene moiety is optionally surrounded by 1, 2 or 3 R. 11 Replace, where each R 11 Independently selected from OH, halogen, C 1-6 Alkyl and =O. In some embodiments, L 1 Selected from -alkylene-N(R) 10 )-、 -alkylene-O- and -alkylene-C(O)-, where Represents the connection point with the DUPA substructure; each R 10 Independently H or alkyl; and each alkylene moiety is optionally surrounded by 1, 2 or 3 R. 11 Replace, where each R 11 Independently selected from OH, halogen, C 1-6 Alkyl and =O.

[0308] In some implementations, L 1 Each alkylene moiety contained herein is independently C 1-12 The alkylene moiety is preferably C 2-6 Alkylene moieties, such as C3, C4, or C5 alkylene moieties, wherein each alkylene moiety is optionally surrounded by 1, 2, or 3 R... 11 Replace, where each R 11 Independently selected from OH, halogen, C 1-6 Alkyl groups and =O (e.g., selected from OH, halogens, C) 1-3 Alkyl and =O). In some embodiments, L 1 Each alkylene moiety contained herein is independently C 2-6 Alkylene moieties, such as C3, C4, or C5 alkylene moieties.

[0309] In some implementation schemes, R 10 Each alkyl moiety is independently C 1-12 Alkyl moiety, such as C 1-6 alkyl moiety or C 1-3 Alkyl moiety. In some embodiments, R 10 It is H or methyl. In some embodiments, R 10 It is H.

[0310] In some implementations, L 1 It is derived from any one of the side chains of lysine, glutamic acid, and aspartic acid.

[0311] In some implementations, L 1 yes -tetramethylene-NH-, where This represents the connection point with the DUPA substructure.

[0312] In some implementations, L 2 It is the connecting part, which improves the binding of the DUPA substructure / derivative to PSMA and / or improves the pharmacokinetics of the DUPA derivative. In some embodiments, L 2 It contains at least one aromatic and / or cycloalkylene moiety, for example, 1 to 4 aromatic or cycloalkylene moieties. In some embodiments, L 2 It comprises 1 to 4 aromatic or cycloalkylene moieties and optionally 1 alkylene moiety. The aromatic and cycloalkylene moieties may be linked to each other in any way, such as through functional groups (e.g., amino, amide, or ester groups) and / or C 1-6 Alkylene moiety.

[0313] In some embodiments, at least one aromatic moiety is selected from C 6-12 Aromatic moiety (e.g., phenyl, biphenyl, and naphthyl). In some embodiments, the aromatic group is divalent (e.g., phenylene). In some embodiments, the aromatic group is monovalent (e.g., biphenyl or naphthyl).

[0314] In some embodiments, at least one cycloalkylene moiety is selected from C 3-10 Cycloalkylene moiety (e.g., C5, C6, or C7 cycloalkylene moiety). In some embodiments, the cycloalkylene moiety is divalent (e.g., cyclohexylene).

[0315] In some implementations, L 2 It contains the divalent structure -CH(CH2-arom)-, where arom is a monovalent C 6-12 Aryl, such as naphthyl or biphenyl. Optionally, L 2 It may also contain one or two additional divalent moieties independently selected from the following: alkylene, cycloalkylene, and arylene moieties, wherein when one additional divalent moieties is present, it may be located before or after the -CH(CH2-arom)- structure, and wherein when two additional divalent moieties are present, one is located before the -CH(CH2-arom)- structure and the other is located after the -CH(CH2-arom)- structure.

[0316] In some implementations, L 2 Included

[0317] -(C(O)) a -(alkylene-N(R) 10 )C(O)-) b -((cyc)-C 1-3 Alkylene-N(R) 10 )) c -

[0318] Each R 10 Independently, it is H or alkyl; cyc is cycloalkylene or aryl; a is 0 or 1; b is 0 or 1; and c is 1, 2, 3, or 4. In some embodiments, particularly where L 1 In embodiments where the terminal group is amino (e.g., -NH-), a is 1. In some embodiments, each R 10 Both are H or C 1-6 Alkyl groups, such as H or C 1-3 Alkyl, such as H or methyl; preferably, each R 10 Both are H. In some embodiments, alkylene-N(R) 10 The alkylene moiety in the C(O)- group is C 1-12 The alkylene moiety is preferably C 2-6 The alkylene moiety, such as a C4 or C5 alkylene moiety. In some embodiments, cyc is a C4 or C5 alkylene moiety. 3-10 Cycloalkylene (e.g., C5, C6, or C7 cycloalkylene) or C 6-12 Aryl group (e.g., phenylene). In some embodiments, cyc is cyclohexylene or phenylene. In some embodiments, C 1-3 The alkylene group is methylene, ethylene, or trimethylene, such as methylene. In some specific examples of these embodiments, L 2 Includes one of the following:

[0319]

[0320] Each n is independently 1, 2, 3, or 4; and the carbonyl end... The preferred representative achieves L through it 2 With L 1 The key to the connection.

[0321] In some implementations, L 2 Included

[0322] -(C(O)) d -((cyc)-C 1-3 Alkylene-N(R) 10 )C(O)-) e -(-CH(CH2-arom)-N(R 10 )) f -(C(O) g (link)-N(R 10 )) h -

[0323] Where d is 0 or 1; cyc is a cycloalkylene or arylene; each R 10Independently H or alkyl; e is 0 or 1; arom is monovalent C. 6-12 Aryl; f is 0 or 1; g is 0 or 1; link is a divalent moiety selected from: alkylene, cycloalkylene, arylene, and combinations thereof (e.g., any binary or ternary combination thereof, e.g., a combination of one aryl moiety and one alkylene moiety, or a combination of one cycloalkylene moiety and two alkylene moiety); and h is 0 or 1; wherein at least one of e and f is 1; and g is 0 when e is 1 and f is 0. In some embodiments, particularly where L 1 In embodiments where the terminal group is an amino group (e.g., -NH-), d is 1. In some embodiments, cyc is C. 3-10 Cycloalkylene (e.g., C5, C6, or C7 cycloalkylene) or C 6-12 Aryl group (e.g., phenylene). In some embodiments, cyc is cyclohexylene or phenylene. In some embodiments, C 1-3 The alkylene group is methylene, ethylene, or trimethylene, for example, methylene. In some embodiments, each R 10 Both are H or C 1-6 Alkyl groups, such as H or C 1-3 Alkyl, such as H or methyl; preferably, each R 10 Both are H. In some embodiments, arom is naphthyl or biphenyl. In some embodiments, link is selected from C. 1-6 Alkylene (e.g., C10) 2-6 Alkylenes (e.g., C3, C4, C5, or C6 alkylenes), C 3-10 Cycloalkylene (e.g., C5, C6, or C7 cycloalkylene), C 6-12 Aryl groups (e.g., phenylene), and combinations thereof (e.g., any binary or ternary combination thereof, such as -methylene-cyclohexylene-methylene- or -phenylene-methylene-).

[0324] In some embodiments, e is 0, f is 1, and h is 0, and optionally, d is 1. In these embodiments, preferably, arom is naphthyl (e.g., 2-naphthyl) or biphenyl; and / or R 10 It is H or methyl (preferably H).

[0325] In some embodiments, e is 0, f is 1, g is 1, and h is 1, and optionally, d is 1. In these embodiments, preferably, (i) arom is naphthyl (e.g., 2-naphthyl) or biphenyl; (ii) each R 10 All are H or methyl (preferably H); or (iii) link is selected from C 2-6 Alkylenes (e.g., C3, C4, C5, or C6 alkylenes), C 3-10Cycloalkylene (e.g., C5, C6, or C7 cycloalkylene), C 6-12 arylene (e.g., phenylene), and combinations thereof (e.g., any binary or ternary combination thereof, such as -methylene-cyclohexylene-methylene- or -phenylene-methylene-); or combinations of any one of (i) to (iii). Some particularly preferred embodiments are as follows: (i) and (ii); (i) and (iii); (ii) and (iii); and (i), (ii) and (iii).

[0326] In some implementations, e is 1, f is 1, g is 1, and h is 1, and optionally, d is 1. In these implementations, preferably, (i) cyc is C. 3-10 Cycloalkylene or C 6-12 (ii) C arylene (preferably, cyc is cyclohexene or phenylene); 1-3 The alkylene group is methylene, ethylene, or trimethylene (e.g., methylene); (iii) each R 10 (iv) arom is naphthyl (e.g., 2-naphthyl) or biphenyl; or (v) link is selected from C 2-6 Alkylenes (e.g., C3, C4, C5, or C6 alkylenes), C 3-10 Cycloalkylene (e.g., C5, C6, or C7 cycloalkylene), C 6-12 arylene (e.g., phenylene), and combinations thereof (e.g., any binary or ternary combination thereof, such as -methylene-cyclohexylene-methylene- or -phenylene-methylene-); or combinations of any one of (i) to (v). Some particularly preferred embodiments are as follows: (i) and (ii); (i) and (iii); (i) and (iv); (i) and (v); (ii) and (iii); (ii) and (iv); (ii) and (v); (iii) and (iv); (iii) and (v); (iv) and (v); (i), (ii) and (iii); (i), (ii) and (iv); (i), (ii) and (v); (i), (iii) and (iv); (i), (iii) and (v); (i), (iv) and (v); (ii), (iii) and (v); (ii), (iv) and (v); (ii) and (iv) and (iii), (iv) and (v).

[0327] In some implementations, e is 1, f is 0, g is 0, and h is 1, and optionally, d is 1. In these implementations, preferably, (i) cyc is C. 3-10 Cycloalkylene or C 6-12 (ii) C arylene (preferably, cyc is cyclohexene or phenylene); 1-3The alkylene group is methylene, ethylene, or trimethylene (e.g., methylene); (iii) each R 10 All are H or methyl (preferably H); or (iv) link is selected from C 2-6 Alkylenes (e.g., C3, C4, C5, or C6 alkylenes), C 3-10 Cycloalkylene (e.g., C5, C6, or C7 cycloalkylene), C 6-12 arylene groups (e.g., phenylene), and combinations thereof (e.g., any binary or ternary combination thereof, such as -methylene-cyclohexylene-methylene- or -phenylene-methylene-); or combinations of any one of (i) to (iv). Some particularly preferred embodiments are as follows: (i) and (ii); (i) and (iii); (i) and (iv); (ii) and (iii); (ii) and (iv); (iii) and (iv); (i), (ii) and (iii); (i), (ii) and (iv); (i), (iii) and (iv); and (ii), (iii) and (iv).

[0328] In some specific examples of these implementation schemes, L 2 Includes one of the following:

[0329]

[0330] Among them, the carbonyl end The preferred representative achieves L through it 2 With L 1 The bond is linked. In some embodiments, the C atom linked to the methylene-naphthyl or methylene-biphenyl group has an S configuration.

[0331] In some implementations, L 3 It exists and is a -C(O)-alkylene group. In some embodiments, the alkylene moiety is C... 1-12 Alkylene, such as C 1-6 Alkylene, for example, C2, C3, C4, or C5 alkylene. In some alternative embodiments, L 3 It does not exist.

[0332] In some implementation schemes,

[0333] (I)L 1 Selected from -alkylene-N(R) 10 )-、 -alkylene-O- and -alkylene-C(O)-, where Represents the connection point with the DUPA substructure; each R 10Independently H or alkyl (preferably H or methyl, more preferably H); and each alkylene moiety is optionally surrounded by 1, 2 or 3 R. 11 Replace, where each R 11 Independently selected from OH, halogen, C 1-6 Alkyl and =O (preferably, L) 1 Each alkylene moiety contained herein is independently C 1-12 The alkylene moiety, more preferably, C 2-6 The alkylene moiety, such as a C3, C4, or C5 alkylene moiety, wherein the alkylene moiety is optionally surrounded by 1, 2, or 3 R... 11 Replace, where each R 11 Independently selected from OH, halogen, C 1-6 Alkyl groups and =O (e.g., selected from OH, halogens, C) 1-3 Alkyl and =O), more preferably, L 1 Each alkylene moiety contained herein is independently C 2-6 (e.g., C3, C4, or C5 alkylene moieties); and

[0334] (II)L 2 Contains at least one aromatic and / or cycloalkylene moiety (preferably at least one selected from phenyl, biphenyl, naphthyl, and cyclohexyl), for example, a divalent structure -CH(CH2-arom)-, where arom is a monovalent carbon. 6-12 Aryl (e.g., naphthyl or biphenyl); and

[0335] (III)L 3 It exists and is -C(O)-C 1-6 Alkylene (preferably, the alkylene portion is a C2, C3, C4, or C5 alkylene portion), or L 3 It does not exist.

[0336] In some implementation schemes,

[0337] (I)L 1 The side chain is derived from any one of lysine, glutamic acid, and aspartic acid, preferably L. 1 yes -tetramethylene-NH-, where Represents the connection point with the DUPA substructure;

[0338] (II)L 2 Includes one of the following:

[0339]

[0340] Among them, the carbonyl end The representative achieves L through it 2 With L 1 The bond (preferably, the C atom bonded to the methylene-naphthyl or methylene-biphenyl group has an S configuration); and

[0341] (III)L 3 It exists and is -C(O)-C 1-6 Alkylene (preferably, the alkylene portion is a C2, C3, C4, or C5 alkylene portion), or L 3 It does not exist.

[0342] In some preferred embodiments, the DUPA derivative comprises one of the following formulas:

[0343]

[0344] Where L 3 It exists and is -C(O)-C 1-6 Alkylene (preferably, the alkylene portion is a C2, C3, C4, or C5 alkylene portion), or L 3 It does not exist; and optionally, the C atom attached to the methylene-naphthyl group has an S configuration.

[0345] When a DUPA derivative is linked to different molecules (especially covalently), the DUPA derivative is preferably the DUPA moiety, i.e., the monovalent group of the DUPA derivative. Preferably, R... 1 The terminal hydrogen atom of the group is removed (in these embodiments, the DUPA portion may include any of the above formulas, where R 1 Yes - (L 1 )-(L 2 ) s -(L 3 ) t Therefore, in some preferred embodiments, the DUPA portion includes one of the following formulas:

[0346]

[0347] Where L 3 It exists and is -C(O)-C 1-6 Alkylene (preferably, the alkylene portion is a C2, C3, C4, or C5 alkylene portion), or L 3 It does not exist; and optionally, the C atom attached to the methylene-naphthyl group has an S configuration.

[0348] It should be understood that any of the above formulas / structures also includes its salts (especially pharmaceutically usable salts), tautomers, solvates (e.g., hydrates), and isotopic labeled forms.

[0349] Type II transmembrane serine protease fibroblast activation protein (FAP) is highly expressed in over 90% of epithelial tumors and is closely associated with various tumor invasions, metastases, and prognoses. Using FAP as a target, several FAP inhibitors (FAPIs) have been developed, most of which possess nanomolar levels of FAP affinity and high selectivity. Such ligands can be used to target different tumors.

[0350] In some implementations, the binding portion of the antigen on the target cell comprises a compound that binds to fibroblast activation protein (FAP), for example, a FAP inhibitor (FAPI).

[0351] According to some implementation schemes, target cell antigens and binding portions thereof are selected to achieve specific or enhanced targeting of certain cells, such as diseased cells, including those involved in or characterized by diseases such as cancer, inflammation, infection, cardiovascular disease (e.g., thrombosis), atherosclerotic lesions, hypoxic sites (e.g., stroke), tumors, cardiovascular disorders, encephalopathy, apoptosis, and angiogenesis. This can be achieved by selecting target cell antigens that are expressed specifically by the cell. For example, cancer antigens, such as those described herein, are expressed in cancer cells but are not expressed in normal non-cancer cells or are expressed in them at low levels.

[0352] The docking compound also contains groups that act as binding partners for antigen receptors on immune effector cells. The antigen receptor-binding portion and the antigen-binding portion on the target cell are linked to each other, preferably by covalent bonds.

[0353] The antigen receptor and the portion of the docking compound that binds to the antigen receptor bind to each other via an ALFA tag and a single-domain antibody that binds to that ALFA tag.

[0354] In some embodiments, the antigen receptor includes an ALFA tag, and the docking compound includes a single-domain antibody that binds to the ALFA tag. In some embodiments, the portion of the antigen receptor that binds to the antigen receptor-binding portion of the docking compound includes an ALFA tag, and the portion of the docking compound that binds to the antigen receptor includes a single-domain antibody that binds to the ALFA tag.

[0355] In some embodiments, the antigen receptor comprises a single-domain antibody that binds to an ALFA tag, and the docking compound comprises an ALFA tag. In some embodiments, the portion of the antigen receptor that binds to the antigen receptor-binding portion of the docking compound comprises a single-domain antibody that binds to an ALFA tag, and the portion of the docking compound that binds to the antigen receptor comprises an ALFA tag.

[0356] In some embodiments, the docking compound comprises a single-domain antibody that binds to an ALFA tag. In some embodiments, the antigen receptor-binding portion of the docking compound comprises a single-domain antibody that binds to an ALFA tag. In some embodiments, the docking compound comprises a bispecific molecule, such as a bispecific peptide, or a bispecific antibody. In some embodiments, the docking compound comprises a single-domain antibody that binds to an ALFA tag and an antibody or antibody fragment that binds to an antigen on a target cell. In some embodiments, the binding portion against the antigen on the target cell comprises a heavy chain variable region (VH) and a light chain variable region (VL) of the antibody. In some embodiments, the binding portion against the antigen on the target cell comprises a single-domain antibody, such as a VHH.

[0357] In some embodiments, the docking compound includes an ALFA tag. In some embodiments, the portion of the docking compound that binds to the antigen receptor includes an ALFA tag. In some embodiments, the docking compound includes an ALFA tag and an antibody or antibody fragment that binds to an antigen on a target cell. In some embodiments, the binding portion against the antigen on the target cell includes a heavy chain variable region (VH) and a light chain variable region (VL) of the antibody. In some embodiments, the binding portion against the antigen on the target cell includes a single-domain antibody, such as a VHH.

[0358] In some embodiments, the docking compound comprises a single peptide chain. In some embodiments, the single peptide chain comprises a portion that binds to an antigen on a target cell, such as an antibody, antibody fragment, or DARPin, and a portion that binds to an antigen receptor, such as a single-domain antibody or tag. In some embodiments, the single peptide chain comprises a portion that binds to an antigen on a target cell, such as an antibody, antibody fragment, or DARPin, and a single-domain antibody that binds to an antigen receptor. In some embodiments, the single peptide chain comprises a portion that binds to an antigen on a target cell, such as an antibody, antibody fragment, or DARPin, and a tag that binds to an antigen receptor. In some embodiments, the antibody fragment is VHH, scFv, or a mixture thereof. In various embodiments, the docking compound comprises one of the following structures (from N-terminus to C-terminus):

[0359] VHH (alpha antigen receptor) - optional adapter - VHH (alpha antigen)

[0360] VHH (α antigen) - Optional linker - VHH (α antigen receptor)

[0361] VHH (alpha antigen receptor) - optional adapter - scFv (alpha antigen)

[0362] scFv (α antigen) - optional linker - VHH (α antigen receptor)

[0363] α-tag - optional adapter - VHH (α-antigen)

[0364] VHH (α antigen) - optional adapter - α tag

[0365] Alpha tag - optional adapter - scFv (alpha antigen)

[0366] scFv (α antigen) - optional adapter - α tag

[0367] The CAR-binding portion of the docking compound described herein is linked to the antigen-binding portion on the target cell by means of a covalent bond. In some embodiments, such a bond may include a linking portion. In some embodiments, the CAR-binding portion of the docking compound is linked to the antigen-binding portion on the target cell by a linking portion comprising a polymer.

[0368] In some embodiments, the polymer is not a polymer of protein amino acids or their D isomers. In some embodiments, the polymer is a hydrophilic polymer. In some embodiments, the polymeric portion of the docking compound helps to impart stealth properties to the docking compound. In some embodiments, the docking compound described herein has a plasma half-life greater than 2 hours, for example, 3 to 10 hours. This feature advantageously allows the docking compound to accumulate at target cells and remain at target cells for a reasonable period of time. As a result, the effectiveness of the targeted delivery described herein is thus increased.

[0369] The term “invisible” is used herein to describe the ability of the compounds and complexes described herein to be undetectable and subsequently isolated and / or degraded by the immune system of the host to which they are applied, or to be barely detected and subsequently isolated and / or degraded, and / or to be detected and subsequently isolated and / or degraded later.

[0370] Macrophages constitute one of the most important components of the immune system and play a major role in eliminating foreign particles, including liposomes and other colloidal particles, from the bloodstream. At the molecular level, particle clearance occurs in two steps: opsonization through the deposition of serum proteins (or "opsonins") on the particle surface, followed by the recognition and capture of opsonized particles by macrophages.

[0371] Modification of particle surfaces using chains of hydrophilic and flexible polymers provides them with spatial protection by preventing opsonins from reaching the particle surface.

[0372] In some embodiments, the polymer used herein is selected from poly(ethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine)), and poly(ethylene glycol) (PEG). POX, polyoxo-zoline Azine (POZ) and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) (including its derivatives).

[0373] In some embodiments, the polymer is designed to spatially stabilize the docking compound by forming a protective hydrophilic layer. In some embodiments, when such a docking compound is administered in vivo, the polymer can reduce the association of the docking compound with serum proteins and / or the resulting uptake by the reticuloendothelial system.

[0374] In some embodiments, PEG is a polymer of linearly or branched ethylene glycol or ethylene oxide, optionally substituted. In some embodiments, PEG is unsubstituted. In some embodiments, PEG is substituted, for example, substituted with one or more alkyl, alkoxy, acyl, hydroxyl, or aryl groups. In some embodiments, the molecular weight of PEG is about 130 to about 50,000; in another embodiment, it is about 150 to about 30,000; in another embodiment, it is about 150 to about 20,000; in another embodiment, it is about 150 to about 15,000; in another embodiment, it is about 150 to about 10,000; in another embodiment, it is about 150 to about 6,000; in another embodiment, it is about 150 to about 5,000; in another embodiment, it is about 150 to about 4,000; in another embodiment, it is about 150 to about 3,000; in another embodiment, it is about 300 to about 3,000; in another embodiment, it is about 1,000 to about 3,000; and in yet another embodiment, it is about 1,500 to about 2,500.

[0375] In some embodiments, the PEG moiety has a molecular weight of 1000 or more. In some embodiments, the PEG moiety comprises 10 or more of the formula (O-CH2-CH2). n Units. In some embodiments, PEG contains 20 to 200 ethylene oxide units, such as about 45 ethylene oxide units.

[0376] In some implementations, PEG contains “PEG2k”, also known as “PEG2000”, with an average molecular weight of about 2,000 Daltons.

[0377] In some implementation schemes, PEG is used. 2000 PEG 3000 and PEG 5000 Used as a polymer.

[0378] In some implementations, pSar contains 2 to 200 sarcosine units, such as 5 to 100 sarcosine units, 10 to 50 sarcosine units, 15 to 40 sarcosine units, such as about 23 sarcosine units.

[0379] In some implementations, pSar includes the structure of the following general formula:

[0380]

[0381] Where s is the number of sarcosine units.

[0382] In some embodiments, the POX and / or POZ polymers comprise 2 to 200, 2 to 190, 2 to 180, 2 to 170, 2 to 160, 2 to 150, 2 to 140, 2 to 130, 2 to 120, 2 to 110, 2 to 100, 2 to 90, 2 to 80, 2 to 70, 5 to 200, 5 to 190, 5 to 180, 5 to 170, 5 to 160, 5 to 150, 5 to 140, 5 Up to 130, 5 to 120, 5 to 110, 5 to 100, 5 to 90, 5 to 80, 5 to 70, 10 to 200, 10 to 190, 10 to 180, 10 to 170, 10 to 160, 10 to 150, 10 to 140, 10 to 130, 10 to 120, 10 to 110, 10 to 100, 10 to 90, 10 to 80, or 10 to 70 POX and / or POZ repeating units.

[0383] In some implementations, the POX and / or POZ polymers comprise the following general formula:

[0384] ,

[0385] Where a is an integer from 1 to 2; R 11 It is an alkyl group, especially C10. 1-3 Alkyl groups, such as methyl, ethyl, isopropyl, or n-propyl, are chosen independently for each repeating unit; and m refers to the number of POX and / or POZ repeating units.

[0386] In some embodiments, the POX and / or POZ polymers are polymers of POX and contain repeating units of the following general formula:

[0387]

[0388] In some embodiments, the POX and / or POZ polymers are polymers of POZ and contain repeating units of the following general formula:

[0389]

[0390] In any of the above embodiments of the formula, m (i.e., the number of repeating units in the polymer) is preferably 2 to 190, for example 2 to 180, 2 to 170, 2 to 160, 2 to 150, 2 to 140, 2 to 130, 2 to 120, 2 to 110, 2 to 100, 2 to 90, 2 to 80, 2 to 70, 5 to 200, 5 to 190, 5 to 180, 5 to 170, 5 to 160, 5 to 15 0, 5 to 140, 5 to 130, 5 to 120, 5 to 110, 5 to 100, 5 to 90, 5 to 80, 5 to 70, 10 to 200, 10 to 190, 10 to 180, 10 to 170, 10 to 160, 10 to 150, 10 to 140, 10 to 130, 10 to 120, 10 to 110, 10 to 100, 10 to 90, 10 to 80, or 10 to 70. In some embodiments, m is 2 to 180, such as 4 to 160, 6 to 140, 8 to 120, or 10 to 100, such as 20 to 80, 30 to 70, or 40 to 50.

[0391] In some embodiments, the POX and / or POZ polymers are copolymers comprising repeating units of the following general formula:

[0392] ,

[0393] The number of repeating units shown on the left in the copolymer is 1 to 199; the number of repeating units on the right in the copolymer is 1 to 199; and the sum of the number of repeating units on the left and the number of repeating units on the right in the copolymer is 2 to 200.

[0394] In some embodiments, the number of left-hand repeating units in the copolymer is 1 to 179, for example 1 to 159, 1 to 139, 1 to 119, or 1 to 99; the number of right-hand repeating units in the copolymer is 1 to 179, for example 1 to 159, 1 to 139, 1 to 119, or 1 to 99; and the sum of the number of left-hand repeating units and the number of right-hand repeating units in the copolymer is 2 to 180, for example 4 to 160, 6 to 140, 8 to 120 or 10 to 100, for example 20 to 80, 30 to 70, or 40 to 50.

[0395] In some of the above implementation schemes, R 11Each occurrence (i.e., in each repeating unit) can be the same alkyl group (e.g., R in each repeating unit). 11 (It can be methyl). In some alternative embodiments, R in at least one repeating unit 11 Unlike R in another repeating unit 11 (For example, for at least one repeating unit, R) 11 It is a specific alkyl group (e.g., ethyl), and for at least one different repeating unit, R 11 These are different specific alkyl groups (e.g., methyl). For example, each R 11 It can be selected from two different alkyl groups (e.g., methyl and ethyl) and not all R 11 They are all the same alkyl group.

[0396] In any of the above implementation schemes, R 11 Preferably methyl or ethyl, more preferably methyl. Therefore, in some embodiments, each R... 11 All are methyl or each R 11 All are ethyl. In some alternative embodiments, for each repeating unit, R 11 Independently selected from methyl and ethyl, wherein R in at least one repeating unit 11 It is a methyl group, and R is present in at least one repeating unit. 11 It is an ethyl group.

[0397] In some embodiments, the polymer comprises poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) or poly-2-(2-(2-methylaminoethoxy)ethoxy)acetic acid (pMAEEA), or derivatives thereof.

[0398] In some implementations, the pAEEA portion or its derivatives comprise the following general formula:

[0399]

[0400] in

[0401] X 2 and X 1 Together they are optionally substituted amides, optionally substituted thioamides, esters or thioesters;

[0402] Y is -CH2-, -(CH2)2-, or -(CH2)3-;

[0403] z is between 2 and 24; and

[0404] n is the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or its derivatives, for example, from 1 to 100.

[0405] In some implementation schemes,

[0406] (i) When X 1 When it is -C(O)-, then X 2 Yes -NR 1 -;

[0407] (ii) When X 1 Yes -NR 1 - when, then X 2 It is -C(O)-;

[0408] (iii) When X 1 When it is -C(S)-, then X 2 Yes -NR 1 -;

[0409] (iv) When X 1 Yes -NR 1 - when, then X 2 It is -C(S)-;

[0410] (v) When X 1 When it is -C(O)-, then X 2 It is -O-; or

[0411] (vi) When X 1 When it is -O-, then X 2 It is -C(O)-;

[0412] (vii) When X 1 When it is -C(S)-, then X 2 It is -O-;

[0413] (viii) When X 1 When it is -O-, then X 2 It is -C(S)-;

[0414] (ix) When X 1 When it is -C(O)-, then X 2 Is -S-; or

[0415] (x) When X 1 When it is -S-, then X 2 It is -C(O)-;

[0416] Where R 1 Is it hydrogen or C? 1-8 Alkyl; preferably

[0417] (i) When X 1 When it is -C(O)-, then X 2 Yes -NR 1 -;

[0418] (ii) When X 1 Yes -NR 1 - when, then X 2 It is -C(O)-;

[0419] (iii) When X 1 When it is -C(S)-, then X 2 Yes -NR 1 -;

[0420] (iv) When X 1 Yes -NR 1 - when, then X 2 It is -C(S)-;

[0421] (v) When X 1 When it is -C(O)-, then X 2 It is -O-; or

[0422] (vi) When X 1 When it is -O-, then X 2 It is -C(O)-;

[0423] Where R 1 Is it hydrogen or C? 1-8 alkyl.

[0424] In some implementation schemes, X 1 It is -C(O)- and X 2 Yes -NR 1 -, where R 1 Is it hydrogen or C? 1-8 Alkyl group. In some embodiments, X 1 It is -C(O)- and X 2 Yes -NR 1 -, where R 1 It is hydrogen or methyl. In some embodiments, X 1 It is -C(O)- and X 2 Yes -NR 1 -, where R 1 It is hydrogen.

[0425] In some implementations, Y is -CH2- or -(CH2)2-.

[0426] In some embodiments, the polymer comprises the following general formula:

[0427]

[0428] in

[0429] R 1 Is it hydrogen or C? 1-8 alkyl;

[0430] z is between 2 and 24; and

[0431] n is the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or its derivatives, for example, from 1 to 100.

[0432] In some embodiments of the above formula, z is 2 to 10. In some embodiments, z is 2 to 7. In some embodiments, z is 2 to 5. In some embodiments, z is 2 or 3. In some embodiments, z is 2.

[0433] In some embodiments, the polymer comprises the following general formula:

[0434]

[0435] in

[0436] R 1 Is it hydrogen or C? 1-8 Alkyl; and

[0437] n is the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or its derivatives, for example, from 1 to 100.

[0438] In some implementations of the above formula, R 1 It is hydrogen or methyl. In some embodiments, R 1 It is hydrogen.

[0439] In some embodiments, the polymer comprises the following general formula:

[0440]

[0441] in

[0442] n is the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA), for example, from 1 to 100.

[0443] In some embodiments of the above formula, n is 5 to 50. In some embodiments, n is 5 to 25. In some embodiments, n is 7 to 14. In some embodiments, n is 10 to 25. In some embodiments, n is 14 to 17. In some embodiments, n is 8 or 14.

[0444] In some implementations, in the chain (main chain) connecting the binding portion for CAR and the binding portion for antigen on target cells, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or its derivatives in all poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moieties or derivatives thereof does not exceed 100, 90, 80, 70, 60, 50, 40, or 30.

[0445] In some embodiments, in the chain (main chain) connecting the binding portion for the CAR and the binding portion for the antigen on the target cell, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or its derivatives in all poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moieties or their derivatives is 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, or at least 15.

[0446] In some embodiments, in the chain (main chain) connecting the binding portion for the CAR and the binding portion for the antigen on the target cell, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or its derivatives in all poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moieties or their derivatives is 2 to 100, 4 to 80, 4 to 70, 4 to 60, 4 to 50, 4 to 40, or 4 to 30.

[0447] In some embodiments, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or its derivatives in all side chains does not exceed 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, or 30.

[0448] In some embodiments, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) or its derivatives in all side chains is at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, at least 26, at least 28, or at least 30.

[0449] In some embodiments, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or its derivatives in all side chains is 10 to 150, 20 to 100, 20 to 80, 20 to 70, 30 to 60, or 30 to 50.

[0450] In some embodiments, in the chain (main chain) connecting the binding portion for the CAR and the binding portion for the antigen on the target cell, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or its derivatives in all poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moieties or their derivatives is 4 to 60, and the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or its derivatives in all side chains is 20 to 100.

[0451] In some embodiments, in the chain (main chain) connecting the binding portion for the CAR and the binding portion for the antigen on the target cell, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or its derivatives in all poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moieties or their derivatives is 4 to 40, and the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or its derivatives in all side chains is 20 to 80.

[0452] In some embodiments, in the chain (main chain) connecting the binding portion for the CAR and the binding portion for the antigen on the target cell, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or its derivatives in all poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moieties or their derivatives is 4 to 30, and the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or its derivatives in all side chains is 20 to 60.

[0453] In some embodiments, the docking compound can be provided by administering a nucleic acid encoding the docking compound to a subject and allowing the subject's cells to express the docking compound. Delivery of the nucleic acid encoding the docking compound to the cells for expression can be achieved using particles containing that nucleic acid. The particles may contain a targeting molecule that binds to a target, such as an antigen on the target cell for expression. In some embodiments, the cells expressing the nucleic acid secrete the docking compound. In some embodiments, the docking compound contains a signal peptide, such as an N-terminal signal peptide, which allows the cells expressing the nucleic acid to secrete the docking compound. In some embodiments, the cells expressing the nucleic acid encoding the docking compound are the same cells to which the immune effector cells are to be delivered. In some embodiments, the cells expressing the nucleic acid encoding the docking compound are different from the cells to which the immune effector cells are to be delivered. In some embodiments, the cells expressing the nucleic acid encoding the docking compound are hepatocytes. In some embodiments, the cells expressing the nucleic acid secrete the docking compound into the bloodstream. In some preferred embodiments, the nucleic acid encoding the docking compound is RNA. RNA-encoded docking compounds are also referred to herein as "RiboDocker".

[0454] Interacting parts on antigen receptors and docking compounds

[0455] The part of the antigen receptor and the docking compound that interact with each other contains an ALFA system, which includes an ALFA tag and an ALFA-specific single-domain antibody (sdAb) (NbALFA-nanobody). The ALFA system is an epitope tag / conjugate system that includes an ALFA tag and an ALFA-specific single-domain antibody (sdAb), NbALFA-nanobody (Götzke, H. et al, Nat Commun 10, 4403 (2019)).

[0456] In some implementations, portions of the antigen receptors on interacting immune effector cells bind nonvalently to portions of the docking compound (the binding portion against the CAR).

[0457] In some implementations, portions of the antigen receptors on interacting immune effector cells bind to portions of the docking compound (the binding portion for CAR) under physiological conditions.

[0458] Humanized antibodies offer the advantage of reduced immunogenicity because they are engineered to be very similar to human antibodies, minimizing the risk of triggering an immune response when administered to patients. Therefore, humanized antibodies have advantages for in vivo applications, such as in imaging techniques. Furthermore, antibodies bound to epitope tags are becoming increasingly interesting for a variety of therapeutic applications (Mitwasi et al., Sci. Rep. 2020; doi:10.1038 / s41598-020-59082-4).

[0459] Antibodies can be humanized from classic VH / VL-based molecules and their single-domain counterparts. A common approach is to graft the CDR onto a humanized acceptor framework to maintain the spatial orientation of the original variable region from which the donor CDR originates. For the humanization of the VHH region, the CDR can be grafted into a compatible human light chain variable region framework. However, sequence grafting can have unexpected and undesirable effects on antibody conformation (Fernández-Quintero et al., Prot.Eng. Des. and Sel., 2019; doi: 10.1093 / protein / gzaa004). Therefore, providing humanized antibodies with at least comparable binding affinity to the original antibody remains challenging.

[0460] When the inventors of this invention attempted to humanize wild-type NbALFA single-domain antibodies, they initially generated seven humanized variants (variants 1 to 7) by grafting CDRs into various humanized acceptor frameworks. However, the resulting single-domain antibodies either failed to express or exhibited complete loss or significant impairment of affinity for the target antigen (see Example 1).

[0461] Having recognized that CDR grafting does not directly lead to the generation of a high-affinity humanized form of NbALFA, the inventors of this application analyzed the antibody structure in detail and tested the effect of retaining residues that might be related to high-affinity binding. Therefore, they unexpectedly discovered that, in addition to CDR grafting, several key residues of wild-type NbALFA should be retained to obtain a humanized form of NbALFA that maintains high affinity for the peptide of SEQ ID NO: 2, or unexpectedly has even improved affinity (see Example 2). Furthermore, the inventors of this application unexpectedly discovered that the resulting humanized form of NbALFA can even have improved thermal stability (see Example 4).

[0462] Not wanting to be bound by theory, the inventors of this application believe that the arginine residue at position 47 of wild-type NbALFA is important for the interaction between the antibody and the C-terminus of the peptide of SEQ ID NO: 2, and therefore believe that retaining Arg 47 of the original Camelidae VHH framework can improve binding behavior. Furthermore, not wanting to be bound by theory, the inventors of this application believe that the methionine residue at position 50 is important for the formation of the hydrophobic bag, which is masked by the peptide of SEQ ID NO: 2 after binding, increasing the free energy of the antibody-antigen interaction. Therefore, retaining Met 50 of the original Camelidae VHH framework is also considered to improve binding behavior. Moreover, not wanting to be bound by theory, the inventors of this application believe that the arginine residue at position 48 of wild-type NbALFA is also important for the binding of the peptide of SEQ ID NO: 2. Therefore, retaining Arg48 of the original Camelidae VHH framework is also considered to improve binding behavior. These three positions, Arg 47, Arg 48, and Met 50, are therefore considered key residues and should be retained in the humanized form of NbALFA. In fact, all variants 8 to 15 tested in Example 2 contain these three key residues, and all variants 8 to 15 have binding affinity at least comparable to wild-type NbALFA.

[0463] Not wanting to be bound by theory, the inventors of this application also believe that His 99 and / or Val 100 may be involved in NbALFA binding. These two amino acid residues are thought to affect the conformation of CDR3.

[0464] Another residue considered to improve binding to the peptide of SEQ ID NO: 2 is Tyr 40 of wild-type NbALFA. Not wishing to be bound by theory, the inventors of this application believe that Tyr 40 forms a hydrogen bond with the R11' side chain of the peptide found in wild-type NbALFA. Retaining Tyr 40 in the humanized form of NbALFA can further improve antigen binding. However, as shown in variant 8, which includes Phe at position 40, binding affinity comparable to wild-type NbALFA can be achieved without the Tyr residue at position 40. However, as shown in variant 9, by replacing Phe at position 40 with Tyr, binding affinity to the peptide of SEQ ID NO: 2 can be further improved.

[0465] On the other hand, the inventors of this application unexpectedly discovered that Met 61 and Glu 64, which form part of CDR2 as determined by Kabat, are not essential for binding to the peptide of SEQ ID NO: 2. These residues can be mutated to their human counterparts, for example, further humanizing the antibody, while retaining a high binding affinity for the peptide of SEQ ID NO: 2.

[0466] Following this principle, the inventors of this application have created a humanized form of NbALFA single-domain antibody with reduced immunogenicity potential, which is preferably used in vivo in human patients, such as for diagnostic or therapeutic applications.

[0467] Furthermore, the inventors of this invention unexpectedly discovered that, in addition to its reduced immunogenicity, several humanized forms of NbALFA can also be bound by Staphylococcal protein A (SpA; see Example 3). Antibodies that can be bound by SpA have the advantage of promoting GMP production of antibodies. Therefore, the binding affinity for SpA is another advantage of some of the humanized antibodies described herein. Unexpectedly, the inventors of this application found that introducing mutations into CDR2 as determined by IMGT, particularly the mutation Ala 60 → Thr, and / or introducing mutations into CDR2 as determined by Kabat, particularly the mutation Gln 67 → Lys, retains a high binding affinity for the peptide of SEQ ID NO: 2 while promoting SpA binding.

[0468] Therefore, this paper describes the sequence containing Epitope-specific binding single-domain antibodies (used in the compositions and methods described herein), wherein the antibody comprises the CDR1 sequence as shown in SEQ ID NO:94 or a sequence having one or two mutations relative to said sequence, the CDR2 sequence as shown in SEQ ID NO:97 or a sequence having one or two mutations relative to said sequence, and the CDR3 sequence as shown in SEQ ID NO:98 or a sequence having one or two mutations relative to said sequence. In some embodiments, the antibody further comprises at positions 47, 48, and / or 50 of the sequence corresponding to SEQ ID NO:1 at at least one of the following amino acid residues: Arg at position 47, Arg at position 48, and / or Met at position 50.

[0469] A single-domain antibody is an antibody whose complementation-determining region is part of a single-domain polypeptide. Examples include, but are not limited to, heavy-chain antibodies, naturally occurring antibodies lacking a light chain, single-domain antibodies derived from conventional four-chain antibodies, modified antibodies, and single-domain scaffolds other than those derived from antibodies. A single-domain antibody can be any antibody in the art, or any future single-domain antibody. Single-domain antibodies can be derived from any species, including but not limited to mice, humans, camels, llamas, goats, rabbits, or cattle. The single-domain antibodies used herein can be derived from naturally occurring antibodies known as heavy-chain antibodies lacking a light chain. For example, such a single-domain antibody is disclosed in WO 94 / 04678. For clarity, this variable domain derived from a naturally occurring heavy-chain antibody lacking a light chain is referred to herein as VHH to distinguish it from the VH of a conventional four-chain immunoglobulin. Such VHH molecules can be derived from antibodies produced in species of the Camelidae family (e.g., camels, dromedary camels, llamas, vicuñas, alpacas, and guanacos). Other species besides Camelidae can also produce naturally occurring heavy chain antibodies lacking the light chain. As an illustrative example, sharks are known to produce naturally occurring heavy chain antibodies lacking the light chain (commonly referred to as IgNARs), which also contain a VHH domain. Furthermore, some species have been genetically modified to artificially produce naturally occurring heavy chain antibodies lacking the light chain, such as certain mouse strains. Additionally, VHHs can be obtained from synthetic libraries. All such VHHs are within the scope of this disclosure. Single-domain antibodies used herein also include humanized forms of single-domain antibodies, such as humanized VHH regions.

[0470] According to this disclosure and as known to those skilled in the art, VHHs are preferably derived from the heavy chain variable domains of immunoglobulins that naturally lack light chains, such as those from the Camelidae family as described in WO 94 / 04678 (and hereinafter referred to as VHH domains or nanobodies). VHH molecules are approximately 10 × smaller than IgG molecules. They are single polypeptides and are very stable, resistant to extreme pH and temperature conditions. Furthermore, they are highly resistant to the action of proteases, which is not the case for conventional antibodies. Moreover, in vitro expression of VHHs or expression in prokaryotic or eukaryotic organisms suitable for recombinant protein expression yields high yields of correctly folded functional VHHs.

[0471] The term "variable" refers to a portion of an immunoglobulin domain that exhibits sequence variability and participates in determining the specificity and binding affinity of a particular antibody (i.e., a "variable domain"). This variability is not uniformly distributed throughout the entire variable domain of the antibody; it is concentrated in subdomains of the variable region in each heavy and light chain. These subdomains are called "hypervariable regions," "HVRs," or "HVs," or "complementarity-determining regions" (CDRs). The more conserved (i.e., non-hypervariant) portions of the variable domain are called "framework" regions (FRs). The naturally occurring variable domains of the heavy and light chains each contain four FRs, which predominantly adopt a β-sheet conformation and are linked by three hypervariable regions that form loops to connect the β-sheet structure and, in some cases, form part of the β-sheet structure. The hypervariable regions in each chain are tightly held together by the FRs and, together with hypervariable regions from the other chain, contribute to the formation of antigen-binding sites (see Kabat et al., see below). Typically, naturally occurring immunoglobulins contain six core domains (CDRs) (see below); three in the VH region (H1, H2, H3) and three in the VL region (L1, L2, L3). Among naturally occurring immunoglobulins, H3 and L3 exhibit the greatest diversity among the six CDRs, with H3 in particular considered to play a unique role in conferring good specificity to immunoglobulins. However, naturally occurring immunoglobulins lacking the light chain contain three CDRs in the VHH region. The constant domains do not directly participate in antigen binding but exhibit various effector functions, such as antibody-dependent antigen-dependent antigen-mediated cytotoxicity and complement activation.

[0472] Unless otherwise expressly or implicitly stated, the CDR sequences of this disclosure follow the hybrid Kabat / IMGT definition. Heavy chain CDR1 follows the hybrid Kabat / IMGT definition, the start of heavy chain CDR1 follows the IMGT definition, and the termination of heavy chain CDR1 follows the Kabat definition. As an illustrative example, the CDR1 of wild-type NbALFA according to IMGT is... According to Kabat's wild-type NbALFA, CDR1 is According to the hybrid Kabat / IMGT definition preferred for the context of this disclosure, the CDR1 sequence of wild-type NbALFA is Heavy chain CDR2 and CDR3 are defined according to the IMGT definition. Similarly, light chain CDR sequences are defined according to the IMGT definition. The reason for the mixed definition of heavy chain CDR1 is to include a longer sequence to ensure that the CDR1 graft contains all important residues. For CDR definitions, see, for example, Dondelinger et al., “Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface / Residue Definition”, 2018; doi: 10.3389 / fimmu.2018.02278 Other criteria exist for defining CDRs, such as those according to Maass 2007 (Journal of Immunological Methods 324 (2007) 13-25). Another criterion for characterizing antigen-binding sites is the hypervariable loop described by Chothia (see, for example, Chothia, et al. (1992); J. MoI. Biol. 227:799-817; and Tomlinson et al. (1995) EMBO J. 14:4628-4638).

[0473] According to the CDR definition described herein, unless otherwise expressly or implicitly stated, the FR regions used herein follow the mixed Kabat / IMGT definition. For the heavy chain variable region, FR1, FR3, and FR4 are defined according to the IMGT definition. For the heavy chain variable region FR2, the start is defined according to the Kabat definition, and the end is defined according to the IMGT definition. Thus, as an illustrative example, FR1 of wild-type NbALFA is preferably defined by amino acid positions 1 to 25 of SEQ ID NO: 1, FR2 of wild-type NbALFA is preferably defined by amino acid positions 39 to 53 of SEQ ID NO: 1, FR3 of wild-type NbALFA is preferably defined by amino acid positions 61 to 98 of SEQ ID NO: 1, and FR4 of wild-type NbALFA is preferably defined by amino acid positions 112 to 122 of SEQ ID NO: 1.

[0474] Each VHH, VH, and VL has three CDRs and four FRs, arranged in the following order from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus): FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy chain and / or light chain contain binding domains that interact with the antigenic epitope.

[0475] The term "epitaxy," also known as "antigenic determinant," refers to a portion of an antigen that specifically binds to an antibody to form a complex. Therefore, the term "epitaxy" includes any molecular or protein determinant capable of specifically binding to an immunoglobulin or T-cell receptor. The binding site (complementary site) of the antibody molecule described herein can specifically bind to / interact with a conformational or sequential epitope that is unique to the target structure. Epitope determinants typically consist of chemically active surface groups of a molecule, such as amino acids or sugar side chains, and typically possess specific three-dimensional structural features and specific charge characteristics. Epitope determinants may contain chemically active surface groups of a molecule, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in some embodiments may possess specific three-dimensional structural features and / or specific charge characteristics. Regarding polypeptide antigens, conformational or discontinuous epitopes are characterized by the presence of two or more discrete amino acid residues that are separate in the primary sequence but assemble into a coherent structure on the molecular surface when the polypeptide folds into its native protein / antigen (Sela, M., Science (1969) 166, 1365-1374; Laver, WG, et al. Cell (1990) 61, 553-556). The two or more discrete amino acid residues constituting an epitope can be present in different parts of one or more polypeptide chains. When the polypeptide chain folds into a three-dimensional structure to form an epitope, these residues aggregate on the molecular surface. In contrast, continuous or linear epitopes consist of two or more discrete amino acid residues that are present in a single linear segment of the polypeptide chain. As an illustrative example, an "environment-dependent" CD3 epitope refers to the conformation of such an epitope. Such environment-dependent epitopes located on the ε chain of CD3 can only form their correct conformation when they are embedded within the rest of the ε chain and held in the correct position by heterodimerization of the ε chain with the CD3γ or δ chain. In contrast, environment-independent CD3 epitopes can be polypeptides or functional fragments of the N-terminus of CD3ε consisting of 1 to 27 amino acid residues. Typically, epitopes can be linear in nature or discontinuous. Therefore, the term “conformational epitope” as used herein refers to a discontinuous epitope formed by the spatial relationships between the amino acids of an antigen, rather than a continuous amino acid sequence. The term “epitaph” also includes antigenic determinants of haptens, which are considered to be small molecules that can act as antigens by exhibiting one or more immunologically recognized epitopes upon binding to larger substances, such as larger molecules, like proteins.

[0476] As used in this article, an "eptopic tag" refers to a segment of amino acids against which specific antibodies or antibody-like proteins can be prepared. Such epitope tags allow for the specific identification and / or tracking of labeled peptides or proteins that may be present in living organisms or cultured cells. Detection of labeled molecules can be achieved using many different techniques. Examples of such techniques include immunohistochemistry, immunoprecipitation, flow cytometry, immunofluorescence microscopy, electron microscopy, ELISA, Western blotting, and affinity chromatography. Epitope tags add a known epitope (antibody binding site) to the target peptide to provide binding to a known and typically high-affinity antibody. Epitope tags can also be used to isolate and / or purify labeled molecules, for example, via drop-down applications.

[0477] As used in this article, the term "mutation" refers to a change in the sequence of a biomolecule, such as the amino acid sequence of a protein or the nucleic acid sequence of DNA or RNA. Mutations include deletions, insertions, and substitutions, and these can apply to individual nucleotides / amino acids as well as larger segments of nucleotide / amino acid sequences.

[0478] As mentioned herein, the antibody described herein contains at least one of the following amino acid residues at positions 47, 48 and / or 50 of the sequence corresponding to SEQ ID NO: 1: Arg at position 47, Arg at position 48, and / or Met at position 50.

[0479] Preferably, the antibody described herein contains an Arg residue at position 47 of the sequence corresponding to SEQ ID NO: 1. Preferably, the antibody described herein contains an Arg residue at position 48 of the sequence corresponding to SEQ ID NO: 1. Preferably, the antibody described herein contains a Met residue at position 50 of the sequence corresponding to SEQ ID NO: 1. Any of these residues is considered to facilitate the binding of NbALFA to the target of SEQ ID NO: 2.

[0480] Preferably, the antibody described herein contains at least two of the following amino acid residues at positions 47, 48, and / or 50 of the sequence corresponding to SEQ ID NO: 1: Arg at position 47, Arg at position 48, and / or Met at position 50. More preferably, the antibody described herein preferably contains the following amino acid residues at positions 47, 48, and 50 of the sequence corresponding to SEQ ID NO: 1: Arg at position 47, Arg at position 48, and Met at position 50.

[0481] In addition to positions 47, 48, and 50, retaining the Tyr residue at position 40 is also considered to improve the affinity of the antibody described herein for SEQ ID NO: 2. While single-domain antibodies lacking the Tyr residue at position 40 can also exhibit comparable binding affinity to SEQ ID NO: 2 compared to wild-type NbALFA, such as variant 8, other variants containing Tyr at this position can show further improved affinity. Therefore, the antibody described herein preferably contains a Tyr residue at position 40 corresponding to SEQ ID NO: 1. Therefore, the antibody described herein preferably contains the following amino acid residues at positions 40, 47, 48, and 50 corresponding to SEQ ID NO: 1: Tyr at position 40, Arg at position 47, Arg at position 48, and Met at position 50.

[0482] Furthermore, it is hypothesized that the His residue at position 99 and / or the Val residue at position 100 may have a positive impact on the binding affinity of the antibody described herein for SEQ ID NO: 2. Therefore, the antibody described herein preferably contains a His residue at position 99 corresponding to SEQ ID NO: 1. Therefore, the antibody described herein preferably contains a Val residue at position 100 corresponding to SEQ ID NO: 1. Therefore, the antibody described herein preferably contains a His residue at position 99 corresponding to SEQ ID NO: 1 and a Val residue at position 100 corresponding to SEQ ID NO: 1. Therefore, the antibody described herein preferably contains the following amino acid residues at positions 47, 48, 50, 99, and 100 corresponding to SEQ ID NO: 1: Arg at position 47, Arg at position 48, Met at position 50, His at position 99, and Val at position 100. Therefore, the antibodies described herein preferably contain the following amino acid residues at positions 40, 47, 48, 50, 99, and 100 of the sequence corresponding to SEQ ID NO: 1: Tyr at position 40, Arg at position 47, Arg at position 48, Met at position 50, His at position 99, and Val at position 100. Other natural amino acids of wild-type NbALFA may also be retained in the antibodies described herein.

[0483] The antibody described herein preferably contains a Val residue at position 5 of the sequence corresponding to SEQ ID NO: 1. However, it is not desirable to be bound by theory, as position 5 is considered to be of less importance for binding the peptide of SEQ ID NO: 2. Furthermore, the antibody described herein preferably contains a Gln residue at position 117 of the sequence corresponding to SEQ ID NO: 1. However, it is not desirable to be bound by theory, as Gln at position 117 is considered of less importance for binding the peptide of SEQ ID NO: 2; however, Gln 117 is considered to improve solubility. Therefore, the antibody described herein preferably contains a Val residue at position 5 of the sequence corresponding to SEQ ID NO: 1 and a Gln residue at position 117 of the sequence corresponding to SEQ ID NO: 1. Preferably, the antibody described herein contains the following amino acid residues at positions 5, 47, 48, 50, and 117 of the sequence corresponding to SEQ ID NO: 1: Val at position 5, Arg at position 47, Arg at position 48, Met at position 50, and Gln at position 117. Preferably, the antibody described herein contains the following amino acid residues at positions 5, 47, 48, 50, 99, 100, and 117 of the sequence corresponding to SEQ ID NO: 1: Val at position 5, Arg at position 47, Arg at position 48, Met at position 50, His at position 99, Val at position 100, and Gln at position 117. Preferably, the antibody described herein contains the following amino acid residues at positions 5, 40, 47, 48, 50, 99, 100, and 117 of the sequence corresponding to SEQ ID NO: 1: Val at position 5, Tyr at position 40, Arg at position 47, Arg at position 48, Met at position 50, His at position 99, Val at position 100, and Gln at position 117. In wild-type NbALFA, Met 61 and Glu 64 form part of CDR2 as determined by Kabat. However, the inventors of this application unexpectedly discovered that these two residues are not essential for binding to the peptide of SEQ ID NO: 2. By mutating these two residues to their sequence counterparts derived from human IGHV3-23, Tyr 61 and / or Asp 64, a high binding affinity to the peptide of SEQ ID NO: 2 can be retained, as shown in variants 13 and 15. The antibodies described herein may also contain a Tyr residue at position 61 of the sequence corresponding to SEQ ID NO: 1. The antibodies described herein may also contain an Asp residue at position 64 of the sequence corresponding to SEQ ID NO: 1.The antibody described herein may also contain a Tyr residue at position 61 of the sequence corresponding to SEQ ID NO: 1 and an Asp residue at position 64 of the sequence corresponding to SEQ ID NO: 1.

[0484] The single-domain antibody described herein preferably comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with or identical to any of SEQ ID NO: 118-125. Preferably, the antibody described herein comprises a sequence having at least 90% sequence identity with any of SEQ ID NO: 118-125. Preferably, the antibody described herein comprises a sequence having at least 95% sequence identity with any of SEQ ID NO: 118-125. Preferably, the antibody described herein comprises a sequence having at least 98% sequence identity with any of SEQ ID NO: 118-125. Preferably, the antibody described herein comprises a sequence having at least 99% sequence identity with any of SEQ ID NO: 118-125. Preferably, the antibody described herein comprises or consists of an amino acid sequence selected from or composed of an amino acid sequence selected from the following:

[0485]

[0486] The single-domain antibody described herein preferably comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with or identical to SEQ ID NO: 118. Preferably, the antibody described herein comprises a sequence having at least 90% sequence identity with SEQ ID NO: 118. Preferably, the antibody described herein comprises a sequence having at least 95% sequence identity with SEQ ID NO: 118. Preferably, the antibody described herein comprises a sequence having at least 98% sequence identity with SEQ ID NO: 118. Preferably, the antibody described herein comprises a sequence having at least 99% sequence identity with SEQ ID NO: 118. Preferably, the antibody described herein comprises or consists of the amino acid sequence shown in SEQ ID NO: 118.

[0487] The single-domain antibody described herein preferably comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with or identical to SEQ ID NO: 119. Preferably, the antibody described herein comprises a sequence having at least 90% sequence identity with SEQ ID NO: 119. Preferably, the antibody described herein comprises a sequence having at least 95% sequence identity with SEQ ID NO: 119. Preferably, the antibody described herein comprises a sequence having at least 98% sequence identity with SEQ ID NO: 119. Preferably, the antibody described herein comprises a sequence having at least 99% sequence identity with SEQ ID NO: 119. Preferably, the antibody described herein comprises or consists of the amino acid sequence shown in SEQ ID NO: 119.

[0488] The single-domain antibody described herein preferably comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with or identical to SEQ ID NO: 120. Preferably, the antibody described herein comprises a sequence having at least 90% sequence identity with SEQ ID NO: 120. Preferably, the antibody described herein comprises a sequence having at least 95% sequence identity with SEQ ID NO: 120. Preferably, the antibody described herein comprises a sequence having at least 98% sequence identity with SEQ ID NO: 120. Preferably, the antibody described herein comprises a sequence having at least 99% sequence identity with SEQ ID NO: 120. Preferably, the antibody described herein comprises or consists of the amino acid sequence shown in SEQ ID NO: 120.

[0489] The single-domain antibody described herein preferably comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with or identical to SEQ ID NO: 121. Preferably, the antibody described herein comprises a sequence having at least 90% sequence identity with SEQ ID NO: 121. Preferably, the antibody described herein comprises a sequence having at least 95% sequence identity with SEQ ID NO: 121. Preferably, the antibody described herein comprises a sequence having at least 98% sequence identity with SEQ ID NO: 121. Preferably, the antibody described herein comprises a sequence having at least 99% sequence identity with SEQ ID NO: 121. Preferably, the antibody described herein comprises or consists of the amino acid sequence shown in SEQ ID NO: 121.

[0490] The single-domain antibody described herein preferably comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with or identical to SEQ ID NO: 122. Preferably, the antibody described herein comprises a sequence having at least 90% sequence identity with SEQ ID NO: 122. Preferably, the antibody described herein comprises a sequence having at least 95% sequence identity with SEQ ID NO: 122. Preferably, the antibody described herein comprises a sequence having at least 98% sequence identity with SEQ ID NO: 122. Preferably, the antibody described herein comprises a sequence having at least 99% sequence identity with SEQ ID NO: 122. Preferably, the antibody described herein comprises or consists of the amino acid sequence shown in SEQ ID NO: 122.

[0491] The single-domain antibody described herein preferably comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with or identical to SEQ ID NO: 123. Preferably, the antibody described herein comprises a sequence having at least 90% sequence identity with SEQ ID NO: 123. Preferably, the antibody described herein comprises a sequence having at least 95% sequence identity with SEQ ID NO: 123. Preferably, the antibody described herein comprises a sequence having at least 98% sequence identity with SEQ ID NO: 123. Preferably, the antibody described herein comprises a sequence having at least 99% sequence identity with SEQ ID NO: 123. Preferably, the antibody described herein comprises or consists of the amino acid sequence shown in SEQ ID NO: 123.

[0492] The single-domain antibody described herein preferably comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with or identical to SEQ ID NO: 124. Preferably, the antibody described herein comprises a sequence having at least 90% sequence identity with SEQ ID NO: 124. Preferably, the antibody described herein comprises a sequence having at least 95% sequence identity with SEQ ID NO: 124. Preferably, the antibody described herein comprises a sequence having at least 98% sequence identity with SEQ ID NO: 124. Preferably, the antibody described herein comprises a sequence having at least 99% sequence identity with SEQ ID NO: 124. Preferably, the antibody described herein comprises or consists of the amino acid sequence shown in SEQ ID NO: 124.

[0493] The single-domain antibody described herein preferably comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with or identical to SEQ ID NO: 125. Preferably, the antibody described herein comprises a sequence having at least 90% sequence identity with SEQ ID NO: 125. Preferably, the antibody described herein comprises a sequence having at least 95% sequence identity with SEQ ID NO: 125. Preferably, the antibody described herein comprises a sequence having at least 98% sequence identity with SEQ ID NO: 125. Preferably, the antibody described herein comprises a sequence having at least 99% sequence identity with SEQ ID NO: 125. Preferably, the antibody described herein comprises or consists of the amino acid sequence shown in SEQ ID NO: 125.

[0494] The “percentage (%) sequence identity” for the amino acid sequences disclosed herein is defined as the percentage of amino acid residues in the candidate sequence that are paired identical to those in the reference sequence (i.e., the antibody molecule of this disclosure) after sequence alignment and the introduction of gaps (if necessary) to achieve maximum percentage sequence identity, and without taking any conserved substitutions as part of the sequence identity. Alignments for determining percentage amino acid sequence identity can be performed in various ways within the art, for example, using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the compared sequences. The same applies to the nucleotide sequences disclosed herein.

[0495] As disclosed herein, the antibodies described herein may contain a CDR1 sequence. Or, relative to the sequence having one or two mutations, the CDR2 sequence. (where X1 is A or T) (SEQ ID NO: 97) or a sequence having one or two mutations relative to the said sequence, and the CDR3 sequence. (SEQ ID NO: 98) or a sequence having one or two mutations relative to the said sequence. Preferably, the antibody described herein may comprise the CDR1 sequence. (SEQ ID NO: 94) or a sequence having one mutation relative to the said sequence, the CDR2 sequence (where X1 is A or T) (SEQ ID NO: 97) or a sequence having one mutation relative to the said sequence, and the CDR3 sequence. (SEQ ID NO: 98) or a sequence having one mutation relative to said sequence. More preferably, the antibody described herein may comprise the CDR1 sequence. CDR2 sequence (where X1 is A or T) (SEQ ID NO: 97), and the CDR3 sequence. (SEQ ID NO: 98).

[0496] The antibodies described herein preferably comprise a CDR2 sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with or identical to any of SEQ ID NO: 95 or 96. Such antibodies described herein may comprise a CDR2 sequence. Or a sequence having one or two mutations relative to the stated sequence, or a CDR2 sequence. Alternatively, a sequence having one or two mutations relative to the stated sequence. Preferably, the antibody described herein comprises the CDR2 sequence of SEQ ID NO: 95 or a sequence having one mutation relative to the stated sequence, or the CDR2 sequence of SEQ ID NO: 96 or a sequence having one mutation relative to the stated sequence. Preferably, the antibody described herein comprises the CDR2 sequence of SEQ ID NO: 95 or SEQ ID NO: 96.

[0497] The antibody described herein may comprise the CDR1 sequence shown in SEQ ID NO: 94 or a sequence having one or two mutations relative to said sequence, the CDR2 sequence shown in SEQ ID NO: 95 or a sequence having one or two mutations relative to said sequence, and the CDR3 sequence shown in SEQ ID NO: 98 or a sequence having one or two mutations relative to said sequence. Preferably, the antibody described herein may comprise the CDR1 sequence shown in SEQ ID NO: 94 or a sequence having one mutation relative to said sequence, the CDR2 sequence shown in SEQ ID NO: 95 or a sequence having one mutation relative to said sequence, and the CDR3 sequence shown in SEQ ID NO: 98 or a sequence having one mutation relative to said sequence. More preferably, the antibody described herein may comprise the CDR1 sequence shown in SEQ ID NO: 94, the CDR2 sequence shown in SEQ ID NO: 95, and the CDR3 sequence shown in SEQ ID NO: 98.

[0498] The antibody described herein may comprise the CDR1 sequence shown in SEQ ID NO: 94 or a sequence having one or two mutations relative to said sequence, the CDR2 sequence shown in SEQ ID NO: 96 or a sequence having one or two mutations relative to said sequence, and the CDR3 sequence shown in SEQ ID NO: 98 or a sequence having one or two mutations relative to said sequence. Preferably, the antibody described herein may comprise the CDR1 sequence shown in SEQ ID NO: 94 or a sequence having one mutation relative to said sequence, the CDR2 sequence shown in SEQ ID NO: 96 or a sequence having one mutation relative to said sequence, and the CDR3 sequence shown in SEQ ID NO: 98 or a sequence having one mutation relative to said sequence. More preferably, the antibody described herein may comprise the CDR1 sequence shown in SEQ ID NO: 94, the CDR2 sequence shown in SEQ ID NO: 96, and the CDR3 sequence shown in SEQ ID NO: 98.

[0499] It should be understood that such antibodies are single-domain antibodies. It should also be understood that antibodies described herein with one or two mutations introduced into one, two, or all three CDR sequences are preferably still able to specifically bind the peptide of SEQ ID NO: 2 with a binding affinity at least comparable to wild-type NbALFA.

[0500] The antibodies described in this article preferably include those with The sequence has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or is the same as the FR1 sequence.

[0501] The antibodies described in this article preferably include those with (where X1 is F or Y) (SEQ ID NO: 102) sequence has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or is the same as the FR2 sequence.

[0502] The antibodies described in this article preferably include those with (where X1 is M or Y, X2 is D or E, X3 is Q or K, X4 is A or S, X5 is N or R, X6 is M or T, and X7 is V or L) (SEQ ID NO: 108) The sequence has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or is the same as the FR3 sequence.

[0503] The antibodies described in this article preferably include those with The sequence has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or is the same as the FR4 sequence.

[0504] Therefore, the antibodies described herein preferably contain FR1, FR2, FR3 and FR4 sequences, each of which has at least 80%, at least 85%, at least 90% or at least 95% sequence identity with or is identical to the sequences of SEQ ID NO: 99, 102, 108 and 109, respectively.

[0505] The antibodies described herein preferably comprise at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with or identical to any of SEQ ID NO: 100 or 101. Such antibodies described herein may comprise... It has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or is identical to the FR2 sequence. Such antibodies described herein may also contain... It has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or is the same as the FR2 sequence.

[0506] The antibodies described herein preferably comprise an FR3 sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with any of SEQ ID NO: 103-107, or identical to such FR3 sequence. Such antibodies described herein may comprise a sequence... The antibody described herein may contain an FR3 sequence that is at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or identical to the FR3 sequence. The antibody described herein may contain an FR3 sequence that is at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or identical to the FR3 sequence. The antibody described herein may contain an FR3 sequence that is at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or identical to the FR3 sequence. The antibody described herein may contain an FR3 sequence that is at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or identical to the FR3 sequence. It has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or is the same as the FR3 sequence.

[0507] The antibodies described herein preferably comprise FR1, FR2, FR3 and FR4 sequences that are at least 80%, at least 85%, at least 90% or at least 95% sequence identity with the sequences shown in SEQ ID NO: 99, 100, 103 and 109, respectively, or that are the same as the sequences shown in SEQ ID NO: 99, 100, 103 and 109, respectively.

[0508] The antibodies described herein preferably comprise FR1, FR2, FR3 and FR4 sequences that are at least 80%, at least 85%, at least 90% or at least 95% sequence identity with the sequences shown in SEQ ID NO: 99, 101, 103 and 109, respectively, or are the same as those sequences.

[0509] The antibodies described herein preferably comprise FR1, FR2, FR3 and FR4 sequences that are at least 80%, at least 85%, at least 90% or at least 95% sequence identity with the sequences shown in SEQ ID NO: 99, 101, 104 and 109, respectively, or that are the same as the sequences shown in SEQ ID NO: 99, 101, 104 and 109, respectively.

[0510] The antibodies described herein preferably comprise FR1, FR2, FR3 and FR4 sequences that are at least 80%, at least 85%, at least 90% or at least 95% sequence identity with the sequences shown in SEQ ID NO: 99, 101, 105 and 109, respectively, or are the same as those sequences.

[0511] The antibodies described herein preferably comprise FR1, FR2, FR3 and FR4 sequences that are at least 80%, at least 85%, at least 90% or at least 95% sequence identity with the sequences shown in SEQ ID NO: 99, 101, 106 and 109, respectively, or are the same as those sequences.

[0512] The antibodies described herein preferably comprise FR1, FR2, FR3 and FR4 sequences that are at least 80%, at least 85%, at least 90% or at least 95% sequence identity with the sequences shown in SEQ ID NO: 99, 101, 107 and 109, respectively, or are the same as those sequences.

[0513] The antibodies described herein preferably comprise the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 94, 97, and 98, and further comprise FR1, FR2, FR3, and FR4 sequences that are at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the sequences shown in SEQ ID NO: 99, 102, 108, and 109, respectively, or are identical to them.

[0514] The antibodies described herein preferably comprise the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 94, 95, and 98, and further comprise FR1, FR2, FR3, and FR4 sequences that are at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the sequences shown in SEQ ID NO: 99, 100, 103, and 109, respectively, or are identical to them.

[0515] The antibodies described herein preferably comprise the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 94, 95, and 98, and further comprise FR1, FR2, FR3, and FR4 sequences that are at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the sequences shown in SEQ ID NO: 99, 101, 103, and 109, respectively, or that are identical to the sequences shown in SEQ ID NO: 99, 101, 103, and 109, respectively.

[0516] The antibodies described herein preferably comprise the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 94, 96, and 98, and further comprise FR1, FR2, FR3, and FR4 sequences that are at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the sequences shown in SEQ ID NO: 99, 101, 103, and 109, respectively, or that are identical to the sequences shown in SEQ ID NO: 99, 101, 103, and 109, respectively.

[0517] The antibodies described herein preferably comprise the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 94, 95, and 98, and further comprise FR1, FR2, FR3, and FR4 sequences that are at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the sequences shown in SEQ ID NO: 99, 101, 104, and 109, or are identical to them, respectively.

[0518] The antibodies described herein preferably comprise the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 94, 96, and 98, and further comprise FR1, FR2, FR3, and FR4 sequences that are at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the sequences shown in SEQ ID NO: 99, 101, 104, and 109, respectively, or are identical to those sequences.

[0519] The antibodies described herein preferably comprise the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 94, 95, and 98, and further comprise FR1, FR2, FR3, and FR4 sequences that are at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the sequences shown in SEQ ID NO: 99, 101, 105, and 109, respectively, or that are identical to the sequences shown in SEQ ID NO: 99, 101, 105, and 109, respectively.

[0520] The antibodies described herein preferably comprise the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 94, 96, and 98, and further comprise FR1, FR2, FR3, and FR4 sequences that are at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the sequences shown in SEQ ID NO: 99, 101, 105, and 109, respectively, or are identical to them.

[0521] The antibodies described herein preferably comprise the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 94, 95, and 98, and further comprise FR1, FR2, FR3, and FR4 sequences that are at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the sequences shown in SEQ ID NO: 99, 101, 106, and 109, respectively, or that are identical to the sequences shown in SEQ ID NO: 99, 101, 106, and 109, respectively.

[0522] The antibodies described herein preferably comprise the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 94, 96, and 98, and further comprise FR1, FR2, FR3, and FR4 sequences that are at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the sequences shown in SEQ ID NO: 99, 101, 106, and 109, respectively, or are identical to them.

[0523] The antibodies described herein preferably comprise the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 94, 95, and 98, and further comprise FR1, FR2, FR3, and FR4 sequences that are at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the sequences shown in SEQ ID NO: 99, 101, 107, and 109, respectively, or are identical to those sequences.

[0524] The antibodies described herein preferably comprise the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 94, 96, and 98, and further comprise FR1, FR2, FR3, and FR4 sequences that are at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the sequences shown in SEQ ID NO: 99, 101, 107, and 109, respectively, or that are identical to the sequences shown in SEQ ID NO: 99, 101, 107, and 109, respectively.

[0525] Preferably, the frame sequence of the antibody is defined by the amino acid positions corresponding to positions 1 to 25, 39 to 53, 61 to 98 and 112 to 122 of SEQ ID NO: 1.

[0526] Preferably, the FR1, FR2, FR3 and / or FR4 regions of the antibody described herein correspond to amino acids 1 to 25, 39 to 53, 61 to 98 and 112 to 122 of SEQ ID NO:1.

[0527] The antibodies described in this article are preferably humanized.

[0528] As used in this article, the term "humanization" refers to the process of modifying a non-human antibody (usually derived from an animal) to maximize the identity of its amino acid sequence with the closest human equivalent. This modification involves genetic engineering that minimizes potential adverse reactions when the antibody is administered to humans while preserving the antibody's ability to recognize and bind to specific antigens. Typically, the degree of antibody humanization is defined by the percentage identity of the V-segment or frame region to its human counterpart.

[0529] The variable regions of both the heavy and light chains are known to contain three complementarity-determining regions (CDRs), which vary depending on the epitope involved and determine binding affinity. These CDRs are flanked by four framework regions (FRs), which are relatively conserved in a given species and presumably provide scaffolding for the CDRs. When preparing non-human antibodies relative to a specific epitope, the variable regions can be "remodeled" or "humanized" by grafting CDRs derived from the non-human antibody onto the FRs present in the human antibody to be modified. Sato,K., et al., (1993) Cancer Res 53:851-856. Riechmann, L., et al., (1988)Nature 332:323-327; Verhoeyen, M., et al., (1988) Science 239:1534-1536;Kettleborough, CA, et al., (1991) Protein Engineering 4:773-3783; Maeda,H., et al., (1991) Human Antibodies Hybridoma 2:124-134; Gorman, SD, etal., (1991) Proc Natl Acad Sci USA 88:4181-4185; Tempest, PR, et al., (1991) Bio / Technology 9:266-271; Co, MS, et al. al., (1991) Proc Natl Acad Sci USA 88:2869-2873; Carter, P., et al., (1992) Proc Natl Acad Sci USA 89:4285-4289; and Co, MS et al., (1992) J Immunol 148:1149-1154 reported the application of this method on various antibodies.

[0530] In some embodiments, the humanized antibody retains all CDR sequences (e.g., a humanized mouse antibody containing all six CDRs from a mouse antibody). In other embodiments, one or more (I, II, III, IV, V, or VI) CDRs of the humanized antibody are altered relative to the original antibody; these are also referred to as one or more CDRs “derived from” one or more CDRs from the original antibody.

[0531] In the context of this invention, and particularly in the context of the humanized form of wtNbALFA, the term "humanized" preferably refers to an antibody that has a higher degree of frame sequence identity than wtNbALFA with a given human pedigree sequence. The given human pedigree sequence is preferably a human pedigree sequence with the highest sequence identity to the frame region of wtNbALFA. More preferably, the given human pedigree sequence is IGHV3-23 as shown in SEQ ID NO: 110 for the V region. 4 and SEQ ID NO: 126 show the IGHJ6 for the J segment. 01.

[0532] The term "germ sequence" refers to an amino acid sequence that represents an unaltered, naturally occurring protein derived from a gene sequence present in the germ cells (sperm or egg cells) of an organism. This sequence serves as basic genetic information, and the term "germ sequence" is used to distinguish proteins derived from the original gene composition from proteins containing any modifications (i.e., mutations) that may have been introduced into the protein sequence for a specific purpose.

[0533] As used herein, “frame identity” refers to the percentage identity between the germline coding frames of the closest hominin lineage (e.g., IGHV3-23 or one of its allotypes) and the frames of humanized or wtNbALFA lines compared to it. The frames are defined as described herein (SEQ ID NO: 99 to 109). Alignments were computed using the Needleman and Wunsch global alignment algorithm, which can be obtained using the Pairwise Sequence Alignment tool (https: / / www.ebi.ac.uk / Tools / psa / emboss_needle / ). A Blosum62 matrix was used as the matrix, with a gap open value of 10, a gap extended value of 0.5, an end gap penalty set to false, an end gap open value of 0, and an end gap extended value of 0.5. These values ​​represent the standard operating parameters of the tool. After running the algorithm, the identity value is given. As used herein, “overall frame identity” refers to the weighted average frame identity, which is calculated as the average identity of FR1 to FR4 normalized by the sequence lengths of the corresponding regions (i.e., 25, 15, 38, and 11 amino acids for FR1, FR2, FR3, and FR4, respectively; see Table 3).

[0534] The antibodies described herein preferably comprise a sequence having at least about 71%, at least about 75%, or preferably at least about 80% overall frame identity with the closest human pedigree sequence. The antibodies described herein may even comprise a sequence having at least about 85% or at least about 90% overall frame identity with the closest human pedigree sequence. The antibodies described herein preferably comprise a sequence similar to IGHV3-23 as shown in SEQ ID NO: 110. 4. Frame regions (for FR1, FR2, and FR3) and IGHJ6 as shown in SEQ ID NO:126 The frame region of 01 (for FR4) has a sequence with at least about 71%, at least about 75%, or preferably at least about 80% overall frame identity. The antibodies described herein may even contain sequences similar to IGHV3-23 as shown in SEQ ID NO: 110. 4. Frame regions (for FR1, FR2, and FR3) and IGHJ6 as shown in SEQ ID NO: 126 The 01 frame region (for FR4) has a sequence with at least about 85% or at least about 90% overall frame identity.

[0535] The term "V-region identity" as used in this paper refers to the percentage identity of FR1, CDR1, FR2, CDR2, FR3 encoded by the closest hominin lineage (e.g., IGHV3-23 or one of its allotypes) with the same FR1, CDR1, FR2, CDR2, FR3 as the humanized or wtNbALFA lineage. For this purpose, the DomainGapAlign tool from IMGT (https: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi) was used. The domain type was set to "V," and the species selection was either "any" or "Homo sapiens," with a Smith-Waterman score greater than 0. An E value of 200 was selected, with a query void penalty of -5 and a reference void penalty of -20. These values ​​represent the standard operating parameters of the tool. The target antibody sequence was inserted in FASTA format. The tool calculated the closest hominin sequences and provided a "V-region identity percentage" value for each of them. The “V-segment identity percentage” of the closest related person sequences is reported as V-segment identity in this paper.

[0536] The antibodies described herein preferably comprise a sequence having at least about 60%, at least about 65%, or preferably at least about 70% V region identity with the closest human germline sequence. The antibodies described herein preferably comprise IGHV3-23 as shown in SEQ ID NO: 110. 4. A sequence having at least about 60%, at least about 65%, or preferably at least about 70% V segment identity.

[0537] The antibodies described herein preferably have an affinity for the epitope tag shown in SEQ ID NO: 2 that is at least equivalent to that of the antibody in SEQ ID NO: 1. As used herein, "at least equivalent" preferably means a K0 of a given antibody. D The K value does not exceed that of the reference antibody (e.g., the antibody of SEQ ID NO: 1). D Approximately 2.5 times that of the given antibody and reference antibody. D It is preferred to use the same method for measurement. Any suitable method in the art for determining K can be used. D The known methods for determining K. D The methods include, but are not limited to, ELISA, surface plasmon resonance (SPR), or biolayer interferometry (BLI). Preferably, the affinity is measured by biolayer interferometry, and more preferably by a method essentially as described in Method 1.

[0538] Preferably, the antibody described herein targets the K-type epitope tag shown in SEQ ID NO: 2. D No more than wtNbALFA for the K of the epitope tag D Approximately 2.5 times. More preferably, the antibody described herein targets the K-type epitope tag shown in SEQ ID NO: 2. D No more than wtNbALFA for the K of the epitope tag D Approximately twice as much. More preferably, the antibody described herein targets the K-type epitope tag shown in SEQ ID NO: 2. D No more than wtNbALFA for the K of the epitope tag D Approximately 1.5 times. More preferably, the antibody described herein targets the K-type epitope tag shown in SEQ ID NO: 2. D No more than wtNbALFA for the K of the epitope tag D Approximately 1.2 times that.

[0539] The antibody described in this article targets the K-type epitope tag shown in SEQ ID NO: 2. D The K value for the epitope tag may not exceed wtNbALFA. D Preferably, the antibody described herein targets the K-type epitope tag shown in SEQ ID NO: 2. D K for the epitope tag can be lower than wtNbALFA.D Preferably, the antibody described herein targets the K-type epitope tag shown in SEQ ID NO: 2. D The K value of the epitope tag can be compared with wtNbALFA. D The antibody is at least about 1.5 times lower than the K-type epitope tag shown in SEQ ID NO: 2. Preferably, the antibody described herein is effective against the K-type epitope tag shown in SEQ ID NO: 2. D The K value of the epitope tag can be compared with wtNbALFA. D At least about 2.0 times lower. Preferably, the antibody described herein targets the K-type epitope tag shown in SEQ ID NO: 2. D The K value of the epitope tag can be compared with wtNbALFA. D It is at least about 2.5 times lower.

[0540] The antibody described in this article targets the K-type epitope tag shown in SEQ ID NO: 2. D K can be used for the epitope tag of wtNbALFA. D Approximately 2.5 times to approximately 100 times lower. The antibody described herein targets the K-type epitope tag shown in SEQ ID NO: 2. D K can be used for the epitope tag of wtNbALFA. D Approximately 2.5 times to approximately 50 times lower. The antibody described herein targets the K-type epitope tag shown in SEQ ID NO: 2. D K can be used for the epitope tag of wtNbALFA. D The antibody described herein is approximately 2.5 times to approximately 40 times lower than the K-type epitope tag shown in SEQ ID NO: 2. D K can be used for the epitope tag of wtNbALFA. D Approximately 2.5 times to approximately 30 times lower. The antibody described herein targets the K-type epitope tag shown in SEQ ID NO: 2. D K can be used for the epitope tag of wtNbALFA. D Approximately 2.5 times to approximately 20 times lower. The antibody described herein targets the K-type epitope tag shown in SEQ ID NO: 2. D K can be used for the epitope tag of wtNbALFA. D Approximately 2.5 times to approximately 15 times lower. The antibody described herein targets the K-type epitope tag shown in SEQ ID NO: 2. D K can be used for the epitope tag of wtNbALFA. D Approximately 2.5 times to approximately 10 times lower. The antibody described herein targets the K-type epitope tag shown in SEQ ID NO: 2. DK can be used for the epitope tag of wtNbALFA. D Approximately 2.5 times to approximately 5 times lower. The antibody described herein targets the K-type epitope tag shown in SEQ ID NO: 2. D K can be used for the epitope tag of wtNbALFA. D Approximately 2.5 times to approximately 4 times lower. The antibody described herein targets the K-type epitope tag shown in SEQ ID NO: 2. D K can be used for the epitope tag of wtNbALFA. D It is about 2.5 times to about 3 times lower than that.

[0541] Preferably, the antibody described herein targets the K-type epitope tag shown in SEQ ID NO: 2. D Antibodies not exceeding SEQ ID NO: 121 against the K of the epitope tag D Approximately 2.5 times. More preferably, the antibody described herein targets the K-type epitope tag shown in SEQ ID NO: 2. D Antibodies not exceeding SEQ ID NO: 121 for K-type epitope tags D Approximately twice as much. More preferably, the antibody described herein targets the K-type epitope tag shown in SEQ ID NO: 2. D Antibodies not exceeding SEQ ID NO: 121 for K-type epitope tags D Approximately 1.5 times. More preferably, the antibody described herein targets the K-type epitope tag shown in SEQ ID NO: 2. D Antibodies not exceeding SEQ ID NO: 121 for K-type epitope tags D Approximately 1.2 times higher. The antibody described herein targets the K-type epitope tag shown in SEQ ID NO: 2. D The antibody may have a K-value not exceeding that of SEQ ID NO: 121 against the epitope tag. D .

[0542] The antibody described in this article targets the K-type epitope tag shown in SEQ ID NO: 2. D The K-type antibody against the epitope tag shown in SEQ ID NO: 121 can be used. D Approximately 2.5 times to approximately 100 times lower. The antibody described herein targets the K-type epitope tag shown in SEQ ID NO: 2. D The K-type antibody against the epitope tag shown in SEQ ID NO: 121 can be used. D Approximately 2.5 times to approximately 50 times lower. The antibody described herein targets the K-type epitope tag shown in SEQ ID NO: 2. DThe K-type antibody can be the one shown in SEQ ID NO: 121 against the epitope tag. D The antibody described herein is approximately 2.5 times to approximately 40 times lower than the K-type epitope tag shown in SEQ ID NO: 2. D The K-type antibody against the epitope tag shown in SEQ ID NO: 121 can be used. D Approximately 2.5 times to approximately 30 times lower. The antibody described herein targets the K-type epitope tag shown in SEQ ID NO: 2. D The K-type antibody against the epitope tag shown in SEQ ID NO: 121 can be used. D Approximately 2.5 times to approximately 20 times lower. The antibody described herein targets the K-type epitope tag shown in SEQ ID NO: 2. D The K-type antibody against the epitope tag shown in SEQ ID NO: 121 can be used. D Approximately 2.5 times to approximately 15 times lower. The antibody described herein targets the K-type epitope tag shown in SEQ ID NO: 2. D The K-type antibody against the epitope tag shown in SEQ ID NO: 121 can be used. D Approximately 2.5 times to approximately 10 times lower. The antibody described herein targets the K-type epitope tag shown in SEQ ID NO: 2. D The K-type antibody against the epitope tag shown in SEQ ID NO: 121 can be used. D Approximately 2.5 times to approximately 5 times lower. The antibody described herein targets the K-type epitope tag shown in SEQ ID NO: 2. D The K-type antibody against the epitope tag shown in SEQ ID NO: 121 can be used. D Approximately 2.5 times to approximately 4 times lower. The antibody described herein targets the K-type epitope tag shown in SEQ ID NO: 2. D The K-type antibody against the epitope tag shown in SEQ ID NO: 121 can be used. D It is about 2.5 times to about 3 times lower than that.

[0543] Preferably, the antibody described herein targets the K-type epitope tag shown in SEQ ID NO: 2. D Antibodies not exceeding SEQ ID NO: 123 against the K of the epitope tag D Approximately 2.5 times. More preferably, the antibody described herein targets the K-type epitope tag shown in SEQ ID NO: 2. D Antibodies not exceeding SEQ ID NO: 123 against the K-type epitope tag DApproximately twice as much. More preferably, the antibody described herein targets the K-type epitope tag shown in SEQ ID NO: 2. D Antibodies not exceeding SEQ ID NO: 123 against the K-type epitope tag D Approximately 1.5 times. More preferably, the antibody described herein targets the K-type epitope tag shown in SEQ ID NO: 2. D Antibodies not exceeding SEQ ID NO: 123 against the K-type epitope tag D Approximately 1.2 times higher. The antibody described herein targets the K-type epitope tag shown in SEQ ID NO: 2. D The antibody may have a K-value not exceeding that of SEQ ID NO: 123 against the epitope tag. D .

[0544] The antibody described in this article targets the K-type epitope tag shown in SEQ ID NO: 2. D The K-type antibody against the epitope tag shown in SEQ ID NO: 123 can be used. D Approximately 2.5 times to approximately 100 times lower. The antibody described herein targets the K-type epitope tag shown in SEQ ID NO: 2. D The K-type antibody against the epitope tag shown in SEQ ID NO: 123 can be used. D Approximately 2.5 times to approximately 50 times lower. The antibody described herein targets the K-type epitope tag shown in SEQ ID NO: 2. D The K-type antibody against the epitope tag shown in SEQ ID NO: 123 can be used. D The antibody described herein is approximately 2.5 times to approximately 40 times lower than the K-type epitope tag shown in SEQ ID NO: 2. D The K-type antibody against the epitope tag shown in SEQ ID NO: 123 can be used. D Approximately 2.5 times to approximately 30 times lower. The antibody described herein targets the K-type epitope tag shown in SEQ ID NO: 2. D The K-type antibody against the epitope tag shown in SEQ ID NO: 123 can be used. D Approximately 2.5 times to approximately 20 times lower. The antibody described herein targets the K-type epitope tag shown in SEQ ID NO: 2. D The K-type antibody against the epitope tag shown in SEQ ID NO: 123 can be used. D Approximately 2.5 times to approximately 15 times lower. The antibody described herein targets the K-type epitope tag shown in SEQ ID NO: 2. D The K-type antibody against the epitope tag shown in SEQ ID NO: 123 can be used.D Approximately 2.5 times to approximately 10 times lower. The antibody described herein targets the K-type epitope tag shown in SEQ ID NO: 2. D The K-type antibody against the epitope tag shown in SEQ ID NO: 123 can be used. D Approximately 2.5 times to approximately 5 times lower. The antibody described herein targets the K-type epitope tag shown in SEQ ID NO: 2. D The K-type antibody against the epitope tag shown in SEQ ID NO: 123 can be used. D Approximately 2.5 times to approximately 4 times lower. The antibody described herein targets the K-type epitope tag shown in SEQ ID NO: 2. D The K-type antibody against the epitope tag shown in SEQ ID NO: 123 can be used. D It is about 2.5 times to about 3 times lower than that.

[0545] The antibody described herein may have an epitope tag of approximately 5 × 10⁻⁶. -10 M or lower, preferably 3×10 -10 M or lower K D This is preferably measured via biolayer interferometry, preferably via a method essentially as described in Method 1. The antibody described herein may also have approximately 2 × 10⁻⁶ epitope tags as shown in SEQ ID NO: 2. -10 M or lower, or 2×10 -10 M or lower K D This is preferably measured by biological layer interferometry, preferably by a method as described in Method 1.

[0546] The antibodies described herein preferably have a higher Tm compared to the antibody of SEQ ID NO: 1.

[0547] As used in this article, the term "Tm" refers to the temperature at which half of all molecules are denatured and half of all molecules retain their conformation. For example, a Tm of 60°C means that at this temperature, 50% of the antibody present in the sample remains undenatured, while the other 50% is denatured. Tm can be measured essentially as described in Method 2.

[0548] The antibodies described herein preferably have a Tm of at least about 62°C, more preferably at least 63°C. The antibodies described herein may also have a Tm of at least about 64°C, more preferably at least 65°C, or even at least about 66°C. The Tm is preferably measured substantially as described in Method 2.

[0549] The antibodies described in this article preferably contain a protein A binding site.

[0550] As used in this article, staphylococcal protein A (SpA) or protein A refers to a molecule derived from the cell wall of the bacterium *Staphylococcus aureus*, which has a strong affinity for immunoglobulins, particularly human IgG. SpA's unique ability to specifically interact with the Fc region of antibodies offers significant potential for various biotechnological applications, including antibody purification, immobilization, and detection in diagnostic and therapeutic settings. SpA affinity purification is currently the most widely used affinity system for antibody purification.

[0551] In the context of this application, it is believed that up to 13 amino acid positions may be involved in SpA recognition: residues at positions 15, 17, 19, 60, 62, 67, 68, 69, 71, 73, 84, 86, and 87 of the sequence corresponding to SEQ ID NO: 1. In particular, residues Gly 15, Ser 17, Arg 19, Thr 60, Tyr 62, Lys 67, Gly 68, Arg 69, Thr 71, Ser 73, Gln 84, Asn 86, and Ser 87 are important for SpA binding. Residues Gly 15, Ser 17, Arg 19, Tyr 62, Gly 68, Arg 69, Thr 71, and Gln 84 are already present in wt NbALFA. wtALFA does not contain residues Ser 73, Asn 86, and Ser 87, but all receptor frameworks used to generate CDR variants 1 to 7 contain them. However, positions 60 and 67 are both located within the CDR2 region (position 67 is located within the CDR2 region as determined by Kabat). Since wtNbALFA contains residues Ala 60 and Gln67, these two residues need to be mutated to improve SpA binding. Although these two residues are located within the CDR2 region or within the CDR2 region as determined by Kabat, the inventors of this application have unexpectedly discovered that mutations introducing Ala 60→Thr and / or Gln67→Lys preserve a high binding affinity to the epitope tag of SEQ ID NO: 2, as shown in variants 10 to 13 and 15. However, mutations introducing Ala 60→Thr and / or Gln 67→Lys can promote SpA binding, as shown in Examples 3 and Table 4.

[0552] The antibodies described herein may contain a Thr residue at position 60 of the sequence corresponding to SEQ ID NO: 1. The antibodies described herein may contain a Lys residue at position 67 of the sequence corresponding to SEQ ID NO: 1.

[0553] The antibody described herein may contain the following amino acid residues at positions 60 and 67 of the sequence corresponding to SEQ ID NO: 1: Thr at position 60 and Lys at position 67.

[0554] The antibodies described herein may contain a Gly residue at position 15 of the sequence. The antibodies described herein may contain a Ser residue at position 17 of the sequence. The antibodies described herein may contain an Arg residue at position 19 of the sequence. The antibodies described herein may contain a Tyr residue at position 62 of the sequence. The antibodies described herein may contain a Gly residue at position 68 of the sequence. The antibodies described herein may contain an Arg residue at position 69 of the sequence. The antibodies described herein may contain a Thr residue at position 71 of the sequence. The antibodies described herein may contain a Ser residue at position 73 of the sequence. The antibodies described herein may contain a Gln residue at position 84 of the sequence. The antibodies described herein may contain an Asn residue at position 86 of the sequence. The antibodies described herein may contain a Ser residue at position 87 of the sequence.

[0555] The antibody described herein may contain at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or preferably all of the following amino acid residues at positions 15, 17, 19, 62, 68, 69, 71, 73, 84, 86, and 87 of the sequence corresponding to SEQ ID NO: 1: Gly at position 15, Ser at position 17, Arg at position 19, Tyr at position 62, Gly at position 68, Arg at position 69, Thr at position 71, Ser at position 73, Gln at position 84, Asn at position 86, and Ser at position 87.

[0556] The antibody described herein may contain at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, or preferably all of the following amino acid residues at positions 15, 17, 19, 60, 62, 67, 68, 69, 71, 73, 84, 86, and 87 of the sequence corresponding to SEQ ID NO: 1: Gly at position 15, Ser at position 17, Arg at position 19, Thr at position 60, Tyr at position 62, Lys at position 67, Gly at position 68, Arg at position 69, Thr at position 71, Ser at position 73, Gln at position 84, Asn at position 86, and Ser at position 87.

[0557] As described herein, the ALFA tag may be part of a CAR or a docking compound (depending on whether the single-domain antibody is located on the docking compound or the CAR, respectively). Preferably, the ALFA tag is part of the docking compound (in which case the single-domain antibody is located on the CAR). In some embodiments, the ALFA tag described herein (for the compositions and methods described herein) comprises the amino acid sequence X1-X2-LE-X5-E-X7-RRRL-X12-X13 (SEQ ID NO:3), wherein X1 is G or S or P or T, X2 is R or G or P, X5 is E or Q, X7 is L or I, X12 is S or T or P or A or D or E, and wherein X13 is P or A or S or K or D or E or no amino acid. In some embodiments, the ALFA tag described herein comprises a peptide represented by any one of SEQ ID NO: 2 to 93. Preferably, the ALFA tag described herein comprises the amino acid sequence of SEQ ID NO:2.

[0558] SEQ ID NO: 3 defines the core structure of the antibody-binding peptide described herein, which may further include up to two additional amino acids at the N-terminus and up to two additional amino acids at the C-terminus. Such additional amino acids at the ends of the core structure of the peptide generally do not necessarily affect the secondary structure of the peptide or the specific binding of the peptide to its specific antibody, but may serve as linker structures in the fusion protein. Therefore, the type and number of additional amino acids may depend on the position of the peptide in the fusion protein and may vary depending on whether the peptide is located at the N-terminus or C-terminus of the polypeptide, or somewhere in between. Thus, the peptide sequence may include amino acids Xa-Xb at the N-terminus of X1, where Xa is D, S, G, M, P, or no amino acid, and Xb is S, D, P, M, R, G, or no amino acid. The peptide may also include amino acids Xy-Xz at the C-terminus of X13, where Xy is G, S, P, D, A, E, K, or no amino acid, and Xz is S, P, or no amino acid.

[0559] In some embodiments, the ALFA tag described herein (for the compositions and methods described herein) comprises the sequence X1-X2-LE-X5-ELRRRL-X12-X13 (SEQ ID NO: 4), wherein X1 is S or T, X2 is R or G, X5 is E or Q, X12 is T or D or E, and X13 is A or D or E or has no amino acid. The ALFA tag may comprise the sequence SRLEEELRRRLTE (SEQ ID NO: 2) or a variant thereof, wherein the variant has 1 to 5 mutations compared to SEQ ID NO: 2, selected from: S1→T, R2→G, E5→Q, T12→D and T12→E, E13→A, E13→D, and E13 deletion. The variant may have the following mutations compared to SEQ ID NO: 2: (a) S1→T and E13→A; (b) R2→G; (c) R2→G and E5→Q; (d) R2→G, E5→Q and E13→A; (e) R2→G, E5→Q, T12→D and E13→A; (f) R2→G, E5→Q, T12→E and E13→A; (g) T12→D and E13→A; (h) T12→E and E13→A; (i) E13→A; (j) E13→D; or (k) E13 deletion. The ALFA tag containing the sequence SEQ ID NO: 4 as its core structure may contain amino acids Xa-Xb at the N-terminus of X1, where Xa is S, G, M, P, or no amino acid, and Xb is R, G, S, P, M, or no amino acid. Xa-Xb can be selected from: P, MP, GR, PG, PS, SP, GP, SP, M, and MS, preferably P or MP. Such peptides may also contain amino acids Xy-Xz at the C-terminus of X13, wherein Xy-Xz is P, D, A, or no amino acid, and Xz is P, S, or no amino acid. Xy-Xz can be selected from: no amino acid, P, DP, A, and AS, preferably no amino acid or P. The ALFA tag may contain a combination of Xa-Xb and Xy-Xz, selected from: (a) MP and P; (b) P and P; and (c) P and no amino acid.

[0560] The ALFA tag described herein (for the compositions and methods described herein) preferably contains the sequence SRLEEELRRRLTE (SEQ ID NO: 2).

[0561] The ALFA tag described herein (for the compositions and methods described herein) may contain a sequence selected from the following:

[0562]

[0563] The ALFA tag described herein (for the compositions and methods described herein) may comprise the sequence X1-X2-LE-X5-E-X7-RRRL-X12-X13 (SEQ ID NO: 31), wherein X1 is G or S or P, X2 is R or G, X5 is E or Q, X7 is L or I, X12 is S or T or P or A, and X13 is P or A or S or no amino acid.

[0564] The ALFA tag described herein (for the compositions and methods described herein) may comprise the sequence GRLEEELRRRLS (SEQ ID NO: 32) or a variant thereof, wherein the variant has 1 to 6 mutations compared to SEQ ID NO: 32, selected from: G1→S, G1→P, R2→G, E5→Q, L7→I, S12→T, S12→P, S12→A, P13 addition, A13 addition, and S13 addition. The variant may have the following mutations compared to SEQ ID NO: 32: (a) G1→S, R2→G, E5→Q, and P13 addition; (b) R2→G, E5→Q, S12→T, and A13 addition; (c) G1→P, R2→G, E5→Q, S12→T, and A13 addition; (d) G1→S, R2→G, E5→Q, S12→T, and P13 addition; (e) G1→S (f) G1→S, R2→G, E5→Q, and S12→T; (g) G1→S, R2→G, E5→Q, S12→T, and A13; (h) G1→S, S12→P, and P13; (i) G1→S, R2→G, E5→Q, S12→T, and P13; (j) E5→Q, L7→I, and P13; (k) P13; or (l) S12→A. The ALFA tag containing sequence SEQ ID NO: 31 as its core structure may contain amino acids Xa-Xb at the N-terminus of X1, where Xa is M, S, P, D, G, or no amino acid, and Xb is S, D, P, or no amino acid. Xa-Xb may be selected from: MS, SD, PD, PS, DS, SP, and GP, preferably MS. Such an ALFA tag may also include amino acids Xy-Xz located at the C-terminus of X13, where Xy is G, P, A, E, K, S, or no amino acid, and Xz is P, S, or no amino acid. Xy-Xz may be selected from: no amino acid, G, P, A, EP, AS, K, and S, preferably no amino acid. The ALFA tag may include a combination of Xa-Xb and Xy-Xz, which is MS and no amino acid.

[0565] The ALFA tag described herein (for the compositions and methods described herein) may contain a sequence selected from the following:

[0566]

[0567] The ALFA tag described herein (used in the compositions and methods described herein) may comprise a peptide having the sequence X1-X2-LE-X5-ELRRRL-X12-X13 (SEQ ID NO: 49) as its core structure, wherein X1 is S, G, or P; X2 is R, G, or P; X5 is E or Q; X12 is S, T, D, or E; and X13 is P, A, D, or no amino acid. The peptide having the sequence SEQ ID NO: 49 as its core structure may comprise amino acids Xa-Xb at the N-terminus of X1, wherein Xa is P, D, S, G, or no amino acid, and Xb is D, S, P, or no amino acid. Such a peptide may also comprise amino acids Xy-Xz at the C-terminus of X13, wherein Xy is G, P, E, D, S, or no amino acid, and Xz is P or no amino acid.

[0568] The ALFA tag described herein (for the compositions and methods described herein) may contain a sequence selected from the following:

[0569]

[0570] The ALFA tag described herein (used in the compositions and methods described herein) may comprise a peptide having the sequence X1-X2-X3-X4-X5-X6-X7-X8-X9-RL-X12-X13 (SEQ ID NO: 66) as its core structure, wherein X1 is G or S, X2 is R or G or A or E, X3 is L or V, X4 is E or Q, X5 is E or Q, X6 is E or Q, X7 is L or I or V, X8 is R or A or Q or E, X9 is R or A or Q or E, X12 is S or T or L or no amino acid, and X13 is K or P or S or no amino acid. The peptide having the sequence SEQ ID NO: 66 as its core structure may comprise amino acids Xa-Xb located at the N-terminus of X1, wherein Xa is D or S or G or M or no amino acid, and Xb is S or D or P or M or no amino acid. Such peptides may also contain amino acids Xy-Xz at the C-terminus of X13, where Xy is G, S, P or no amino acid, and Xz is S or no amino acid.

[0571] The ALFA tag described herein (for the compositions and methods described herein) may contain a sequence selected from the following:

[0572]

[0573] In some embodiments, the ALFA tag described herein (for the compositions and methods described herein) comprises the amino acid sequence: -AA0-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-AA10-AA11-AA12-AA13-AA14-, wherein the amino acids AA0, AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, AA10, AA11, AA12, AA13, and AA14 are as follows:

[0574] AA0 indicates Pro or is missing;

[0575] AA1 can be Ser, Gly, Thr, or Pro;

[0576] AA2 can be Arg, Gly, Ala, Glu, or Pro;

[0577] AA3 is Leu, Ile, or Val;

[0578] AA4 is either Glu or Gln;

[0579] AA5 is either Glu or Gln;

[0580] AA6 is either Glu or Gln;

[0581] AA7 is Leu, Ile, or Val;

[0582] AA8 can be Arg, Ala, Gln, or Glu;

[0583] AA9 can be Arg, Ala, Gln, or Glu;

[0584] AA10 is Arg;

[0585] AA11 is Leu;

[0586] AA12 is Thr, Ser, Asp, Glu, Pro, Ala, or missing;

[0587] AA13 is Glu, Lys, Pro, Ser, Ala, Asp, or is missing; and

[0588] AA14 indicates Pro or missing.

[0589] In some implementations, the ALFA label contains a selection from... , , and sequence.

[0590] In some embodiments, the ALFA tag comprises a cyclic amino acid sequence: -AA0-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-AA10-AA11-AA12-AA13-AA14-, wherein the side chains of any two (X1, X2) of amino acids AA0, AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, AA10, AA11, AA12, AA13, and AA14 are covalently linked, and wherein the amino acids AA0, AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, AA10, AA11, AA12, AA13, and AA14 that are not X1 and X2 are as follows:

[0591] AA0 indicates Pro or is missing;

[0592] AA1 can be Ser, Gly, Thr, or Pro;

[0593] AA2 can be Arg, Gly, Ala, Glu, or Pro;

[0594] AA3 is Leu, Ile, or Val;

[0595] AA4 is either Glu or Gln;

[0596] AA5 is either Glu or Gln;

[0597] AA6 is either Glu or Gln;

[0598] AA7 is Leu, Ile, or Val;

[0599] AA8 can be Arg, Ala, Gln, or Glu;

[0600] AA9 can be Arg, Ala, Gln, or Glu;

[0601] AA10 is Arg;

[0602] AA11 is Leu;

[0603] AA12 is Thr, Ser, Asp, Glu, Pro, Ala, or missing;

[0604] AA13 is Glu, Lys, Pro, Ser, Ala, Asp, or is missing; and

[0605] AA14 indicates Pro or missing.

[0606] In some implementations, X1 and X2 are separated by 2 or 3 amino acids.

[0607] In some implementations, AA5 is X1 and AA9 is X2, AA5 is X1 and AA8 is X2, AA9 is X1 and AA13 is X2, AA6 is X1 and AA9 is X2, AA9 is X1 and AA12 is X2, AA10 is X1 and AA13 is X2, AA6 is X1 and AA10 is X2, or AA4 is X1 and AA8 is X2.

[0608] In some implementations, the ALFA tag comprises a cyclic amino acid sequence selected from the following:

[0609] The side chains of amino acid residues X1 and X2 are covalently linked;

[0610] AA0 indicates Pro or is missing;

[0611] AA1 can be Ser, Gly, Thr, or Pro;

[0612] AA2 can be Arg, Gly, Ala, Glu, or Pro;

[0613] AA3 is Leu, Ile, or Val;

[0614] AA4 is either Glu or Gln;

[0615] AA5 is either Glu or Gln;

[0616] AA6 is either Glu or Gln;

[0617] AA7 is Leu, Ile, or Val;

[0618] AA8 can be Arg, Ala, Gln, or Glu;

[0619] AA9 can be Arg, Ala, Gln, or Glu;

[0620] AA12 is Thr, Ser, Asp, Glu, Pro, Ala, or missing;

[0621] AA13 is Glu, Lys, Pro, Ser, Ala, Asp, or is missing; and

[0622] AA14 indicates Pro or missing.

[0623] In some embodiments, X1 and X2 of the peptides disclosed herein are covalently linked by an amide, disulfide, thioether, ether, ester, thioester, thioamide, alkylene, alkenylene, alkyne and / or 1,2,3-triazole.

[0624] In some embodiments, the cyclized amino acid sequence described herein is generated by linking the amino group of the side chain of one of X1 and X2 to the carboxyl group of the side chain of the other of X1 and X2 via an amide bond. The side chain amino group of an amino acid having a pendant amino group (e.g., lysine or a lysine derivative) and the side chain carboxyl group of an acidic amino acid (e.g., aspartic acid, glutamic acid, or a derivative thereof) can be used to generate the cyclized amino acid sequence via an amide bond.

[0625] In some embodiments, the cyclized amino acid sequence described herein is generated by linking the side chain thiol group of one of X1 and X2 to the side chain thiol group of the other of X1 and X2 via a disulfide bond. Thiol-containing amino acids include cysteine ​​and other thiol-containing amino acids (such as Pen).

[0626] In some implementations, X1 and X2 are independently selected from the following: Glu, DGlu, Asp, DAsp, Lys, DLys, hLys, DhLys, Orn, DOrn, Dab, DDab, Dap, DDap, Cys, DCys, hCys, DhCys, Pen, and DPen, provided that when X1 is Glu, DGlu, Asp, or DAsp, X2 is Lys, DLys, hLys, DhLys, O rn, DOrn, Dab, DDab, Dap, or DDap; when X1 is Lys, DLys, hLys, DhLys, Orn, DOrn, Dab, DDab, Dap, or DDap, X2 is Glu, DGlu, Asp, or DAsp; and when X1 is Cys, DCys, hCys, DhCys, Pen, or DPen, X2 is Cys, DCys, hCys, DhCys, Pen, or DPen.

[0627] In some embodiments, X1 is Glu and X2 is Lys. In some embodiments, -cyclo(Glu--------Lys)-, -c(Glu-------Lys)-, -cyclo(E-------K)-, -c(E---------K)-, -E------K-cyclo, or -cycloE----cycloK- includes the following structures:

[0628] .

[0629] In some implementations, X1 is Lys and X2 is Glu. In some implementations, the ring (Lys------Glu)-, -c(Lys------Glu)-, -ring (K------E)-, -c(K-------E)-, -K-----E-ring or ring K------ring E- includes the following structures:

[0630] .

[0631] In some implementations, X1 is Cys and X2 is Cys. In some implementations, -ring(Cys------Cys)-, c(Cys------Cys)-, -ring(C------C)-, -c(C--------C)-, -C----C-ring or -ringC-----ringC- includes the following structures:

[0632] .

[0633] Some specific cyclic amino acid sequences of the general formula identified above include, for example:

[0634] In some embodiments, the cyclic peptide is linked to a 3-mercaptopropionyl moiety via the α-amine portion of the leftmost amino acid in the cyclic peptide. In some embodiments, the rightmost amino acid in the cyclic peptide comprises an amide.

[0635] In some implementations, the cyclic amino acid sequence is selected from one of the following:

[0636] In some embodiments, the cyclic amino acid sequence is -Ser-Arg-Leu-Glu-cyclic (Glu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-. In other embodiments, the cyclic amino acid sequence is -Ser-Arg-Leu-Glu-cyclic (Asp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-. In still other embodiments, the cyclic amino acid sequence is -Ser-Arg-Leu-Glu-cyclic (Glu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-. In yet still other embodiments, the cyclic amino acid sequence is -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclic (Lys-Arg-Leu-Thr-Glu)-.

[0637] Cyclic peptides can have different cyclic bridging moieties that form ring structures. Preferably, the chemically stable bridging moieties are contained within the ring structure, such as amide, lactone, ether, thioether, disulfide group, alkylene, alkenyl, or 1,2,3-triazole groups. The following are some examples illustrating the variability of the bridging moieties in peptides:

[0638]

[0639] In one aspect, this disclosure provides a complex in which a genetically modified immune effector cell expressing a chimeric antigen receptor (CAR) binds to a docking compound. Thus, the genetically modified immune effector cell expressing a chimeric antigen receptor (CAR) and the docking compound both contain portions that interact with each other.

[0640] This article describes different embodiments of the combined genetically modified immune effector cells expressing chimeric antigen receptors (CARs) and the docking compound.

[0641] In some embodiments, the docking compound comprises an ALFA tag. In these embodiments, the portion of the antigen receptor on the immune effector cell that binds to the docking compound comprises an NbALFA-nanobody (NbALFA). In some embodiments, the docking compound may have a structure selected from: ALFA tag × anti-target cell antigen DARPin, ALFA tag × anti-target cell antigen VHH, and ALFA tag × anti-target cell antigen scFv.

[0642] In some embodiments, the single-domain antibody (used in the compositions and methods described herein) is characterized by the following:

[0643] Project 1: A single-domain antibody that specifically binds to an epitope tag containing the sequence SRLEEELRRRLTE (SEQ ID NO: 2), wherein the antibody comprises the CDR1 sequence as shown in SEQ ID NO: 94, the CDR2 sequence as shown in SEQ ID NO: 97, and the CDR3 sequence as shown in SEQ ID NO: 98; wherein the antibody further comprises the following amino acid residues at positions 47, 48, and 50 corresponding to the sequence in SEQ ID NO: 1: Arg at position 47, Arg at position 48, and Met at position 50; and wherein the antibody comprises a sequence having at least 90% sequence identity with any one of SEQ ID NO: 118 to 125.

[0644] Project 2: The antibody described in Project 1, wherein the antibody is humanized.

[0645] Item 3: The antibody of any one of the preceding items, wherein the antibody comprises a sequence having at least about 70% V segment identity with the closest human lineage sequence.

[0646] Item 4: The antibody described in Item 3, wherein the human lineage sequence is IGHV3-23 as shown in SEQ ID NO: 110. 4.

[0647] Item 5: The antibody according to any one of the preceding items, wherein the antibody has an affinity for the epitope tag shown in SEQ ID NO: 2 that is at least equivalent to the affinity of the antibody in SEQ ID NO: 1, wherein at least equivalent preferably means that the K of the antibody is... D K not exceeding SEQ ID NO: 1 D 2.5 times, which is preferably measured by biological layer interferometry, preferably by a method as described in Method 1.

[0648] Item 6: The antibody according to any one of the preceding items, wherein the antibody is K-type of the epitope tag shown in SEQ ID NO: 2. D Approximately 5 × 10 -10 M or lower, preferably 3×10 -10 M or lower, which is preferably measured by biological layer interferometry, preferably by a method as described in Method 1.

[0649] Item 7: The antibody of any one of the preceding items, wherein the antibody has a higher Tm compared with the antibody of SEQ ID NO: 1.

[0650] Item 8: The antibody of any one of the preceding items, wherein the antibody has a Tm of at least about 63°C, wherein the Tm is preferably measured substantially as described in Method 2.

[0651] Item 9: The antibody according to any one of the preceding items, wherein the antibody contains a His residue at position 99 of the sequence corresponding to SEQ ID NO: 1.

[0652] Item 10: The antibody according to any one of the preceding items, wherein the antibody contains a Val residue at position 100 of the sequence corresponding to SEQ ID NO: 1.

[0653] Item 11: The antibody according to any one of the preceding items, wherein the antibody contains a Val residue at position 5 of the sequence corresponding to SEQ ID NO: 1.

[0654] Item 12: The antibody of any one of the preceding items, wherein the antibody contains a Tyr residue at position 40 of the sequence corresponding to SEQ ID NO: 1.

[0655] Item 13: The antibody according to any one of the preceding items, wherein the antibody contains a Gln residue at position 117 of the sequence corresponding to SEQ ID NO: 1.

[0656] Item 14: An antibody according to any one of the preceding items, wherein the antibody comprises an FR1 sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with, or is identical to, the sequence of SEQ ID NO: 99.

[0657] Item 15: An antibody according to any one of the preceding items, wherein the antibody comprises an FR2 sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with, or is identical to, the sequence of SEQ ID NO: 102.

[0658] Item 16: An antibody according to any one of the preceding items, wherein the antibody comprises an FR3 sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with, or is identical to, the sequence of SEQ ID NO: 108.

[0659] Item 17: An antibody according to any one of the preceding items, wherein the antibody comprises an FR4 sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with, or being identical to, the sequence of SEQ ID NO: 109.

[0660] Item 18: The antibody according to any one of the preceding items, wherein the antibody comprises FR1, FR2, FR3 and FR4 sequences, which have at least 80%, at least 85%, at least 90% or at least 95% sequence identity with, or are identical to, the sequences shown in SEQ ID NO: 99, 102, 108 and 109, respectively.

[0661] Item 19: The antibody according to any one of the preceding items, wherein the frame sequence of the antibody is defined by the amino acid positions corresponding to positions 1 to 25, 39 to 53, 61 to 98 and 112 to 122 of SEQ ID NO: 1.

[0662] Item 20: The antibody described in any one of the preceding items, wherein the antibody comprises a protein A binding site.

[0663] Item 21: The antibody according to any one of the preceding items, wherein the antibody contains a Thr residue at position 60 of the sequence corresponding to SEQ ID NO: 1.

[0664] Item 22: The antibody according to any one of the preceding items, wherein the antibody contains a Lys residue at position 67 of the sequence corresponding to SEQ ID NO: 1.

[0665] Item 23: The antibody of any one of the preceding items, wherein the antibody contains the following amino acid residues at positions 60 and 67 of the sequence corresponding to SEQ ID NO: 1: Thr at position 60 and Lys at position 67.

[0666] Item 24: The antibody of any one of the preceding items, wherein the antibody contains a Tyr residue at position 61 of the sequence corresponding to SEQ ID NO: 1.

[0667] Item 25: An antibody according to any one of the preceding items, wherein the antibody contains an Asp residue at position 64 of the sequence corresponding to SEQ ID NO: 1.

[0668] Item 26: An antibody according to any one of the preceding items, wherein the antibody comprises the CDR2 sequence shown in SEQ ID NO: 95 or 96.

[0669] Item 27: An antibody according to any one of the preceding items, wherein the antibody comprises an FR2 sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with, or is identical to, any one of SEQ ID NO: 100, 101.

[0670] Item 28: An antibody according to any one of the preceding items, wherein the antibody comprises an FR3 sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with, or being identical to, any one of SEQ ID NO: 103 to 107.

[0671] Item 29: An antibody according to any one of the preceding items, wherein the antibody comprises a sequence having at least 95% sequence identity with any one of SEQ ID NO: 118 to 125.

[0672] Item 30: An antibody according to any one of the preceding items, wherein the antibody comprises the sequence shown in any one of SEQ ID NO: 118 to 125.

[0673] In some embodiments, the antibody described in any one of items 1 to 30 is used in combination with an ALFA tag comprising the amino acid sequence X1-X2-LE-X5-E-X7-RRRL-X12-X13 (SEQ ID NO: 3), wherein X1 is G or S or P or T, X2 is R or G or P, X5 is E or Q, X7 is L or I, X12 is S or T or P or A or D or E, and X13 is P or A or S or K or D or E or no amino acid.

[0674] In some embodiments, the antibody described in any one of items 1 to 30 is used in combination with an ALFA tag comprising the sequence X1-X2-LE-X5-ELRRRL-X12-X13 (SEQ ID NO: 4), wherein X1 is S or T, X2 is R or G, X5 is E or Q, X12 is T or D or E, and X13 is A or D or E or has no amino acid.

[0675] In some embodiments, the antibody described in any one of items 1 to 30 is used in combination with an ALFA tag containing the sequence SRLEEELRRRLTE (SEQ ID NO: 2).

[0676] In some embodiments, the antibody described in any one of items 1 to 30 is used in combination with an ALFA tag comprising a sequence selected from:

[0677]

[0678] In some embodiments, the antibody described in any one of items 1 to 30 is used in combination with an ALFA tag comprising the sequence X1-X2-LE-X5-E-X7-RRRL-X12-X13 (SEQ ID NO: 31), wherein X1 is G or S or P, X2 is R or G, X5 is E or Q, X7 is L or I, X12 is S or T or P or A, and X13 is P or A or S or has no amino acids.

[0679] In some embodiments, the antibody described in any one of items 1 to 30 is used in combination with an ALFA tag comprising the sequence GRLEEELRRRLS (SEQ ID NO: 32) or a variant thereof, wherein the variant has 1 to 6 mutations selected from the group consisting of G1→S, G1→P, R2→G, E5→Q, L7→I, S12→T, S12→P, S12→A, P13 addition, A13 addition, and S13 addition compared to (SEQ ID NO: 32).

[0680] In some embodiments, the antibody described in any one of items 1 to 30 is used in an ALFA tag assembly, the ALFA tag comprising a sequence selected from:

[0681]

[0682] Immune effector cells

[0683] The immune effector cells used in conjunction with the methods and reagents described herein specifically include immune effector cells such as: cells with lysis potential, particularly lymphocytes, and preferably T cells, especially cytotoxic lymphocytes, preferably selected from cytotoxic T cells, natural killer (NK) cells, and lymphokine-activated killer (LAK) cells. Once activated, each of these cytotoxic lymphocytes triggers the destruction of the target cell. For example, cytotoxic T cells trigger the destruction of the target cell by any one or two of the following: First, upon activation, the T cell releases cytotoxins such as perforin, granzyme, and granulysin. Perforin and granulysin create pores in the target cell, while granzyme enters the cell and triggers a caspase cascade in the cytoplasm, which induces apoptosis (programmed cell death). Second, apoptosis can be induced by Fas-Fas ligand interactions between the T cell and the target cell. Although heterologous or allogeneic cells can be used, the cells used in conjunction with this disclosure are preferably autologous cells.

[0684] In the context of this disclosure, the term "effective function" includes any function mediated by components of the immune system that results in, for example, killing diseased cells such as tumor cells, or inhibiting tumor growth and / or development, including inhibiting tumor dissemination and metastasis. Preferably, in the context of this disclosure, effector function is a T cell-mediated effector function. In helper T cells (CD4+) + In the case of T cells, such functions include releasing cytokines and / or activating CD8. + Lymphocytes (CTLs) and / or B cells, and in the case of CTLs, this includes, for example, eliminating cells (i.e. cells characterized by expressing antigens) through apoptosis or perforin-mediated cell lysis, producing cytokines such as IFN-γ and TNF-α, and specifically cytolytic killing of target cells expressing antigens.

[0685] In the context of this disclosure, the terms "immune effector cells" or "immune reactive cells" refer to cells that perform effector functions during an immune response. In some embodiments, "immune effector cells" are capable of binding antigens and mediating immune responses, said antigens being presented, for example, in the context of MHC on the cell or expressed on the cell surface. For example, immune effector cells include T cells (cytotoxic T cells, helper T cells, tumor-infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages, and dendritic cells. Preferably, in the context of this disclosure, "immune effector cells" are T cells, preferably CD4+. + and / or CD8 + T cells, CD8 are the most preferred. + T cells. The term "immune effector cells" also includes cells that can mature into immune cells (such as T cells, especially helper T cells or cytolytic T cells) in response to appropriate stimuli. Immune effector cells contain CD34. + Hematopoietic stem cells, immature and mature T cells, and immature and mature B cells. When exposed to antigens, the differentiation of T cell precursors into cytolytic T cells is analogous to clonal selection in the immune system.

[0686] In some implementations, the genetically modified immune effector cells are CAR-expressing immune effector cells.

[0687] The immune effector cells used in this study may express endogenous antigen receptors, such as T-cell receptors or B-cell receptors, or may lack endogenous antigen receptor expression.

[0688] "Lymphoid cells" are cells that, optionally after appropriate modification (e.g., after transfer of antigen receptors such as TCRs or CARs), are capable of generating an immune response (e.g., a cellular immune response), or are precursor cells of such cells, and include lymphocytes (preferably T lymphocytes), lymphoblasts, and plasma cells. Lymphoid cells can be immune effector cells as described herein. Preferred lymphoid cells are T cells that can be modified to express antigen receptors on their cell surface. In some embodiments, lymphoid cells lack endogenous expression of T cell receptors.

[0689] The terms "T cell" and "T lymphocyte" are used interchangeably in this article, and they include T helper cells (CD4+). + T cells) and cytotoxic T cells (CTLs), including cytotoxic T cells (CD8+). + T cells).

[0690] T cells belong to the leukocyte group called lymphocytes and play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types, such as B cells and natural killer cells, by the presence of specialized receptors on their cell surface called T cell receptors (TCRs). The thymus is the main organ responsible for T cell maturation. Several distinct T cell subsets have been identified, each with unique functions.

[0691] T helper cells assist other white blood cells in the immune process, including the maturation of B cells into plasma cells and the activation of cytotoxic T cells and macrophages, as well as other functions. These cells are also known as CD4 cells. + T cells are activated because they express the CD4 glycoprotein on their surface. Helper T cells are activated when class II MHC molecules expressed on the surface of antigen-presenting cells (APCs) present peptide antigens to them. Once activated, they rapidly divide and secrete small proteins called cytokines, which regulate or assist in active immune responses.

[0692] Cytotoxic T cells destroy virus-infected cells and tumor cells, and are also involved in transplant rejection. These cells are also known as CD8 cells. + T cells are formed because they express the CD8 glycoprotein on their surface. These cells recognize their targets by binding to antigens that are associated with class I MHC, which are present on the surface of almost every cell in the body.

[0693] Regulatory T cells, or Tregs, are a subset of T cells that regulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune diseases. Tregs are immunosuppressive and typically inhibit or downregulate the induction and proliferation of effector T cells. Tregs express the biomarkers CD4, FoxP3, and CD25.

[0694] As used herein, the term "naïve T cell" refers to a mature T cell that is distinct from activated T cells or memory T cells and has not been exposed to its homologous antigens in the peripheral environment. Naïve T cells are typically characterized by surface expression of L-selectin (CD62L), the absence of activation markers CD25, CD44, or CD69, and the absence of the memory CD45RO isotype.

[0695] As used herein, the term "memory T cells" refers to a subset or subgroup of T cells that have previously been exposed to and responded to their homologous antigens. Upon a second exposure to the antigen, memory T cells can replicate to produce a faster and stronger immune response compared to the first immune response. Memory T cells can be CD4+. + or CD8 + And it is usually expressed as CD45RO.

[0696] As used in this article, the term "T cell" also includes cells that can mature into T cells upon appropriate stimulation.

[0697] Most T cells possess a T cell receptor (TCR) that exists as a complex of several proteins. The actual T cell receptor consists of two separate polypeptide chains, produced by independent T cell receptor α and β (TCRα and TCRβ) genes, and are referred to as the α-TCR chain and the β-TCR chain. γδ T cells (gamma delta T cells) represent a small subset of T cells that possess a unique T cell receptor (TCR) on their surface. However, in γδ T cells, the TCR consists of one γ chain and one δ chain. This group of T cells is much less common than αβ T cells (comprising only 2% of all T cells).

[0698] All T cells originate from hematopoietic stem cells in the bone marrow. Hematopoietic progenitor cells, derived from hematopoietic stem cells, reside in the thymus and proliferate through cell division to produce a large number of immature thymocytes. The earliest thymocytes express neither CD4 nor CD8, and are therefore classified as double-negative (CD4+). - CD8 - ) cells. As they progress through development, they become double-positive (CD4+) cells. + CD8 + ) thymocytes, and eventually mature into single-positive (CD4+) cells. + CD8 - or CD4 - CD8 + Thymocytes are then released from the thymus into the peripheral tissues.

[0699] T cells can typically be prepared in vitro or ex vivo using standard procedures. For example, commercially available cell isolation systems can be used to isolate T cells from bone marrow, peripheral blood, or fractions of bone marrow or peripheral blood in mammals (e.g., patients). Alternatively, T cells can be derived from related or unrelated humans, non-human animals, cell lines, or cultures. Samples containing T cells can be, for example, peripheral blood mononuclear cells (PBMCs).

[0700] As used in this article, the term "NK cell" or "natural killer cell" refers to a subset of peripheral blood lymphocytes defined by the expression of CD56 or CD16 and the absence of T cell receptors.

[0701] As provided in this article, NK cells can also differentiate from stem cells or progenitor cells.

[0702] Chimeric antigen receptor (CAR)

[0703] T cells typically possess T cell receptors (TCRs) that recognize target cells by binding to antigens presented in the context of the MHC. As used herein, the term "T cell receptor" or "TCR" refers to the protein receptor on T cells, which is composed of heterodimers of alpha (α) and beta (β) chains, although in some cells the TCR consists of gamma and delta (γδ) chains. Each α, β, γ, and δ chain consists of two Ig-like domains: a variable (V) domain that confers antigen recognition via a complementarity-determining region (CDR), followed by a constant (C) domain anchored to the cell membrane via a linker peptide and a transmembrane (TM) region. The TM region associates with the invariant subunit of the CD3 signaling apparatus. Each V domain has three CDRs. These CDRs interact with a complex formed by the binding of an antigenic peptide to a protein encoded by the major histocompatibility complex (MHC).

[0704] T cells can be engineered to express chimeric antigen receptors (CARs) that target (preferably in an MHC-dependent manner) virtually any antigen. For example, a patient's T cells can be genetically engineered (genetically modified) to express CARs that specifically target antigens on the patient's tumor cells. Adoptive cell transfer therapy using CAR-modified T cells expressing chimeric antigen receptors is a promising anticancer treatment.

[0705] As used herein, the term "CAR" (or "chimeric antigen receptor") is synonymous with the terms "chimeric T-cell receptor" and "artificial T-cell receptor" and refers to an artificial receptor comprising a single molecule or molecular complex that recognizes (i.e., binds) a target structure (e.g., an antigen) (e.g., by binding to the antigen via an antigen-binding domain) and can confer specificity to immune effector cells (e.g., T cells) expressing the CAR on their cell surface. Such cells do not necessarily need to process and present antigens to recognize target cells, but may preferably specifically recognize any antigen. Preferably, the recognition of the target structure by the CAR causes the immune effector cell expressing the CAR to be activated. A CAR may comprise one or more protein units containing one or more domains as described herein. The term "CAR" does not include T-cell receptors.

[0706] CARs typically contain target-specific binding elements (also known as antigen-binding portions or antigen-binding domains), which are usually part of the extracellular domain of the CAR.

[0707] In some embodiments, the antigen-binding domain comprises an antigen-specific immunoglobulin heavy chain variable region (VH) and an antigen-specific immunoglobulin light chain variable region (VL). In some embodiments, the immunoglobulin is an antibody. In some embodiments, the heavy chain variable region (VH) and the corresponding light chain variable region (VL) are linked by a peptide linker. The antigen-binding portion in the CAR may be an scFv. In some embodiments, the antigen-binding domain comprises a VHH domain. The target-specific binding element or extracellular domain of the CAR described herein may comprise a single-domain antibody or ALFA tag that binds an ALFA tag as described herein. In some embodiments, the target-specific binding element or extracellular domain of the CAR described herein comprises a single-domain antibody that binds an ALFA tag as described herein.

[0708] The CAR is preferably designed to include a transmembrane domain fused to the extracellular domain of the CAR. In some embodiments, the transmembrane domain does not naturally associate with one of the domains in the CAR. In some embodiments, the transmembrane domain naturally associates with one of the domains in the CAR. In some embodiments, the transmembrane domain is modified by amino acid substitution to prevent such a domain from binding to the transmembrane domains of the same or different surface membrane proteins, thereby minimizing its interaction with other members of the receptor complex. The transmembrane domain may be derived from a natural or synthetic source. In the case of a natural source, the domain may be derived from any membrane-binding protein or transmembrane protein. Transmembrane regions that are particularly useful herein may be derived from (i.e., at least containing a transmembrane region): the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. Alternatively, the transmembrane domain can be synthetic, in which case it will primarily contain hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan, and valine will be present at each end of the synthetic transmembrane domain.

[0709] In some instances, CARs contain hinged domains that form connections between transmembrane and extracellular domains.

[0710] The cytoplasmic domains or other intracellular signaling domains of a CAR are responsible for activating at least one normal effector function in the immune cells in which the CAR has been implanted. The term "effector function" refers to a specific function of the cell. For example, the effector function of a T cell can be cytolytic activity or helper activity, including the secretion of cytokines. Therefore, the term "intracellular signaling domain" refers to a portion of a protein that transduces effector signals and directs the cell to perform a specific function. Although the entire intracellular signaling domain can usually be used, in many cases it is not necessary to use the entire strand. Regarding the use of a truncated portion of an intracellular signaling domain, such a truncated portion can be used in place of the complete strand, provided that it transduces effector signals. Therefore, the term "intracellular signaling domain" is intended to include any truncated portion of an intracellular signaling domain sufficient to transduce effector signals.

[0711] It is known that the signal generated by the TCR alone is insufficient to fully activate T cells, and secondary or co-stimulatory signals are also required. Therefore, it can be said that T cell activation is mediated by two different types of cytoplasmic signaling sequences: those that induce antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences) and those that function in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences).

[0712] In some implementations, the CAR includes a primary cytoplasmic signaling sequence derived from CD3-ζ. Furthermore, the cytoplasmic domain of the CAR may include a CD3-ζ signaling domain that binds to a co-stimulatory signaling region.

[0713] The properties of the co-stimulatory domains are limited to their ability to enhance cell proliferation and survival after the CAR binds to the target moiety. Suitable co-stimulatory domains include CD28, CD137 (4-1BB) (a member of the tumor necrosis factor receptor (TNFR) superfamily), CD134 (OX40) (a member of the TNFR receptor superfamily), and CD278 (ICOS) (a co-stimulatory molecule of the CD28 superfamily expressed on activated T cells). Those skilled in the art will understand that sequence variants of these mentioned co-stimulatory domains can be used without adversely affecting the present disclosure, wherein said variants have the same or similar activity as the domains they mimic. Such variants will have at least about 80% sequence identity with the amino acid sequence of the domain from which they are derived. In some embodiments, the CAR construct comprises two co-stimulatory domains. While some specific combinations include all possible variations of the four mentioned domains, some specific examples include CD28+CD137 (4-1BB) and CD28+CD134 (OX40).

[0714] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR can be linked together in a random or specific order. Optionally, short oligopeptide or polypeptide linkers (preferably 2 to 10 amino acids in length) can form the linkers. Glycine-serine duplexes provide particularly suitable linkers.

[0715] In some embodiments, the CAR includes a signal peptide that guides nascent proteins into the endoplasmic reticulum. In some embodiments, the signal peptide precedes the antigen-binding domain. In some embodiments, the signal peptide is derived from an immunoglobulin, such as IgG.

[0716] CARs can contain the aforementioned domains (collectively in the form of a fusion protein). Such fusion proteins typically contain a target-specific binding domain linked from the N-terminus to the C-terminus, one or more co-stimulatory domains, and a signal transduction sequence. However, CARs are not limited to this arrangement, and other arrangements are also acceptable and include binding domains, signal transduction domains, and one or more co-stimulatory domains. It should be understood that because the binding domain must freely bind to the target, the arrangement of the binding domain in the fusion protein will generally enable the display of that region outside the cell. Similarly, because the co-stimulatory and signal transduction domains are used to induce the activity and proliferation of cytotoxic lymphocytes, the fusion protein will typically display both domains inside the cell.

[0717] In some implementations, the CAR molecule comprises:

[0718] i) Target binding domain;

[0719] ii) Transmembrane domains; and

[0720] iii) Intracellular domains comprising signal transduction domains (e.g., CD3-ζ signal transduction domains), which optionally combine with one or more co-stimulatory domains, such as intracellular domains comprising 4-1BB co-stimulatory domains.

[0721] In some embodiments, the target-binding domain comprises a single-domain antibody that binds an ALFA tag as described herein. In some embodiments, the target-binding domain comprises an ALFA tag. In some embodiments, the transmembrane domain comprises a transmembrane domain selected from the following proteins:

[0722] In some embodiments, the transmembrane domain comprises a CD8α transmembrane domain. In some embodiments, the target-binding domain is connected to the transmembrane domain via a hinge domain. In some embodiments, the hinge domain is a CD8α hinge domain.

[0723] In some implementations, the CAR molecule comprises:

[0724] i) Target binding domain;

[0725] ii) CD8α hinge structural domain;

[0726] iii) CD8α transmembrane domain; and

[0727] iv) Intracellular domains containing the 4-1BB co-stimulatory domain and the CD3-ζ signaling domain.

[0728] Genetic modification of immune effector cells

[0729] The immune effector cells described herein are cells that express antigen receptors (e.g., CARs) and are targeted by docking compounds. Immune effector cells are typically cells that have been genetically modified, either in vitro or in vivo, to express antigen receptors. Immune effector cells can be provided to a subject, for example, by administering immune effector cells (e.g., genetically modified immune effector cells) to the subject or by generating genetically modified immune effector cells in the subject.

[0730] Nucleic acids encoding antigen receptors can be delivered to immune effector cells (such as T cells, especially CD8+ T cells) in vitro or in vivo, thereby generating immune effector cells that are genetically modified to express antigen receptors.

[0731] In some embodiments, genetically modified immune effector cells expressing a chimeric antigen receptor (CAR) are provided to a subject by administration of a nucleic acid (e.g., RNA) encoding the antigen receptor. Delivery of the nucleic acid encoding the antigen receptor to the immune effector cells for expression can be achieved using particles containing said nucleic acid. The particles may contain a targeting molecule that binds to a target (e.g., an antigen) on the immune effector cells. In some embodiments, the target includes CD3, such as CD3e, CD4, or CD8. In some embodiments, genetically modified immune effector cells expressing a chimeric antigen receptor (CAR) are provided to a subject by administration of genetically modified immune effector cells expressing a chimeric antigen receptor (CAR). In some embodiments, a pre-formed complex is provided to the subject by administration, wherein a docking compound binds to the genetically modified immune effector cells expressing a chimeric antigen receptor (CAR).

[0732] The genetic modification of immune effector cells to express antigen receptors described in this article includes: virus-free DNA transfection, virus-free RNA transfection (e.g., mRNA transfection), transposon-based systems, and virus-based systems. Virus-free DNA transfection carries a low risk of insertional mutagenesis. Transposon-based systems integrate transgenes more efficiently than plasmids that do not contain integrative elements. Virus-based systems include those using gamma retroviruses and lentiviral vectors. Gamma retroviruses are relatively easy to produce, efficiently and permanently transduce cells (e.g., T cells), and have shown initial safety in primary human T cells from an integrative perspective. Lentiviral vectors also efficiently and permanently transduce cells (e.g., T cells), but are more expensive to manufacture. They may also be safer than retrovirus-based systems.

[0733] In some embodiments, T cells or T cell progenitor cells are transfected in vitro or in vivo with nucleic acids encoding antigen receptors. In some embodiments, a combination of in vitro and in vivo transfection may be used. In some embodiments, the T cells or T cell progenitor cells are derived from the subject to be treated. In some embodiments, the T cells or T cell progenitor cells are derived from a different subject than the subject to be treated.

[0734] In some implementations, CAR T cells can be generated in vivo using T cell-targeting particles (e.g., nanoparticles), and thus generated almost instantaneously. After binding to T cells, these particles can be internalized. Their contents, such as nucleic acids encoding antigen receptors, or plasmid DNA encoding CARs, can be directed to the T cell nucleus by, for example, containing peptides with microtubule-associated sequences (MTAS) and nuclear localization signals (NLS). The presence of transposons (e.g., CAR gene expression cassettes) flanking the nucleic acid encoding the antigen receptor and separate nucleic acids (e.g., plasmids) encoding overactive transposases allows the nucleic acid encoding the antigen receptor (e.g., a CAR vector) to be efficiently integrated into the chromosome.

[0735] Another possibility is to intentionally place peptide / polypeptide coding sequences, such as antigen receptor coding sequences (e.g., CAR coding sequences), at specific loci using the CRISPR / Cas9 method. For example, an existing T cell receptor (TCR) can be knocked out, and a CAR can be knocked in and placed under the dynamic regulatory control of the endogenous promoter that originally regulates TCR expression.

[0736] Therefore, in addition to nucleic acids encoding antigen receptors, particles can also deliver gene-editing tools such as CRISPR / Cas9 (or related technologies) or transposon systems such as sleeping beauty or piggy bags. Such tools for genome integration / editing (e.g., transposases, gene-editing tools like CRISPR / Cas9) can be delivered as proteins or encoding nucleic acids (DNA or RNA). However, delivery of mRNA is also an option for inducing transient expression of antigen receptors (such as CARs).

[0737] In some implementations, cells genetically modified to express antigen receptors are stably or transiently transfected with nucleic acids encoding the antigen receptors. Thus, the nucleic acids encoding the antigen receptors are integrated into or not integrated into the cell's genome.

[0738] In some implementations, cells genetically modified to express antigen receptors are deprived of the activity of expressing endogenous T cell receptors and / or endogenous HLA.

[0739] In some embodiments, the cells described herein may be autologous, allogeneic, or syngeneic for the subject of treatment. In some embodiments, this disclosure envisions removing the cells from the patient and subsequently re-delivering the cells to the patient. In some embodiments, this disclosure does not envision removing the cells from the patient. In the latter case, all steps of genetic modification of the cells can be performed in vivo.

[0740] Cells for targeted delivery

[0741] According to this disclosure, immune effector cells are specifically delivered to target cells by targeting targets on target cells (e.g., antigens on target cells).

[0742] In some implementations, the target cell antigen is a structure present on the surface of the target cell, such as a protein, such as a cell surface antigen, including a cell surface receptor.

[0743] Terms such as "expressed on cell surface," "associated with cell surface," or "cell surface molecule" mean that a molecule (e.g., a receptor or antigen) is located on and associated with the cell's plasma membrane, wherein at least a portion of the molecule faces the extracellular space of the cell and can be accessed from the outside of the cell by, for example, binding molecules (e.g., antibodies) located outside the cell. In this case, the portion is preferably at least 4, preferably at least 8, preferably at least 12, more preferably at least 20 amino acids. The association can be direct or indirect. For example, the association can be through one or more transmembrane domains, one or more lipid anchors, or through interaction with any other protein, lipid, carbohydrate, or other structure that may be present on the outer layer of the cell membrane. For example, a molecule associated with the cell surface can be a transmembrane protein having an extracellular portion, or it can be a protein that associates with the cell surface by interacting with another protein that is a transmembrane protein.

[0744] The term "cell surface" or "the surface of the cell" is used according to its normal meaning in the art and therefore includes the extracellular space that can be approached and bound by proteins and other molecules. If an antigen is located on the surface of a cell and can be approached and bound by an antigen-specific antibody, for example, added to the cell, then the antigen is expressed on the surface of the cell. In one embodiment, the antigen expressed on the cell surface is an integrated membrane protein having an extracellular portion that is recognized by binding molecules (e.g., antibodies).

[0745] In the context of this invention, the term "extracellular portion" or "extracellular domain" refers to the extracellular space of a molecule (e.g., a protein) facing the cell and preferably accessible from the outside of the cell by binding molecules (e.g., antibodies) located outside the cell.

[0746] In some implementations, the antigen on the target cell may be present on the diseased cell.

[0747] Antigens on target cells can be upregulated during disease, such as infection or cancer. In diseased tissue, biomarkers can differ from those in healthy tissue, offering unique possibilities for treatment, especially targeted therapy.

[0748] In some implementations, the antigens on the target cells are disease-related antigens, such as tumor antigens, viral antigens, or bacterial antigens. This allows for the targeting of diseased cells using the methods and reagents described herein.

[0749] The term "disease-associated antigen" is used in its broadest sense to refer to any antigen associated with a disease. Disease-associated antigens can be associated with infection by microorganisms (usually microbial antigens) or with cancer (usually tumors).

[0750] In some embodiments, the antigen on the target cells is a tumor antigen. In the context of this disclosure, the term "tumor antigen" or "tumor-associated antigen" refers to a protein that is specifically expressed under normal conditions in a limited number of tissues and / or organs or at a specific developmental stage. For example, a tumor antigen may be specifically expressed under normal conditions in gastric tissue (preferably in the gastric mucosa), in reproductive organs (e.g., in the testes), in trophoblastic tissue (e.g., in the placenta), or in germline cells, and is expressed or abnormally expressed in one or more tumors or cancerous tissues. In this context, "limited number" preferably means no more than 3, more preferably no more than 2. Tumor antigens in the context of this disclosure include, for example, differentiation antigens, preferably cell type-specific differentiation antigens, i.e., proteins that are specifically expressed under normal conditions at a specific differentiation stage in a specific cell type; cancer / testis antigens, i.e., proteins that are specifically expressed under normal conditions in the testes and sometimes in the placenta; and germline-specific antigens. In the context of this disclosure, tumor antigens preferably associate with the cell surface of cancer cells and are preferably not expressed or only poorly expressed in normal tissues. Preferably, the tumor antigen or its aberrant expression identifies cancer cells. In the context of this disclosure, the tumor antigen expressed by cancer cells in a subject (e.g., a patient with cancer) is preferably a self-protein in said subject. In a preferred embodiment, the tumor antigen in the context of this disclosure is specifically expressed under normal conditions in non-essential tissues or organs (i.e., tissues or organs that do not cause death when damaged by the immune system), or in body organs or structures that the immune system cannot or can hardly reach. Preferably, the amino acid sequence of the tumor antigen is identical between tumor antigens expressed in normal tissues and tumor antigens expressed in cancerous tissues.

[0751] Examples of tumor antigens include p53, ART-4, BAGE, β-linkin / m, Bcr-abL, CAMEL, CAP-1, CASP-8, CDC27 / m, CDK4 / m, CEA, cell surface proteins of the claudin family (e.g., CLAUDIN-6, CLAUDIN-18.2, and CLAUDIN-12), c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gap100, HAGE, HER-2 / neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR / FUT, and MAGE-A (preferably MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8). -A8, MAGE-A9, MAGE-A10, MAGE-A11 or MAGE-A12), MAGE-B, MAGE-C, MART-1 / Melan-A, MC1R, myosin / m, MUC1, MUM-1, -2, -3, NA88-A, NF1, NY-ESO-1, NY-BR-1, p190 minor BCR-abL, Pm1 / RARa, PRAME, protease 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, SURVIVIN, TEL / AML1, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, TPTE and WT. Particularly preferred tumor antigens include CLAUDIN-18.2 (CLDN18.2) and CLAUDIN-6 (CLDN6).

[0752] In some implementations, the target cell antigen is a structure, such as a protein, present on the surface of the target cell, like a cell surface antigen or cell surface receptor, whose presence or quantity is characteristic of a particular cell type compared to other cell types. This allows targeting specific cell types characterized by increased presence or quantity using the methods and reagents described herein.

[0753] Bonding components and binders

[0754] This disclosure describes binding portions or binders (e.g., antibodies or antibody derivatives). Furthermore, this disclosure describes bispecific or multispecific binders, such as bispecific molecules comprising first and second binding domains, wherein the first binding domain is capable of binding to an antigen on a target cell, and the second binding domain is capable of binding to an antigen receptor on an immune effector cell.

[0755] As used herein, the term "binding agent" refers to any substance capable of binding to a desired antigen. In some embodiments, the binding agent is or comprises: an antibody, an antibody fragment, or any other binding protein, or any combination thereof.

[0756] As used herein, the term "binding moiety" refers to any portion, group, or domain capable of binding to a desired antigen. In some embodiments, the binding moiety is or includes the following: an antibody, an antibody fragment, or any other binding protein, or any combination thereof.

[0757] As used herein, the term "antigen" is a molecule capable of being bound by a binding moiety or binder (e.g., an antibody). Antigens may additionally induce humoral and / or cellular immune responses, leading to the production of B-lymphocytes and / or T-lymphocytes. Antigens may have one or more epitopes (B-cell and T-cell epitopes).

[0758] The term "immunoglobulin" refers to a class of structure-associated glycoproteins composed of two pairs of polypeptide chains (a pair of light (L) low molecular weight chains and a pair of heavy (H) chains), all four chains interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized. See, for example, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. RavenPress, NY (1989)). In short, each heavy chain typically consists of a heavy chain variable region (abbreviated as V in this paper). H (or VH) and heavy chain constant region (abbreviated as C in this article) H The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. The hinge region is the area between the CH1 and CH2 domains of the heavy chain and is highly flexible. The disulfide bonds in the hinge region are the part where the two heavy chains interact in the IgG molecule. Each light chain typically consists of a light chain variable region (abbreviated as V in this article). L or VL) and light chain constant region (abbreviated as C in this article) L The light chain constant region is typically composed of a single CL domain. The VH and VL regions can be further subdivided into hypervariable regions (or highly variable regions in the sequence or structurally defined ring form), also known as complement-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL typically consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol). 196, 901-917 (1987)).

[0759] As used herein, the term "antibody" (Ab) refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative thereof that has the ability to bind to an antigen, preferably specifically. In some embodiments, binding occurs under normal physiological conditions and has a significant half-life, such as at least about 30 minutes, at least about 45 minutes, at least about one hour, at least about two hours, at least about four hours, at least about eight hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 days or more, or any other relevant functionally defined time period (e.g., sufficient to induce, promote, enhance, and / or modulate the physiological response associated with antibody-antigen binding). Variable regions of the heavy and light chains of an immunoglobulin molecule contain binding domains that interact with the antigen. As used herein, the term antibody includes not only monospecific antibodies but also multispecific antibodies that contain multiple (e.g., two or more, or three or more) distinct antigen-binding regions. The constant region of an antibody (Ab) mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system (e.g., C1q, the first component in the classical complement activation pathway). As noted above, unless otherwise stated or clearly contradicted by the context, the term antibody as used herein includes fragments of antibodies as antigen-binding segments (i.e., those retaining the ability to specifically bind antigens) and antibody derivatives (i.e., constructs derived from antibodies). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of antigen-binding fragments encompassed within the term "antibody" include (i) Fab' or Fab fragments (monovalent fragments consisting of VL, VH, CL, and CH1 domains, or monovalent antibodies as described in WO2007 / 059782 (Genmab); (ii) F(ab')2 fragments (bivalent fragments comprising two Fab fragments linked by disulfide bonds through a hinge region); (iii) Fd fragments consisting essentially of VH and CH1 domains; (iv) Fv fragments consisting essentially of the VL and VH domains of a single antibody arm; and (v) dAb fragments (Ward et al., Nature). 341 , 544-546 (1989)), which is basically composed of VH domains and is also called a domain antibody (Holt et al; Trends Biotechnol. 2003 Nov; 21(11):484-90); (vi) Cameloids or nanobody molecules (Revets et al; Expert Opinion Biol Ther. 2005 Jan; 5 (1):111-24) and (vii) separate complementarity-determining regions (CDRs). Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be linked together using recombination methods via synthetic linkers, allowing them to be made into single protein chains where the VL and VH regions pair to form monovalent molecules (called single-chain antibodies or single-chain Fvs, scFv; see, for example, Bird et al., Science). 242 ,423-426 (1988) and Huston et al., PNAS USA 85 , 5879-5883 (1988). Unless otherwise stated or clearly indicated by the context, such single-chain antibodies are encompassed within the term antibody. Although such fragments are generally included within the meaning of antibody, they are collectively and independently unique features of this disclosure, exhibiting distinct biological properties and utilities. In the context of this disclosure, these and other available antibody fragments, as well as bispecific forms of such fragments, are further discussed herein. It should also be understood that, unless otherwise indicated, the term antibody also includes polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like peptides, such as chimeric antibodies and humanized antibodies, and antibody fragments (antigen-binding fragments) that retain the ability to bind specifically to antigens, provided by any known technique (e.g., enzyme cleavage, peptide synthesis, and recombinant techniques).

[0760] The terms “Fab,” “Fab region,” “Fab section,” or “Fab fragment” are understood to define a subset containing V. H C H 1. V L and C LA polypeptide of an immunoglobulin domain. Fab can refer to the region in its standalone state or within the context of the antibody molecule according to the invention and the full-length immunoglobulin or immunoglobulin fragment. Typically, the Fab region comprises the entire light chain of the antibody. The Fab region can be used to define an “arm” of the immunoglobulin molecule. It contains the epitope-binding portion of the Ig. The Fab region of a naturally occurring immunoglobulin can be obtained as a proteolytic fragment by partial papain digestion. The “F(ab’)2 moiety” is a proteolytic fragment of an immunoglobulin digested with partial pepsin. The “Fab’ moiety” is the product produced by reducing the disulfide bonds of the F(ab’)2 moiety. The terms “Fab,” “Fab region,” “Fab moiety,” or “Fab fragment” as used herein may also include a hinge region defining the C-terminal end of the antibody arm. This hinge region corresponds to the C-terminal portion of the full-length immunoglobulin. H A hinge region exists at the C-terminus of the 1 domain, where the arms of the antibody molecule can be used to define Y. The term hinge region is used in this art because immunoglobulins have a degree of flexibility in this region.

[0761] An “Fv” or “Fv fragment” consists only of the VL and VH domains of an immunoglobulin “single arm”. Therefore, an “Fv” is the smallest antibody fragment containing a complete antigen recognition and binding site. A “double-stranded” Fv fragment consists of a dimer formed by the tight, non-covalent association of a heavy chain variable domain and a light chain variable domain. Single-stranded Fv (scFv) types include the VH and VL domains of immunoglobulins, which are present in a single polypeptide chain, where they are covalently linked to each other by a flexible peptide linker. Typically, in scFv fragments, the variable domains of the light and heavy chains associate in a dimer structure similar to that in double-stranded Fv types. In a single-stranded Fv fragment, there may be either a variable domain of the light chain at the N-terminus of a single polypeptide chain, followed by a linker, and a variable domain of the heavy chain at the C-terminus of the polypeptide chain; or vice versa, there may be a variable domain of the heavy chain at the N-terminus and a variable domain of the light chain at the C-terminus, with a peptide linker between them. Peptide linkers can be any flexible linker known in the art, such as linkers composed of glycine and serine residues. Alternatively, domain association between the VH and VL domains can be stabilized by introducing disulfide bonds into conserved framework regions (see Reiter et al. Stabilization of the Fv fragments in recombinant immunotoxins by disulfide bonds engineered into conserved framework regions, Biochemistry 1994, 33, 6551-5459). Such scFv fragments are also called disulfide-stabilized scFv fragments (ds-scFv).

[0762] The term "Fc region" or "Fc fragment" is used herein to define the C-terminal region of the immunoglobulin heavy chain, including the native sequence Fc region and variant Fc regions. The Fc portion mediates the effector functions of antibodies, such as activation of the complement system and activation of immune effector cells (e.g., NK cells) carrying Fc receptors. In human IgG molecules, the Fc region is generated by papain cleavage targeting the N-terminus of Cys226. Although the boundaries of the Fc region of the immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is generally defined as extending from an amino acid residue at position Cys226 or from Pro230 to its C-terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during the production or purification of the antibody molecule, or by recombinant modification of the nucleic acid encoding the heavy chain of the antibody molecule. Thus, compositions of complete antibodies may comprise antibody groups with all K447 residues removed, antibody groups without K447 residue removal, and antibody groups consisting of mixtures containing and without K447 residues. Suitable natural sequence Fc regions for the antibodies described herein include mammalian (e.g., human or mouse) IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. The Fc region contains two or three constant domains, depending on the antibody class. In embodiments where the immunoglobulin is IgG, the Fc region has CH2 and CH3 domains.

[0763] Antibodies can have any isotype. As used herein, the term "isotype" refers to a class of immunoglobulins (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) encoded by the heavy chain constant region gene. When a specific isotype, such as IgG1, is mentioned herein, the term is not limited to a specific isotype sequence, such as a specific IgG1 sequence, but is used to indicate that an antibody is sequence-closer to that isotype, such as IgG1, rather than other isotypes. Thus, an IgG1 antibody, for example, can be a sequence variant of a naturally occurring IgG1 antibody, including variations in the constant region.

[0764] In various embodiments, the antibody is an IgG1 antibody (more particularly IgG1,κ or IgG1,λ isotypes (i.e. IgG1,κ,λ)), an IgG2a antibody (e.g. IgG2a,κ,λ), an IgG2b antibody (e.g. IgG2b,κ,λ), an IgG3 antibody (e.g. IgG3,κ,λ), or an IgG4 antibody (e.g. IgG4,κ,λ).

[0765] When used in the context of antibodies, the term "full-length" means that an antibody is not a fragment, but rather contains all the domains of a particular isotype that are normally found in nature, such as the VH, CH1, CH2, CH3, hinge, VL, and CL domains of an IgG1 antibody.

[0766] When an antibody or antibody molecule / fragment recognizes its target antigen in a complex mixture of proteins and / or macromolecules, it is said to bind "specifically" to the antigen. Typically, an antibody is able to specifically interact with and / or bind to its target, but substantially not to another (preferably unrelated) epitope or antigen. If antibodies cross-compete such that only one antibody can bind to an epitope at a given time point—that is, one antibody inhibits the binding or regulatory effect of another antibody—it is said that the antibody "binds to the same epitope." However, antibodies or antibody molecule / fragment that specifically bind to a particular target can cross-react with similar structures (e.g., closely related variants of the target they specifically bind to).

[0767] Typically, bindings considered specific can also have high affinity, for example, when the binding affinity is higher than 10. -6 M (as for K) D In particular, the binding affinity can be approximately 10. -8 Up to 10 -11 M(K) D ), or about 10 -9 Up to 10 -11 M or even higher. Therefore, the affinity is in the picomolar range (Kd = 9.9 × 10⁻⁶). -10 M to 10 -12 Antibody molecules of type M are also covered in this invention. If necessary, non-specific binding at the binding site can be reduced by changing the binding conditions without substantially affecting specific binding.

[0768] The term "DARPin" refers to a engineered ankyrin repeat protein. DARPin is based on naturally occurring ankyrin repeat proteins but contains one or more amino acid mutations that can affect, for example, its binding affinity to target molecules, its cell surface expression, etc. DARPin preferably contains 2 to 3 ankyrin repeat modules, flanked by N- and C-capped repeats. Each ankyrin repeat module contains approximately 33 amino acid residues.

[0769] Ankyrin repeats were first identified in 1987 through sequence comparisons among four such proteins in *Saccharomyces cerevisiae*, *Drosophila melanogaster*, and *Caenorhabditis elegans*. Breeden and Nasmyth reported multiple copies of a repeat unit of approximately 33 residues in the sequences swi6p, cddOp, notch, and lin-12 (Breeden et al., *Nature* 329, 651-654 (1987)). Subsequently, 24 copies of this repeat unit were found in ankyrin, leading to its naming as ankyrin repeats (Lux et al., *Nature* 344, 36-42 (1990)). Since then, this repeat unit has been identified in hundreds of proteins from various organisms and viruses (Bork, *Proteins* 17(4), 363-74 (1993)). These proteins are located in the nucleus, cytoplasm, or extracellular space. This is consistent with the fact that the ankyrin repeat domains of these proteins are independent of disulfide bridges and therefore independent of the oxidation state of the environment. The number of repeat units in each protein ranges from 2 to more than 20. The tertiary structure of the ankyrin repeat unit shares a characteristic fold (Sedgwick and Smerdon, Trends Biochem Sci. 24(8), 311-6 (1999)) consisting of a β-hairpin followed by two antiparallel α-helices, ending with a loop connecting the repeat unit to the next repeat unit. The domains constructed from the ankyrin repeat units are formed by stacking the repeat units into an extended and curved structure. Proteins containing ankyrin repeat domains often contain additional domains. While the latter domains have variable functions, the function of the ankyrin repeat domain is most commonly binding to other proteins. When analyzing the repeat units of these proteins, target-interacting residues are predominantly found in the β-hairpin and the exposed portion of the first α-helix. Thus, these target-interacting residues form a large contact surface on the ankyrin repeat domain. The contact surface is exposed on a frame constructed from stacked units of α-helix 1, α-helix 2 and rings.

[0770] DARPins that bind to a specific target can be identified by screening DARPin combination libraries and selecting those that have the desired binding properties to the target. Such screening methods are described, for example, in Muench et al., Molecular Therapy, 16(4), 686-693, 2011. For example, ribosome display or phage display methods can be used to select target-specific DARPins from different libraries.

[0771] The term "repetitive protein" refers to a (poly)peptide / protein containing one or more repeating domains. In one embodiment, the repeating protein contains up to four repeating domains. In one embodiment, the repeating protein contains up to three repeating domains. In one embodiment, the repeating protein contains up to two repeating domains. In the most preferred embodiment, the repeating protein contains one repeating domain.

[0772] The domains of a repeating protein can be linked to each other directly or through a (poly)peptide linker. The term "(poly)peptide linker" refers to an amino acid sequence capable of linking two protein domains. Such linkers include, for example, variable-length glycine-serine linkers and are known to those skilled in the art.

[0773] The term "repetitive domain" refers to a protein domain comprising two or more consecutive repeating units (modules). In one embodiment, the repeating units are structural units having the same or similar folding structures, and are preferably closely stacked to preferably produce a supercoiled structure with a common hydrophobic core.

[0774] The term "structural unit" refers to a locally ordered portion of a (poly)peptide, formed by three-dimensional interactions between two or more secondary structural segments close to each other along the (poly)peptide chain. Such structural units contain structural motifs.

[0775] The term "structural motif" refers to the three-dimensional arrangement of secondary structural elements present in at least one structural unit. Structural motifs are well known to those skilled in the art. The defined three-dimensional arrangement may not be achievable by the structural unit alone; however, its continuous arrangement as repeating modules within a repeating structural domain leads to mutual stability of adjacent units, which can result in a superspiral structure.

[0776] The term "repetitive module" refers to the repetitive amino acid sequence of a repetitive protein, which is derived from the repetitive units of naturally occurring proteins. Each repetitive module contained in a repetitive domain is derived from one or more repetitive units of a family of naturally occurring repetitive proteins, such as ankylosing spondylins.

[0777] The term "repeating module group" refers to the total number of repeating modules present in a repeating structure domain. Such a "repeating module group" in a repeating structure domain contains two or more consecutive repeating modules and may contain only one type of repeating module in two or more copies, or two or more different types of modules, each present in one or more copies. For example, a repeating module group containing three repeating modules may consecutively contain repeating module 1, repeating module 2, and repeating module 3 from end N to end C.

[0778] Different repeating structural domains can have the same number of repeating modules in each repeating structural domain, or the number of repeating modules in each repeating structural domain can be different.

[0779] Preferably, the repeating modules included in a group are homologous repeating modules. In the context of this disclosure, the term "homologous repeating module" refers to a repeating module in which more than 70% of the framework residues are homologous. Preferably, more than 80% of the framework residues of the repeating module are homologous. Most preferably, more than 90% of the framework residues of the repeating module are homologous. Computer programs such as Fasta, Blast, or Gap for determining the percentage of homology between peptides are known to those skilled in the art.

[0780] The term "repetitive unit" refers to an amino acid sequence containing one or more naturally occurring protein sequence motifs, wherein the "repetitive unit" exists in multiple copies and exhibits a common, defined folding topology across all said motifs, which determines the protein's folding. Such a repetitive unit comprises framework residues and interacting residues.

[0781] An example of such repeating units is the ankyrin repeating unit. Naturally occurring proteins containing two or more such repeating units are called "naturally occurring repeating proteins." When compared with each other, the amino acid sequences of the individual repeating units of a repeating protein can have a large number of mutations, substitutions, additions, and / or deletions, while still largely retaining the general pattern or motif of the repeating unit.

[0782] The term "repetitive sequence motif" or "repetitive common sequence" refers to an amino acid sequence inferred from one or more repeating units. Such a repetitive sequence motif includes framework residue positions and target-interacting residue positions. The framework residue positions correspond to the positions of framework residues within the repeating unit. The target-interacting residue positions correspond to the positions of target-interacting residues within the repeating unit. Such a repetitive sequence motif includes fixed positions and random positions. The term "fixed position" refers to an amino acid position in the repetitive sequence motif, where the position is set to a specific amino acid. Typically, such a fixed position corresponds to a framework residue position.

[0783] The term "random position" refers to an amino acid position in a repeating sequence motif, where two or more amino acids are permitted at that position. Typically, such random positions correspond to the positions of target-target interacting residues. However, some positions of framework residues can also be random.

[0784] The term "folded topology" refers to the tertiary structure of the repeating unit. The folded topology will be determined by amino acid segments forming at least a partial α-helix or β-sheet, or by amino acid segments forming a linear polypeptide or ring, or by any combination of α-helices, β-sheets, and / or linear polypeptides / rings.

[0785] The term "continuous" refers to an arrangement in which the modules are arranged in series.

[0786] In repeating proteins, there are at least 2, usually 6 or more, 10 or more, or 20 or more repeating units, typically about 2 to 6 repeating units. In most cases, repeating proteins are structural and / or adhesion proteins found in prokaryotes and eukaryotes (including vertebrates and invertebrates).

[0787] In most cases, the repeating units will exhibit high sequence identity (the amino acid residues at the corresponding positions are the same) or sequence similarity (the amino acid residues are different, but have similar physicochemical properties), and some of these amino acid residues may be key residues that are highly conserved in different repeating units present in naturally occurring proteins.

[0788] However, as long as a common folding topology is maintained, high sequence variability resulting from amino acid insertions and / or deletions and / or substitutions between different repeating units present in naturally occurring proteins will be possible.

[0789] The term "framework residue" refers to an amino acid residue of a repeating unit or a corresponding amino acid residue of a repeating module that contributes to the folding topology, i.e., it contributes to the folding of the repeating unit (or module) or it contributes to the interaction with adjacent units (or modules). Such contribution can be an interaction with other residues in the repeating unit (module) or an effect on the polypeptide framework imagery present in amino acid segments that form linear polypeptides or rings, or in α-helices or β-sheets.

[0790] The term "target-interacting residue" refers to an amino acid residue of a repeating unit or a corresponding amino acid residue of a repeating module that contributes to the interaction with a target substance. Such a contribution can be a direct interaction with the target substance or an effect on other directly interacting residues, for example by stabilizing the conformation of the (poly)peptide of the repeating unit (module) to allow or enhance the interaction of the directly interacting residue with the target.

[0791] A “target” can be a single molecule, such as a nucleic acid molecule, a (poly)peptide protein, a carbohydrate, or any other naturally occurring molecule, including any part of such a single molecule or a complex of two or more such molecules. In particular, a target can be a molecule on immune effector cells, especially CD8.

[0792] In one embodiment, the repeat modules are directly linked. In the context of this invention, the term "directly linked" refers to repeat modules that are arranged as direct repeats in a repeating protein without any inserted amino acid sequences.

[0793] In another embodiment, the repeating modules are linked via (poly)peptide linkers. Thus, the repeating modules can be indirectly linked via (poly)peptide linkers as insert sequences separating the individual modules. The "insert sequence" can be any amino acid sequence that allows the individual modules to be linked without interfering with the folded topology or module stacking. Preferably, the insert sequence is a short (poly)peptide linker of fewer than 10, and even more preferably fewer than 5 amino acid residues.

[0794] In...

Claims

1. A system, which includes: (i) genetically modified immune effector cells to express chimeric antigen receptors (CARs), or nucleic acids encoding CARs, said nucleic acids being used to genetically modify immune effector cells to express said CARs; and (ii) A compound comprising a binding moiety targeting a CAR and a binding moiety targeting an antigen on a target cell, or a nucleic acid encoding said compound, The CAR and the binding portion targeting the CAR comprise a binding system comprising an ALFA tag and a single-domain antibody that binds to the ALFA tag. The antibody comprises a CDR1 sequence as shown in SEQ ID NO: 94 or a sequence having one or two mutations relative to the sequence, a CDR2 sequence as shown in SEQ ID NO: 97 or a sequence having one or two mutations relative to the sequence, and a CDR3 sequence as shown in SEQ ID NO: 98 or a sequence having one or two mutations relative to the sequence; The antibody further comprises the following amino acid residues at positions 47, 48, and 50 of the sequence corresponding to SEQ ID NO: 1: Arg at position 47, Arg at position 48, and Met at position 50; And the antibody described herein contains a sequence having at least 90% sequence identity with any of SEQ ID NO: 118-125.

2. The system of claim 1, wherein the immune effector cells comprise T cells.

3. The system of claim 1 or 2, wherein the immune effector cells express the CAR on their cell surface.

4. The system of any one of claims 1 to 3, wherein the CAR includes a portion that is coupled to the coupling portion for the CAR.

5. The system of any one of claims 1 to 4, wherein the portion of the CAR that binds to the binding portion for the CAR is contained in the extracellular portion of the CAR.

6. The system of any one of claims 1 to 5, wherein the portion of the CAR that binds to the CAR-targeting portion comprises an ALFA tag, and the CAR-targeting portion comprises the antibody.

7. The system of any one of claims 1 to 5, wherein the portion of the CAR that binds to the CAR-targeting binding portion comprises the antibody, and the CAR-targeting binding portion comprises an ALFA tag.

8. The system of any one of claims 1 to 7, wherein the antibody comprises a sequence Epitope tag specific binding.

9. The system of any one of claims 1 to 8, wherein the antibody is humanized and / or wherein the antibody comprises a sequence having at least about 70% V segment identity with the closest human lineage sequence.

10. The system of any one of claims 1 to 9, wherein the antibody has a higher Tm compared to the antibody of SEQ ID NO:

1.

11. The system of any one of claims 1 to 10, wherein the antibody contains a His residue at position 99 of the sequence corresponding to SEQ ID NO: 1, a Val residue at position 100 of the sequence corresponding to SEQ ID NO: 1, and / or a Tyr residue at position 40 of the sequence corresponding to SEQ ID NO:

1.

12. The system of any one of claims 1 to 11, wherein the antibody comprises FR1, FR2, FR3 and FR4 sequences having at least 80%, at least 85%, at least 90% or at least 95% sequence identity with or identical to the sequences shown in SEQ ID NO: 99, 102, 108 and 109, respectively.

13. The system of any one of claims 1 to 12, wherein the antibody comprises a protein A binding site.

14. The system of any one of claims 1 to 13, wherein the antibody comprises a sequence having at least 95% sequence identity with any of SEQ ID NO: 118-125, or wherein the antibody comprises a sequence shown in any of SEQ ID NO: 118-125.

15. The system of any one of claims 1 to 14, wherein the ALFA tag contains The amino acid sequence, wherein X1 is G or S or P or T, X2 is R or G or P, X5 is E or Q, X7 is L or I, X12 is S or T or P or A or D or E, and X13 is P or A or S or K or D or E or has no amino acid.

16. The system of any one of claims 1 to 15, wherein the binding portion for CAR and the binding portion for antigen on target cells are connected by a connecting portion.

17. The system of claim 16, wherein the connecting portion comprises a polymer.

18. The system of claim 16 or 17, wherein the connecting portion comprises a non-peptide portion.

19. The system of any one of claims 16 to 18, wherein the connecting portion comprises a polymer selected from: poly(ethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine)), poly... POX, polyoxo-zoline Azine (POZ), and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA), its derivatives and combinations thereof.

20. The system of claim 16 or 17, wherein the connecting portion comprises a peptide portion.

21. The system of any one of claims 1 to 20, wherein the binding portion against an antigen on a target cell comprises a portion selected from: an antibody binding to the antigen, an antibody derivative binding to the antigen, and a ligand of the antigen.

22. The system of claim 21, wherein the antibody derivative binding to the antigen comprises an antibody fragment.

23. The system of any one of claims 1 to 22, wherein the binding portion against the antigen on the target cell comprises a peptide portion.

24. The system of any one of claims 1 to 17 and 20 to 23, wherein the compound comprises a fusion polypeptide comprising the CAR-binding portion and the antigen-binding portion on the target cell.

25. The system of claim 21, wherein the ligand of the antigen comprises DUPA or FAPI.

26. The system of any one of claims 1 to 25, wherein the target cell is a diseased cell.

27. The system of any one of claims 1 to 26, wherein the target cell is a cancer cell.

28. The system of any one of claims 1 to 27, wherein the antigen on the target cell comprises a cell surface antigen.

29. The system of any one of claims 1 to 28, wherein the antigen on the target cell comprises a tumor antigen.

30. The system of any one of claims 1 to 29, wherein the nucleic acid encoding the CAR comprises DNA.

31. The system of any one of claims 1 to 17, 20 to 24 and 26 to 30, wherein the nucleic acid encoding the compound comprises RNA.

32. The system of any one of claims 1 to 31, wherein the binding of a complex comprising the following components to cells expressing the antigen results in the killing of cells expressing the antigen: (i) the genetically modified immune effector cells expressing chimeric antigen receptors (CARs); and (ii) The compound comprising a binding portion for CAR and a binding portion for antigens on target cells.

33. A method for treating a subject suffering from a disease, disorder, or condition characterized by cells expressing antigens, comprising: (i) Providing the subject with genetically modified immune effector cells that express chimeric antigen receptors (CARs); as well as (ii) Providing the object with a compound comprising a binding moiety against a CAR and a binding moiety against the antigen, The CAR and the binding portion targeting the CAR comprise a binding system comprising an ALFA tag and a single-domain antibody that binds to the ALFA tag. The antibody comprises a CDR1 sequence as shown in SEQ ID NO: 94 or a sequence having one or two mutations relative to the sequence, a CDR2 sequence as shown in SEQ ID NO: 97 or a sequence having one or two mutations relative to the sequence, and a CDR3 sequence as shown in SEQ ID NO: 98 or a sequence having one or two mutations relative to the sequence; The antibody further comprises the following amino acid residues at positions 47, 48, and 50 of the sequence corresponding to SEQ ID NO: 1: Arg at position 47, Arg at position 48, and Met at position 50; And the antibody described herein contains a sequence having at least 90% sequence identity with any of SEQ ID NO: 118-125.

34. The method of claim 33, wherein the immune effector cells comprise T cells.

35. The method of claim 33 or 34, wherein the immune effector cells express the CAR on their cell surface.

36. The method of any one of claims 33 to 35, wherein the CAR includes a portion that is coupled to the coupling portion for the CAR.

37. The method of any one of claims 33 to 36, wherein the portion of the CAR that binds to the binding portion for the CAR is contained in the extracellular portion of the CAR.

38. The method of any one of claims 33 to 37, wherein the portion of the CAR that binds to the CAR-targeting portion comprises an ALFA tag, and the CAR-targeting portion comprises the antibody.

39. The method of any one of claims 33 to 37, wherein the portion of the CAR that binds to the CAR-targeting binding portion comprises the antibody, and the CAR-targeting binding portion comprises an ALFA tag.

40. The method of any one of claims 33 to 39, wherein the antibody comprises a sequence Epitope tag specific binding.

41. The method of any one of claims 33 to 40, wherein the antibody is humanized and / or wherein the antibody comprises a sequence having at least about 70% V segment identity with the closest human lineage sequence.

42. The method of any one of claims 33 to 41, wherein the antibody has a higher Tm compared to the antibody of SEQ ID NO:

1.

43. The method of any one of claims 33 to 42, wherein the antibody contains a His residue at position 99 of the sequence corresponding to SEQ ID NO: 1, a Val residue at position 100 of the sequence corresponding to SEQ ID NO: 1, and / or a Tyr residue at position 40 of the sequence corresponding to SEQ ID NO:

1.

44. The method of any one of claims 33 to 43, wherein the antibody comprises FR1, FR2, FR3 and FR4 sequences having at least 80%, at least 85%, at least 90% or at least 95% sequence identity with or identical to the sequences shown in SEQ ID NO: 99, 102, 108 and 109, respectively.

45. The method of any one of claims 33 to 44, wherein the antibody comprises a protein A binding site.

46. ​​The method of any one of claims 33 to 45, wherein the antibody comprises a sequence having at least 95% sequence identity with any of SEQ ID NO: 118-125, or wherein the antibody comprises a sequence shown in any of SEQ ID NO: 118-125.

47. The method of any one of claims 33 to 46, wherein the ALFA tag comprises The amino acid sequence, wherein X1 is G or S or P or T, X2 is R or G or P, X5 is E or Q, X7 is L or I, X12 is S or T or P or A or D or E, and X13 is P or A or S or K or D or E or has no amino acid.

48. The method of any one of claims 33 to 47, wherein the binding portion for CAR and the binding portion for antigen on target cells are connected by a connecting portion.

49. The method of claim 48, wherein the connecting portion comprises a polymer.

50. The method of claim 48 or 49, wherein the connecting portion comprises a non-peptide portion.

51. The method of any one of claims 48 to 50, wherein the connecting portion comprises a polymer selected from: poly(ethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine)), poly... POX, polyoxo-zoline Azine (POZ), and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA), its derivatives and combinations thereof.

52. The method of claim 48 or 49, wherein the connecting portion comprises a peptide portion.

53. The method of any one of claims 33 to 52, wherein the binding portion against the antigen comprises a portion selected from: an antibody binding to the antigen, an antibody derivative binding to the antigen, and a ligand of the antigen.

54. The method of claim 53, wherein the antibody derivative binding to the antigen comprises an antibody fragment.

55. The method of any one of claims 33 to 54, wherein the binding portion against the antigen comprises a peptide portion.

56. The method of any one of claims 33 to 49 and 52 to 55, wherein the compound comprises a fusion polypeptide comprising the CAR-binding portion and the antigen-binding portion.

57. The method of claim 53, wherein the ligand of the antigen comprises DUPA or FAPI.

58. The method of any one of claims 33 to 57, wherein the cell expressing the antigen is a diseased cell.

59. The method of any one of claims 33 to 58, wherein the cell expressing the antigen is a cancer cell.

60. The method of any one of claims 33 to 59, wherein the antigen comprises a cell surface antigen.

61. The method of any one of claims 33 to 60, wherein the antigen comprises a tumor antigen.

62. The method of any one of claims 33 to 61, wherein the binding of a complex comprising the following components to cells expressing the antigen results in the killing of cells expressing the antigen: (i) the genetically modified immune effector cells expressing chimeric antigen receptors (CARs); and (ii) The compound comprising a binding portion for CAR and a binding portion for antigen.

63. The method of any one of claims 33 to 62, wherein the method comprises administering the genetically modified immune effector cells expressing CAR to the subject.

64. The method of any one of claims 33 to 62, wherein the method comprises generating the genetically modified immune effector cells expressing CAR in the subject.

65. The method of any one of claims 33 to 62 and 64, wherein the method comprises administering a nucleic acid encoding the CAR to the object.

66. The method of claim 65, wherein the nucleic acid encoding the CAR comprises DNA.

67. The method of any one of claims 33 to 66, wherein the method comprises applying the compound to the object.

68. The method of any one of claims 33 to 49, 52 to 56 and 58 to 66, wherein the method comprises producing the compound in the object.

69. The method of any one of claims 33 to 49, 52 to 56, 58 to 66 and 68, wherein the method comprises administering a nucleic acid encoding the compound to the object.

70. The method of claim 69, wherein the nucleic acid encoding the compound comprises RNA.

71. The method of claim 69 or 70, wherein the method comprises causing cells of the object to express the compound such that the compound is associated with cells expressing the antigen and the binding portion against the CAR can be bound by the immune effector cells.

72. The method of claim 71, wherein cells expressing the compound are transfected with nucleic acids encoding the compound.

73. The method of claim 71 or 72, wherein the cell expressing the compound secretes the compound.

74. The method of any one of claims 71 to 73, wherein the cells expressing the compound express the compound to release it into the bloodstream.

75. The method of any one of claims 65 to 74, wherein the nucleic acid encoding the CAR and / or the nucleic acid encoding the compound is administered as a particulate formulation, for example, formulated as lipid nanoparticles.

76. The method of any one of claims 33 to 75, wherein the disease, disorder, or ailment is cancer.

77. Chimeric antigen receptor (CAR), which contains a single-domain antibody that binds to an ALFA tag. The antibody comprises a CDR1 sequence as shown in SEQ ID NO: 94 or a sequence having one or two mutations relative to the sequence, a CDR2 sequence as shown in SEQ ID NO: 97 or a sequence having one or two mutations relative to the sequence, and a CDR3 sequence as shown in SEQ ID NO: 98 or a sequence having one or two mutations relative to the sequence; The antibody further comprises the following amino acid residues at positions 47, 48, and 50 of the sequence corresponding to SEQ ID NO: 1: Arg at position 47, Arg at position 48, and Met at position 50; And the antibody described herein contains a sequence having at least 90% sequence identity with any of SEQ ID NO: 118-125.

78. The CAR of claim 77, wherein the antibody comprises a sequence Epitope tag specific binding.

79. The CAR of claim 77 or 78, wherein the antibody is humanized and / or wherein the antibody comprises a sequence having at least about 70% V segment identity with the closest human lineage sequence.

80. The CAR of any one of claims 77 to 79, wherein the antibody has a higher Tm compared to the antibody of SEQ ID NO:

1.

81. The CAR of any one of claims 77 to 80, wherein the antibody contains a His residue at position 99 of the sequence corresponding to SEQ ID NO: 1, a Val residue at position 100 of the sequence corresponding to SEQ ID NO: 1, and / or a Tyr residue at position 40 of the sequence corresponding to SEQ ID NO:

1.

82. The CAR of any one of claims 77 to 81, wherein the antibody comprises FR1, FR2, FR3 and FR4 sequences having at least 80%, at least 85%, at least 90% or at least 95% sequence identity with or identical to the sequences shown in SEQ ID NO: 99, 102, 108 and 109, respectively.

83. The CAR of any one of claims 77 to 82, wherein the antibody comprises a protein A binding site.

84. The CAR of any one of claims 77 to 83, wherein the antibody comprises a sequence having at least 95% sequence identity with any of SEQ ID NO: 118-125, or wherein the antibody comprises a sequence shown in any of SEQ ID NO: 118-125.

85. The CAR of any one of claims 77 to 84, wherein the antibody is contained in the extracellular domain of the CAR.

86. A nucleic acid encoding a CAR as described in any one of claims 77 to 85.

87. The nucleic acid of claim 86, wherein it is DNA.

88. A host cell comprising the nucleic acid of claim 86 or 87 and / or expressing the CAR of any one of claims 77 to 85.

89. The host cell of claim 88, comprising immune effector cells.

90. The host cell of claim 89, wherein the immune effector cell comprises a T cell.

91. The host cell of any one of claims 88 to 90, wherein it is genetically modified to contain the nucleic acid and / or express the CAR.

92. The host cell of any one of claims 88 to 91, wherein the CAR is expressed on its cell surface.

93. A compound comprising a single-domain antibody that binds to an ALFA tag and a binding moiety against an antigen on a target cell. The antibody comprises a CDR1 sequence as shown in SEQ ID NO: 94 or a sequence having one or two mutations relative to the sequence, a CDR2 sequence as shown in SEQ ID NO: 97 or a sequence having one or two mutations relative to the sequence, and a CDR3 sequence as shown in SEQ ID NO: 98 or a sequence having one or two mutations relative to the sequence; The antibody further comprises the following amino acid residues at positions 47, 48, and 50 of the sequence corresponding to SEQ ID NO: 1: Arg at position 47, Arg at position 48, and Met at position 50; And the antibody described herein contains a sequence having at least 90% sequence identity with any of SEQ ID NO: 118-125.

94. The compound of claim 93, wherein the antibody comprises a sequence... Epitope tag specific binding.

95. The compound of claim 93 or 94, wherein the antibody is humanized and / or wherein the antibody comprises a sequence having at least about 70% V segment identity with the closest human lineage sequence.

96. The compound of any one of claims 93 to 95, wherein the antibody has a higher Tm compared to the antibody of SEQ ID NO:

1.

97. The compound of any one of claims 93 to 96, wherein the antibody contains a His residue at position 99 of the sequence corresponding to SEQ ID NO: 1, a Val residue at position 100 of the sequence corresponding to SEQ ID NO: 1, and / or a Tyr residue at position 40 of the sequence corresponding to SEQ ID NO:

1.

98. The compound of any one of claims 93 to 97, wherein the antibody comprises FR1, FR2, FR3 and FR4 sequences having at least 80%, at least 85%, at least 90% or at least 95% sequence identity with or identical to the sequences shown in SEQ ID NO: 99, 102, 108 and 109, respectively.

99. The compound of any one of claims 93 to 98, wherein the antibody comprises a protein A binding site.

100. The compound of any one of claims 93 to 99, wherein the antibody comprises a sequence having at least 95% sequence identity with any of SEQ ID NO: 118-125, or wherein the antibody comprises a sequence shown in any of SEQ ID NO: 118-125.

101. The compound of any one of claims 93 to 100, wherein the antibody is connected to the binding portion against the antigen on the target cell via a connecting portion.

102. The compound of claim 101, wherein the connecting portion comprises a polymer.

103. The compound of claim 101 or 102, wherein the linking portion comprises a non-peptide portion.

104. The compound of any one of claims 101 to 103, wherein the linking portion comprises a polymer selected from: poly(ethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine)), poly(ethylene glycol) (PEG), poly(N-methylglycine ... POX, polyoxo-zoline Azine (POZ), and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA), its derivatives and combinations thereof.

105. The compound of claim 101 or 102, wherein the linking portion comprises a peptide moiety.

106. The compound of any one of claims 93 to 105, wherein the binding portion against an antigen on a target cell comprises a portion selected from: an antibody binding to the antigen, an antibody derivative binding to the antigen, and a ligand of the antigen.

107. The compound of claim 106, wherein the antibody derivative binding to the antigen comprises an antibody fragment.

108. The compound of any one of claims 93 to 107, wherein the binding portion against the antigen on the target cell comprises a peptide portion.

109. The compound of any one of claims 93 to 102 and 105 to 108, wherein the compound comprises a fusion polypeptide comprising the antibody and the binding portion against the antigen on the target cell.

110. The compound of claim 106, wherein the ligand of said antigen comprises DUPA or FAPI.

111. The compound of any one of claims 93 to 110, wherein the target cell is a diseased cell.

112. The compound of any one of claims 93 to 111, wherein the target cell is a cancer cell.

113. The compound of any one of claims 93 to 112, wherein the target cell antigen comprises a cell surface antigen.

114. The compound of any one of claims 93 to 113, wherein the antigen on the target cell comprises a tumor antigen.

115. A nucleic acid encoding a compound according to any one of claims 93 to 102, 105 to 109 and 111 to 114.

116. The nucleic acid of claim 115, wherein it is RNA.

117. A host cell comprising the nucleic acid of claim 115 or 116 and / or expressing the compound of any one of claims 93 to 102, 105 to 109 and 111 to 114.

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