D-domain containing polypeptides and uses thereof

Abstract

Provided are non-antibody structural scaffold-based D-domain (DD)-containing polypeptides (DDpp) that specifically bind to BCMA, CD123, AFP, AFPp26, CS1, or HER2 target proteins.SOLUTION: To provide a DDpp binding to a target protein having a specific sequence, a fusion protein (chimeric antibody receptor or the like) containing the DDpp, a nucleic acid encoding these proteins, a vector and a host cell containing the nucleic acid, and a method for diagnosing and treating diseases using the protein, the nucleic acid, the vector, the cell or the pharmaceutical composition.SELECTED DRAWING: None

Description

[Background technology]

[0001] Antibody-based reagents have accelerated the pace of biological research and development. Antibody compositions are used in pharmaceuticals. It is one of the most important and successful therapeutic and diagnostic agents used in industry. However, due to cost, time and efficacy considerations, there is momentum towards the development of alternative affinity reagents. has been increasing.

[0002] A variety of non-antibody conjugation formats are emerging for applications historically achieved with antibodies. Although many successful cases have been reported for amorphous linear peptides, By imposing structural constraints on the column—usually by introducing disulfide bonds—further definition can be achieved. This constraint is due to the more favorable thermodynamics and residue structure of fixed shape complementarity. surface presentation of (e.g., hydrophobic amino acids) (which would otherwise be buried and therefore provides higher affinity and higher specificity through targeting (which may become non-target) (Ladner,Trends in Biotech.13(10):426-430 (1995)). Conversely, formats containing disulfide bonds typically have intradomain or Prone to improper pairing of cysteines between domains, which can lead to poor expression and product yield This can lead to adverse effects on productivity and product quality.

[0003] Structures found in protein subdomains provided another source of structural constraints. Cutin type III repeats (adnectins), z-proteins (affibodies), knottins , lipocalins (anticalins) and ankyrin repeats (DARPins) , which generated antibody-like affinities for a variety of different targets (Hey et al., Trends s in Biotech.23(10):514-422(2005)). The main regions are typically framework and complementarity determining regions (CDRs) found in antibody variable domains. Two CDR-like features: a structural framework and library that confer high thermodynamic stability. These include residues or loops that form the basis for displaying the variability of the peptide. Summary of the Invention

[0004] Novel target binding compositions, particularly such compositions that include alternative binding scaffolds (e.g., non-antibody scaffolds), are also disclosed. There remains a substantial unmet need for drugs. Reagents of particular interest include, for example, , substantially reduced production costs and / or comparable or superior assay performance compared to antibodies. The present disclosure provides a method for the preparation of antibodies based on non-antibody scaffolds, which may be characterized by medicinal, diagnostic and / or therapeutic properties. In some embodiments, novel target-binding D domain (DD) polypeptides are provided. D domain polypeptides (DDpp) are characterized by high target binding affinity and a non-antibody structural framework. In some embodiments, DDpp acts as a signaling agent for specific cells (e.g., diseased cells). and can be conveniently used as a targeted therapeutic agent (e.g., immune cells) to prevent off-target These are target-specific binding polypeptides that can reduce or eliminate the effect of some In embodiments, the provided DDpp binds to cellular or soluble factors involved in disease. It is used as a therapeutic agent.

[0005] In some embodiments, the present disclosure provides a protein comprising a D domain (DD) target binding domain. The DD protein (DDpp) was provided, and the DD protein was a marker for BCMA (SEQ ID NO: 7), CD123 (SEQ ID NO: 8). ), CS1 (SEQ ID NO: 965), HER2 (SEQ ID NO: 967), AFP (SEQ ID NO: 9), A target molecule selected from the group consisting of AFP p26 (SEQ ID NO: 10), or a fragment thereof In some embodiments, the DDpp is monovalent or multivalent. In some embodiments, the DDpp is monospecific or multispecific. In some embodiments, the DDpp is monospecific and multivalent. p is multispecific and multivalent. Similar to the methods of making and using fusion proteins, Fusion proteins containing one or more DDs are also provided. Nucleic acids encoding the nucleic acids, as well as host cells containing the nucleic acids, are also provided. Non-limiting examples of such uses include: , including but not limited to, targeted analysis, and diagnostic and therapeutic applications.

[0006] In a further embodiment, the present disclosure provides a protein comprising a D domain (DD) target binding domain. providing a protein (DDpp) wherein the DD (a) specifically binds to BCMA (SEQ ID NO: 7); (b) a CD123 (sequence number 11 to 305 or 306) containing the amino acid sequence of SEQ ID NO: 11 to 305; 8) and specifically binds to the amino acid sequence of SEQ ID NO: 307 to 739 or 740. (c) specifically binds to AFP (SEQ ID NO: 9) or a fragment thereof and contains the sequence DD containing the amino acid sequence of numbers 741 to 874 or 886 to 895; (d) AFP p26 (SEQ ID NO: 10) and SEQ ID NO: 741 to 874, or 886 to 8 (e) CS1 (SEQ ID NO: 965) or a fragment thereof; DD containing the amino acid sequence of SEQ ID NO: 896 to 909 or 910 and (f) a sequence encoding a polypeptide that specifically binds to HER2 or a fragment thereof and is selected from the group consisting of SEQ ID NOs: 911 to 919. DD comprising 49 or 950 amino acid sequences. Mutants (a) to (f) retain the ability to specifically bind to their respective targets. Also provided are proteins comprising the heterologous polypeptide. In some embodiments, the heterologous polypeptide is fused to a full-length antibody or an antibody thereof. In some embodiments, the DD comprises the amino-terminal end of a full-length antibody heavy chain. the amino terminus of a full-length antibody light chain; the carboxyl terminus of a full-length antibody heavy chain; or a full-length antibody In another embodiment, the DD is fused to the carboxyl terminus of the antibody fragment that is the Fc. In a further embodiment, the heterologous polypeptide is fused to a segment comprising (i) a transmembrane domain (ii) a membrane-binding domain; (iii) human serum albumin or a fragment thereof (iv) AFP or a fragment thereof; (v) AFP p26 or a fragment thereof (vi) the extracellular domain of the receptor or a fragment thereof; and (vii) the intracellular domain of the receptor. The group consisting of the extracellular domain of a receptor (e.g., a nuclear protein) or a fragment thereof In some embodiments, the protein comprises a member selected from the group consisting of BCMA (sequence No. 7) or CD123 (SEQ ID NO: 8) or CD19 (SEQ ID NO: 3) or CS1 ( A heterologous polypeptide comprising the extracellular domain of SEQ ID NO: 965, or a fragment of the extracellular domain. In some embodiments, the protein comprises a peptide such as CD19, CD20, CD 22, HVEM, BTLA, DR3, CD37; TSLPR, IL7R, and gp96 an extracellular domain or a fragment of an extracellular domain of a receptor selected from the group consisting of In some embodiments, the protein comprises a heterologous polypeptide comprising a serum protein. and heterologous polypeptides containing antigenic portions of proteins (e.g., AFP, and AFP p26). In some embodiments, the protein is an intracellular protein (e.g., a nuclear protein). In some embodiments, the protein comprises a heterologous polypeptide comprising an antigenic portion of In further embodiments, the label is an enzyme label, a fluorescent label, a luminescent label, a biolabel, or a In a further embodiment, the tag is selected from the group consisting of a bioluminescent label, and a biotin moiety. In some embodiments, the protein is conjugated to a therapeutic or cytotoxic agent. The antigen binds to one or more major histocompatibility complex (MHC) class I or class II complexes. It contains a heterologous polypeptide to which it is bound.

[0007] In some embodiments, the DD of the DDpp specifically binds to BCMA. In one embodiment, the DD comprises a BCMA protein having an amino acid sequence consisting of SEQ ID NO:7. In a further embodiment, DDpp specifically binds to SEQ ID NOs: 11 to 305, and and 306. In another embodiment, the BCMA binding DDpp is an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 to 305, and 306. In some embodiments, DDpp comprises multiple target binding domains (e.g., In some embodiments, DDpp includes 2, 3 In some embodiments, the antibody comprises one, four, five, or more than five DDs that bind to BCMA. In this case, DDpp contains 2, 3, 4, 5, or more than 5 DDs with the same sequence. In some embodiments, the DDpp comprises 2, 3, 4, 5, or more than 5 B In some embodiments, the DDpp comprises a DD that binds to a different epitope of CMA. , which contains a DD that specifically binds to BCMA, and two that bind to BCMA or a different target antigen. , 3, 4, 5, or more than 5 additional distinct DD or target binding domains ( In some embodiments, the DDpp further comprises a BCMA specific antibody. It contains a heterologous binding DD and binds to one or more antigens expressed on the surface of B cells. Further comprising one or more additional DD or other target binding domains. In some embodiments, the DDpp comprises one or more DDs that specifically bind to BCMA. one or more additional DDs or other target-binding binding domains that bind to multiple cancer antigens In some embodiments, the DDpp further comprises 2, 3, 4, 5, or In a further embodiment, the DDpp specifically binds to more than two, three, or more different targets. Specifically binds to 4, 5, or more than 5 different cancer antigens. In this study, DDpp was expressed on the surface of two, three, four, five, or more than five cancer cells. In some embodiments, the DDpp specifically binds to different cancer antigens. Specifically binds to five or more cancer antigens expressed on the surface of different cancer cells .

[0008] In some embodiments, the DDpp is a fusion tag comprising a DD that specifically binds BCMA. In some embodiments, the DDpp fusion protein is a protein selected from the group consisting of SEQ ID NO: 7 The DD specifically binds to the BCMA protein and has an amino acid sequence comprising: In this embodiment, the DDpp is selected from the group consisting of SEQ ID NOs: 11 to 305, and 306. In another embodiment, the BCM is a fusion protein comprising a DD containing an amino acid sequence that is A-binding DDpp fusion proteins are selected from the group consisting of SEQ ID NOs: 11 to 305, and 306. In some embodiments, the DDpp fusion protein comprises a variant of a selected amino acid sequence. The protein comprises a full-length antibody or a portion (fragment) of an antibody. , DDpp fusion proteins can be used to express full-length IgG antibodies (e.g., IgG1, IgG2, IgG In a further embodiment, the DDpp comprises a commercially approved Therapeutic antibodies (e.g., rituximab, ofatumumab, ocrelizumab, veltuzumab, M EDI-551, epratuzumab, belimumab, tabalumab, AMG-557, MEDI In other embodiments, the BCMA-binding DDpp comprises Fc It is a fusion protein.

[0009] In some embodiments, the DDpp fusion protein is operably linked to a serum protein. In some embodiments, the DDpp fusion protein comprises a BCMA-binding DD. , SEQ ID NOs: 11 to 305, and 306. In other embodiments, the BCMA-binding DDpp fusion protein is selected from the group consisting of SEQ ID NOs: 11-305, and and 306. In one embodiment, the DDpp fusion protein comprises human serum albumin or a fragment thereof. In some embodiments, the DDpp fusion protein is AFP or AFP p26, or or a fragment thereof. In some embodiments, the DDpp fusion protein comprises A FP (e.g., SEQ ID NO: 9), or a fragment thereof. The pp fusion protein contains AFP p26 (SEQ ID NO: 10), or a fragment thereof. nothing.

[0010] In some embodiments, the DDpp fusion protein comprises one or more target-binding DDpps. pp and p29 proteins (e.g., SEQ ID NOs: 10, 968, 969, 970, 971 , 972, 973, or 974). Fusion proteins containing such p29 sequences have surprisingly been shown herein to produce long serum half-length peptides. In some embodiments, the soluble DDpp fusion protein Quality is at least 1 hour, at least 2 hours, at least 4 hours, at least 8 hours, At least 16 hours, at least 32 hours, at least 64 hours, or more In some embodiments, the soluble fusion protein has a plasma half-life in vivo. at least 1 hour, at least 2 hours, at least 4 hours, at least 8 hours, at least 16 hours, at least 32 hours, at least 64 hours, or longer than 65 hours hours, or 1-10 hours, 2-10 hours, 4-10 hours, 6-10 hours, or 6- In some embodiments, the soluble DDpp fusion has an in vivo plasma half-life of 9 hours. The protein may be incubated for at least 1 hour, at least 2 hours, at least 4 hours, or for at least 1 hour in humans. at least 8 hours, at least 16 hours, at least 32 hours, at least 64 hours, or is longer than 65 hours, or 1-10 hours, 2-10 hours, 4-10 hours, 6-10 hours hours, or 6-9 hours in vivo plasma half-life.

[0011] In some embodiments, the BCMA-binding DDpp fusion protein binds to the extracellular domain of the receptor. In some embodiments, the DDpp fusion protein comprises a The material contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 to 305, and 306. In another embodiment, the BCMA-binding DDpp fusion protein comprises a DD comprising SEQ ID NO: 1. 1 to 305, and 306. In another embodiment, the CD123-binding DDpp fusion protein binds to CD123 (e.g., In some embodiments, the extracellular domain of the polypeptide of the present invention is a polypeptide of the present invention. , BCMA-binding DDpp fusion proteins bind to CD19, CD20, CD22, HVEM, Consists of BTLA, DR3, CD37, CS1, TSLPR, IL7R, and gp96 The antibody comprises the extracellular domain of a receptor selected from the group, or a fragment thereof.

[0012] In a further embodiment, the BCMA-binding DDpp fusion protein is an intracellular protein ( In some embodiments, the DD The pp fusion protein is selected from the group consisting of SEQ ID NOs: 11 to 305, and 306. In another embodiment, the BCMA-binding DDpp fusion protein comprises a DD comprising the amino acid sequence The quality is a mutation of an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 to 305, and 306. In some embodiments, the BCMA-binding DDpp fusion protein is a serum tag. Proteins (e.g., HSA, AFP, and AFP p26), receptors (e.g., BCM A, CD123, CS1, and CD19) extracellular domains, or intracellular proteins (e.g., nuclear protein) fragments, 5-500, 5-400, 5-300 , 5~200, 5~100, 5~50, 10~500, 10~400, 10~300, 1 It consists of 0 to 200, 10 to 100, or 10 to 50 amino acid residues.

[0013] In some embodiments, the DD of the DDpp specifically binds to CD123. In some embodiments, the DDpp is a CD123 tag having an amino acid sequence consisting of SEQ ID NO:8. In a further embodiment, DDpp specifically binds to the protein of SEQ ID NO: 307-7 39, and 740. In other embodiments, The CD123-binding DDpp is selected from the group consisting of SEQ ID NOs: 307 to 739, and 740. In some embodiments, the DDpp comprises a variant of the amino acid sequence that is targeted to multiple targets. In some embodiments, the DD The pp contains 2, 3, 4, 5, or more than 5 DDs that bind to CD123. In some embodiments, the DDpp comprises two, three, four, five, or more than five identical sequences. In some embodiments, the DDpp comprises 2, 3, 4, 5, or The antibody comprises more than five DDs that bind to different epitopes of CD123. In this state, the DDpp comprises a DD that specifically binds to CD123 and binds to BCMA or a different Two, three, four, five, or more than five additional different DDs or targets that bind to the target antigen In some embodiments, the DD further comprises a specific binding domain (e.g., an scFv). pp contains a DD that specifically binds to CD123 and is expressed on the surface of B cells. The antibody may contain one or more additional DDs or other target-binding domains that bind to multiple antigens. In some embodiments, the DDpp specifically binds to CD123. DD, and one or more additional DDs or In some embodiments, the DDpp further comprises other target binding domains. In further embodiments, the antibody specifically binds to 4, 5, or more than 5 different targets. DDpp specifically binds to two, three, four, five, or more than five different cancer antigens. In some embodiments, the DDpp comprises 2, 3, 4, 5, or more than 5 Specifically binds to different cancer antigens expressed on the surface of cancer cells. In some embodiments, DDpp was expressed on the surface of two, three, four, five, or more than five different cancer cells. It specifically binds to cancer antigens.

[0014] In some embodiments, the DDpp is a fusion comprising a DD that specifically binds to CD123. In some embodiments, the DD is a protein. In some embodiments, DDpp specifically binds to a CD123 protein having the formula: The present invention relates to a method for producing a human medicament comprising the steps of: In another embodiment, the CD123-binding DDpp fusion protein is selected from the group consisting of SEQ ID NOs: 307-7 39, and 740. In embodiments, the DDpp fusion protein is a full-length antibody or a portion of an antibody (fragment In some embodiments, the DDpp fusion protein comprises a full-length IgG antibody (e.g., For example, IgG1, IgG2, IgG2, or IgG4). , DDpp is a commercially approved therapeutic antibody (e.g., rituximab, ofatumumab, Ocrelizumab, veltuzumab, MEDI-551, epratuzumab, belimumab, tabacum Other embodiments include: ramucirumab, AMG-557, MEDI-570, and NN8828. In this embodiment, the CD123-binding DDpp is an Fc fusion protein.

[0015] In some embodiments, the DDpp fusion protein is operably linked to a serum protein. In some embodiments, the DDpp fusion protein comprises a CD123-binding DD. The present invention relates to a method for producing a human medicament comprising the steps of: In another embodiment, the CD123-binding DDpp fusion protein is selected from the group consisting of SEQ ID NOs: 307-7 39, and 740. In an embodiment, the DDpp fusion protein comprises human serum albumin or a fragment thereof. In some embodiments, the DDpp fusion protein comprises AFP or AFP p 26, or a fragment thereof. In some embodiments, the DDpp fusion protein The substance comprises AFP (e.g., SEQ ID NO: 9), or a fragment thereof. The DDpp fusion protein is AFP p26 (SEQ ID NO: 10), or a fragment thereof. Includes ent.

[0016] In some embodiments, the CD123-binding DDpp fusion protein binds to the extracellular domain of the receptor. In some embodiments, the CD123-binding domain comprises a CD123-binding domain or a fragment thereof. The pp fusion protein is selected from the group consisting of SEQ ID NOs: 307-739, and 740. In another embodiment, the CD123-binding DDpp fusion protein comprises an amino acid sequence A variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 307 to 739 and 740 In a further embodiment, the CD123-binding DDpp fusion protein comprises BCMA ( the extracellular domain of CD123 (SEQ ID NO: 7) or CD123 (SEQ ID NO: 8), or a fragment thereof In a further embodiment, the CD123-binding DDpp fusion protein comprises BCMA (SEQ ID NO: 7), or CD123 (SEQ ID NO: 8), or CS1 (SEQ ID NO: 965) In some embodiments, the CD123 The binding DDpp fusion proteins are CD19, CD20, CD22, HVEM, BTLA, Selected from the group consisting of DR3, CD37, CS1, TSLPR, IL7R, and gp96 The antibody comprises the extracellular domain of the receptor to be treated or a fragment thereof.

[0017] In a further embodiment, the CD123-binding DDpp fusion protein is an intracellular protein (e.g., nucleoprotein) or fragments thereof. In some embodiments, The D123-binding DDpp fusion protein consists of SEQ ID NOs: 307 to 739 and 740. In another embodiment, the CD123-binding DDpp fusion protein comprises an amino acid sequence selected from the group consisting of: The conjugated protein is an amino acid sequence selected from the group consisting of SEQ ID NOs: 307 to 739, and 740. This includes variants of the amino acid sequence.

[0018] In some embodiments, the CD123-binding DDpp fusion protein is a serum protein (e.g., HSA, AFP, and AFP 26), receptors (e.g., BCMA, CS1 , CD123, and CD19), or the extracellular domains of intracellular proteins (e.g., nucleoprotein) fragments, 5-500, 5-400, 5-300, 5-20 0, 5-100, 5-50, 10-500, 10-400, 10-300, 10-200 , 10 to 100, or 10 to 50 amino acid residues.

[0019] In some embodiments, the DD of the DDpp specifically binds to CS1. In embodiments, the DDpp is a CS1 protein having an amino acid sequence consisting of SEQ ID NO: 965. In a further embodiment, DDpp specifically binds to proteins. In another embodiment, the CS 1-binding DDpp is an amino acid selected from the group consisting of SEQ ID NOs: 896 to 909, and 910 In some embodiments, DDpp comprises multiple target binding domains. In some embodiments, the DDpp comprises: 2, 3, 4, 5, or more than 5 DDs that bind to CS1. In the form, DDpp is a DD having two, three, four, five, or more than five identical sequences. In some embodiments, the DDpp comprises 2, 3, 4, 5, or more than 5 In some embodiments, the DDpp comprises a DD that binds to a different epitope of CS1. contains a DD that specifically binds to CS1, and two that bind to BCMA or a different target antigen. , 3, 4, 5, or more than 5 additional distinct DD or target binding domains ( In some embodiments, the DDpp further comprises a CS1 specific antibody. 1, which contains a DD that specifically binds to one or more antigens expressed on the surface of B cells; Further comprising one or more additional DD or other target binding domains. In this embodiment, the DDpp comprises a DD that specifically binds to CS1 and one or more The compound may further comprise one or more additional DDs or other target-binding domains that bind to cancer antigens. In some embodiments, the DDpp comprises 2, 3, 4, 5, or more than 5 In a further embodiment, the DDpp specifically binds to different targets. Specifically binds to five or more than five different cancer antigens. DDpp can be used to identify two, three, four, five, or more than five different proteins expressed on the surface of cancer cells. In some embodiments, the DDpp specifically binds to a cancer antigen. , or more than five different cancer antigens expressed on the surface of cancer cells.

[0020] In some embodiments, the DDpp is a fusion protein comprising a DD that specifically binds to CS1. In some embodiments, DD is a protein. In some embodiments, DDpp specifically binds to a CS1 protein having the formula: It comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 896-909, and 910. In another embodiment, the CS1-binding DDpp fusion protein is selected from the group consisting of SEQ ID NOs: 896-909, and and 910. In the present invention, DDpp fusion proteins contain full-length antibodies or portions (fragments) of antibodies. In some embodiments, the DDpp fusion protein is a full-length IgG antibody (e.g., I In a further embodiment, the DDp p is a commercially approved therapeutic antibody (e.g., rituximab, ofatumumab, ocreglitazone, uzumab, veltuzumab, MEDI-551, epratuzumab, belimumab, tabalumab, In other embodiments, the compounds include: The CS1-binding DDpp is an Fc fusion protein.

[0021] In some embodiments, the DDpp fusion protein is operably linked to a serum protein. In some embodiments, the DDpp fusion protein comprises a CS1-binding DD. It comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 896-909, and 910. In another embodiment, the CS1-binding DDpp fusion protein is selected from the group consisting of SEQ ID NOs: 896-909, and and 910. In a further embodiment, In one embodiment, the DDpp fusion protein comprises human serum albumin or a fragment thereof. In some embodiments, the DDpp fusion protein is AFP or AFP p26, or or a fragment thereof. In some embodiments, the DDpp fusion protein comprises A FP (e.g., SEQ ID NO: 9), or a fragment thereof. The pp fusion protein contains AFP p26 (SEQ ID NO: 10), or a fragment thereof. nothing.

[0022] In some embodiments, the CS1-binding DDpp fusion protein binds to the extracellular domain of the receptor. In some embodiments, the CS1-binding DDpp fusion comprises a CS1-binding DDpp fusion or a fragment thereof. The protein is selected from the group consisting of SEQ ID NOs: 896-909, and 910. In another embodiment, the CS1-binding DDpp fusion protein comprises the sequence of SEQ ID NO: 89. 6 to 909, and 910. In another embodiment, the CS1-binding DDpp fusion protein is selected from the group consisting of BCMA (SEQ ID NO: 7), C D123 (SEQ ID NO: 8), or the extracellular domain of CS1 (SEQ ID NO: 965), or In some embodiments, the CS1-binding DDpp fusion protein comprises a fragment of , CD19, CD20, CD22, HVEM, BTLA, DR3, CD37, CS-1, The extracellular domain of a receptor selected from the group consisting of TSLPR, IL7R, and gp96. or fragments thereof.

[0023] In a further embodiment, the CS1-binding DDpp fusion protein binds to an intracellular protein (e.g., In some embodiments, the CS1 The conjugated DDpp fusion protein is selected from the group consisting of SEQ ID NOs: 896-909, and 910. In another embodiment, the CS1-binding DDpp fusion protein comprises an amino acid sequence selected from the group consisting of: The amino acid sequence of the variant selected from the group consisting of SEQ ID NOs: 896 to 909 and 910. Includes variants.

[0024] In some embodiments, the CS1-binding DDpp fusion protein binds to a serum protein (e.g., receptors (e.g., BCMA, CS1, C the extracellular domains of proteins (e.g., CD123, and CD19), or intracellular proteins (e.g., nuclear transcription factors Contains fragments of 5-500, 5-400, 5-300, 5-200, 5~100, 5~50, 10~500, 10~400, 10~300, 10~200, 1 It consists of 0 to 100 or 10 to 50 amino acid residues.

[0025] In some embodiments, the DD of the DDpp is specific for AFP or a fragment thereof. In some embodiments, DDpp binds to the amino acid sequence of SEQ ID NO: 9 or or a fragment thereof. DDpp is selected from the group consisting of SEQ ID NOs: 741 to 874 and 886 to 895. In another embodiment, DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874, and variants of the amino acid sequence selected from the group consisting of 886-895. In this embodiment, the DDpp comprises multiple target binding domains (e.g., dimers, trimers, etc. In some embodiments, the DDpp comprises 2, 3, 4, 5, or more than 5 In some embodiments, the D Dpp contains 2, 3, 4, 5, or more than 5 DDs with the same sequence. In some embodiments, the DDpp comprises 2, 3, 4, 5, or more than 5 AFP variants. In some embodiments, the DD binds to an epitope comprising , DDpp comprises a DD that specifically binds to AFP or a fragment thereof, AFP, AFP fragments, or 2, 3, 4, 5, or 5 that bind to different target antigens and further comprising an additional, different DD or target-binding binding domain (e.g., scFv) beyond the target. In some embodiments, DDpp specifically binds to AFP or a fragment thereof. The antibody binds to one or more antigens expressed on the surface of B cells and contains a DD that binds to one or more antigens expressed on the surface of B cells. The antibody further comprises a plurality of additional DD or other target binding domains. In the form, the DDpp comprises a DD that specifically binds to AFP and one or more cancer antigens. and further comprising one or more additional DD or other target binding domains that bind to the target. In some embodiments, the DDpp comprises 2, 3, 4, 5, or more than 5 different In a further embodiment, the DDpp specifically binds to a target comprising 2, 3, 4, 5, or more than five different cancer antigens. pp is a set of 2, 3, 4, 5, or more than 5 different cancer antigens expressed on the surface of cancer cells. In some embodiments, the DDpp specifically binds to the antigen. specifically binds to more than five cancer antigens expressed on the surface of different cancer cells.

[0026] In some embodiments, the DDpp specifically binds to AFP, or a fragment thereof. In some embodiments, the DD is a fusion protein comprising a DD linked to the nucleotide sequence of SEQ ID NO: 9. or a fragment thereof. In a further embodiment, DDpp is selected from the group consisting of SEQ ID NOs: 741 to 874 and 886 to 888. 95. In another embodiment, the AFP-binding DD pp is an amino acid sequence selected from the group consisting of SEQ ID NOs: 741 to 874, and 886 to 895. In some embodiments, the fusion protein comprises a full-length antibody or comprises an AFP-binding DD operably linked to a portion (fragment) of an antibody. In this embodiment, the DDpp fusion protein is a full-length IgG antibody (e.g., IgG1, I In a further embodiment, the DDpp comprises a commercially available Clinically approved therapeutic antibodies (e.g., rituximab, ofatumumab, ocrelizumab, venom) Rutuzumab, MEDI-551, epratuzumab, belimumab, tabalumab, AMG-5 57, MEDI-570, and NN882). In some embodiments, AFP The conjugated DDpp is an Fc fusion protein.

[0027] In some embodiments, the DDpp fusion protein is operably linked to a serum protein. Some of the experiments included DDs that specifically bind to the ligated AFP or AFP fragments. In embodiments, the DDpp fusion protein is selected from the group consisting of SEQ ID NOs: 741-874, and 886-89. 5. In another embodiment, the AFP-binding DDp p is an amino acid selected from the group consisting of SEQ ID NOs: 741 to 874, and 886 to 895 In a further embodiment, the DDpp fusion protein comprises a variant of the human serum albumin (SAA) sequence. It includes albumin or a fragment thereof.

[0028] In some embodiments, the DDpp fusion protein comprises an AFP or an AFP fragment. The extracellular domain of the receptor or a fragment thereof may further comprise a DD that specifically binds to the receptor. In some embodiments, the DDpp fusion protein comprises any of the sequences set forth in SEQ ID NOs: 741-87. 4, and 886-895. In the example, the AFP-bound DDpp is composed of SEQ ID NOs: 741 to 874 and 886 to 895. In some embodiments, the DDpp fusion comprises a variant of an amino acid sequence selected from the group The protein is a marker for the extracellular domain of BCMA (SEQ ID NO: 7) or CD123 (SEQ ID NO: 8). In some embodiments, the DDpp fusion protein comprises a DDpp fusion protein, or a fragment thereof. BCMA (SEQ ID NO: 7), or CD123 (SEQ ID NO: 8), or CS1 (SEQ ID NO: No. 965), or a fragment thereof. , DDpp fusion proteins bind to CD19, CD20, CD22, HVEM, BTLA, D R3, CD37, TSLPR, IL7R, and gp96 The extracellular domain of the body or a fragment thereof.

[0029] In some embodiments, the DDpp fusion protein comprises an AFP or an AFP fragment. and a DD that specifically binds to an intracellular protein (e.g., a nuclear protein) or In some embodiments, the DDpp fusion protein further comprises a fragment of the sequence An amino acid sequence selected from the group consisting of sequence numbers 741 to 874 and 886 to 895 In another embodiment, the AFP-binding DDpp comprises SEQ ID NOs: 741-874, and 88. The present invention also includes variants of the amino acid sequence selected from the group consisting of 6-895.

[0030] In some embodiments, the AFP-binding DDpp fusion protein binds to a serum protein (e.g., receptors (e.g., BCMA, CS1, CD123, and CD19) Contains extracellular domains or fragments of intracellular proteins (e.g., nuclear proteins) 5-500, 5-400, 5-300, 5-200, 5-100, 5-50, 10- 500, 10-400, 10-300, 10-200, 10-100, or 10-50 It consists of amino acid residues.

[0031] In some embodiments, the DD of the DDpp specifically binds to AFP p26. In some embodiments, DDpp has an amino acid sequence consisting of SEQ ID NO: 10 or a fragment thereof. In some embodiments, the DDp specifically binds to AFP p26. p is an amino acid selected from the group consisting of SEQ ID NOs: 741 to 874, and 886 to 895 In a further embodiment, DDpp comprises the sequences of SEQ ID NOs: 741 to 874, and 8 86-895. In this case, DD specifically binds to AFP p26 having the amino acid sequence of SEQ ID NO: 10. However, it does not specifically bind to AFP having the amino acid sequence of SEQ ID NO: 9. In some embodiments, the DDpp comprises multiple target binding domains (e.g., dimers, trimers, In some embodiments, the DDpp comprises 2, 3, 4, 5, or 5 In some embodiments, the DDpp comprises more than , 2, 3, 4, 5, or more than 5 DDs with the same sequence. In an embodiment, the DDpp comprises 2, 3, 4, 5, or more than 5 variants of AFP p26. In some embodiments, the DDpp comprises a DD that binds to an epitope comprising AFP. Contains a DD that specifically binds to p26, and binds to AFP p26 or a different target antigen 2, 3, 4, 5, or more than 5 additional distinct DD or target binding domains In some embodiments, the DDpp further comprises an AFP p 26, which specifically binds to one or more antigens expressed on the surface of B cells. further comprising one or more additional DD or other target-binding binding domains that bind In some embodiments, the DDpp comprises a DD that specifically binds to AFP p26. and one or more additional DDs or other targeting bonds that bind to one or more cancer antigens. In some embodiments, the DDpp further comprises two, three, four, five or more binding domains. In a further embodiment, the DDp specifically binds to five or more different targets. p specifically binds to 2, 3, 4, 5, or more than 5 different cancer antigens. In some embodiments, the DDpp is present on two, three, four, five, or more than five of the surfaces of the cancer cells. In some embodiments, DDpp specifically binds to different cancer antigens expressed on the surface. , 2, 3, 4, 5, or more than 5 different cancer antigens expressed on the surface of cancer cells. Heterogenously bonded.

[0032] In some embodiments, the DDpp comprises a DD that specifically binds to AFP p26. In some embodiments, DD is an amino acid sequence consisting of SEQ ID NO: 10. In some embodiments, the AFP p26 specifically binds to the AFP p26 having the DDpp The fusion protein is selected from the group consisting of SEQ ID NOs: 741-874, and 886-895. In another embodiment, the AFP p26-binding DDpp fusion protein comprises an amino acid sequence The protein is an amino acid sequence selected from the group consisting of SEQ ID NOs: 741 to 874, and 886 to 895. In some embodiments, the DD of the DDpp fusion protein comprises an AFP sequence. specifically binds to p26, but specifically binds to AFP having the amino acid sequence of SEQ ID NO:9 In a further embodiment, DDpp does not bind to SEQ ID NOs: 741 to 874, and 8 A fusion protein comprising an amino acid sequence selected from the group consisting of 86 to 895. In an embodiment, DDpp is selected from the group consisting of SEQ ID NOs: 741-874, and 886-895. In another embodiment, the AFP p26-binding D Dpp is an Fc fusion protein.

[0033] In some embodiments, the DDpp fusion protein specifically binds to AFP p26. In another embodiment, the DDpp fusion protein comprises a DD that acts on a serum protein. In some embodiments, the DDpp fusion protein comprises an AFP p26-binding DD operably linked to the AFP p26. The fusion protein is selected from the group consisting of SEQ ID NOs: 741-874, and 886-895. In another embodiment, the AFP p26-binding DDpp fusion protein comprises an amino acid sequence The substance is an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874, and 886-895. In a further embodiment, the DDpp fusion protein comprises a variant of the human serum albumin (SAA) sequence. It includes albumin or a fragment thereof.

[0034] In some embodiments, the DDpp fusion protein comprises an AFP p26-binding DD, In some embodiments, the antibody further comprises the extracellular domain of the receptor or a fragment thereof. , DDpp fusion protein consists of SEQ ID NOs: 741 to 874 and 886 to 895 In another embodiment, the AFP p26-binding DDpp The fusion protein is selected from the group consisting of SEQ ID NOs: 741-874, and 886-895. In some embodiments, the DDpp fusion protein comprises a variant of the amino acid sequence of is the extracellular domain of BCMA (SEQ ID NO: 7) or CD123 (SEQ ID NO: 8), or In some embodiments, the DDpp fusion protein comprises a BCM A (SEQ ID NO: 7), CD123 (SEQ ID NO: 8), or CS1 (SEQ ID NO: 965) cells In some embodiments, the DDpp fusion tag comprises the ectodomain, or a fragment thereof. Proteins include CD19, CD20, CD22, HVEM, BTLA, DR3, CD37, The extracellular domain of a receptor selected from the group consisting of TSLPR, IL7R, and gp96. or fragments thereof.

[0035] In a further embodiment, the DDpp fusion protein comprises an AFP p26-binding DD and It further includes intracellular proteins (e.g., nuclear proteins) or fragments thereof. In some embodiments, the DDpp fusion protein is selected from the group consisting of SEQ ID NOs: 741-874, and 886. In some embodiments, the AF P p26-binding DDpp fusion proteins are shown in SEQ ID NOs: 741-874 and 886-888. 95. In another embodiment, the AFP The p26-binding DDpp fusion protein binds to serum proteins (e.g., HSA), receptors ( For example, the extracellular domains of BCMA, CS1, CD123, and CD19, or Fragments of intracellular proteins (e.g., nuclear proteins) are included, and 5-500, 5-40 0, 5-300, 5-200, 5-100, 5-50, 10-500, 10-400, 1 Consists of 0-300, 10-200, 10-100, or 10-50 amino acid residues .

[0036] In some embodiments, the DD of the DDpp specifically binds to HER2. In this embodiment, the DDpp is a HER2 tag having an amino acid sequence consisting of SEQ ID NO: 967. In a further embodiment, DDpp specifically binds to the protein of SEQ ID NO: 911-912. 49, and 950. In other embodiments, The HER2-binding DDpp is selected from the group consisting of SEQ ID NOs: 911-949, and 950. In some embodiments, the DDpp comprises variants of the amino acid sequence that bind to multiple targets. In some embodiments, the DDp comprises a fusion domain (e.g., a dimer, a trimer, etc.). p includes 2, 3, 4, 5, or more than 5 DDs that bind to HER2. In some embodiments, the DDpp has 2, 3, 4, 5, or more than 5 identical sequences. In some embodiments, the DDpp comprises 2, 3, 4, 5, or 5 In some embodiments, the DDs bind to different epitopes of HER2. , DDpp, which contains a DD that specifically binds to HER2, BCMA or a different target antigen 2, 3, 4, 5, or more than 5 additional different DD or target binding bonds that bind to the target. In some embodiments, the DDpp further comprises a binding domain (e.g., an scFv). One or more antibodies expressed on the surface of cancer cells containing DD that specifically binds to HER2 and one or more additional DD or other target-binding binding domains that bind to the target. In some embodiments, the DDpp comprises a DD that specifically binds to HER2, One or more additional DDs that bind to one or more cancer antigens or other target binding In some embodiments, the DDpp further comprises 2, 3, 4, 5, In a further embodiment, the DDpp specifically bind to two, three, four, five, or more than five different cancer antigens. In some embodiments, the DDpp is present on 2, 3, 4, 5, or more than 5, of the surface of the cancer cell. In some embodiments, DDpp specifically binds to different cancer antigens expressed on Specific for two, three, four, five, or more than five cancer antigens expressed on the surface of different cancer cells actively binds.

[0037] In some embodiments, the DDpp is a fusion tag comprising a DD that specifically binds to HER2. In some embodiments, DD is a protein consisting of the amino acid sequence of SEQ ID NO:967. In some embodiments, DDpp specifically binds to the HER2 protein having a The present invention relates to a method for producing a human hamster ovary comprising administering to a human a mammal a human having an hamster ovary, ... In other embodiments, the HER2-binding DDpp fusion protein is selected from the group consisting of SEQ ID NOs: 911-94. 9, and 950. In embodiments, the DDpp fusion protein is a full-length antibody or a portion (fragment) of an antibody. In some embodiments, the DDpp fusion protein comprises a full-length IgG antibody (e.g., In a further embodiment, the antibody may be an IgG1, IgG2, IgG2, or IgG4. The DDpp comprises a commercially approved therapeutic antibody. In another embodiment, the HER2-binding DDpp pp is an Fc fusion protein.

[0038] In some embodiments, the DDpp fusion protein is operably linked to a serum protein. In some embodiments, the DDpp fusion protein comprises a HER2-binding DD. , comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 911 to 949, and 950 In another embodiment, the HER2-binding DDpp fusion protein is selected from the group consisting of SEQ ID NOS: 911-949. and 950. In one embodiment, the DDpp fusion protein comprises human serum albumin or a fragment thereof. In some embodiments, the DDpp fusion protein is AFP or AFP p26 or a fragment thereof. In some embodiments, the DDpp fusion protein comprises , AFP (e.g., SEQ ID NO: 9), or a fragment thereof. The DDpp fusion protein may comprise AFP p26 (SEQ ID NO: 10), or a fragment thereof. Includes:

[0039] In some embodiments, the HER2-binding DDpp fusion protein binds to the extracellular domain of the receptor. In some embodiments, the HER2-binding DDpp The fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 911-949, and 950. In another embodiment, the HER2-binding DDpp fusion protein comprises the amino acid sequence SEQ ID NO: and variants of the amino acid sequence selected from the group consisting of Nos. 911 to 949 and 950. In a further embodiment, the HER2-binding DDpp fusion protein is a fusion protein comprising BCMA (SEQ ID NO: 1). 7), the extracellular domain of CD123 (SEQ ID NO: 8), or CS1 (SEQ ID NO: 965); or a fragment thereof. In some embodiments, the HER2-binding DDpp fusion tag Proteins include CD19, CD20, CD22, HVEM, BTLA, DR3, CD37, CS-1, TSLPR, IL7R, and gp96. It comprises the extracellular domain or a fragment thereof.

[0040] In a further embodiment, the HER2-binding DDpp fusion protein is an intracellular protein ( In some embodiments, the HE comprises a nucleotide sequence (e.g., a nucleoprotein) or a fragment thereof. The R2-binding DDpp fusion protein is selected from the group consisting of SEQ ID NOs: 911 to 949 and 950. In another embodiment, the HER2-binding DDpp fusion protein comprises an amino acid sequence selected from the group consisting of: The protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 911-949 and 950. Contains column variants.

[0041] In some embodiments, the HER2-binding DDpp fusion protein is a serum protein ( For example, HSA, AFP, and AFP 26), receptors (e.g., BCMA, CS1, CD123, and CD19), or the extracellular domains of intracellular proteins (e.g., nuclear Protein) fragments, 5-500, 5-400, 5-300, 5-200 , 5~100, 5~50, 10~500, 10~400, 10~300, 10~200, It consists of 10 to 100 or 10 to 50 amino acid residues.

[0042] In some embodiments, the DDpp fusion protein comprises a full-length antibody. In an embodiment, the DDpp is a fusion protein comprising a full-length antibody that specifically binds to a cancer antigen. In a further embodiment, the DDpp is a fusion protein comprising a full-length antibody, The DDpp fusion protein is believed to be expressed by the cancer in the subject to which it is administered. It specifically binds to cancer antigens.

[0043] In some embodiments, the disclosed DDpp (e.g., DDpp fusion protein) comprises a label. Labels that can be used to label DDpp include, but are not limited to, enzyme labels, These include fluorescent labels, luminescent labels, and bioluminescent labels. In some embodiments, the label is a bioluminescent label. In some embodiments, the label is a streptavidin moiety. In some embodiments, the label is a His tag or a FLAG tag. In the present study, the label may be luciferase, green fluorescent protein, red fluorescent protein, or any of its Other similar drugs.

[0044] In other embodiments, the DDpp fusion protein is bound to a solid support. In embodiments, the solid support is a bead, a slide, a chip, gelatin, or agarose. The compound is selected from the group consisting of

[0045] In some embodiments, the DDpp (e.g., DDpp fusion protein) is a liposome. In some embodiments, the DDpp is covalently attached to the liposome. In some embodiments, the DDpp is a fusion protein. In one embodiment, the DDpp is CAR. In a further embodiment, the DDpp is a covalently bonded The liposomes are bound by ionic bonds rather than by fusion.

[0046] In some embodiments, the target-binding DDpp is a therapeutic or cytotoxic agent (e.g., a chemotherapeutic agent). The compound is bound to a chemotherapeutic or radiotherapeutic agent.

[0047] In a further embodiment, the present disclosure provides a DD disclosed herein (e.g., SEQ ID NOs: 11-9). Chimeric antibodies containing a target-binding domain containing a 49 or 950 amino acid sequence In some embodiments, the DD binds to BCMA and It comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 to 305, and 306. In some embodiments, the DD binds to CD123 and is selected from the group consisting of SEQ ID NOs: 307-739, and 740. In some embodiments, DD comprises an amino acid sequence selected from the group consisting of: , which binds to AFP and is selected from the group consisting of SEQ ID NOs: 741 to 874, and 886 to 895 In some embodiments, the DD comprises an amino acid sequence that binds to AFP p26. , an amino acid sequence selected from the group consisting of SEQ ID NOs: 741 to 874, and 886 to 895 In some embodiments, a CAR comprises a target binding domain, a transmembrane domain, and and an intracellular signaling domain. In some embodiments, the CAR transmembrane domain In some embodiments, the CAR comprises a 41BB or CD28 transmembrane domain. is a domain of the human T cell receptor alpha, beta, or zeta chain; human 41BB domain a human CD28 domain; a human CD28 domain; and any combination thereof. In some embodiments, the CAR intracellular signaling domain comprises Transduction domains include CD27, CD28, 41BB, OX40, CD30, CD40, and PD 1, lymphocyte function-associated antigen 1 (LFA1), CD2, CD7, LIGHT, NKG2C, Ligands that specifically bind to B7-H3, CD83, and any combination thereof In some embodiments, the intracellular domain of a costimulatory molecule selected from the group consisting of: The CAR may comprise a second target binding domain having the same or a different target than the DD target binding domain. In some embodiments, the CAR is expressed in an immune cell. In some embodiments, the immune effector cells are T cells (CAR-T cells) or natural T cells. In some embodiments, the CAR is a human killer (NK) cell (CAR-NK cell). , bound to liposomes.

[0048] In some embodiments, the CAR is a targeted (e.g., B) antigen expressed on the surface of a cancer cell. 2, 3, 4, 5, or more than 5 specific binding sites for CMA or CD123 DD and / or other binding domains (e.g., scFv). In this state, the CAR specifically binds to a second, different target expressed on the surface of the cancer cell. , 2, 3, 4, 5, or more than 5 DD or other binding domains (e.g., s In a further embodiment, the administered CAR comprises a second, different cancer cell or specifically binds to a second, different target expressed by vascular endothelial cells. , five, or more than five DD or other binding domains (e.g., scFv). This includes:

[0049] Nucleic acids encoding the disclosed DDpp (e.g., nucleic acid DDpp fusion proteins) are also provided. Additionally, vectors containing nucleic acids encoding DDpp (e.g., DDpp fusion proteins) are - (e.g., plasmids, viral vectors, and non-viral vectors) and nucleic acids and a host cell comprising the vector. In some embodiments, the vector is a nuclear It also contains a nucleotide sequence that regulates the expression of a polypeptide encoded by the nucleic acid molecule. In certain embodiments, the vector comprises an inducible promoter sequence. The vector may contain one or more additional standard components for expression of a protein-encoding nucleic acid. (e.g., promoter, packaging components, etc.). In some embodiments, , the vector is a lentiviral vector.

[0050] The present disclosure also provides host cells comprising nucleic acid molecules encoding the target-binding DDpps disclosed herein. In some embodiments, the host cell (e.g., a cell line of cells) is a host cell as described herein. The disclosed DD (e.g., DD having the amino acid sequence of SEQ ID NO: 11 to 949, or 950) In some embodiments, the host is genetically modified to express a protein comprising Expression of DDpp by the cells allows for the production and isolation of DDpp. In this study, expression was observed in DDpp expressed on the surface and / or integrated into the membrane of the host cell. In some embodiments, the host cell is a cell that is capable of expressing viral particles or a bacterial, yeast, yeast, or yeast cell. In other embodiments, the host cell is a mammalian cell. In one embodiment, the mammalian cell is an immune cell. In one embodiment, the host cell is a human immune cell. In some embodiments, the human immune cells are T cells. In other embodiments, the human immune cells are T cells. In some embodiments, the human immune cells are natural killer (NK) cells. Immune cells present DDpp on their cell surface.

[0051] The present disclosure further provides host cells expressing proteins comprising the DD disclosed herein. In some embodiments, the host cell is provided with a chimeric antigen comprising a DD disclosed herein. In some embodiments, the CAR expresses a receptor (CAR). 5, and 306; SEQ ID NOs: 307 to 739, and 740; SEQ ID NOs: 741 to 874, and and 886 to 895; SEQ ID NOs: 896 to 909 and 910; or SEQ ID NOs: 911 to 9 49, and 950, and a DD containing an amino acid sequence selected from the group consisting of a transmembrane domain. In some embodiments, the CAR comprises a target binding domain containing an intracellular domain (signature In some embodiments, the CAR immune cell further comprises a T In some embodiments, the CAR immune cells are NK cells. In some embodiments, the CAR immune cell is not a T cell or an NK cell. The host cell may then express the first CAR to the same or a different target (the same or different target) as the first CAR expressed by the host immune cell. specific for a different epitope on the same target, or a second target expressed by cancer cells A second CAR polypeptide having a DD or other binding domain (e.g., scFv) that binds and immune cells further comprising the polypeptide.

[0052] Mammalian cells that produce membrane-bound virus-like particles (VLPs) are also provided, The cells express a D domain polypeptide (DDpp) fused to a chimeric antigen receptor (CAR). The fusion protein is generated by genetically modifying the vector to express a fusion protein containing V Depending on the embodiment, the mammalian The VLPs produced by the cells are suitable for use as immunogens for antibody production.

[0053] Pharmaceutical compositions comprising proteins containing the DD disclosed herein, nucleic acids encoding the proteins Also provided are nucleic acids, vectors containing the nucleic acids, and host cells containing the nucleic acids and / or vectors. Similarly, the disclosed target-binding DDpp (e.g., DD-Fc and DD-CAR) pp fusion protein), nucleic acid molecule, vector, and host cell. Kits including the methods (e.g., therapeutic kits, diagnostic kits, kits for research use, etc.) are also provided.

[0054] The DDpp provided herein can be used to treat, but is not limited to, diseases of interest in vitro or in vivo. Targets (e.g., BCMA, CD123, CS1, HER2, AFP, and AFP p2 6, peptide tags, and alpha-fetoprotein, human serum albumin (HSA), or or serum proteins such as immunoglobulins) The ability to bind heterologously and to bind proteins such as DDpp fusion proteins to one or more for linking or binding to additional moieties (e.g., solid supports), and / or other The DDpp provided herein has the ability to serve as a reactive site for modification of Additionally, additional desirable properties useful for manufacturing, formulation, and biological, diagnostic, and therapeutic applications are also disclosed. It may also have a property and / or function.

[0055] In some embodiments, the DDpp is a target molecule (e.g., a BCM) in a sample containing the target. A, CD123, CS1, HER2, AFP, or AFP p26) In one embodiment, the present disclosure provides a method for detecting and / or quantifying a target protein in a sample. (e.g., BCMA, CD123, CS1, HER2, AFP, or AFP p26) The present invention provides a method for detecting a nucleic acid sequence comprising: (a) a sample; and (b) detecting a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11 to 949 and 95. 0, and D including DD that specifically binds to the target. and Dpp under conditions favoring specific binding of Dpp to the target, (b) detecting the presence of the complex and / or the capture target. In some embodiments, the DDpp is immobilized on a solid support. In some embodiments, the DD is selected from the group consisting of SEQ ID NOs: 11-305 or 306. and DDpp is a BCMA or BCMA-containing fusion protein in the sample. Used to bind to, detect, and / or quantify proteins. In embodiments, DD is selected from the group consisting of SEQ ID NOs: 307-739 or 740. DDpp is a fusion protein containing CD123 in a sample. It is used to bind to, detect, and / or quantify proteins. In this embodiment, the DD is selected from the group consisting of SEQ ID NOs: 741-874 or 886-895. DDpp comprises an amino acid sequence that identifies AFP p26 or AFP p26 in a sample. It is used to bind to, detect, and / or quantify fusion proteins containing the In some embodiments, the DD is selected from the group consisting of SEQ ID NOs: 896-909 or 910. DDpp contains the amino acid sequence selected from CS1 or a fusion protein containing CS1 in the sample. It is used to bind to, detect, and / or quantify proteins. In this embodiment, DD is an amino acid sequence selected from the group consisting of SEQ ID NOs: 911 to 949 or 950. DDpp contains a nucleotide sequence that identifies HER2 or a fusion protein containing HER2 in the sample. The binding is used to detect and / or quantify the binding.

[0056] Targeted targeting (e.g., BCMA, CD123, CS1, HER2, A Also provided is a method for quantifying AFP (or AFP p26), the method comprising: (a) detecting a sample; 11 to 949, and 950, The DDp is immobilized on the substrate and contains a DD that specifically binds to the target. and contacting the target / DDpp complex under conditions favorable for specific binding of the target / DDpp complex to the target / DDpp complex. and (b) detecting the presence of the target / DDpp complex and / or the capture target. and quantitative detection of the product determines the amount of target or the amount of fusion protein containing the target in the sample. These quantities can be correlated in various ways.

[0057] In a further embodiment, the provided DDpp is used in protein analysis. In some embodiments, the DDpp is conjugated to a detectable agent and / or tag. In some embodiments, DDpp is selected from the group consisting of SEQ ID NOs: 11-305, and 306; SEQ ID NO: 307 ~739, and 740; SEQ ID NOs: 741-874 and 886-895; SEQ ID NO: 89 6 to 909 and 910; or from the group consisting of SEQ ID NOs: 911 to 949, and 950 In another embodiment, the DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-305. , and 306; SEQ ID NOs: 307 to 739, and 740; or SEQ ID NOs: 741 to 747 4 and 886-895; SEQ ID NOs: 896-909 and 910; or SEQ ID NO: 911 949, and 950. In some embodiments, the DDpp is conjugated to a detectable agent. The agent comprises a chromogen. In another embodiment, the detectable agent comprises a fluorescent dye. In some embodiments, the detectable agent comprises a radionuclide. In some embodiments, the DDpp is coupled to a detectable agent by covalent bonding. In a further embodiment, the DDpp is a multimer. In some embodiments, the DDpp is attached to a tag. In some embodiments, the tag is a polyhistidine The tag is a member selected from the group consisting of a nucleotide tag, a nucleotide tag, and a FLAG tag. In further embodiments, the DDpp comprises a combination of tags (e.g., a polyhistidine tag and In some embodiments, the DDpp is covalently linked to a FLAG tag. In some embodiments, the DDpp is a fusion protein. In some embodiments, the DDpp is multimeric.

[0058] In a further embodiment, the DDpp is bound to a solid support or a tag. In embodiments, the solid support may be a chromatography bead, a resin, a glass slide, a chip, or the like. , gelatin, or agarose.

[0059] Methods of using DDpp in diagnostic and therapeutic applications are also provided. provides a method for treating a disease or disorder, the method comprising targeting a therapeutic target of interest (e.g., BCMA, Therapeutics that specifically bind to CD123, CS1, HER2, AFP, or AFP p26 A therapeutically effective amount of a DDpp (e.g., a DDpp fusion protein) is administered to a subject in need thereof. In some embodiments, the disease or disorder is cancer, B-cell malignancy, or Cancer, a disease or disorder of the immune system, or an infectious disease. Also provided are methods for treating a disease or disorder comprising administering simultaneously. In some embodiments, the disease or disorder is breast cancer or multiple myeloma. or ovarian cancer.

[0060] The target-binding DDpp disclosed herein have uses, including diagnostic and therapeutic uses. In some embodiments, DDpp is used in therapeutic settings, for example, in the treatment of cancer (e.g., solid or hematologic tumors). These compounds are useful for the treatment and / or diagnosis of diseases such as leukemia (cancer).

[0061] In some embodiments, the present disclosure provides a method of treating a subject for cancer, the method comprising: A therapeutically effective amount of: a DD disclosed herein (e.g., a DD disclosed in Table 1, or SEQ ID NO: 11 to DD having an amino acid sequence of 949 or 950 (i.e., DD pp); a nucleic acid encoding the protein; a vector containing the nucleic acid or the nucleic acid or vector In some embodiments, the method further comprises administering to a subject host cells containing the vector. comprises a DD amino acid sequence that specifically binds to BCMA. The DD of Dpp specifically binds to BCMA having the amino acid sequence of SEQ ID NO: 7. In a further embodiment, the DDpp is selected from the group consisting of SEQ ID NOs: 11 to 305 and 306. In some embodiments, the DDpp comprises an amino acid sequence selected from the group consisting of: In some embodiments, the DD of DDpp comprises a DD amino acid sequence that specifically binds to specifically binds to CD123 having the amino acid sequence of SEQ ID NO: 8. In embodiments, the DDpp is selected from the group consisting of SEQ ID NOs: 307-739, and 740. In some embodiments, the DDpp comprises an amino acid sequence that specifically binds to CS1. In some embodiments, the DD of DDpp comprises a DD amino acid sequence matching SEQ ID NO: In a further embodiment, the present invention specifically binds to CS1 having an amino acid sequence consisting of 965. DDpp is an amino acid selected from the group consisting of SEQ ID NOs: 896 to 909, and 910 In some embodiments, the DDpp comprises a DD that specifically binds to HER2. In some embodiments, the DD of the DDpp comprises the amino acid sequence of SEQ ID NO: 967. In a further embodiment, the HER2 specifically binds to a HER2 having an amino acid sequence comprising: p is an amino acid sequence selected from the group consisting of SEQ ID NOs: 911 to 949, and 950 In some embodiments, the DDpp comprises a DD amino acid sequence that specifically binds to AFP. In some embodiments, DD of DDpp comprises the amino acid sequence of SEQ ID NO:9. In a further embodiment, DDpp specifically binds to AFP having the sequence of SEQ ID NO: 7. The amino acid sequence is selected from the group consisting of 41 to 874 and 886 to 895. In some embodiments, the DDpp is an AFP having an amino acid sequence consisting of SEQ ID NO:10. In a further embodiment, DDpp specifically binds to p26. 74, and 886-895. In an embodiment, the DD of the DDpp specifically binds to AFP p26 but is selected from SEQ ID NO: 9 In a further embodiment, the DD pp is an amino acid sequence selected from the group consisting of SEQ ID NOs: 741 to 874, and 886 to 895. Contains the amino acid sequence.

[0062] In some embodiments, the present disclosure provides a method of treating a subject with a B-cell malignancy, comprising: The method includes administering an effective amount of: a DD disclosed herein (e.g., a DD disclosed in Table 1, or a DD of sequence a protein comprising the amino acid sequence of numbers 11 to 949 or 950 (DD); a nucleic acid encoding a protein; a vector containing said nucleic acid or a vector containing said nucleic acid or vector In some embodiments, the B cell malignancy is a lymphoma. lymphomas (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma (NHL)), leukemia, and and myeloma. In some embodiments, the B-cell malignancy is selected from the group consisting of acute myeloma, lymphocytic leukemia, chronic lymphocytic leukemia, follicular lymphoma, mantle cell lymphoma, and Selected from the group consisting of diffuse large B-cell lymphoma, plasmacytoma, and multiple myeloma It is selected.

[0063] In some embodiments, the present disclosure provides a method of treating a subject for cancer, the method comprising administering a steroid hormone to a subject. administering to the subject immune cells comprising a CAR-antigen receptor (CAR), the CAR comprising the sequence DD having an amino acid sequence selected from the group consisting of numbers 11 to 949 and 950 a target binding domain comprising the target and a transmembrane domain, In a further embodiment, the target of interest is expressed by cancer cells. In one embodiment, the DD specifically binds to BCMA having an amino acid sequence consisting of SEQ ID NO:7. In a further embodiment, DD is selected from the group consisting of SEQ ID NOs: 11 to 305, and 306. In some embodiments, DD comprises an amino acid sequence selected from SEQ ID NO: 8. In a further embodiment, DD specifically binds to CD123 having the amino acid sequence The present invention relates to a method for producing a human medicament comprising the steps of: In some embodiments, the DD comprises a C having an amino acid sequence consisting of SEQ ID NO: 965. In a further embodiment, the DD specifically binds to S1. and 910. In some embodiments, D D specifically binds to HER2 having the amino acid sequence of SEQ ID NO: 967. In one embodiment, DD is selected from the group consisting of SEQ ID NOs: 911-949, and 950. In some embodiments, the DD comprises an amino acid sequence consisting of SEQ ID NO: 10. In a further embodiment, DD specifically binds to AFP p26 having the amino acid sequence , an amino acid sequence selected from the group consisting of SEQ ID NOs: 741 to 874, and 886 to 895 In some embodiments, the CAR comprises an intracellular domain. In this state, the intracellular domain comprises a signaling domain, and upon administration to a subject with cancer, the intracellular domain The binding domain binds to target molecules expressed by cancer cells (e.g., BCMA and CD123). This specific binding allows immune cells to exert a cytotoxic effect on cancer cells. In some embodiments, immune cells are induced to produce a cytotoxic signal that can In some embodiments, the immune cells are T cells. In other embodiments, the immune cells are NK cells. In a further embodiment, the administered CAR immune cells are not T cells or NK cells. The study aims to investigate whether a combination of different CAR immune cell types (e.g., NK cells and T cells) can be administered to a subject. In some embodiments, the immune cells are administered intravenously. In this study, a combination of different CAR immune cell types is administered intravenously to a subject. In some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is breast cancer or ovarian cancer. is.

[0064] In some embodiments, the present disclosure provides a method of treating a subject with a B-cell malignancy, comprising: The method includes administering to a subject immune cells comprising a chimeric antigen receptor (CAR), CAR is an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 to 949, and 950 a targeting molecule comprising a polypeptide that specifically binds to a target expressed by a cancer cell, a binding domain; a transmembrane domain; and an intracellular domain. In some embodiments, The target binding domain specifically binds to BCMA and has the amino acid sequence of SEQ ID NO:7. In a further embodiment, the target binding domain is selected from the group consisting of SEQ ID NOs: 11-305, and 30 6. In some embodiments, the target binding domain comprises an amino acid sequence selected from the group consisting of: The antibody specifically binds to CD123 having the amino acid sequence of SEQ ID NO: 8. In further embodiments, the target binding domain is selected from the group consisting of SEQ ID NOs: 307-739, and 740. In some embodiments, the target binding domain comprises an amino acid sequence selected from the group consisting of: specifically binds to CS1 having the amino acid sequence of SEQ ID NO: 965. In embodiments, the target binding domain is selected from the group consisting of SEQ ID NOs: 896-909, and 910. In some embodiments, the CAR comprises an amino acid sequence selected from the following: In a further embodiment, the intracellular domain comprises a signaling domain and is Upon administration to a subject, the target binding domain binds to the target (e.g., , BCMA, CS1, and CD123) and stimulates immune response through targeted binding. cells to produce cytotoxic signals that exert a cytotoxic effect on malignant B cancer cells. In some embodiments, the B cell malignancy is a lymphoma (e.g., hodgkin's lymphoma). A group consisting of Hodgkin's lymphoma (Hodgkin's lymphoma and non-Hodgkin's lymphoma (NHL)), leukemia, and myeloma In some embodiments, the B-cell malignancy is selected from acute lymphoblastic leukemia, chronic lymphocytic leukemia, and / or leukemia. Lymphocytic leukemia, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma The tumor is selected from the group consisting of lymphoma, plasmacytoma, and multiple myeloma. In some embodiments, the B cell malignancy is multiple myeloma. In other embodiments, the immune cells are NK cells. The administered CAR immune cells are not T cells or NK cells. A combination of different CAR immune cell types (e.g., NK cells and T cells) is administered to the subject. In some embodiments, the immune cells are administered intravenously. A combination of different CAR immune cell types is administered intravenously to a subject.

[0065] In some embodiments, the present disclosure provides a method of treating a subject with a B cell-associated disorder. The method includes administering an effective amount of: a DD disclosed herein (e.g., an amino acid sequence disclosed in Table 1); or a DD having an amino acid sequence of SEQ ID NO: 11 to 949, or 950) a protein comprising the protein; a nucleic acid encoding the protein; a vector comprising the nucleic acid or the nucleic acid Alternatively, it involves administering host cells containing the vector to a subject.

[0066] In some embodiments, the present disclosure provides a method of treating a subject with an immune system disorder. The method includes administering an effective amount of: a DD disclosed herein (e.g., an amino acid sequence disclosed in Table 1); or a DD having an amino acid sequence of SEQ ID NO: 11 to 949, or 950) a protein comprising the protein; a nucleic acid encoding the protein; a vector comprising the nucleic acid or the nucleic acid Alternatively, the method includes administering to a subject a host cell containing the vector. The immune system disorder is an autoimmune disease such as rheumatoid arthritis.

[0067] In some embodiments, the present disclosure provides for the use of a composition for treating cancer, The composition may comprise a DD disclosed herein (e.g., a DD having an amino acid sequence disclosed in Table 1, or a protein comprising the amino acid sequence of SEQ ID NO: 11 to 949 or 950 (DD) a nucleic acid encoding said protein; a vector containing said nucleic acid or said nucleic acid or vector In some embodiments, the cancer is a B-cell malignancy. In this embodiment, the B-cell malignancy is a lymphoma (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma). The cancer is selected from the group consisting of non-Hodgkin lymphoma (NHL), leukemia, and myeloma. In embodiments, the B-cell malignancy is selected from acute lymphocytic leukemia, chronic lymphocytic leukemia, follicular leukemia, and the like. lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, plasmacytoma, and and multiple myeloma. In some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is breast cancer or ovarian cancer.

[0068] In some embodiments, the present disclosure provides for the use of a composition to treat a B-cell malignancy. The compositions are provided, and the compositions comprise a DD disclosed herein (e.g., a DD having an amino acid sequence disclosed in Table 1). or a DD having an amino acid sequence of SEQ ID NO: 11 to 949, or 950). a protein containing the protein; a nucleic acid encoding the protein; a vector containing the nucleic acid; or or a host cell containing the vector. In some embodiments, the B cell malignancy is lymphomas (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma (NHL)), leukemia, and and myeloma. In some embodiments, the B-cell malignancy is selected from the group consisting of acute myeloma, Lymphocytic leukemia, chronic lymphocytic leukemia, follicular lymphoma, mantle cell lymphoma, diffuse large intestine a tumor selected from the group consisting of malignant large B-cell lymphoma, plasmacytoma, and multiple myeloma; In some embodiments, the B-cell malignancy is multiple myeloma.

[0069] In some embodiments, the present disclosure provides for the use of a composition to treat a B-cell related disorder. The compositions are provided, and the compositions comprise a DD disclosed herein (e.g., a DD having an amino acid sequence disclosed in Table 1). or a DD having an amino acid sequence of SEQ ID NO: 11 to 949, or 950). a protein containing the protein; a nucleic acid encoding the protein; a vector containing the nucleic acid; or or a host cell containing the vector.

[0070] In some embodiments, the present disclosure provides for the use of the composition to treat disorders of the immune system. The composition comprises a DD (e.g., a DD having an amino acid sequence disclosed in Table 1) disclosed herein. DD, or DD having the amino acid sequence of SEQ ID NO: 11 to 949, or 950) a protein; a nucleic acid encoding the protein; a vector containing the nucleic acid; or In some embodiments, the immune system disorder is a rheumatoid arthritis disorder, or a host cell containing the vector. Autoimmune diseases such as rheumatoid arthritis.

[0071] In a further embodiment, the present disclosure provides chimeric antigen receptor (CAR)-based therapeutics for the treatment of cancer. The present invention provides the use of immune cells comprising a CAR, the CAR consisting of SEQ ID NOs: 11 to 949, and 950. and a target molecule expressed by a cancer cell, the target molecule having an amino acid sequence selected from the group consisting of: a target binding domain comprising a polypeptide that binds to the target; a transmembrane domain; and an intracellular domain. the intracellular domain comprises a signaling domain and, upon administration to a subject with cancer, binds to the target. The binding domain specifically binds to a target expressed by cancer cells, and upon target binding, This allows immune cells to generate cytotoxic signals that produce cytotoxic effects against cancer cells. In some embodiments, the immune cells are T cells or natural killer (N In some embodiments, the immune cells are T cells. In some embodiments, the immune cells are natural killer (NK) cells. In a further embodiment, the immune cell is not a T cell or an NK cell. A combination of types (e.g., T cells and NK cells) is used. In some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is breast cancer or ovarian cancer. .

[0072] In a further embodiment, the present disclosure provides chimeric antigen receptor (CAR) antibodies for the treatment of B-cell malignancies. The present invention provides the use of immune cells comprising a CAR, the CAR being selected from the group consisting of SEQ ID NOS: 11 to 949, and 9 50, and a target (e.g., BCMA, CS 1, or targets expressed by malignant B cells, such as CD123. peptides, consisting of SEQ ID NOs: 11 to 306, 307 to 740, and 896 to 910. a target binding domain comprising an amino acid sequence selected from the group consisting of: a transmembrane domain; and a cell the intracellular domain comprises a signaling domain, and when administered to a subject with cancer The target binding domain specifically binds to a target expressed by malignant B cells and binds to the target. Upon target binding, immune cells release cytotoxic signals that produce a cytotoxic effect against malignant B cells. In some embodiments, the immune cells are T cells or nucleic acids. In some embodiments, the immune cells are T cells. In some embodiments, the immune cells are natural killer (NK) cells. In some embodiments, the immune cells are not T cells or NK cells. Combinations of different CAR immune cell types (e.g., T cells and NK cells) are used. In some embodiments, the B-cell malignancy is multiple myeloma.

[0073] In a further embodiment, the present disclosure provides chimeric antigen receptor (CAR) antibodies for the treatment of disorders of the immune system. The present invention provides the use of immune cells comprising a CAR, the CAR being selected from the group consisting of SEQ ID NOS: 11 to 949, and 9 50, wherein the amino acid sequence is selected from the group consisting of: a target-binding domain comprising a polypeptide that specifically binds to the target; a transmembrane domain; and an intracellular domain, the intracellular domain comprising a signal transduction domain and an immunoglobulin. Upon administration to a subject with an immune disorder, the target binding domain binds to the target expressed by the targeted immune cells. The targeted binding allows the recombinant immune cells to specifically bind to the targeted immune cells. These cells are induced to produce cytotoxic signals that produce cytotoxic effects. In this embodiment, the immune cells are T cells or natural killer (NK) cells. In some embodiments, the immune cells are not T cells or NK cells. In some embodiments, the immune cells are natural killer (NK) cells. is not a T cell or an NK cell. In further embodiments, a different CAR immune cell type (e.g., For example, a combination of T cells and NK cells is used.

[0074] The methods summarized above and / or described herein may be performed by a medical practitioner. Describes specific acts; but does not preclude the teaching of those acts by another party. It should be understood that the term "target-specific binding polypeptide" can also include An act such as "administration of T cells containing a target-specific binding polypeptide-CAR" is considered to be an act such as "administration of T cells containing a target-specific binding polypeptide-CAR." "Instructions for administering T cells comprising:

[0075] In some embodiments, the present disclosure provides: [1] A D domain (DD) target binding domain, which is a member selected from the group consisting of: Proteins containing: (a) a polypeptide that specifically binds to BCMA and has the amino acid sequence of SEQ ID NO: 11 to 305 or 306; DD containing columns, (b) specifically binds to CD123 and has an amino acid sequence of SEQ ID NO: 307 to 739 or 740; DD containing the acid sequence, (c) AFP specifically binds to an antibody having any of SEQ ID NOs: 741 to 874 or 886 to 895. DD containing amino acid sequence, (d) AFP p26 specifically binds to SEQ ID NOs: 741 to 874, or 886 to 8 DD containing a 95 amino acid sequence, (e) A polypeptide that specifically binds to CS1 and has the amino acid sequence of SEQ ID NO: 896 to 909 or 910. DD containing columns, (f) a nucleic acid sequence that specifically binds to HER2 and is selected from the group consisting of amino acids 911 to 949 and 950; The DD containing the array. [2] The protein according to [1], wherein the DD is fused to a heterologous polypeptide. [3] The method according to [2], wherein the heterologous polypeptide comprises a full-length antibody or an antibody fragment. Proteins listed. [4]. The heterologous polypeptide comprises a member selected from the group consisting of: The protein described in (a) transmembrane domain, (b) membrane-binding domain, (c) human serum albumin or a fragment thereof; (d) AFP or a fragment thereof; (e) AFP p26 or a fragment thereof, and (f) The extracellular domain of the receptor or a fragment thereof. [5].DD is the amino terminus of the full-length antibody heavy chain; the amino terminus of the full-length antibody light chain; fused to the carboxyl terminus of an antibody heavy chain; or to the carboxyl terminus of a full-length antibody light chain; [3] The protein described in [3]. [6] The protein according to [3], wherein the heterologous polypeptide is Fc. [7]. The heterologous polypeptide comprises BCMA, CD123, CS1, and CD19. The present invention relates to a method for treating a leukemia, a leukemia, or a leukemia comprising administering to a patient a therapeutically effective amount of a leukemia or a leukemia virus, comprising administering to a patient a therapeutically effective amount of a leukemia virus, an leukemia virus, or a leukemia virus. The protein described in [2]. [8]. A labeled protein according to any one of [1] to [7]. [9]. The label may be an enzyme label, a fluorescent label, a luminescent label, a bioluminescent label, or a biotin moiety. The protein according to [9], selected from the group consisting of:

[10] . Any one of [1] to

[10] , conjugated to a therapeutic or cytotoxic agent. The protein described.

[11] . A target-binding domain comprising a protein according to any one of [1] to [5]. A chimeric antigen receptor (CAR) comprising:

[12] . It contains a target-binding domain, a transmembrane domain, and an intracellular signaling domain.

[11] The CAR according to

[11] .

[13] . A CAR described in

[0011] or

[12] , wherein the transmembrane domain comprises a 41BB or CD28 transmembrane domain.

[14] . The intracellular signaling domains of human T cell receptors alpha, beta, and is the zeta chain domain; human 41BB domain; human CD28 domain; and these Any combination of any one of

[11] to

[13] is selected from the group consisting of CAR.

[15] . The intracellular signaling domains are CD27, CD28, 41BB, and OX40 , CD30, CD40, PD1, lymphocyte function-associated antigen 1 (LFA1), CD2, CD7 , LIGHT, NKG2C, NKG2D, B7-H3, CD83-specific binding ligands and any combination thereof. A CAR according to any one of

[11] to

[14] , including a main.

[16] . A second target binding domain with the same or different target as the DD target binding domain. The protein according to any one of [1] to

[15] , further comprising a binding domain.

[17] . An isolated nucleic acid encoding a protein according to any one of [1] to

[16] . acid.

[18] . A vector comprising the nucleic acid according to

[17] .

[19] . Nucleic acids are nucleotides that regulate the expression of proteins encoded by the nucleic acids. The vector according to

[18] , wherein the vector is operably linked to the sequence.

[20] . The vector according to

[19] , which is a lentiviral vector.

[21] . The nucleic acid according to

[17] or any one of

[18] to

[21] . A host cell containing the vector.

[22] . A gene that expresses a protein according to any one of [1] to

[16] . Child-modified, cells.

[23] . The cell according to

[21] or

[0022] , which is a T cell or a natural killer (NK) cell.

[24] . The protein according to any one of [1] to

[16] , or the nucleus according to

[17] . Acid, the vector according to

[18] ,

[19] or

[20] , or the vector according to

[21] to

[23] A pharmaceutical composition comprising any one of the cells.

[25] A kit comprising the protein according to any one of [1] to

[16] .

[26] . A method of treating a subject for cancer, comprising:

[18] The protein according to any one of [1] to

[16] , the nucleic acid according to

[17] ,

[19] or

[20] , or the vector described in any one of

[21] to

[23] . or administering to a subject an effective amount of the pharmaceutical composition according to

[24] . .

[26] . Cancer is lymphoma (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma (N B-cell malignancies selected from the group consisting of leukemia, plasmacytoma, and myeloma. The method according to

[26] .

[28] . B-cell malignancies include chronic lymphocytic leukemia, follicular lymphoma, and mantle cell lymphoma. from lymphoma, and diffuse large B-cell lymphoma, plasmacytoma, and multiple myeloma The method according to

[27] , wherein the compound is selected from the group consisting of:

[27] . Cancers include chronic myeloid leukemia, acute myeloid leukemia, leukemia, plasmacytoma, and bone marrow tumors. The method according to

[26] , wherein the myeloid malignancy is selected from the group consisting of myeloma.

[29] B-cell-related disorders (e.g., monoclonal gammopathy of undetermined significance) A method for treating a subject with MGUS, comprising administering an effective amount of any one of [1] to

[16] .

[17] , the nucleic acid according to

[18] ,

[19] or

[20] the vector, the cell according to any one of

[21] to

[23] , or the cell according to

[24] A method comprising administering a pharmaceutical composition to a subject.

[30] A method for treating a subject with an immune system disorder, comprising: an effective amount of a protein according to any one of [1] to

[0016] ; a nucleic acid according to

[17] ; or a compound according to

[18] ,

[19] , or or the vector according to

[20] , the cell according to any one of

[21] to

[23] , or A method comprising administering the pharmaceutical composition described in

[24] to a subject.

[31] . Disorders of the immune system are autoimmune diseases such as rheumatoid arthritis, as described in

[30] . How to do it.

[32] . A method of treating a subject with cancer, comprising administering to a subject an immunosuppressant comprising a chimeric antigen receptor (CAR). and administering to a subject cells, wherein the CAR is selected from the group consisting of SEQ ID NOs: 11-949, and 950. a target molecule having an amino acid sequence selected from the group consisting of: a target binding domain comprising a DD, which may specifically bind to a target molecule; a transmembrane domain; and an intracellular domain, the intracellular domain comprising a signaling domain, The target binding domain specifically binds to a target expressed by cancer cells and inhibits the target. Upon binding, immune cells generate cytotoxic signals that produce a cytotoxic effect on cancer cells. A method in which one is induced to achieve something.

[33] The method according to

[32] , wherein the immune cells are T cells.

[34] The method according to

[32] , wherein the immune cells are NK cells.

[35] . The method of

[32] , wherein the administration is intravenous.

[36] . Use of a composition for treating cancer, the composition comprising any one of [1] to

[16] .

[0020] The present invention includes a protein according to any one of

[17] , a nucleic acid according to

[18] , a vector according to

[19] , or a cell according to any one of

[21] to

[23] . ,use.

[37] . The use according to

[36] , wherein the cancer is a B-cell malignancy.

[38] . B-cell malignancies include lymphomas (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma).

[37] , selected from the group consisting of non-Hodgkin lymphoma (NHL), leukemia, and myeloma. Use of the above.

[39] . B-cell malignancies include chronic lymphocytic leukemia, follicular lymphoma, and mantle cell lymphoma. and diffuse large B-cell lymphoma,

[38] Use as described.

[40] . Use of a composition for treating a B-cell-related disorder, the composition comprising: [1]

[16] to

[17] , the nucleic acid according to

[17] , the vector according to

[18] ,

[0019] or

[20] , the cell according to

[21] to

[23] , or a use comprising the pharmaceutical composition described in

[24] .

[41] . Use of a composition for treating a disorder of the immune system, the composition comprising [1] to

[16] The protein according to any one of

[16] , the nucleic acid according to

[17] , the vector according to

[18] ,

[0019] or

[20] , or the cell according to any one of

[21] to

[23] . Or use comprising the pharmaceutical composition described in

[24] .

[42] . Disorders of the immune system, such as autoimmune diseases like rheumatoid arthritis, are described in

[41] . Use of.

[43] . The use of immune cells containing chimeric antigen receptors (CARs) for the treatment of cancer. Thus, the CAR is an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 to 949, and 950. and optionally, a DD containing a nucleotide sequence that specifically binds to a target expressed by cancer cells. a target binding domain; a transmembrane domain; and an intracellular domain, The intracellular domain comprises a signaling domain, and upon administration to a subject with cancer, the target binding domain Specific binding to targets expressed by cancer cells, and upon target binding, immune cells Induced to produce cytotoxic signals that produce a cytotoxic effect on cancer cells ,use.

[44] . The immune cells are T cells or natural killer (NK) cells.

[43] Use as described in. [Brief explanation of the drawings]

[0076] [Figure 1] (Figures 1A-1B) DDpp, as part of a fusion protein (e.g., an antibody-DDpp fusion protein), confers a new binding specificity to another molecule (e.g., a full-length antibody). DDpp-antibody fusions were created using an RSV-specific antibody (SYN) and a non-targeting peptide (DD) of SEQ ID NO: 1 or the CD137-specific DDpp (bb10) (SEQ ID NO: 876). DDpp is fused to the N-terminus (bb10-SYN and DD-SYN) or C-terminus (SYN-bb10 and SYN-DD). All four antibody fusions bind to RSV (Figure 1A). However, fusion of bb10 to the N-terminus (bb10-SYN) or C-terminus (SYN-bb10) of the antibody heavy chain confers a new CD137-binding specificity to a normally monospecific antibody (Figure 1B). [Figure 2-1](Figures 2A-2F) CD123-DDpp-CAR T cells produce cytokines in response to target binding. Figures 2A and 2B show data related to interferon gamma (IFNγ) production by CD123-DDpp-CAR-expressing T cells when co-cultured with the CD123-negative tumor K562 and the CD123-positive tumor BDCM, respectively. Figures 2C and 2D show similar data measuring interleukin-2 (IL2) production by CD123-DDpp-CAR T cells when co-cultured with K562 and BDCM, respectively. Figures 2E and 2F show similar target-driven cytokines (IFNγ, IL2) by PD-L1-DDpp CAR T cells when co-cultured with PDL1-expressing tumor cells (SUDHL-1). [Figure 2-2] See description of Figure 2-1. [Figure 3] (Figures 3A-3B) DDpp-CAR-expressing T cells do not undergo over-depletion to a greater extent than scFv. Figure 3A shows the expression of three exhaustion markers (LAG3, PD1, and TIM3) on T cells expressing various DDpp-CARs at levels similar to the expression of these markers on scFv-CAR 32716 (3278 (Du X1, Ho M, Pastan I. 2007. New immunotoxins targeting CD123, a stem cell antigen on acute myeloid leukemia cells. J Immunother. 30(6):607-13). Figure 3B shows flow cytometry data demonstrating similar exhaustion marker expression on DDpp-CAR T cells (expressing CD123-targeting cg06 DDpp) compared to CAR T cells expressing a CD123-specific scFv (32716). [Figure 4] (Figure 4A-4C) When co-cultured with target-expressing tumor cells (BDCM), DDpp-CAR-expressing T cells (CD123-targeted) undergo degranulation. Figures 4A-4C show results obtained by culturing T cells alone (Figure 4A) or in the presence of K562 cells (Figure 4B) or BDCM cells (Figure 4C). [Figure 5-1](Figures 5A-5D) DDpp-CAR-expressing T cells mediate target-specific tumor cytotoxicity. Figure 5A shows data related to % killing of CD123-negative K562 tumor cells by CD123-DDpp-CAR T cells. Figure 5B shows % killing when CD123-targeted DDpp-CAR T cells were co-cultured with CD123-positive BDCM cells. Data from Figures 5A and 5B were generated using T cells from a first donor blood sample. Figures 5C and 5D show similar data from collected T cells from a second donor. [Figure 5-2] See description of Figure 5-1. [Figure 6] (Figures 6A-6E) Bispecific DDpp-CAR T cells. Figure 6A shows the % of T cells expressing CD123-targeted DDpp-CAR (cg06). Figure 6B shows the % of T cells expressing PDL1-targeted DDpp-CAR (pb04). Figure 6C shows the % of T cells expressing bispecific CD123-PDL1-targeted DDpp-CAR (expressed with cg06 DDpp distal to the T cell membrane compared to pb04 DDpp). Figure 6D shows the % of T cells expressing bispecific PDL1-CD123-targeted DDpp-CAR (expressed with pb04 DDpp distal to the T cell membrane compared to cg06 DDpp). Figure 6E shows data related to the increased intracellular signaling of bispecific DDpp. [Figure 7](Figures 7A-7B) Dual-binding domain adaptor proteins promote enhanced signaling by CAR-expressing Jurkat NFAT-luciferase reporter cells compared with single-binding domain adaptor proteins. In Figure 7A, 50,000 reporter cells previously transduced with AFP (p26 domain)-linked CAR (af03) were cultured for 5 hours in the presence of the CD123-specific Cg06 adaptor (Cg06-p26) or Cg06-dual adaptor protein (Cg06-p26-Cg06) in the presence of 50,000 CD123+ MOLM13 or CD123-deficient MOLM13 cells, and then luciferase activity was assessed. CD123-deficient cells were generated using CRISPR / Cas9 genetic engineering technology. In Figure 7B, 50,000 reporter cells previously transduced with AFP (p26 domain)-conjugated CAR (af03) were cultured for 5 hours in the presence or absence of 50,000 BCMA+ U266 cells in the presence of the BCMA-specific Bc40-adapter (Bc40-p26) or Bc40-dual adaptor protein (Bc40-p26-Bc40), and luciferase activity was then assessed. [Figure 8] A BCMA-targeted DDpp-CAR containing bc40 DDpp (sequence number 164) eradicates the BCMA-expressing tumor, U226, in a mouse model of B-cell cancer. DETAILED DESCRIPTION OF THE INVENTION

[0077] Any section headings used herein are for organizational purposes only and do not limit the scope of the present disclosure. should not be construed as limiting the subject matter.

[0078] Definition of Terms When embodiments are described herein with the term "comprising" always includes "consisting of" and / or "essentially consisting of" Another described in terms of "consisting essentially of" Similar embodiments are provided as well. However, the term "invention" used as a transitional phrase in the claims Each case should be interpreted separately and in its appropriate legal and factual context. (e.g., "comprising" is considered a more open-ended phrase) "consisting of" is considered more exclusive, while "from" is considered more exclusive. "Consisting essentially of" is one of these. (considered to be intermittent).

[0079] As used herein, the singular forms "a," "an," and "the" are used unless expressly stated otherwise. Includes multiple references unless otherwise noted.

[0080] The term "and / or" as used in expressions such as "A and / or B" is intended to be illustrative and not restrictive. is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" used in expressions such as "A, B and / or C" "or" encompasses each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone) ;B(alone); and C(alone).

[0081] The terms "protein" and "polypeptide" are used interchangeably herein and refer to peptides. means a biological polymer containing units derived from amino acids linked via tetramide bonds; A protein can be composed of two or more polypeptide chains.

[0082] When used interchangeably herein, the terms "antibody" or "immunoglobulin" refer to a complete Full-length antibodies and antigen-binding fragments or single chains thereof, effector domains, salvage An antibody fragment containing any functional domain of an antibody, such as a receptor-binding epitope, or a portion thereof. A typical antibody contains at least two fragments interconnected by disulfide bonds. It contains two heavy (H) chains and two light (L) chains. Each heavy chain contains a heavy chain variable region (abbreviated as VH). ) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and Each light chain consists of a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region consists of one domain, Cl. The VH and VL regions are more conserved. Complementarity-determining regions (CDRs) interspersed with regions called framework regions (FWs) Each VH and VL has three CDRs and a hypervariable region called a It consists of four FWs, arranged in the following order from the amino terminus to the carboxy terminus: FW1, C DR1, FW2, CDR2, FW3, CDR3, FW4. The variable regions of the heavy and light chains are The constant region of an antibody contains a binding domain that interacts with an antigen. effector cells) and host tissues containing the first component of the classical complement system (C1q). or may mediate the binding of immunoglobulins to factors. Examples of antibodies of the present disclosure include the exemplary antibodies , scFv, and combinations thereof, for example: DDpp is covalently attached to the N-terminus of the heavy and / or light chains of a typical whole (full-length) antibody. (e.g., via a peptide bond or via a chemical linker) or full-length antibodies It is intercalated into the heavy and / or light chains of the antibody.

[0083] The term "antibody fragment" refers to a portion of an intact antibody, e.g., an antigen-binding fragment. ment or a single chain thereof, an effector domain, a salvage receptor binding epitope, or means any functional domain of an antibody, such as a portion thereof. Examples of antibody fragments include: Non-limiting examples include Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, and Examples of antibodies include monoclonal antibodies, single chain antibodies, and multispecific antibodies formed from antibody fragments. As used herein, an "antibody fragment" refers to an antibody fragment that contains an antigen-binding site or epitope-binding fragment. In one embodiment, the DDpp fusion protein comprises an effector domain or In one embodiment, the DDpp fusion protein comprises a salvage receptor binding enzyme. It comprises a pitope or a part thereof.

[0084] As used herein, the term "Fc region" or simply "Fc" refers to an Fc region that is a region of an immunoglobulin a carboxyl group of an immunoglobulin chain constant region, preferably an immunoglobulin heavy chain constant region, or a portion thereof; For example, the Fc region of an immunoglobulin is understood to mean (1) a CH1 domain; (1) the main, CH2, and CH3 domains; (2) the CH1 and CH2 domains Main, (3) CH1 domain and CH3 domain, (4) CH2 domain and CH 3 domains, or (5) a combination of two or more domains and an immunoglobulin hinge region Thus, in various embodiments, Fc may comprise the fragments of IgA, IgD, and IgG. The last two constant region immunoglobulin domains and the last three of IgE and IgM The constant region immunoglobulin domains of the ribosomal protein, as well as flexible human ribosomal domains N-terminal to these domains, For IgA and IgM, Fc may include the J chain. For IgG, In this case, Fc is the region between immunoglobulin domains Cγ2 and Cγ3 and between Cγ1 and Cγ2. In a preferred embodiment, the immunoglobulin Fc region comprises at least one of the following: It contains a globulin hinge region, a CH2 domain, and a CH3 domain, and preferably a CH1 domain. In one embodiment, the class of immunoglobulin from which the heavy chain constant region is derived is Ig G (Igγ) (γ subclasses 1, 2, 3, or 4). IgA (Igα), IgD (Igδ), IgE (Igε) and IgM (Igμ) Although the boundaries of the Fc region vary, the human IgG heavy chain Fc region typically begins at residue C2 26 or p260 at its carboxyl terminus, and the numbering is abat(Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public National Institutes of Health, Bethesda, Md. (1991) The Fc region can be used alone or in a full-length antibody or antibody It can refer to a fragment of this region, or this region in the context of an Fc fusion protein. However, in the EU index numbering, they are at positions 270, 272, 312, 315, and 3 Polymorphisms have been observed at a number of different Fc positions, including 56 and 358, and therefore There may be slight differences between the sequence and the prior art sequences. Suitable immunoglobulin heavy chains The selection of constant regions is described in U.S. Patent Nos. 5,541,087 and 5,726,044. Each of these patents is incorporated herein by reference in its entirety. from specific immunoglobulin classes and subclasses to achieve specific results. The selection of a particular immunoglobulin heavy chain constant region sequence is within the skill level of the art. Some DNA constructs encoding immunoglobulin Fc regions are considered to be within the scope of the present invention. , preferably including at least a portion of the hinge domain, and preferably including at least one The CH3 domain of the Fc gamma region or any of IgA, IgD, IgE, or IgM In addition, amino acid substitutions or modifications within the immunoglobulin heavy chain constant region Deletions such as these may be useful in practicing the methods and compositions disclosed herein. Amino acid substitutions were introduced into the H2 region to generate Fc mutants with reduced affinity for Fc receptors. (Cole, J. Immunol. 159:3613 (1997)) .

[0085] "Antibody-dependent cellular cytotoxicity" or "ADCC" is a type of antibody that binds to Fc receptors (FcRs) (e.g., NADH). Nonspecific cells expressing IL-1 (NK cells, neutrophils, and macrophages) The cytotoxic cells recognize the bound antibodies on the target cells and then lyse (or ADCC refers to a cell-mediated reaction that results in the target molecule being targeted (or causing other cytotoxic effects). To assess activity, U.S. Pat. No. 5,500,362 or U.S. Pat. No. 5,821,337 any in vitro ADCC assay known in the art, such as those described in Useful effector cells for such assays include, but are not limited to: However, it includes peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of a molecule of interest can be assayed using methods such as those described in, e.g., Clynes et al. et al., PNAS 95:652-656 (1998) The assay can be performed in vivo using a DNA library.

[0086] As used herein, a "single chain variable fragment," or "scFv," antibody is A form of antibody (e.g., a nucleotide sequence) that contains only the variable regions of the heavy and light chains joined by a linker peptide. In one embodiment, the DDpp fusion protein is a DD Includes pp and scFv.

[0087] The term "linker" refers to a linker between a DDpp and another polypeptide in a DDpp fusion protein. The term "DD" refers to a peptide or other chemical linkage located at two or more positions. Suitable linkers for attaching pp will be apparent to those skilled in the art, and non-limiting examples include: is described herein.

[0088] As used herein, the term "operably linked" means that each molecule so that each molecule retains at least some level of functional activity that it had alone (each molecule retaining functional activity). (holds) indicates that two molecules bind. In some embodiments, if the other molecule has at least some level of its functional activity, It is operably linked to another molecule. Operably linked means two non-functional molecules. It can also mean the connection of molecules. Two molecules can be connected either directly or through an indirect They can be "operably linked" even if directly (e.g., via a linker).

[0089] The terms "specifically bind" or "have selective affinity for," "bind," or "binding" are used interchangeably and refer to the ability of a binding agent, such as DDpp, to bind to a target epitope, more frequently than when using other substances containing proteins unrelated to the target molecule. , more rapidly, for a longer period of time, with greater affinity, or some combination of the above. It means reacting with or binding to an epitope, protein, or target molecule. Due to sequence identity between homologous proteins in different species, specific binding is difficult to achieve in some instances. In embodiments, the binding agents include those that recognize proteins or targets in more than one species. Specific binding occurs due to homology within sequence regions of specific polypeptides of different proteins. In certain embodiments, the antibody may comprise a binding agent that recognizes more than one protein or target. a binding substance that specifically binds to a first target may also specifically bind to a second target, or Thus, "specific binding" does not necessarily mean exclusive binding, e.g., binding to a single target. It does not necessarily require (although it can include) binding. Therefore, binding agents are In embodiments, the antibody may specifically bind to more than one target. In certain embodiments, the multiple targets include: They can be bound by the same antigen-binding site on the binding agent.

[0090] A "target" is a DDpp, such as a DDpp fusion protein, or an antibody or antibody variable domain. Any component that can be bound by other components of the DDpp fusion protein, such as a fusion fragment. It means a molecule or a combination of molecules.

[0091] The terms "epitope" and "antigenic determinant" are used interchangeably herein and refer to a specific Any molecule that can be recognized and specifically bound by a compound (e.g., DDpp or antibody). target genes (e.g., BCMA, CD123, AFP, or AFP p26) If the molecule being recognized is a polypeptide, the epitope is a portion of the protein. Contiguous and non-contiguous amino acids juxtaposed by tertiary folding of the protein formed from chemically active surface groups (such as carbohydrates) of other molecules. Epitopes formed from amino acids are usually preserved upon protein denaturation, but tertiary forms Epitopes formed by binding are usually lost upon protein denaturation. A peptide usually contains at least three amino acids, more commonly fewer, in a unique spatial conformation. Both contain 5 or 8-10 amino acids.

[0092] As used herein, a "peptide tag" is a peptide tag that confers function to the resulting fusion. a peptide that is part of or attached (e.g., by genetic engineering) to another protein Peptide tags are usually short compared to the proteins to which they are fused. For example, peptide tags may in some embodiments be four or more amino acids in length, e.g. For example, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more amino acids in length In some embodiments, the DDpp is a fusion protein containing a peptide tag. In embodiments, the DDpp specifically binds to the peptide tag. Numerous peptide tags with utility are known in the art. bound by a protein fusion protein or DDpp (e.g., a DDpp fusion protein) Examples of peptide tags that can be components of a target include, but are not limited to, HA (hemagglutinin), c-myc, herpes simplex virus glycoprotein D (gD), T7, GST, GFP, M BP, Strep-tag, His-tag, Myc-tag, TAP-tag and FLAG (registered trademark) (Mark) (Eastman Kodak, Rochester, NY). Similarly, antibodies against tag epitopes can be used in Western blots of cells, ELISA assays, and other assays. and immunostaining techniques known in the art to detect the fusion protein. This allows for the detection and localization of

[0093] When used in reference to biological materials such as nucleic acid molecules, polypeptides, and host cells, The term "native" means that which is found in nature and has not been modified by man. When used in reference to biological material, the terms "non-natural" or "synthetic" refer to materials that are not naturally occurring. It refers to substances not found in nature and that have been modified by humans.

[0094] As used herein, a "modification" to the sequence of a reference sequence includes any modification of the reference sequence (e.g., , DD) as disclosed herein) and / or substitutions, deletions, insertions, and / or deletions of the sequences at the corresponding amino acid positions. Or additions are included.

[0095] A "substitution" relative to a sequence of a reference sequence refers to the substitution of a particular amino acid at the corresponding amino acid position of the reference sequence. It refers to the replacement of an amino acid residue with a different amino acid residue.

[0096] A "conservative" amino acid substitution is one in which one amino acid residue is substituted with another amino acid residue having a similar side chain. A family of amino acid residues with similar side chains is called a substitution. It has been demonstrated in the art that these side chains include basic side chains (e.g., lysine, ketone (K), arginine (R), histidine (H)), acidic side chains (e.g., aspartic acid (D), glutamic acid (E), uncharged polar side chains (e.g., glycine (G), asparagine (A), N, Glutamine (Q), Serine (S), Threonine (T), Tyrosine (Y), Cysteine ​​(C), catenin (C)), non-polar side chains (e.g., alanine (A), valine (V), leucine (L) , isoleucine (I), proline (P), phenylalanine (F), methionine (M), tryptophan (W), β-branched side chains (e.g., threonine (T), valine (V), isoforms leucine (I), and aromatic side chains (e.g., tyrosine (Y), phenylalanine (F ), tryptophan (W), histidine (H). For example, the phenylalanine In certain embodiments, the substitution of arginine with arginine is a conservative substitution. The target substitution is the target molecule to which it binds (e.g., BCMA, CD123, AFP, or AF P p26) resulting in altered or unchanged specific binding of DDpp. In embodiments, conservative substitutions in the sequence of DDpp include substitutions for the binding target. Nucleotides that do not inhibit Dpp binding and that confer, alter, or maintain selective binding affinity Methods for identifying amino acid conservative and non-conservative substitutions are well known in the art. It is known in the field (e.g., Brummell, Biochem. 32:1180- 1187(1993);Kobayashi,Protein Eng.12(10): 879-884(1999); and Burks, PNAS 94:412-417( 1997).

[0097] A "non-conservative" amino acid substitution is one in which one amino acid residue is substituted with another amino acid residue having a dissimilar side chain. In one embodiment, the non-conservative substitutions in the sequence of DDpp are , the target to which it binds (e.g., BCMA, CD123, AFP, or AFP p2 6) results in specific binding of DDpp containing substitutions of the following: A conservative substitution does not abrogate binding of the DDpp containing the substitution to the binding target.

[0098] "Unnatural amino acids," "amino acid analogs," and "non-standard amino acid residues" are used herein. The unnatural amino acids that can be substituted for DDpp provided herein are: In one embodiment, the unnatural amino acid can replace proline. 4-hydroxyproline, which can replace lysine; 5-hydroxylysine, which can replace histidine; 3-methylhistidine can be substituted, homoserine can be substituted for serine, and lysine can be substituted Additional unnatural amino acids that can be substituted with DDpp disclosed herein are ornithine. Examples include, but are not limited to, the D-isomers of common amino acids, 2,4-diaminobutyric acid, α-aminobutyric acid, Aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-Ahx, 6 -Aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocysteine Toluline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine , cyclohexylalanine, β-alanine, lanthionine, dehydroalanine, γ-amino Fluoro- and β-methyl amino acids, such as selenocysteine, pyrrolidine, and lysine. Which designer amino acids, Cα-methyl amino acids, and Nα-methyl amino acids, or The additional unnatural amino acids include, for example, For example, 4-aminobutyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 4-amino 3-hydroxy-6-methylheptanoic acid, 2-thienylalanine, and / or alanine As discussed herein, some embodiments may include D-isomers of amino acids. In this embodiment, the unnatural amino acid or amino acid analogue may be one or more amino acids from the sequence. The deletion may include:

[0099] The terms "polynucleotide" and "nucleic acid," used interchangeably herein, refer to any refers to a long polymeric form of nucleotide, ribbon nucleotide or deoxynucleotide. These terms include DNA, RNA, cDNA (complementary DNA), and mRNA (messenger RNA). senescent RNA), rRNA (ribosomal RNA), shRNA (small hairpin RNA) , snRNA (small nuclear RNA), snoRNA (small nucleolar RNA), miRNA (microRNA), genomic DNA, synthetic DNA, synthetic RNA, and / or tRNA These include, but are not limited to:

[0100] As used herein, the term "naked DNA" refers to DNA that is suitable in the proper orientation for expression. The DDp disclosed herein is cloned into an expression vector (e.g., a plasmid). DNA encoding a protein such as ribosomal protein (e.g., CAR) (e.g., histone-free DNA) A) is used to maintain and / or express DNA encoding DDpp. Viral vectors that can be used include, but are not limited to, SIN lentiviral vectors, retroviral vectors, Rovirus vectors, foamy virus vectors, adenovirus vectors, adeno-associated virus vectors AAV vectors, hybrid vectors, and / or plasmid transformants transposon (e.g., Sleeping Beauty transposon system), or This includes integrase-based vector systems. Other vectors that can be used are described herein or in the art. It is known.

[0101] As used herein, the terms "vector," "cloning vector," and An "expression vector" is a vehicle by which a host cell can be transformed and the expression of introduced sequences can be promoted. The nucleic acid sequence (e.g., the disclosed DDpp code) may be modified to facilitate expression (e.g., transcription and translation) of the nucleic acid sequence. a sequence) can be maintained or propagated in a host cell (e.g., a cloning vector); or A vector is a vehicle that can be introduced into a host cell. Vectors include plasmids, phages, and This includes Ilus, etc.

[0102] A "host cell" includes any cell that can be the recipient of nucleic acid encoding the disclosed DDpp, or Host cells include, but are not limited to, individual cells or cell cultures that were recipients. but includes virus particles, phagemids, bacteria, yeast, plant, animal, and mammalian cells A host cell includes progeny of a single host cell, whether naturally occurring, accidental, or are completely identical (in morphology or The host cell may be in vivo, in vitro, or This includes cells transfected or infected with nucleic acids encoding the disclosed DDpp ex vivo. In some instances, the host cell is a host cell, e.g., in a phage display or CAR T cell. The disclosed DDpp can be expressed and displayed on its surface. or translations are included.

[0103] As used herein, the terms "solid support," "support," "matrix," and " The term "resin" is used interchangeably and refers to, but is not limited to, any column (or column material). materials), beads, test tubes, microtiter dishes, solid particles (e.g., agarose or Sepharose), microchips (e.g., silicon, silicon-glass, or or gold chips), or membranes (e.g., biological or filter membranes), In contrast, DDpp, antibodies, or other proteins directly or indirectly (e.g., can be bound (e.g., via another antibody or other binding partner intermediate such as protein A) For example, they may be coupled, linked, or glued together, or among these, DDpp or or antibodies may be embedded (e.g., via receptors or channels). Reagents and methods for binding peptides to solid supports (e.g., matrices, resins, plastics, etc.) Methods and techniques for cleaving solid supports are well known in the art. Suitable solid supports include, but are not limited to: Chromatographic resin or matrix (e.g., Sepharose-4FF agarose beads) size), well walls or floors of plastic microtiter dishes, silica-based bars Iochip, polyacrylamide, agarose, silica, nitrocellulose, paper, plastic PP, nylon, metal, and combinations thereof. The compositions may be non-covalently or covalently bound using reagents and techniques known in the art. In one embodiment, the DDpp can be immobilized on a support material by bonding. bound to a chromatographic material.

[0104] As used herein, the terms "pharmaceutically acceptable" or "physiologically acceptable" "Capable" and its grammatical variations are used when they refer to compositions, carriers, diluents, and reagents. In this case, the terms are used interchangeably and are used to refer to therapeutically prohibited and undesirable symptoms such as nausea, dizziness, and acute gastric peristalsis. This demonstrates that the substance can be administered to humans without producing any adverse physiological effects.

[0105] "Modulate" means the regulation or control of magnitude, frequency, extent, or activity. In related aspects, such modulation can be positively modulated (e.g., frequency, degree, or activity) or adversely modulated (e.g., decreased frequency, degree, or activity) In some embodiments, the positive or negative modulation is determined by measuring the expression level of the cells prior to administration of the therapeutic agent. In a further embodiment, the function of the serotonin receptor is compared to the function of the serotonin receptor, tissue, or organ. The upward regulation is relative to the function of a normal, healthy cell, tissue, or organ.

[0106] An "effective amount" of a DDpp fusion protein provided herein, etc. The DDpp is associated with a target to which the DDpp (e.g., DDpp fusion protein) binds. specifically demonstrated to produce an observable change in the level of one or more biological activities In certain embodiments, the alteration is an amount sufficient to increase the level of target activity. In other embodiments, the alteration decreases the level of the target activity. The term "therapeutically effective amount" can be determined empirically and routinely for the purposes indicated. , "treating" a disease or disorder in a subject (mammal) (e.g., reducing the symptoms of the disease or disorder) ) means an amount of DDpp or other therapeutic agent, such as a DDpp fusion protein, that is effective to The term "therapeutically effective amount" also refers to the dosage and duration necessary to achieve the desired prophylactic result. This means an effective amount between

[0107] "Patient," "subject," "animal," and "mammal" are used interchangeably and refer to human patients and mammals such as mammals and non-human primates, as well as laboratory animals such as rabbits, rats, and mice Other animals include all vertebrates, such as chickens, amphibians, and As used herein, "mammal" includes mammals and non-mammals, such as mammals and reptiles. " includes, but is not limited to, humans and chimpanzees and other ape and monkey species. non-human primates; farm animals such as cattle, sheep, pigs, goats and horses al); domestic animals such as dogs and cats; mice Any member of the mammalian class, including rodents, laboratory animals, including rats and guinea pigs In certain embodiments, the patient is a human. The term refers to a patient of a particular age or Therefore, adult and neonatal / pup subjects, and fetuses / pups Both male and female animals are intended to be included within the scope of this term.

[0108] As used herein, "treat," "treatment," and and "treating" refers to both therapeutic treatment and preventative or prophylactic treatment. The purpose of this is to identify the symptoms, complications, or biochemical changes of a disease, condition, or disorder. prevent or delay (reduce or delay) the development of a disease, condition, or disorder The purpose of treatment is to alleviate or prevent or inhibit their further progression. "treatment" targets the pathology and prevents symptoms after the onset of a disease, condition, or disorder. (prevent or delay the onset of disease or prevent the development of clinical or asymptomatic symptoms) or therapeutic suppression or alleviation, prevention of a disease state, or pursuit of a beneficial result or the likelihood of progression of an individual's condition, even if treatment is ultimately unsuccessful. Subjects in need of treatment include those already with the condition, as well as subjects prone to developing the condition or subjects in whom the condition is to be prevented. Treatment may involve the use of DDpp fusion proteins alone or in combination with additional therapeutic agents. can be done.

[0109] "Cancer," "tumor," or "malignancy" are used interchangeably and refer to the uncontrolled growth of cells. Abnormal proliferation of infected cells locally or via the bloodstream and lymphatic system to other parts of the body Many of these are characterized by their diffusion (transition) into other tissues and by a number of distinctive structural and / or molecular features. As used herein, "tumor" refers to any of the diseases, whether malignant or benign. All neoplastic cell growth and proliferation, and all pre-cancerous and cancerous cells and tissues, regardless of age. "Cancerous tumors" or "malignant cells" refer to tissues that have specific structural characteristics and lack differentiation. The DDpp fusion proteins provided herein are understood to be cells that are capable of invading and metastasizing. Cancers that can be treated with proteins include blood tumors, such as leukemia and lymphoma, or solid tumors. In certain embodiments, the cancer being treated is leukemia or lymphoma. Cancer types treated with Bpp include, but are not limited to, carcinoma, blastoma, and sarcoma, and and certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignant tumors, e.g. Examples of cancers and tumors that can be treated with DDpp include sarcomas, carcinomas, and melanomas. Types include, but are not limited to, breast cancer, lung cancer, brain cancer, bone cancer, liver cancer, kidney cancer, colon cancer, head and neck cancer Cancers that cause cancer include ovarian cancer, hematopoietic cancers (e.g., leukemia), and prostate cancer. In embodiments, cancers and tumors that may be treated with DDpp include breast cancer and ovarian cancer. Other types of cancers and tumors that can be treated with DDpp-containing antibodies are described herein. These methods are either conventional or known in the art.

[0110] The terms "tumor antigen" or "cancer antigen" are used interchangeably herein. and cancer antigens are classified as tumor-specific antigens (TSA), cancer-specific antigens (CSA), tumor-associated antigens (TSA), and TSAs can be tumor-associated antigens (TAAs) or cancer-associated antigens (CAAs). TSAs are specific to tumor cells and are expressed by the body. TAAs are antigens that do not occur on other cells in the body. Due to the dynamic nature of tumors, in some cases tumor cells , may express unique antigens at specific stages, and even on non-tumor cells Therefore, the inclusion of a particular marker, such as a TAA, indicates that it is a T It does not preclude it from being considered SA.

[0111] As used herein, "target cells" are cells that are involved in a disease and that are targeted by a DDpp-containing composition. Other target cells include those that can be targeted by the disclosed DDpp. Target cells include any cell in a subject (e.g., a human or animal) that is infected with the DDpp fusion tag. The target may be a cell expressing or overexpressing a target that is specifically bound by the protein.

[0112] As used herein, the term "effector cell" refers to a cell that expresses one or more FcRs. These cells express at least FcR and perform effector functions. Preferably, the cells express ATP1F3 and carry out ADCC effector function. Examples of human leukocytes include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, and mononuclear cells. In particular embodiments, PBMCs and neutrophils are included. Effector cells may be derived from their natural source, e.g., K cells, as described herein. Alternatively, it can be isolated from blood or PBMCs, as known in the art. In some embodiments, the effector cells are human effector cells.

[0113] The term "effector function" refers to the differentiated function of a differentiated cell. Effector functions include, for example, helper activity, including cytolytic activity or secretion of cytokines. It could be.

[0114] As used herein, the term "immune cells" includes antigen-presenting cells, B cells, basophils, , cytotoxic T cells, dendritic cells, eosinophils, granulocytes, helper T cells, white blood cells, lymphocytes, Macrophages, mast cells, memory cells, monocytes, natural killer cells, neutrophils, phagocytes Mammalian immune cells, including but not limited to phagocytes, plasma cells, and T cells It means cells of the virus system.

[0115] The terms "T cell" and "T lymphocyte" are used interchangeably and are used herein Examples include naive T cells, central memory T cells, and F These include, but are not limited to, vector memory T cells, or combinations thereof. do not have.

[0116] As used herein, the term "immune response" refers to all types of immune responses, including innate immunity, humoral immunity, and cellular immunity. These include immune responses, inflammatory responses, acquired (acquired) immunity, autoimmunity, and / or hyperactive immunity. Immunity includes, but is not limited to:

[0117] As used herein, the term "transduction" refers to the transfer of exogenous genes to a cell using a viral vector. As used herein, the term "gene transfer" refers to the introduction of a gene into a cell. refers to the introduction of foreign nucleic acid into a cell using recombinant DNA techniques. "Transformation" refers to the transfer of "exogenous" (e.g., exogenous, extracellular, or otherwise non-endogenous) nuclear A nucleic acid (DNA or RNA) sequence is introduced into a host cell, and the host cell expresses the introduced nucleic acid. To produce a substance, such as a protein or enzyme, encoded by a desired introduced coding sequence. An introduced nucleic acid sequence also refers to a "cloned" or "foreign" gene or sequence. They can also be called start, stop, promoter, signal, secretion, or the genetic machinery of a cell. The nucleic acid sequence may include regulatory or control sequences, such as other sequences used by the The introduced nucleic acid (e.g., DNA or RNA) may contain a non-functional sequence that does not have a function. A host cell that receives and expresses a gene encoding the gene is "transformed" and is called a "transformant" or " The DNA or RNA introduced into a host cell is from the same genus or species as the host cell. may be from any source, including cells of the same species, or cells of a different genus or species, and may also be non-naturally occurring.

[0118] "Cell surface receptors" receive signals and transmit such signals across the plasma membrane of a cell. The term "cell surface receptors" refers to molecules and complexes of molecules capable of transmitting signals. Examples of receptors include activated integrin receptors, e.g., activated αvβ3 integrin receptors on metastatic cells. As used herein, a "cell surface receptor" refers to a target (e.g., a phospholipid receptor). For example, BCMA, CD123, CS1, HER2, AFP, or AFP p26 The term "receptor" also encompasses molecules expressed on the cell surface that contain DDpp that can bind to the receptor. They bind to or interact with molecules (e.g., ligands) and promote the release of the ligands on cells. In some embodiments, a receptor interacts with a cell-associated protein that mediates the action of a receptor. The interacting molecules are bioactive molecules. Membrane-bound cell surface receptors are usually multidomain They are characterized by a structure that includes a ligand-binding domain, a transmembrane domain, and a common synaptic domain. They contain intracellular effector domains involved in signal transduction.

[0119] As used herein, the term "CS1" refers to an NK cell receptor that regulates immune function. , which is also expressed on B cells, T cells, dendritic cells, NK-T cells, and monocytes CS1 is overexpressed in multiple myeloma and is a promising target for immunotherapy of multiple myeloma. It has been successful. Malaer & Mathew, Am J Cancer Res. 7 (8):1637-1641(2017). CS1 also binds to SLAM7, a protein 19 Also known as A, CRACC, and CD319. The term "CS1" refers to the mutant A CS1 is a transmembrane protein, including isoforms, homologs, orthologs, and paralogs. There are several alternatively spliced ​​isoforms. is a 22 amino acid residue N-terminal signal sequence (MAGSPTCLTLIYILWQLT GSAA, SEQ ID NO: 964) and 226 N-terminal residues (SEQ ID NO: 965) The amino acid sequence of human CS1, including the ectodomain, is available under GenBank accession number NP_06700 4 (SEQ ID NO: 966). In some embodiments, the amino acid sequence of human CS1 is , GenBank accession numbers NP_001269517, NP_001269518, NP_ 001269519, NP_001269520, NP_001269521, NP_0 01269522, NP_001269523, NP_001269524, or NP It has _001269525.

[0120] As used herein, "chimeric antigen receptor" or "CAR" or "CAR "(s)" refers to the ability of a cell (e.g., naive T cells, central memory) to express an antigen or target specificity. T cells, such as memory T cells and effector memory T cells; NK cells; NKT cells or CARs are also called artificial T cells. receptor, also known as chimeric T cell receptor or chimeric immune receptor.

[0121] D domain polypeptide (DDpp) Unless otherwise indicated, the practice of the disclosed compositions and methods may involve techniques including molecular biology (recombinant techniques, tissue engineering, cultures, and cell transformation), microbiology, cell biology, biochemistry, and immunology Such techniques are generally within the skill of the art. , according to manufacturer's specifications or as commonly practiced, by known procedures such as those described below. or routine modifications thereof, or as described herein. :Sambrook et al.(Molecular Cloning: A Lab oratory Manual.Cold Spring Harbor Labora tory Press, Cold Spring Harbor, NY (1989) );PCR Technology:Principles and Applications ions for DNA Amplification(ed.HAErlich , Freeman Press, NY, NY, 1992); tide Synthesis(Gait,ed.,1984);Animal Cel l Culture(Freshney,ed.,1987);Handbook of Experimental Immunology (Weir et al., eds. .;Gene Transfer Vectors for Mammalian Ce lls(Miller, ed., 1987);Current Protocols i n Molecular Biology(Ausubel.,ed.,1987);P CR Protocols: A Guide to Methods and Appl ications(Innis, ed., Academic Press, San Di ego, Calif., 1990); Mattila, et al., Nucleic Acids Res.19:967(1991);Eckert, et al., PCR Methods and Applications 1:17(1991); PCR (McPherson, ed., IRL Press, Oxford); PCR:The Polymerase Chain Reaction,(Mullis,ed.,1 994);Harlow,Antibodies:A Laboratory Manu al,(Cold Spring Harbor Laboratory Press, 2nd ed.1988) and Kontermann, ed., “The Anti body Engineering Lab Manual”(Springer Ve rlag, Heidelberg / New York, 2000);Current P rotocols in Immunology (Coligan, ed., 1991) ;The Immunoassay Handbook(Wild,ed.,Stock ton Press NY, 1994); and Methods of Immunol logical analysis (Masseyeff., ed., Weinheim: VCH Verlags gesellschaft mbH,1993); and Ge nnaro,et al.2000,Remington:the Science a nd Practice of Pharmacy,20th Ed.Lipincott t Williams and Wilkins:Baltimore,Md. specific Unless definitions are given, analytical chemistry, synthetic organic chemistry, and medicinal chemistry as described herein The nomenclature used in connection with these tests and techniques is well known in the art. Furthermore, standard techniques for chemical synthesis, chemical analysis, recombinant production, It can be used raw, purified, for pharmaceutical preparation, formulation, delivery, and patient treatment.

[0122] In various embodiments, the present disclosure provides a method for detecting BCMA, CD123, CS1, HER2, AFP, and AFP p26. In some embodiments, the DD of the DDpp is selected from the group consisting of SEQ ID NOs: 11-949, and 950. In some embodiments, the DDp p is (a) a polypeptide that specifically binds to BCMA and is selected from the group consisting of amino acids 11 to 305, or 306; (b) a DD comprising a sequence of SEQ ID NOs: 307 to 739; (c) a DD containing a 740 amino acid sequence; (d) a DD specifically binding to AFP or a fragment thereof; DD containing the amino acid sequence of SEQ ID NO: 741 to 874 or 886 to 895; ) AFP p26 specifically binds to the antibody of SEQ ID NO: 741 to 874 or 886 to 895 (e) a DD containing the amino acid sequence of CS1 (SEQ ID NO: 965) or a fragment thereof; a DD heterologously linked to the nucleotide sequence of SEQ ID NO: 896 to 909 or 910; (f) specifically binds to HER2 or a fragment thereof, and is selected from SEQ ID NOs: 911 to 949; or a D domain (DD) selected from the group consisting of DDs comprising 950 amino acid sequences. Variants (a) to (f) that retain the ability to specifically bind to their respective targets. Proteins including variants are also provided.

[0123] In a further embodiment, DD of DDpp is selected from the group consisting of SEQ ID NOs: 11 to 949 and 950. a DD reference sequence selected from the group consisting of: In some embodiments, the sequence of variant DD is SEQ ID NO: 11 to SEQ ID NO: 12. 949, and 950. %, 80%, 85%, 87%, 89%, 90%, 92%, 94%, 96%, or 98 % sequence identity of the variant DD sequence to the reference DD sequence. The antibody retains the ability to specifically bind to the target.

[0124] In some embodiments, the DD of the DDpp is selected from the group consisting of SEQ ID NOs: 11-305 and 306. a variant of a BCMA-binding DD reference sequence selected from the group consisting of: In some embodiments, the sequence of the mutant DD is SEQ ID NO: 11 to 305, and 306. or 75%, 80%, 85%, 87%, 89%, 90%, 92%, 94%, 96%, or contains the amino acid sequence of a variant with 98% sequence identity, and variant DD is a variant of BCMA. It retains the ability to bind specifically.

[0125] In some embodiments, the DD of DDpp is selected from the group consisting of SEQ ID NOs: 307-739, and 740. and a variant of the CD123-binding DD reference sequence selected from the group consisting of: In some embodiments, the sequence of the mutant DD is For a reference DD sequence selected from the group consisting of sequence numbers 307 to 739 and 740, At least 75%, 80%, 85%, 87%, 89%, 90%, 92%, 94%, 96% or a variant having 98% sequence identity, and variant DD comprises the amino acid sequence of C It retains the ability to specifically bind to D123.

[0126] In some embodiments, the DD of the DDpp is selected from the group consisting of SEQ ID NOs: 896-909, and 910. a variant of the CS1-binding DD reference sequence selected from the group consisting of: In some embodiments, the sequence of mutant DD is SEQ ID NO:8. At least one of the following sequences is selected from the group consisting of 96 to 909 and 910: or 75%, 80%, 85%, 87%, 89%, 90%, 92%, 94%, 96%, or contains the amino acid sequence of a variant with 98% sequence identity, and variant DD is specific to CS1. It retains the ability to bind heterologously.

[0127] In some embodiments, the DD of the DDpp is selected from the group consisting of SEQ ID NOs: 911-949, and 950. a variant of a HER2-binding DD reference sequence selected from the group consisting of: In some embodiments, the sequence of mutant DD is SEQ ID NO: At least one nucleotide sequence is selected from the group consisting of 911 to 949 and 950. At most 75%, 80%, 85%, 87%, 89%, 90%, 92%, 94%, 96%, or a variant amino acid sequence having 98% sequence identity, and variant DD is a variant of HER It retains the ability to specifically bind to 2.

[0128] In some embodiments, the DD of DDpp is selected from the group consisting of SEQ ID NOs: 741-874, and 886. A variant of the AFP-binding DD reference sequence selected from the group consisting of 1 to 895, In some embodiments, the sequence of the mutant DD is A reference DD sequence selected from the group consisting of sequence numbers 741 to 874 and 886 to 895. Vs. at least 75%, 80%, 85%, 87%, 89%, 90%, 92%, 94% , 96%, or 98% sequence identity of the amino acid sequence of the variant, variant D D retains the ability to specifically bind to AFP.

[0129] In some embodiments, the DD of DDpp is selected from the group consisting of SEQ ID NOs: 741-874, and 886. A variant of the AFP p26 binding DD reference sequence selected from the group consisting of In some embodiments, the mutant retains the ability to specifically bind to FP p26. The sequence of DD is selected from the group consisting of SEQ ID NOs: 741 to 874, or 886 to 895. At least 75%, 80%, 85%, 87%, 89%, 90% of the reference DD sequence , 92%, 94%, 96%, or 98% sequence identity of the amino acid sequence of the variant wherein mutant DD retains the ability to specifically bind AFP p26.

[0130] In certain embodiments, the identity between the variant DD (query) sequence and the reference DD sequence is , also known as global sequence alignment, Brutlag et al. Comp.Ap p. Biosci. 6:237-245(1990) based on the FASTD algorithm B computer program. FASTDB amino acid alignment. The preferred parameters used are: matrix PAM 0, k tuples = 2, mismatch Chi penalty = 1, Joining penalty = 20, Randomization group length = 0, Cut Offscore = 1, window size = sequence length, gap penalty = 5, gap size penalty The window size was 500 or the length of the target amino acid sequence, whichever was shorter. In embodiments, the reference DD sequence is not due to an internal deletion but rather due to an N- or C-terminal deletion. If the variant DD is shorter than the query sequence, FASTDB Considering the fact that the program does not consider N- and C-terminal truncations of the reference DD sequence, The results are then manually corrected. For reference sequences truncated at the N- and C-terminus, the % identity is calculated based on the N- and C-terminus of the reference sequence. This is corrected by calculating the number of residues in the query sequence that are The percentage of total bases in the column that do not match / align with the corresponding target residue. The decision to align a gene is determined by the results of FASTDB sequence alignment. The % of identity is then subtracted from the % identity and run against the FASTDB program described above using the specified parameters. The final percent identity score is calculated by the program to arrive at a final percent identity score. This is the purpose of the embodiment.

[0131] In some embodiments, DDpp is a corresponding one of SEQ ID NOs: 11-949, or 950. A reference DD is a sequence that contains two or more sequence modification categories (i.e., substitutions, deletions, insertions, and deletions). and variant DDs containing different amino acid sequences in each of the Targets of reference DD (e.g., BCMA, CD123, CS1, HER2, AFP, and A For example, the sequence of the mutant DD is similar to that of the reference DD. The sequence may contain a combination of amino acid deletions, insertions and substitutions compared to the sequence of In embodiments, the sequence of the variant DD is selected from the group consisting of SEQ ID NOs: 11 to 949, or 950. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 meshes compared to the row In some embodiments, the sequence of the mutant DD is SEQ ID NO: 11-949. , or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 compared to the 950 reference DD sequences or more than 10 conservative amino acid substitutions. In some embodiments, variant D The sequence of D is 1, 2, 3 or 4 times different from the reference DD sequence of SEQ ID NO: 11 to 949 or 950. , 4, 5, 6, 7, 8, 9, 10, or more than 10 non-conservative amino acid substitutions In some embodiments, the sequence of the mutant DD is SEQ ID NO: 11-949, or 950 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 10 compared to the reference DD sequence In some embodiments, the variant D The sequence of D is 1, 2, 3 or 4 times different from the reference DD sequence of SEQ ID NO: 11 to 949 or 950. , 4, 5, 6, 7, 8, 9, 10, or more than 10 amino acid deletions. In some embodiments, the sequence of the mutant DD is a reference D of SEQ ID NO: 11-949, or 950. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 addresses in a D sequence The mutant DD contains an amino acid insertion and retains the ability to bind to the target of the reference DD. , the amino terminus, carboxy terminus of the corresponding reference DD of SEQ ID NO: 866, and 867, or mutant DD, which has amino acid residues deleted from both the amino and carboxy termini In some embodiments, the sequence of the mutant DD is SEQ ID NO: 11 to 15. 1, 2, 3, 4, 5, 6, 7, or 949 or 950 from the amino terminus of the reference DD sequence The mutant DD contains sequences with deletions of 8, 9, 10, 11, or 12 amino acid residues. In some embodiments, the mutant DD retains the ability to bind to the target of the reference DD. The sequence of SEQ ID NO: 11 to 949 or 950 is from the carboxy terminus of the reference DD, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues deleted The variant DD contains the sequence, and retains the ability to bind to the target of the reference DD. In embodiments, the sequence of the variant DD is selected from the group consisting of SEQ ID NOs: 11 to 949, or 950. containing 1-5, 1-10, or 1-15 amino acid residues deleted from the amino terminus of the sequence. The variant DD sequence is composed of 1-5, 1-10, or 1-15 carboxylates of the reference DD sequence. The mutant DD has amino acid residues deleted from the C-terminus and exhibits the ability to bind to the target of the reference DD. holds.

[0132] DDpp fusion protein "Fusion proteins," "chimeric polypeptides," "containing heterologous polypeptides" "Chimeric protein," "chimeric antigen," and DDpp are defined as chimeric proteins consisting of at least two polypeptides. and optionally, two polypeptides, e.g., produced by recombinant processes. The two polypeptides are operably linked into one continuous polypeptide by a linker. The peptides may be operably linked directly or indirectly.

[0133] The "DDpp fusion proteins" provided herein are intended to be fusion proteins that bind to a target (e.g., BCMA ( SEQ ID NO: 7), CD123 (SEQ ID NO: 8), CS1 (SEQ ID NO: 965), HER2 (SEQ ID NO: No. 967), AFP (SEQ ID NO: 9), AFP p26 (SEQ ID NO: 10), or a fragment thereof The DDpp comprises at least one DDpp disclosed herein that specifically binds to the DDpp fragment. In one embodiment, the DDpp fusion protein comprises one DDpp.

[0134] In some embodiments, the DDpp fusion protein comprises one or more target-binding DDpps. pp and p26 proteins (e.g., SEQ ID NOs: 10, 968, 969, 970, 971 , 972, 973, or 974). Fusion proteins containing such p26 sequences have surprisingly been shown herein to produce long serum half-length peptides. In some embodiments, the soluble DDpp fusion protein Quality is at least 1 hour, at least 2 hours, at least 4 hours, at least 8 hours, At least 16 hours, at least 32 hours, at least 64 hours, or more In some embodiments, the soluble fusion protein has a plasma half-life in vivo. at least 1 hour, at least 2 hours, at least 4 hours, at least 8 hours, at least 16 hours, at least 32 hours, at least 64 hours, or longer than 65 hours hours, or 1-10 hours, 2-10 hours, 4-10 hours, 6-10 hours, or 6- In some embodiments, the soluble DDpp fusion has an in vivo plasma half-life of 9 hours. The protein may be incubated for at least 1 hour, at least 2 hours, at least 4 hours, or for at least 1 hour in humans. at least 8 hours, at least 16 hours, at least 32 hours, at least 64 hours, or is longer than 65 hours, or 1-10 hours, 2-10 hours, 4-10 hours, 6-10 hours hours, or 6-9 hours in vivo plasma half-life.

[0135] In some embodiments, the present disclosure provides a method for the production of a p26 protein (e.g., SEQ ID NO: 10, 96 8, 969, 970, 971, 972, 973, or 974) Methods for Altering the In Vivo Half-Life (e.g., in Mice or Humans) of Soluble Fusion Proteins In some embodiments, the soluble p26 fusion protein comprises one or more In some embodiments, the p26 soluble fusion protein comprises a target-binding DDpp. The reduced activity is due to substitution of one or more amino acid residues normally found in the human p26 protein. or by deleting, or by adding, one not normally found in the human p26 protein. Alternatively, it may be lengthened or shortened by inserting multiple amino acid residues. In this study, the p26 sequence of the soluble fusion protein was determined to increase the in vivo half-life of the soluble fusion protein. To lengthen or shorten, 1, 2, 3, 5, 5, 10, or 1-20, 1-10, 3 ~10, or 3-5 amino acid substitutions (conservative and / or non-conservative substitutions), deletions, and / or by making an insertion. In a particular embodiment, the p26 sequence is The amino acid residue corresponding to glutamine (Gln, Q) at position 217 of No. 10 is another amino acid. In a further embodiment, the substitution is Gln217Pro. In this embodiment, the p26 sequence of the soluble fusion protein is To extend or shorten the half-life, select 1-150, 1-100, 1-50, 1-25, Alternatively, the amino acid sequence is modified by deleting 1 to 10 amino acid residues. The p26 sequence of the soluble fusion protein regulates the interaction of FeRn with the soluble fusion protein. To increase or decrease, 1, 2, 3, 5, 5, 10, or 1-20, 1-10, 3-10, or 3-5 amino acid substitutions (conservative and / or non-conservative substitutions), deletions , and / or by making insertions.

[0136] Multimeric DDpp fusion protein In one embodiment, the DDpp fusion protein comprises two or more DDpps, The DDpps have the same or different specificities. The protein is a DDpp fusion protein that targets multiple targets and / or repeat epitopes on the same target. The same or different DDs can be used to bind to the same or different epitopes. In some embodiments, the DDpp fusion protein comprises at least In some embodiments, the DDpp may comprise 2, 3, 4, or 5 or more than 5 DDpp. The DDpp fusion protein may contain 1-3, 1-4, 1-5, or more than 5 different D In some embodiments, the DDpp fusion protein comprises at least two, three , 4, 5, or more than 5 different DDpps. The protein may be a monomeric DDpp (i.e., containing one DDpp) or a multimeric DDpp p (i.e., two or more DD In some embodiments, the use of multimeric DDpp can be enhanced. In a further embodiment, the multimeric DDpp results in synergistic (e.g., synergistic) target binding. Targeting of two or more targets using a single DDpp construct (e.g., bispecific, trispecific, etc.) The binding of two or more identical DDpps offers distinct advantages over monovalent compositions. Multivalency resulting in increased avidity (e.g., increased avidity, target clustering, and receptor activation) The binding of two or more different DDpps allows for the binding of different antigens to two or more target antigens. This results in multivalent and multispecific molecules with the potential to bind multiple or simultaneously.

[0137] Multimeric DDpp fusion proteins are DDpp homomultimers (i.e., optionally with a linker). (e.g., homodimers, homodimers, homodimers, etc.) homotrimers, homotetramers, etc.) or DDpp heteromultimers (i.e., at least two Multimers may be present (containing two or more DDpps in which different DDpp proteins are present). The number of monomers DDpp included in the composition may vary depending on the embodiment, and may be at least This may be determined, at least in part, by the expression system in which DDpp is produced. In some embodiments, the fusion protein comprises about 5 to about 10 DDpp subunits, about 10 to about 20 DDpp subunits, Approximately 15 subunits, approximately 15 to approximately 20 subunits, approximately 20 to approximately 25 subunits or a polynucleotide of about 25 to about 30 subunits (including intermediate numbers and endpoints of the numbers listed) Furthermore, the multiple tandem components of the DDpp fusion protein may be the same or In some DDpp fusions, the DDpp may be a monomer. as a dimer or a homomultimer or heteromer, e.g., a homodimer or heterodimer , homotrimers or heterotrimers, homotetramers or heterotetramers.

[0138] DDpp fusion proteins can be "monospecific" or "multispecific." specificity" (e.g., bispecific, trispecific, or even multispecific) DDpp fusion A conjugated protein is a protein that is composed of one or more different molecules (e.g., proteins, solid support structures, etc.). It recognizes and binds to two or more different epitopes present on the surface of a target antigen (e.g., a nucleotide sequence).

[0139] In some embodiments, two or more DDs are fused together as a multivalent DDpp. The DDs of a multivalent DDpp may be the same or different. and optionally linked by one or more linkers. DDpp homodimers (i.e., DDpp containing two identical DDs), DDpp Homodimers (i.e., DDpp containing three or more identical DDs), DDpp heterodimers (i.e., DDpp containing two different DDs), and DDpp heteromultimers (i.e., (i.e., a DDpp containing three or more DDs, where at least two DDs are different).

[0140] In some embodiments, two or more DDs are linked or combined by a multimerization domain. The DDs of the multivalent DD complex are linked by chemical linkage to form a multivalent DD complex. or may be different. Thus, the present disclosure includes any DD described herein. DD homodimer complexes, optionally linked by one or more linkers complexes (i.e., DD complexes containing two identical DDs), DD homomultimeric complexes (i.e. , DD complexes containing three or more identical DDs), DD heterodimeric complexes (i.e., two DD complexes (i.e., containing three or more different DDs), and DD heteromultimeric complexes (i.e., A DD complex containing DDs, where at least two DDs are different).

[0141] In one embodiment, the multispecific DDpp fusion protein is a single target (e.g., B CMA, CD123, CS1, HER2, AFP, or AFP p26) Also contains at least two DDpp binding to two different epitopes. In this state, the DDpp fusion is bispecific and expressed on the surface of two different cell types. In one embodiment, the bispecific DDpp fusion protein specifically binds to two different targets. The protein specifically binds to a target on a cancer cell and a target on an immune effector cell. In this embodiment, the bispecific DDpp fusion protein binds to a target expressed on a cancer cell (e.g., B CMA, CS1, CD123, and CD19) and markers expressed on the surface of T lymphocytes In one embodiment, the bispecific DDpp fusion tag specifically binds to a target (e.g., CD3). The protein specifically binds to BCMA and CS1.

[0142] In a further embodiment, the multispecific DDpp fusion protein comprises one enzyme on the target. At least one DDpp that specifically binds to a target antigen, and a different DDpp that specifically binds to the same target antigen. At least one domain or sequence (e.g., For example, antibody fragments such as scFv (antibody fragments or domains). In an embodiment, the multispecific DDpp fusion protein specifically targets an epitope on the target. At least one DDpp that binds to a target antigen and specifically binds to an epitope on a different target antigen. at least one domain or sequence that confers binding function, e.g., an antibody fragment or domains (e.g., scFv). In one embodiment, the multispecific DDpp fusion The protein comprises at least one DDpp that specifically binds to an epitope on a target protein; and at least one domain that specifically binds to an epitope on a different target on the same cell. In another embodiment, the DDpp fusion protein comprises at least one and at least one other DDpp or domain sequence that confers function; For example, they include antibody fragments or domains that specifically bind to solid supports.

[0143] In a further embodiment, the multimeric DDpp fusion comprising two or more DDpps further comprises , fused to other heterologous proteins (or subdomains thereof), This confers multivalency and multispecificity to the fusion partner. Examples include, but are not limited to, antibodies, antibody subdomains (e.g., scFv or Fc domains), ), serum albumin, serum albumin subdomains, cell surface receptors, T cell receptors ( TCR) α chain, T cell receptor β chain, cell surface receptor subdomains, peptides, tag (e.g., FLAG or myc), fibronectin type III repeat, z-domain The number and location of DDpp and their respective The position within each fusion protein can vary. For example, DDpp is located at one of the fusion partners. The heterologous subunits can be located at one or all termini and / or at the heterologous subunits within the DDpp fusion partner. They may be interspersed within the knit.

[0144] In a further embodiment, the DDpp fusion protein comprises DDpp and an additional domain. In some embodiments, the DDpp fusion protein comprises a polypeptide sequence comprising Dpp and antibodies, antibody fragments (e.g., antigen-binding domains or portions thereof, e.g., scFv), effector domain or part thereof, FcRn-binding domain or part thereof and Fc or a portion thereof), serum proteins (e.g., albumin or a portion thereof) , cytokines, growth factors, hormones, imaging agents, labeling agents, and peptide tags. In some embodiments, the DDpp fusion protein comprises a member selected from the group consisting of immunoglobulins, The Fc domain of a human Fc domain (e.g., a human Fc domain) or a portion thereof. In the present invention, the Fc domain is a variable human Fc domain.

[0145] In some embodiments, the DDpp is fused to a heterologous polypeptide. In some embodiments, the heterologous polypeptide comprises a full-length antibody or antibody fragment thereof. In some embodiments, DD is the amino terminus of the full-length antibody heavy chain; the amino terminus of the full-length antibody light chain. fused to the carboxyl terminus of a full-length antibody heavy chain; or fused to the carboxyl terminus of a full-length antibody light chain In another embodiment, the DD is fused to an antibody fragment that is an Fc. In some embodiments, the heterologous polypeptide comprises: (i) a transmembrane domain; (ii) a membrane-binding domain; (iii) human serum albumin or a fragment thereof; (iv) AFP or a fragment thereof (v) AFP p26 or a fragment thereof; (vi) the extracellular domain of the receptor (vii) an intracellular receptor (e.g., a nuclear protein) ) or a fragment thereof. In some embodiments, DDpp is an extracellular domain of a cell surface receptor, or an extracellular This includes heterologous polypeptides comprising fragments of the domain.

[0146] In some embodiments, the DDpp of the DDpp fusion protein is a larger, multi- Incorporated into domain molecular complexes (e.g., monomeric or multimeric DDpp fusion proteins) ), thereby imparting the functional properties of the incorporated DDpp to the resulting fusion protein. In some embodiments, the DDpp fusion protein comprises DDpp and an antibody, antibody fragment, serum protein (e.g., human serum albumin) or serum protein Protein fragment, or cell surface receptor, alpha chain of T cell receptor (TCR), T cell beta chain of a cell receptor, cytokine, growth factor, hormone, or enzyme, or fragment thereof Multidomain and / or multifunctional complexes, including polypeptide sequences derived from the The incorporation of DD into another polypeptide can be accomplished using techniques known in the art. recombinant fusion to another polypeptide (or other desired polypeptide), conjugation to another chemical moiety, and This can be routinely achieved by covalent chemical linkage to a desired chemical compound. The p-fusion protein may contain other optional components, such as a linker and other components described herein. can further include:

[0147] DDpp fusion protein as CAR In addition to incorporating DD into soluble multidomain proteins, the present invention also provides a method for incorporating DD into membrane-bound fusion proteins. at least one DDpp designed to confer binding specificity to the conjugated protein. This provides a means to generate cell-bound DDpp that can be expressed by any cell type. It can be expressed by

[0148] In one embodiment, the DDpp receptor fusion protein is a chimeric antigen receptor (CAR), or or DDpp-CAR, which contains an extracellular targeting domain and a transmembrane domain. In another embodiment, the DDpp-CAR comprises an extracellular targeting domain, a transmembrane domain, and and a cytoplasmic domain, the cytoplasmic domain containing the signal transduction domain. In certain embodiments, the DDpp-CAR extracellular domain comprises one or more DDpp Each DDpp constitutes a specific binding domain with the same or different specificity. In some embodiments, the target-specific domain may be any of the target-specific domains disclosed herein, including, but not limited to, Examples include BCMA, CD123, CS1, HER2, AFP, and AFP p26 In one embodiment, the method is directed to one or more cancer or tumor antigens, such as DDpp -The intracellular domain (e.g., cytoplasmic domain) of CAR is identical to the intracellular domain of the CD3 zeta chain In another embodiment, the intracellular signaling domain of DDpp comprises a CD3 kinase. In a further embodiment, the DDpp-CAR comprises a portion of the intracellular domain of the DDpp-CAR chain. The intracellular domain of CD3 zeta chain and the costimulatory signaling region are The costimulatory signaling region comprises all or part of the intracellular domain of a costimulatory molecule. Refers to a part of DDpp-CAR. Costimulatory molecules are necessary for efficient responses of lymphocytes to antigens. These are cell surface molecules other than the essential antigen receptors or their ligands. Some of these molecules capable of conferring stimulatory properties to the AR are known in the art and are In addition, these intracellular signals can be incorporated into Dpp-CAR by conventional methods. Truncations or mutations to the receptor transduction and costimulatory domains are incorporated to enhance receptor signaling. In a preferred embodiment, the T cells are stably transduced with DDp In such an embodiment, the DDpp-C is genetically modified to express p-CAR. The cytoplasmic domain of AR binds the CD28 and / or 41BB signaling domains alone. or any other desired cytoplasm useful in connection with the disclosed embodiments. In one embodiment, the cytoplasmic domain of DDpp-CAR can be combined with The antibody can be designed to further include the signaling domain of the CD3 zeta chain. DDpp-CAR contains an extracellular targeting domain and a transmembrane domain that penetrates the cell membrane. an extracellular protein linker (such as found in T cells or NK cells) having and a cytoplasmic domain, and may optionally contain multiple signaling modules. In some embodiments, the DDpp-CAR may also include an epitope tag. In some embodiments, the cytoplasmic domain of DDpp-CAR binds to, but is not limited to, CD3 enzyme. Contains the 41BB and CD28 signaling modules and combinations thereof. obtain.

[0149] In a further embodiment, the present disclosure provides a chimeric antigen receptor (CAR), the CAR comprising: A DDpp comprising a transmembrane domain and an intracellular signaling domain as disclosed herein. In some embodiments, the intracellular signaling domain is a human CD3 zeta domain. , a 41BB domain, a CD28 domain, and / or any combination thereof. Depending on the embodiment, the costimulatory signaling region may be selected from the group consisting of, for example, CD27 , CD28, 41BB, OX40, CD30, CD40, PD1, lymphocyte function-associated antigen 1 (LFA1), CD2, CD7, LIGHT, NKG2C, B7H3, CD83 specific and any combination thereof. In some embodiments, the targeting domain of the CAR comprises an intracellular domain of the targeting molecule. Multiple binding domains (e.g., DD or one or more) containing additional target-binding polypeptides The targeting region contains a target binding domain (multiple DDs and scFvs). Also provided is an isolated nucleic acid encoding a CAR comprising the polypeptide.

[0150] The present disclosure also provides a cell comprising a nucleic acid sequence encoding a CAR, the CAR comprising at least Partially targeted (e.g., BCMA, CD123, CS1, HER2, AFP, and an antigen-binding domain consisting of the disclosed DDpp that binds to AFP p26; In some embodiments, the CAR comprises a tumor domain, a tumor suppressor domain, and a signaling domain. specifically bind to tumor antigens (thus functioning to deliver CAR-expressing cells to tumors) In some embodiments, the tumor antigen is associated with a hematological tumor. In some embodiments, the tumor antigen is BCMA. In some embodiments, the tumor antigen is CD123. In some embodiments, the tumor antigen is CS1. In further embodiments, the tumor antigen is In some embodiments, the tumor antigen is HER2. In some embodiments, both solid and hematological tumors are targeted. CAR-expressing cells can be T cells, natural killer (NK) cells, or other immune cells. In some embodiments, the CAR-expressing cells (T cells, NK cells, The polypeptide binds to its corresponding tumor cell, either a tumor cell or other cell type, It exhibits anti-tumor immunity.

[0151] Extracellular domain Depending on the desired antigen to be targeted, the DDpp-CAR can be specific for the antigen target of interest. For example, BCMA should be targeted. If the antigen of interest is a BCMA-binding DDpp, one or more BCMA-binding DDpps can be used in combination with the DDpp-CAR. In addition, DDpp-CARs can be constructed by incorporating two or more D Dpp can be included to confer multispecificity or multivalency to the DDpp-CAR. In some embodiments, the DDpp-CAR comprises a BCMA-binding DDpp. In some embodiments, the DDpp-CAR comprises a CS1-linked DDpp. DDpp-CAR contains a BCMA-binding DDpp and a CS1-binding DDpp.

[0152] DDp incorporated into the extracellular domain of a DDpp receptor (e.g., DDpp-CAR) The choice of p depends on the intrinsic properties of the cells to be targeted. For example, DDpp-CAR They can specifically bind to cell surface proteins, such as receptors, on the same or different cells. In this embodiment, the DDpp-CAR specifically binds to a soluble molecule such as an immunoglobulin. In another embodiment, the target of interest bound by DDpp-CAR is a virus, Associated with bacterial and parasitic infections, diseases and disorders of the immune system (e.g., autoimmune diseases) Includes targets.

[0153] In other embodiments, the DDpp-CAR binds to a cell surface marker on the target cell that is associated with cancer. In some embodiments, the DD pp-CAR can be used to express tumor antigens (e.g., TAAs or other antigens described herein or in the art). The present invention relates to a method for targeting and binding to a tumor antigen (e.g., a tumor antigen or other tumor antigens known in the art). Provided are methods for producing DDpp-CARs, human T cells and natural killer their use in generating chimeric cells such as T cells, and adoptive transfer of these chimeric T cells. Use in immunotherapy.

[0154] In the context provided herein, a "tumor antigen" refers to an antigen common to a particular hyperproliferative disorder, such as cancer. The tumor antigen specifically bound by the DDpp in the DDpp-CAR is the In one embodiment, the DDpp in the DDpp-CAR is a tumor-specific antibody. TSAs bind specifically to tumor-associated antigens (TSA) or tumor-associated antigens (TAA). TAA-associated antigens are unique to tumor cells and do not occur on other cells in the body. Instead, they are expressed on normal cells under conditions that do not induce a state of immune tolerance to the antigen. Expression of antigens on tumors can occur under conditions that allow the immune system to respond to the antigen. TAAs are expressed on normal cells during fetal development when the immune system is immature and unable to respond. It can be an antigen that is present in a very low level on normal cells, but is present in tumor cells. It may be an antigen that is expressed at a much higher level above.

[0155] In some embodiments, the DDpp in the antigen-binding portion of the DDpp-CAR is BCMA In some embodiments, the antibody specifically binds to CS1, HER2, or CD123. DDpp specifically binds to the BCMA protein having the amino acid sequence of SEQ ID NO:7. In a further embodiment, the DDpp specifically binds to BCMA and is selected from the group consisting of SEQ ID NOs: 11 to 14. 305, and 306. In this embodiment, DDpp binds to the CD123 protein having the amino acid sequence of SEQ ID NO:8. In a further embodiment, DDpp specifically binds to CD123 and The amino acid sequence is selected from the group consisting of sequences 307 to 739 and 740. In some embodiments, the DDpp comprises a CS having an amino acid sequence consisting of SEQ ID NO:965. In a further embodiment, DDpp specifically binds to CS1. Binds to an amino acid sequence selected from the group consisting of SEQ ID NOs: 896 to 909 and 910 In some embodiments, the DDpp specifically binds to CS1 and BCMA. In some embodiments, the DDpp has an amino acid sequence consisting of SEQ ID NO: 967. In a further embodiment, the DDpp specifically binds to the HER2 protein. and specifically binds to the antibody selected from the group consisting of SEQ ID NOs: 911 to 949, and 950. In a further embodiment, the antigen-binding portion of DDpp-CAR comprises the amino acid sequence M, BTLA, DR3, CD19, CD20, and CD22. Combine with.

[0156] In one embodiment, the DDpp in the DDpp-CAR is specific for a tumor antigen associated with a malignant tumor. In one embodiment, the DDpp of the DDpp-CAR specifically binds to a B-cell lymphoma-specific Idiotypic immunoglobulins; B cell differentiation such as CD19, CD20, and CD37 antigens; TSLPR and IL7R on myeloid cells, and heat shock on multiple myeloma cells The antibody binds to an antigen selected from the group consisting of the peptides of the polypeptides of the invention and the polypeptides of the invention.

[0157] In some embodiments, the DDpp in the antigen-binding portion of the DDpp-CAR is AFP, In some embodiments, the AFP specifically binds to p26, or a fragment thereof. DDpp is an AFP having an amino acid sequence consisting of SEQ ID NO: 9 or a fragment thereof. In some embodiments, DDpp specifically binds to a protein selected from SEQ ID NO: 10 and SEQ ID NO: 11. AFP specific to p26 protein having an amino acid sequence consisting of: In a further embodiment, DDpp binds to SEQ ID NOs: 741-874, and 88. In some embodiments, the amino acid sequence is selected from the group consisting of: 6 to 895. In a further embodiment, the antigen-binding portion of the DDpp-CAR further binds to a tumor antigen. The antigen-binding portion of DDpp-CAR binds to BCMA, CD123, CS1, HER2, and H Targets selected from VEM, BTLA, DR3, CD19, CD20, and CD22 Further combine.

[0158] Transmembrane domain As used herein, the term "transmembrane domain" (TMD) refers to the domain of DDpp-C. It refers to the region of a cell surface-expressed DDpp fusion protein, such as AR, that spans the cell membrane. In some embodiments, the transmembrane domain of DDpp-CAR is a transmembrane protein (e.g., transmembrane regions of a type I transmembrane protein, an artificial hydrophobic sequence, or a combination thereof Other transmembrane domains will be apparent to those skilled in the art and are included in the alternatives provided herein. It may be used in connection with the embodiment.

[0159] DDpp receptors (e.g., DDpp-CAR) are expressed in the extracellular domain of the DDpp receptor. As mentioned above, the extracellular domain and Fusion of the nucleotide and transmembrane domains can be achieved with or without a linker. In embodiments, a transmembrane domain that is naturally associated with one of the domains in DDpp-CAR. In a specific embodiment, the transmembrane domain in DDpp-CAR is a CD8 transmembrane domain. In some cases, the transmembrane domain of DDpp-CAR is a CD In some embodiments, the transmembrane domain comprises a nucleotide sequence similar to that of other surface membrane proteins. The polypeptides are selected or modified by amino acid substitutions to enhance or inhibit binding to proteins.

[0160] The transmembrane domain can be derived from natural or synthetic sources. If the source is natural, The domain can be derived from any membrane-bound or transmembrane protein. Transmembrane regions of particular use for the following groups: α, β, or ζ chains of the T cell receptor; CD28, C D3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154 The polypeptide may be derived from a member selected from the group consisting of (i.e., containing at least the transmembrane domain). Alternatively, the transmembrane domain can be synthetic, in which case the DDpp-CAR transmembrane domain is the primary In a further embodiment, the transmembrane domain comprises hydrophobic residues such as leucine and valine. The synthetic transmembrane domain is equipped with a triplet of phenylalanine, tryptophan and phenylalanine at each end. Contains valine.

[0161] As used herein, an "extracellular spacer domain" (ESD) refers to an antigen-specific It refers to the hydrophilic region between the targeting region and the transmembrane domain. In some embodiments, DDpp-CAR comprises an extracellular spacer domain. AR does not contain an extracellular spacer domain. The extracellular spacer domain contains a limited number of However, the Fc fragment of an antibody or its fragment or derivative, an antibody or its a fragment or derivative of a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an artificial spacer sequences, or combinations thereof. Additional examples include, but are not limited to, CD8a hinge, and may be as small as Gly3. Artificial spacers made from polypeptides or CHI of IgG (e.g., human IgG4) and a CH3 domain. In some embodiments, the extracellular spacer domain The fragments are (i) the hinge, CH2 and CH3 regions of IgG4, (ii) the hinge of IgG4, (iii) the hinge region and CH2 of IgG4, (iv) the hinge region of CD8a, and (v) (vi) the hinge region of IgG1, or (vi) any one or more of the hinge region and CH2 region of IgG1. Other extracellular spacer domains will be apparent to those skilled in the art and are included in the alternatives provided herein. It may be used in connection with alternative embodiments.

[0162] In some embodiments, short oligo- or polypeptides of about 1-100 amino acids in length A linker is used to link any of the domains of DDpp-CAR together. The glycine and serine (or The linker amino acid sequence can be composed of flexible residues such as α, β ... The composition minimizes the immunogenic potential of the DDpp-CAR or DDpp fusion protein. It can be ensured that two adjacent domains do not sterically interfere with each other. In some embodiments, a longer linker can be used if desired. , which is 2–10 amino acids long and contains the transmembrane domain and cytoplasmic signaling domain of DDpp-CAR. In a further embodiment, the linker preferably forms a bond between the ~15 amino acids in length, or 15-20 amino acids in length, or 20-30 amino acids in length, or 30-60 amino acids in length, or 60-100 amino acids in length (or as specified) In a further embodiment, the linker is a glycine-serine doublet. A further embodiment is the human T cell surface glycoprotein CD8 alpha chain. (For example, amino acid positions 138-182 of SwissProt accession number P01732 CD8 A further embodiment uses a fragment of the hinge region from the alpha chain. CD8 that has been further modified by amino acid substitutions to improve current function or immunogenicity A further embodiment uses a fragment of the hinge region. Further embodiments use fragments of the A fragment of the CD28 extracellular domain that has been further modified by amino acid substitution was used. do.

[0163] Intracellular domain As used herein, "intracellular signaling domain" (ISD) or "intracellular signaling domain" (ISD) refers to a The "protein domain" transmits effector function signals and allows the cell to carry out its specialized functions. This refers to the part of DDpp-CAR that directs the cytoplasmic domain of DDpp-CAR. In (i.e., the intracellular signaling domain), to express DDpp-CAR A gene involved in the activation of at least one normal effector function of the genetically modified immune cells. The term "effector function" refers to the differentiated function of a cell. Helper functions include, for example, cytolytic activity and helper activity including cytokine secretion. Thus, the term "intracellular signaling domain" refers to a domain that transmits an effector function signal. and the DDpp-CAR protein, which instructs the cell to carry out its differentiated functions. It usually refers to the entire intracellular signaling domain corresponding to the native receptor. Although it is possible to use the entire chain, in many cases it is not necessary to use the entire chain. To the extent that truncated portions of the transduction domain are available, they may be used to mediate effector function signals. Such truncated portions can be used in place of the complete chain, so long as they transmit the The signal transduction domain thus provides intracellular signal transduction sufficient to transmit an effector function signal. It is intended to include any truncated portion of the signaling domain. The intracellular signaling domain in DDpp-CAR binds to the T cell receptor (TCR) A co-receptor that functions in concert to initiate signal transduction after cytoplasmic sequences and antigen receptor binding. The present invention also includes the sequences of the receptors, or any derivatives or variants of these sequences that retain functional capabilities. Examples of domains that transmit effector function signals include, but are not limited to, T cell The ζ chain of the receptor complex or any of its homologs (e.g., the η chain, FcsRly and β chain, MB1 (Iga) chain, B29 (Ig) chain, etc.), human CD3 zeta chain, CD3 poly peptides (Δ, δ, and ε), syk family tyrosine kinases (Syk, ZAP7 0, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.), and Other molecules involved in T cell transduction include CD2, CD5, and CD28. do.

[0164] Signals generated by the TCR alone are insufficient for full T cell activation; It is known that T cell activation requires a co-stimulatory signal. Different types of cytoplasmic signaling sequences: TCR (primary cytoplasmic signaling sequences) The sequence that initiates antigen-dependent primary activation and the sequence that initiates antigen-independent secondary or costimulatory signals. (secondary cytoplasmic signaling sequences) that function to provide It can be said that.

[0165] The primary cytoplasmic signaling sequences are designed to promote and mediate primary activation of the TCR complex. The primary cytoplasmic signaling sequences that act to suppress the Signaling motifs may include immunoreceptor tyrosine-based activation motifs (ITAMs). ) is known as

[0166] In the embodiments provided, the primary cytoplasmic signaling sequence for a particular use is Examples of ITAMs include TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, and CD3ε. , CD22, CD79a, CD79b, and CD66d. The cytoplasmic signaling molecules in CAR bind to CD3 zeta-derived cytoplasmic signaling molecules. It is particularly preferred that the cascade comprises a column.

[0167] As used herein, a "costimulatory domain" (CSD) is a molecule that mediates the proliferation of memory cells, CAR or DDpp-CAR portion that enhances survival and / or development. p-CAR can contain one or more costimulatory domains. Each costimulatory domain For example, CD28, CD137 (41BB), CD134 (OX40), Dap10 , CD27, CD2, CD5, ICAM-1, LFA-1(CD11a / CD18), L ck, TNFR-I, TNFR-II, Fas, CD30, and CD40, or Any one of the members of the TNFR superfamily selected from the following combinations: It contains one or more costimulatory domains. It may contain other costimulatory domains (e.g., derived from other proteins). The following statements (or equivalents) may be used in connection with alternative embodiments that will be apparent to those skilled in the art and are encompassed by this disclosure. It can be used.

[0168] In a preferred embodiment, the cytoplasmic domain of DDpp-CAR is a CD3 zeta signaling Any other desirable cytoplasmic transduction domain useful alone or in conjunction with DDpp-CAR. For example, the cytoplasmic domain of DDpp-CAR binds to the CD3 enzyme. The co-stimulatory signaling region may comprise a co-stimulatory signaling region. The term refers to the part of the CAR that contains the intracellular domain of the co-stimulatory molecule. These are cell surface molecules other than antigen receptors or their ligands that are required for an efficient response to antigens. Examples of such molecules include CD27, CD28, 41BB (CD 137), OX40, CD30, CD40, PD1, ICOS, lymphocyte function-associated antigen 1 (LFA1), CD2 , CD7, LIGHT, NKG2C, B7H3, TIM1, and LAG3 .

[0169] A polypeptide linker can be placed between adjacent elements of the DDpp-CAR. For example, The linker may be between adjacent DDpps, between a DDpp and a transmembrane domain, or between the transmembrane domains. It may be located between the DDpp domain and the cytoplasmic domain or between adjacent cytoplasmic domains. - The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR are randomly spaced from one another. Optionally, a short linker, preferably 2 to 10 amino acids, may be used. A linker of amino acid length can form the bond. A glycine-serine doublet is particularly preferred. Provide a linker.

[0170] In a further embodiment, the DDpp fusion protein is a DD disclosed herein (e.g., A target binding domain (DD) containing the amino acid sequence of SEQ ID NO: 11 to 949 or 950. In some embodiments, the DD is a chimeric antigen receptor (CAR) comprising a BCM. A, and an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 to 305, and 306. In some embodiments, the DD binds to CD123 and comprises a sequence of SEQ ID NOs: 307-7 39, and 740. In the present invention, DD binds to CS1 and is selected from the group consisting of SEQ ID NOs: 896 to 909, and 910. In some embodiments, the DD comprises an amino acid sequence selected from the group consisting of: It comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 911-949, and 950. In some embodiments, the DD binds to AFP and is selected from the group consisting of SEQ ID NOs: 741-874, and 8 In some embodiments, the amino acid sequence is selected from the group consisting of: 86 to 895. DD binds to AFP p26 and is selected from SEQ ID NOs: 741-874 and 886-895 In some embodiments, the CAR comprises an amino acid sequence selected from the group consisting of: It contains a binding domain, a transmembrane domain, and an intracellular signaling domain. In embodiments, the CAR transmembrane domain comprises a 41BB or CD28 transmembrane domain. In some embodiments, the CAR is a human T cell receptor alpha, beta, or zeta chain domain; human 41BB domain; human CD28 domain; and any combination thereof In some embodiments, the intracellular signaling domain is selected from the group consisting of: In morphology, the CAR intracellular signaling domains are CD27, CD28, 41BB, and OX 40, CD30, CD40, PD1, lymphocyte function-associated antigen 1 (LFA1), CD2, C Ligands that specifically bind to D7, LIGHT, NKG2C, B7-H3, and CD83, and and any combination thereof. In some embodiments, the CAR comprises a DD target binding domain that is the same as or different from the DD target binding domain. In some embodiments, the target binding domain further comprises a second target binding domain having a target comprising CA R comprises a first target binding domain that binds to CS1 and a second target binding domain that binds to BCMA. In some embodiments, the CAR is expressed in an immune cell. In this embodiment, the immune effector cells are T cells (CAR-T cells) or natural killer cells. In some embodiments, the CAR is a human NK cell. In some embodiments, the CAR is expressed on the surface of the cancer cell. 2. Specific binding to a target (e.g., BCMA, CS1, or CD123) , 3, 4, 5, or more than 5 DD and / or other binding domains (e.g. In a further embodiment, the CAR comprises a second Fv expressed on the surface of the cancer cell. Two, three, four, five, or more than five DDs or In a further embodiment, the administered The CAR expressed by a second, different cancer cell or vascular endothelial cell expresses a second, different target. Two, three, four, five, or more than five DDs or other binding sites that specifically bind to a target It further comprises a binding domain (e.g., scFv).

[0171] Additional DDpp fusion proteins In some embodiments, DDpp is a fragment of 5-500 amino acids of the extracellular domain of a cell surface receptor. , 5~400, 5~300, 5~200, 5~100, 5~50, 10~500, 10~ 400, 10-300, 10-200, 10-100, or 10-50 amino acids In some embodiments, the heterologous polypeptide comprises a fragment comprising: is the extracellular domain of BCMA (SEQ ID NO: 7) or CD123 (SEQ ID NO: 8), or In some embodiments, the heterologous polypeptide comprises a fragment of the extracellular domain. DDpp is BCMA (SEQ ID NO: 7) or CD123 (SEQ ID NO: 8) or CS1 (SEQ ID NO: 965), or a heterologous fragment of the extracellular domain In some embodiments, DDpp comprises a polypeptide selected from the group consisting of CD19, CD20, CD 22, HVEM, BTLA, DR3, CD37; TSLPR, IL7R, and gp96 an extracellular domain or a fragment of an extracellular domain of a receptor selected from the group consisting of and a heterologous polypeptide comprising:

[0172] In some embodiments, the protein is a serum protein or an antigen of a serum protein. Heterologous polypeptides, including fragments (e.g., AFP, and AFP p26) In some embodiments, DDpp comprises 5-500 amino acids of the extracellular domain of a cell surface receptor. , 5~400, 5~300, 5~200, 5~100, 5~50, 10~500, 10~ 400, 10-300, 10-200, 10-100, or 10-50 serum proteins In some embodiments, the heterologous polypeptides include fragments of the protein consisting of amino acids. In this state, the protein may be an intracellular protein (e.g., a nuclear protein) or an intracellular protein. In some embodiments, DDpp comprises a heterologous polypeptide comprising an antigenic portion of a protein. , 5-500, 5-400, 5-300, and 5-200 of the extracellular domain of cell surface receptors , 5~100, 5~50, 10~500, 10~400, 10~300, 10~200, Fragments of 10-100 or 10-50 amino acids of intracellular proteins In some embodiments, the DDpp comprises a heterologous polypeptide comprising SEQ ID NO: 10, Heterologous polypeptides having the sequences 968, 969, 970, 971, 972, 973, or 974 Contains lipids.

[0173] In some embodiments, the DDpp fusion protein is selected from the group consisting of BCMA, CD123, CS1, specifically binds to HER2, AFP, and / or AFP p26 and Further binding to additional targets. Targets specifically bound by DDpp fusion proteins. The target can be any molecule to which it is desirable for DDpp to bind. For example, The targets specifically bound by the fusion protein are BCMA, CD123, CS1, HE R2, AFP, and / or AFP p26, and further, the method for manufacturing, prescribing, treating, diagnosing, Additional targets of either diagnostic or prognostic relevance or value may be included. Although examples are provided herein, they are intended to be illustrative and not limiting. Additional targets bound by the DDpp fusion protein include: It may be naturally occurring or synthetic. Additional targets may include extracellular or intracellular components, possibly Soluble factors (e.g., enzymes, hormones, cytokines, growth factors, toxins, venoms, contaminants, etc.) It may be a protein (e.g., a protein of interest), or a transmembrane protein (e.g., a cell surface receptor). In this embodiment, the target protein bound by the DDpp fusion protein is a human protein. In one embodiment, the DDpp (e.g., DDpp fusion protein) is a human protein. Targeted and its monkeys (e.g., cynomolgus monkeys), mice, rabbits, hamsters and / or or binds to the rabbit orthologue.

[0174] In one embodiment, the DDpp fusion protein is selected from the group consisting of BCMA, CD123, CS1, HER2 , AFP, and / or AFP p26, and specifically binds to serum proteins. In one embodiment, the DDpp fusion protein is a fusion protein comprising a fusion protein selected from the group consisting of serum albumin (e.g., human serum albumin) and DDpp fusion protein. amine (HSA), thyroxine-binding protein, transferrin, fibrinogen, and a serum protein selected from immunoglobulins (e.g., IgG, IgE, and IgM). Without being bound by theory, it is believed that the carrier protein of DDpp Conjugation to the protein confers improved pharmacodynamic properties to DDpp (or its fusions). This property is believed to include, but is not limited to, DDpp lacking a protein binding sequence. Compared to fusion proteins, improved tumor targeting, tumor penetration, intratumoral spread, and enhanced (See, e.g., WO 01 / 45746. the contents of which are incorporated herein by reference in their entirety).

[0175] Antibody-based DDpp fusion proteins In some embodiments, the DDpp fusion protein is a full-length antibody or a flag-binding fragment of an antibody. In some embodiments, the DDpp fusion protein comprises a cleavage domain or subdomain. , including full-length IgG antibodies (e.g., IgG1, IgG2, IgG2, or IgG4) In a further embodiment, the DDpp fusion protein is a full-length DDpp fusion protein that specifically binds to a cancer antigen. In a further embodiment, the DDpp comprises a commercially approved therapeutic antibody (e.g. , rituximab, ofatumumab, ocrelizumab, veltuzumab, MEDI-551, Epratuzumab, belimumab, tabalumab, AMG-557, MEDI-570, and In another embodiment, the DDpp is an Fc fusion protein. In some embodiments, the Fc protein comprises a variable human Fc domain.

[0176] In some embodiments, the DDpp fusion protein is a full-length antibody or a flag-binding fragment of an antibody. domains or subdomains (e.g., IgG1 antibodies, IgG3 antibodies, antibody variable regions, CD R3, scFv, Fc, FcRn-binding subdomains, and other antibody subdomains) The DDpp proteins may be linked to each other and / or to one or more antibody termini, anti- operatively linked to a DDpp fusion It can form proteins.

[0177] The antibody component of the DDpp fusion protein may be any suitable full-length immunoglobulin or antibody. fragments (e.g., antigen-binding domains and / or effector domains) or In one embodiment, the DDpp-antibody fusion protein may be a conventional antibody. The structural and functional properties of the monoclonal antibody are retained. In this form, the DDpp-antibody fusion protein retains epitope binding properties but advantageously also incorporate one or more additional target binding specificities via DDpp fusions. Antibodies that can be used in pp fusions include, but are not limited to, monoclonal, multispecific, human The immunoglobulins provided herein include humanized, humanized, primatized, and chimeric antibodies. A phospho or antibody molecule is any type of immunoglobulin molecule (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, Ig IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclasses. In some embodiments, the antibody is an Fc-optimized antibody. The antibody may be derived from any species, including birds and mammals. It may be derived from or derived from animal sources, or may be artificially produced. The antibody component of the Dpp-antibody fusion protein may be naturally derived or recombinantly engineered (e.g., In certain embodiments, the method may be the result of various techniques (display, xeno-mouse, and synthetic technologies). The antibody component of the antibody-DDpp fusion has an extended half-life and inhibits antibody-dependent cellular cytotoxicity (ADCC). ), and / or enhance or reduce complement-dependent cytotoxicity (CDC) activity. In one embodiment, the antibody is human, murine, donkey, rabbit, goat, guinea pig, camel, lamb, or any other mammal. In certain embodiments, the antibody is human.

[0178] The constant region is generally understood to mediate several effector functions, for example: The binding of the C1 component of complement to antibodies activates the complement system. Complement activation is important in the regulation of immune responses and the lysis of cellular pathogens. It is also involved in hypersensitivity. Furthermore, antibodies bind to cells via the Fc region, and The Fc receptor site of IgG binds to the Fc receptor (FcR) on the cell. Various classes, including IgE (η receptors), IgA (α receptors), and IgM (μ receptors) There are many Fc receptors specific for each antibody. Binding of antibodies to Fc receptors on the cell surface However, phagocytosis and destruction of antibody-coated particles, excretion of immune complexes, and antibody-coated particles by killer cells Lysis of target cells (called antibody-dependent cellular cytotoxicity (ADCC)), release of inflammatory mediators, It mediates many important and diverse biological responses, including regulation of fetal development, placental transfer, and immunoglobulin production. Induce.

[0179] In certain embodiments, the DDpp-Fc fusion protein has an altered effector function. This in turn affects the biological profile of the administered DDpp-Fc fusion protein. For example, deletion or inactivation of constant region subdomains (point mutations or In other cases, the use of a modified antibody (by other means) can reduce Fc receptor binding of the circulating modified antibody. The common region modifications inhibit complement fixation and therefore shorten the serum half-life and non-specific activity of the conjugated cytotoxin. Further modifications of the constant region may be used to improve antigen specificity or antibody flexibility. Disulfide bonds or oligonucleotides that increase affinity for the nucleotides, allowing for enhanced localization. Similarly, modifications to the constant region according to the present disclosure may be made without departing from the spirit or scope of the invention. This can be readily achieved using biochemical or molecular engineering techniques.

[0180] In some embodiments, the DDpp-Fc fusion protein comprises one or more For example, in some embodiments, the DDpp-Fc fusion protein The quality of the antibody is determined by its antibody-dependent cellular cytotoxicity (ADCC) activity and / or complement-dependent cytotoxicity (CDC) activity. In certain embodiments, the DDpp-Fc fusion protein does not have Fc receptor agonist activity. and / or does not bind to complement factors. In certain embodiments, the DDpp-Fc fusion protein The protein does not have effector function. ADCC and / or CDC activity and Fc receptor activity. Examples of genetic modifications of the Fc sequence that reduce or eliminate receptor and / or complement factor binding are The assays and procedures for testing the It is known in the art.

[0181] In some embodiments, the DDpp-Fc fusion protein directly binds the CH3 domain. The constructs are genetically engineered to fuse to the hinge region of each engineered antibody. a peptide spacer between the hinge region and the modified CH2 and / or CH3 domains For example, the CH2 domain is deleted and the remaining CH3 domain (altered or unmodified) is attached to the hinge region using a 5-20 amino acid spacer. Such spacers can be used, for example, to control the length of the constant domains. The joint elements remain free and accessible, or the hinge parts remain flexible. Amino acid spacers are added to ensure that the immunization It may be demonstrated that the construct is virulent and induces an undesirable immune response against the construct. In certain embodiments, any spacer added to the construct is a modified DDpp-Fc fusion. To maintain the desired biochemical properties of the protein, it must be relatively non-immunogenic or can be completely removed.

[0182] In a further embodiment, the DDpp-Fc fusion protein comprises a minority of the amino acid sequence in the constant region. or by partial deletion or substitution of a single amino acid. Mutation of a single amino acid in a selected region of the polypeptide may be sufficient to substantially reduce Fc binding. Similarly, one or more proteins that control effector functions (e.g., complement C1Q binding) The constant region domain may be deleted completely or partially. The deletion is believed to improve select properties (e.g., serum half-life) of the DDpp-Fc fusion protein. At the same time, other desired functions associated with the corresponding constant region domain can remain. In some embodiments, the constant region of the DDpp-Fc fusion protein is modified by one or more amino acid mutations or substitutions that improve the This inhibits the activity provided by conserved binding sites (e.g., Fc binding). It is possible to obtain modified DDpp-Fc fusion proteins and their immunogenicity. The present disclosure also provides a method for the preparation of a cytotoxin, a label, or a carbohydrate moiety, which can substantially maintain the activity profile of the cytotoxin. Increased or decreased effector function or binding site for one or more of the Addition of one or more amino acids to the constant region to improve a desired property, such as providing In such embodiments, the DDpp-Fc fusion protein comprises a selected constant region. It may be desirable to insert or repeat a particular sequence from a regional domain.

[0183] In some embodiments, the DDpp is an antibody fragment or subdomain (e.g., , scFv, diabody, EP 404,097; WO 93 / 11116 1; 14 / 028776; and Holliger et al., PNAS 90:6444-6448 (1993), the contents of each of which are incorporated herein by reference. The antibody fragment or subunit is operably linked to the antibody fragment or subunit. The antibody domain can be any fragment or domain of an antibody. See Nos. 04 / 058820, 99 / 42077 and 05 / 017148. No. 6,239,999, the contents of each of which are incorporated herein by reference in their entirety. For example, a DDpp fusion protein may be used to attach one or more effector functions to DDpp. The DDpp fusion protein is then conferred with the ability to bind to one or more Fc receptors. antibody effector domain or antibody effector domain derivative to be attached to the protein In some embodiments, the DDpp-antibody fusion protein can be an antibody or In a further embodiment, the DDpp-antibody comprises an antigen-binding fragment thereof. The fusion protein utilizes the effector functions provided by the CH2 and CH3 domains. an immunoglobulin comprising one or more CH2 and / or CH3 domains of an antibody having The DDp comprises an effector domain that provides effector function and is encompassed by the present invention. Other sequences in the p fusion will be apparent to those skilled in the art and will be selected by conventional methods. DDpp fusion proteins encompassed herein based on desired effector function. It can be designed as follows.

[0184] In one embodiment, the DDpp fusion is an anti- In a further embodiment, the antibody or antibody fragment is a disease-associated antibody. In one embodiment, the DDpp fusion protein specifically binds to a cancer antigen. In another embodiment, the DDpp fusion protein comprises an antibody or antibody fragment that , pathogens (e.g., bacterial cells (tuberculosis, smallpox, anthrax)), viruses (e.g., HIV) , parasites (e.g., malaria, leishmaniasis), fungal infections, mold, mycoplasma In another embodiment, the antibody or antibody fragment specifically binds to a prion antigen. The DDpp fusion protein is expressed in pathogens (e.g., bacterial cells (tuberculosis, smallpox, anthrax)) , viruses (e.g., HIV), parasites (e.g., malaria, leishmaniasis), Antibodies or antibodies that specifically bind to fungal, mold, mycoplasma, or prion antigens In another embodiment, the DDpp fusion protein is a fusion protein for treating a disease of the immune system or The antibody or antibody fragment specifically binds to an antigen associated with the disorder.

[0185] In a preferred embodiment, a DDpp fusion protein comprising an antibody fragment or domain is The antibody fragment or fragments retain the activity of the parent antibody. DDpp fusion proteins containing the domain can induce complement-dependent cytotoxicity. In embodiments, DDpp fusion proteins comprising antibody fragments or domains are used to inhibit antibody-dependent It can induce antibody-dependent cell cytotoxicity (ADCC).

[0186] Thus, in some embodiments, the DDpp fusion protein is antibody fragments that confer biological or biochemical properties of immunoglobulins to the target antigen. In some embodiments, the antibody fragment comprises one or more DDpp Ability to non-covalently dimerize, tumor site, compared to deleted DDpp fusion proteins The antibody confers properties selected from the ability to localize to a specific site, and an extended serum half-life. In this embodiment, the DDpp fusion protein is a corresponding antibody in the absence of bound DDpp. In certain embodiments, the DDpp fusion protein is at least as stable as the DDpp fusion protein. The DDpp fusion protein is more stable than the corresponding antibody without the DDpp. Proteins can be measured using established methods, for example, ELISA techniques. In this embodiment, the DDpp fusion protein is expressed in whole blood (in vivo or ex vivo) at 37°C. at least about 10 hours, at least about 15 hours, at least about 20 hours, About 24 hours, at least about 25 hours, at least about 30 hours, or at least about 35 hours , at least about 40 hours, at least about 45 hours, at least about 48 hours, at least about 50 hours, at least about 55 hours, at least about 60 hours, at least about 65 hours, at least At least about 70 hours, at least about 72 hours, at least about 75 hours, at least about 80 hours for at least about 85 hours, at least about 90 hours, at least about 95 hours, or less The compound is stable for at least about 100 hours, including any time between the times listed. In one embodiment, the DDpp fusion comprises at least a portion of one or more constant region domains: compared to an immunoglobulin fragment having a corresponding unmodified immunoglobulin sequence. reduced or increased effector function, the ability to dimerize non-covalently, and localize to tumor sites. Desirable properties include enhanced ability to localize, reduced serum half-life, or extended serum half-life. corresponding to an immunoglobulin domain or fragment that has been modified to confer the properties of The immunoglobulin comprises an immunoglobulin effector domain or a domain that affects half-life. The modifications of these constant region domains can be amino acid substitutions, insertions, or deletions.

[0187] In one embodiment, the DDpp fusion protein is an antibody-dependent protein against the DDpp fusion protein. immunoglobulin effector domains or immunoglobulins that confer antibody-dependent cell cytotoxicity (ADCC) In a further embodiment, the amino acid sequence of a D Dpp fusion proteins are immunoglobulin effector proteins that have been modified to enhance ADCC. The sequence of the activator domain (see, e.g., Bruhns, Blood 113:3716- 3725(2009);Shields,J.Biol.Chem.276:6591- 6604(2001);Lazar,PNAS 103:4005-4010(2006 );Stavenhagen,Cancer Res.67:8882-8890(20 07);Horton, Cancer Res. 68:8049-8057(2008) ;Zalevsky,Blood 113:3735-3743(2009);Bruc kheimer, Neoplasia 11:509-517(2009); International Publication No. 06 / 020114;Strohl,Curr.Op.Biotechnol.20: 685-691 (2009); and WO 04 / 074455. (Each of these documents is incorporated herein by reference in its entirety.) ADCC The immunoglobulin fragment contained in the amino acid sequence of the DDpp fusion protein enhances Examples of genetic modifications include IgG1-S298A, E333A, K334A; IgG1-S 239D, I332E; IgG1-S239D, A330L, I332E; IgG1-P 247I, A339D or Q; IgG1-D280H, K290S and S298D or or V together or K290S alone; IgG1-F243L, R292P, Y300 L; IgG1-F243L, R292P, Y300L, P396L; and IgG1-F 243L, R292P, Y300L, V305I, P396L (number of residues in the Fc region) The rings are from the EU index of Kabat et al. Sequences of proteins of Immunological I Interest, 1991, 5th Edition, the contents of which are incorporated herein by reference in their entirety. an immunoglobulin effector having one or more modifications corresponding to It contains a domain sequence.

[0188] In another embodiment, the DDpp fusion protein is modified to reduce ADCC. The immunoglobulin effector domain sequence (e.g., Idusogie e t al.,J.Immunol.166:2571-2575(2001);Sazi nsky et al.,PNAS 105:20167-20172(2008);D avis et al.,J.Rheumatol.34:2204-2210(200 7);Bolt et al.,Eur.J.Immunol.23:403-411( 1993);Alegre et al.,Transplantation 57:1 537-1543(1994);Xu et al.,Cell Immunol.20 0:16-26(2000);Cole et al.,Transplantatio n 68:563-571(1999);Hutchins et al.,PNAS 92:11980-11984(1995);Reddy et al., J. Immu nol.164:1925-1933(2000);WO 97 / 11971;Country International Patent Publication No. 07 / 106585; U.S. Patent Application Publication No. 2007 / 0148167A1; McEarchern et al.,Blood 109:1185-1192(20 07);Strohl,Curr.Op.Biotechnol.20:685-691 (2009); and Kumagai et al., J. Clin. Pharmaco 1.47:1489-1497 (2007). (The contents of which are incorporated herein by reference in their entirety.) DDpp reduces ADCC Genetic modification of immunoglobulin fragment sequence contained in the amino acid sequence of the fusion protein Examples include IgG1-K326W, E333S; IgG2-E333S; IgG1-N2 97A;IgG1-L234A, L235A;IgG2-V234A, G237A;Ig G4-L235A, G237A, E318A; IgG4-S228P, L236E; Ig G2-118-260;IgG4- 261-447;IgG2-H268Q, V309 L, A330S, A331S; IgG1-C220S, C226S, C229S, p26 8S; IgG1-C226S, C229S, E233P, L234V, L235A; also IgG1-L234F, L235E, P331S (residue numbering is by Kabat The EU index is based on the data from Kabat et al., Sequences of proteins of Immunological Interest,19 91 5th Edition, the contents of which are incorporated herein by reference in their entirety. Immunoglobulin effector domain sequences having one or more corresponding modifications are included. can be.

[0189] In a further embodiment, the DDpp fusion protein is Immunoglobulin effector domains that confer antibody-dependent cellular phagocytosis (ADCP) or comprises the amino acid sequence of an immunoglobulin effector domain derivative. In this study, DDpp fusion proteins were modified to enhance antibody-dependent cellular phagocytosis (ADCP). The sequence of an immunoglobulin effector domain that has been altered (e.g., Shield s et al., J. Biol. Chem. 276:6591-6604 (2001) ;Lazar et al.,PNAS 103:4005-4010(2006);S tavenhagen et al.,Cancer Res.67:8882-889 0(2007);Richards et al.,Mol.Cancer Ther. 7:2517-2527(2008);Horton et al., Cancer R es.68:8049-8057(2008);Zalevsky et al.,Bl ood 113:3735-3743(2009);Bruckheimer et a l., Neoplasia 11:509-517(2009); International Publication No. 06 / 02 No. 0114;Strohl,Curr.Op.Biotechnol.20:685-6 91 (2009); and WO 04 / 074455. The contents of each of these references are incorporated herein by reference in their entirety. The immunoglobulin fragment contained in the amino acid sequence of the DDpp fusion protein Examples of genetic modifications include IgG1-S298A, E333A, K334A; IgG1-S2 39D, I332E; IgG1-S239D, A330L, I332E; IgG1-P2 47I, A339D or Q; IgG1-D280H, K290S and S298D or V together or K290S alone; IgG1-F243L, R292P, Y300L ;IgG1-F243L, R292P, Y300L, P396L;IgG1-F243L , R292P, Y300L, V305I, P396L; and IgG1-G236A, S 239D, I332E (residue numbering is from the EU index of Kabat et al. (Kabat et al., Sequences of proteins f Immunological Interest, 1991 5th Edition, The content of the present invention may include one or more modifications corresponding to the above-mentioned Immunoglobulin effector domain sequences having mutations are included.

[0190] In another embodiment, the DDpp fusion protein is modified to reduce ADCP. The sequences of immunoglobulin effector domains that are involved in the t al.,PNAS 105:20167-20172(2008);Davis e t al.,J.Rheumatol.34:2204-2210(2007);Bol t et al.,Eur.J.Immunol.23:403-411(1993); Alegre et al.,Transplantation 57:1537-15 43(1994);Xu et al.,Cell Immunol.200:16-2 0(2000);Cole et al.,Transplantation 68:5 63-571(1999);Hutchins et al.,PNAS 92:119 80-11984(1995);Reddy et al.,J.Immunol.16 4:1925-1933(2000); International Publication No. 97 / 11971; International Publication No. 07 / 106585; U.S. Patent Application Publication No. 2007 / 0148167A1; McEarc hern et al.,Blood 109:1185-1192(2007);St rohl,Curr.Op.Biotechnol.20:685-691(2009) ; and Kumagai et al., J. Clin. Pharmacol. 47:1 489-1497 (2007). The contents of each of these documents are available at (The entire contents of which are incorporated herein by reference.) In one example, a DDpp fusion protein may be One or more of the following modifications that reduce ADCC: IgG1-N297A; IgG1-L2 34A, L235A; IgG2-V234A, G237A; IgG4-L235A, G2 37A, E318A; IgG4-S228P, L236E; IgG2-EU sequence 118- 260; IgG4-EU sequence 261-447; IgG2-H268Q, V309L, A3 30S, A331S; IgG1-C220S, C226S, C229S, p268S; I gG1-C226S, C229S, E233P, L234V, L235A; and IgG 1-L234F, L235E, P331S (residue numbering follows the EU numbering scheme of Kabat et al. Index (Kabat et al., Sequences of p roteins of Immunological Interest,1991 No. 5th Edition, the contents of which are incorporated herein by reference in their entirety) It may contain a fragment or a domain.

[0191] In a further embodiment, the DDpp fusion protein is Immunoglobulin effector domains or immunoglobulins that confer complement-dependent cytotoxicity (CDC) In a further embodiment, the amino acid sequence of an immunoglobulin effector domain derivative is The DDpp fusion protein has been modified to enhance complement-dependent cytotoxicity (CDC). The immunoglobulin effector domain sequence (e.g., Idusogie e t al.,J.Immunol.166:2571-2575(2001);Stro hl, Curr. Op. Biotechnol. 20:685-691(2009); and Natsume et al., Cancer Res. 68: 3863-3872 (2008), each of which is incorporated herein by reference in its entirety. For example, DDpp fusion proteins may contain one or more fusion proteins that enhance CDC. The following modifications were made: IgG1-K326A, E333A; IgG1-K326W, E333S , IgG2-E333S (residue numbering follows the EU index of Kabat et al.) (Kabat et al., Sequences of proteins of Immunological Interest, 1991 5th Edition, this literature The contents of which are incorporated herein by reference in their entirety) can include a domain.

[0192] In a further embodiment, the DDpp fusion protein binds to FcγR Immunoglobulin effector domains or immunoglobulins that confer the ability to bind to IIb receptors In a further embodiment, the amino acid sequence comprises a derivative of a globulin effector domain. DDpp fusion proteins engineered to enhance inhibitory binding to the FcγRIIb receptor These sequences include sequences of immunoglobulin effector domains that have been reported (e.g., Chu et al. al., Mol. Immunol. 45:3926-3933 (2008) The DDpp fusion protein inhibits FcγRIIb receptor binding. An example of an immunoglobulin fragment genetic modification involving an amino acid sequence is IgG1-S2 67E, L328F.

[0193] In another embodiment, the DDpp fusion protein is modified to reduce CDC. and the sequences of immunoglobulin effector domains (see, for example, WO 97 / 119 71; U.S. Patent Application Publication No. 07 / 106585; U.S. Patent Application Publication No. 2007 / 0148167A1 No.;McEarchern et al.,Blood 109:1185-1192( 2007);Hayden-Ledbetter et al., Clin. Cance r 15:2739-2746(2009);Lazar et al.,PNAS 1 03:4005-4010(2006);Bruckheimer et al.,Ne oplasia 11:509-517(2009);Strohl,Curr.Op. Biotechnol.20:685-691(2009); and Sazinsky See, e.g., et al., PNAS 105:20167-20172 (2008). (The contents of each of these documents are incorporated herein by reference in their entirety). In one example, the DDpp fusion protein may contain one or more of the following modifications that reduce CDC: IgG1-S239D, A330L, I332E; IgG2-118-260; IgG4 -261-447;IgG2-H268Q, V309L, A330S, A331S;Ig G1-C226S, C229S, E233P, L234V, L235A; IgG1-L2 34F, L235E, P331S; and IgG1-C226S, p260S (residues Na The index numbering is that of the EU index by Kabat et al. .,Sequences of proteins of Immunological Interest, 1991, 5th Edition, the contents of which are incorporated herein by reference in their entirety. The present invention may include antibody fragments or domains containing the amino acid sequences incorporated herein.

[0194] The half-life of IgG is mediated by pH-dependent binding to its neonatal receptor FcRn. In certain embodiments, the DDpp fusion protein comprises a neonatal receptor for the DDpp fusion. Immunoglobulin effector domains or immunoglobulins that confer the ability to bind to FcRn In certain embodiments, the DDp comprises the amino acid sequence of a purine effector domain derivative. The fusion protein is an immunoglobulin that has been modified to enhance binding to FcRn. It contains a functional domain containing the sequence of the FcRn binding domain (e.g., Petkova et al.,Int.Immunol.18:1759-1769(2006);Da ll'Acqua et al., J. Immunol. 169:5171-5180( 2002);Oganesyan et al.,Mol.Immunol.46:17 50-1755(2009);Dall'Acqua et al.,J.Biol.C hem.281:23514-23524(2006),Hinton et al., J.Immunol.176:346-356(2006);Datta-Mannan et al.,Drug Metab.Dispos.35:86-94(2007) ;Datta-Mannan et al.,J.Biol.Chem.282:170 9-1717(2007); WO 06 / 130834; Strohl, Curr Op. Biotechnol. 20:685-691 (2009); and Yeung See et al., J. Immunol. 182:7663-7671 (2009). The contents of each of these documents are incorporated herein by reference in their entirety. (Can be).

[0195] In a further embodiment, the DDpp fusion protein exhibits selective activity against FcRn at pH 6.0. immunoglobulins that have been engineered to have selective affinity for IgG but no affinity at pH 7.4 It contains a functional domain containing the sequence of a purin effector domain, e.g., a DDpp fusion The protein may contain one or more of the following modifications that extend half-life: IgG1-M252Y, S 254T, T256E; IgG1-T250Q, M428L; IgG1-H433K, N 434Y; IgG1-N434A; and IgG1-T307A, E380A, N434 A (residue numbering is that of the EU index of Kabat et al. (Kabat et al., Sequences of proteins of Immunol (Ogical Interest, 1991 5th ed.), the contents of which are incorporated by reference. (the entirety of which is incorporated herein) can be done.

[0196] In another embodiment, the DDpp fusion protein is designed to reduce binding to FcRn. The sequences of immunoglobulin effector domains that have been modified into ova et al., Int. Immunol. 18:1759-1769 (2006 );Datta-Mannan et al.,Drug Metab.Dispos. 35:86-94(2007);Datta-Mannan et al., J.Bio l.Chem.282:1709-1717(2007);Strohl,Curr.O p.Biotechnol.20:685-691(2009); and Vaccar o et al.,Nat.Biotechnol.23:1283-1288(200 5), the contents of each of which are incorporated herein by reference in their entirety. For example, DDpp fusion proteins may contain one or more half-life-reducing conjugates. The following modifications were made: IgG1-M252Y, S254T, T256E; H433K, N434 F, 436H; IgG1-I253A; and IgG1-P257I, N434H and D376V, N434H (residue numbering is based on the EU index of Kabat et al.) (Kabat et al., Sequences of proteins of Immunological Interest, 1991 5th Edition, this literature The contents of which are incorporated herein by reference in their entirety) can include a domain.

[0197] In another embodiment, the DDpp fusion protein is 49, 252, 254, 255, 256, 258, 265, 267, 268, 269, 2 70, 272, 276, 278, 280, 283, 285, 286, 289, 290, 2 92, 293, 294, 295, 296, 298, 301, 303, 305, 307, 3 09, 312, 315, 320, 322, 324, 326, 327, 329, 330, 3 31, 332, 333, 334, 335, 337, 338, 340, 360, 373, 3 76, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 4 34, 435, 437, 438, and 439 (e.g., modified to contain at least one substitution in its sequence corresponding to a position in the Fc gamma sequence (e.g., Fc gamma). The amino acid sequence corresponding to the immunoglobulin effector domain (Fc domain) The numbering of residues in the region follows the EU numbering system of Kabat et al. et al., Sequences of proteins of Immunolo of the National Institute of Standards and Technology, 1991, 5th Edition; the contents of this publication are incorporated by reference. (which is incorporated herein in its entirety). In certain embodiments, the DDpp fusion protein The quality is such that at least one residue corresponding to position 434 is selected from A, W, Y, F and H. In another embodiment, the sequence of the immunoglobulin effector domain derivative is a sequence of residues that are selected from the group consisting of methyl, ... In the DDpp fusion protein, the following substitutions were made: S298A / E333A / K33 4A. In this form, the DDpp fusion protein is an immunoglobulin having a substitution corresponding to K322A. In another embodiment, the DDpp fusion protein comprises a derivative of the ATPase effector domain. Substitutes K246H, H268D, E283L, S324G, S239D and I33 Immunoglobulin effector flags with one or any combination of 2E In yet another embodiment, the DDpp fusion protein comprises a sequence of a D2 Immunoglobulin effector domain derivatives with substitutions corresponding to 65A / N297A Contains an array of

[0198] In certain embodiments, the DDpp fusion protein can be prepared by techniques known in the art. Immunoglobulins that have been glycosylated or mutated to enhance effector function using It contains the sequence of a phospho-effector domain, e.g., the constant region domain contained in DDpp. Inactivation of the sequence (by point mutation or other means) results in the loss of the circulating DDpp fusion protein. It may reduce Fc receptor binding, thereby enhancing tumor localization. Constant region modifications consistent with the provided embodiments slow complement binding and therefore serum half-life. Further modifications of the constant region may shorten the sequence and potentially reduce nonspecific binding of the conjugated cytotoxin. Mutations can be used to increase antigen specificity or antibody flexibility, thereby enhancing localization. The disulfide bond or oligosaccharide moiety may be modified to allow for tumor localization, The resulting physiological profile, such as distribution and serum half-life, and other biochemical modifications The agonistic effect can be readily measured and quantified using known immunological techniques without undue experimentation. It can be quantified.

[0199] In some embodiments, the immune effector cells are immunoglobulin or other peptides. Cell surface receptors for peptide-binding molecules, e.g., receptors for immunoglobulin constant regions and receptors, including a class of receptors commonly referred to as "Fc receptors" ("FcRs"). Contains specific ability to interact with restricted subsets of immunoglobulin heavy chain isotypes. FcRs with different affinities, or FcRs that interact with the Fc domain with different affinities, and / or Contains FcRs that can be expressed on restricted subsets of immune effector cells under certain conditions Many FcRs have been structurally and / or functionally characterized, and are well known in the art. It is known in the literature (e.g., Kijimoto-Ochichai et al., Ce ll Mol.Life.Sci.59:648(2002);Davis et al .,Curr.Top.Microbiol.Immunol.266:85(2002 );Pawankar,Curr.Opin.Allerg.Clin.Immunol .1:3(2001);Radaev et al.,Mol.Immunol.38: 1073(2002);Wurzburg et al.,Mol.Immunol.3 8:1063(2002);Sulica et al.,Int.Rev.Immun ol.20:371(2001);Underhill et al.,Ann.Rev .Immunol.20:825(2002);Coggeshall,Curr.Di r.Autoimm.5:1(2002);Mimura et al.,Adv.Ex p.Med.Biol.495:49(2001);Baumann et al.,A dv.Exp.Med.Biol.495:219(2001);Santoso et al.,Ital.Heart J.2:811(2001);Novak et a l.,Curr.Opin.Immunol.13:721(2001);Fossat i et al.,Eur.J.Clin.Invest.31:821(2001)) , the contents of each of which are incorporated herein by reference in their entirety.

[0200] Cells capable of mediating ADCC are examples of immune effector cells. These cells include natural killer cells, tumor-infiltrating T lymphocytes (TILs), and cytotoxic T lymphocytes (CTLs). These include T lymphocytes, and granulocyte cells, such as cells involved in allergic reaction mechanisms. Thus, immune effector cells include, but are not limited to, various differentiation stages within the myeloid and lymphoid lineages. Cells of hematopoietic origin, including cells of the first stage, as well as T lymphocytes, B lymphocytes, NK cells, monocytes, and macrophages phages, dendritic cells, neutrophils, basophils, eosinophils, mast cells, platelets, red blood cells, and and precursors, progenitor cells (e.g., hematopoietic stem cells), as well as the resting, activated, and may express (or not express) one or more types of functional cell surface FcR, including the mature and non-mature forms. Other immune effector cells include those that mediate immune functions. Cells of non-hematopoietic origin that can be used to stimulate the growth of cells, such as endothelial cells, keratinocytes, fibroblasts, osteoclasts, Immune effector cells may also include cytotoxic and inflammatory cells. mitotic or cytostatic events, or endocytic, phagocytic, or pinocytic events Cells that mediate venting, or cells that result in the induction of apoptosis, or microbial immunity cells that result in the neutralization of bacterial or microbial infections, or in the treatment of allergic, inflammatory, hypersensitivity, and and / or cells that mediate autoimmune reactions.

[0201] DDpp fusion proteins with extended half-lives The disclosed DDpp can be fused or conjugated to a second peptide domain, which can increase the half-life of the DDpp. Extend or increase its stability.

[0202] In one embodiment, the DDpp comprises one or more amino acids that facilitate the synthesis, handling, or use of the peptide. The polypeptide may further comprise one or more amino acids, including, but not limited to, those that increase the solubility of the polypeptide. Preferably, one or two lysines at the N-terminus and / or C-terminus are used to enhance the affinity of the amino acid sequence. Suitable fusion proteins include, but are not limited to, fusion proteins that are not normally recognized as part of a protein sequence. one or more polypeptides, polypeptide fragments, or amino acids that do not In one embodiment, the fusion peptide comprises two or more DDpp-linked proteins. The entire amino acid sequence of the peptide above, or alternatively, portions (fractions) of two or more peptides In some embodiments, peptides (e.g., protein S-binding peptides) are included. ) is, for example, operably linked to one or more of the following: a marker protein peptides that facilitate purification, peptide sequences that facilitate the formation of multimeric proteins, or a fragment of any of the foregoing. Suitable fusion partners include, but are not limited to, histidine, PEG, PEG-1, PEG-2, PEG-3, PEG-4, PEG-5, PEG-6, PEG-7, PEG-8, PEG-9, PEG-10, PEG-11, PEG-12, PEG-13, PEG-14, PEG-15, PEG-16, PEG-17, PEG-18, PEG-19, PEG-20, PEG-21, PEG-22, PEG-23, PEG-24, PEG-25, PEG-26, PEG-27, PEG-28, PEG-29, PEG-21, PEG-29, PEG-25, PEG-26, PEG-27, PEG-28, PEG-29, PEG-29, PEG-29, PEG-29, PEG Examples of such tags include the gene tag, FLAG tag, strep tag, and mic tag.

[0203] In some embodiments, the DDpp comprises one or more polypeptides that extend the half-life of the polypeptide. The half-life can be increased, for example, by increasing the molecular weight of the DDpp to reduce renal clearance. and / or incorporate binding domains for FcRn-mediated recycling processes. In one embodiment, the DDpp can be extended by incorporating an albumin polypeptide or or fragments thereof (e.g., human serum albumin (HSA)), or chemically In certain embodiments, fused or chemically conjugated albumin fragments The albumin fragment may contain 10%, 25%, 50%, or 75% of the full-length albumin protein. In further or alternative embodiments, DDpp binds to albumin when administered in vivo. The albumin-binding domain or fatty acid is fused or conjugated to the albumin-binding domain or fatty acid. An example of a conjugation domain is the moiety derived from 4-(p-iodophenyl)butanoic acid, " albu-tag" (Dumelin et al., Angew Chem. I nt.Ed Engl.47:3196-3201(2008)).

[0204] In one embodiment, DDpp is a transferrin polypeptide or a fragment thereof. (e.g., human transferrin) or is fused or chemically conjugated to the antibody. In this embodiment, the fused or chemically conjugated transferrin fragments are similar to the full-length transferrin. 10%, 25%, 50%, or 75% of the phosphoprotein. In an embodiment, the DDpp is a trans-peptide that binds to albumin when administered in vivo. It is fused or conjugated to a ferrin-binding domain.

[0205] In some embodiments, DDpp is a proline-alanine-serine multimer (PASy lation;XL-Protein GmbH), non-exact repetition Peptide sequence (XTENylation, rPEG), homopolymer of glycine residues (H APylation), elastin-like repeat sequence (ELPylation; e.g., rice See US Patent Application No. 61 / 442,106, the contents of which are incorporated herein by reference in their entirety. ), artificial GLK (GLK fusion; Huang et al., Eur. J .Pharm.Biopharm.72:435-41(2010)), or human CG fused or chemically conjugated to a CTP peptide (CTP fusion) derived from the beta subunit can be.

[0206] Additional DDpp fusion proteins In some embodiments, the DDpp fusion protein is selected from the group consisting of BCMA, CD123, CS1, Specific binding to HER2, AFP, and / or AFP p26. Disease-associated antigens , cancer, and / or specific cell types (e.g., hyperproliferative cells), and / or pathogens ( For example, bacterial cells (e.g., tuberculosis, smallpox, and anthrax), viruses (e.g., HIV ), parasites (e.g., malaria and leishmaniasis), fungal infections, mold, mycoplasma The antigen may be an antigen characteristic of plasma, a prion antigen, or an antigen associated with a disorder of the immune system. In a further embodiment, the DDpp fusion protein is conjugated to a therapeutic or cytotoxic agent. do.

[0207] In additional embodiments, the DDpp fusion protein is capable of detecting, multimerizing, or identifying interaction partners. or one or more nucleotides that facilitate characterization of DDpp activity. A representative chemical moiety is biotin. Other moieties suitable for conjugation to include, but are not limited to, photosensitizers, dyes, fluorescent dyes, radioactive dyes, etc. These include photonuclides, radionuclide-containing conjugates, enzymes, toxins, and cytotoxic agents. For example, Photofrin, Visudyne, Levlan, Hoscan, Metovix, Hex Vicks (registered trademark, Cysview™), Laserphyrin, Antolin, Photo Kroll, Photosens, Photorex, Lumacan, Cevira, Vis onac, BF-200ALA, and Amfinex. In this state, histidine tag, FLAG tag, Strep tag, or mic tag is used as DD It is bound to pp.

[0208] In another embodiment, the DDpp fusion protein is a fusion protein that binds to BCMA, CD123, CS1, HE The DD binds to R2, AFP, AFP p26 or a fragment thereof. The peptide tag can be used to detect and / or bind to a target molecule containing the peptide tag. In one embodiment, the DDpp fusion protein is a member of the group: A peptide selected from a histidyl (His6) tag, a mic tag, and a FLAG tag Other peptide tags may be used as described herein or is known in the art.

[0209] DDpp fusion proteins with epitope tags In some embodiments, the DDpp fusion protein comprises a peptide epitope tag. In some embodiments, the peptide tag is a hexahistidyl (His6) tag, a midi tag, or a nucleotide tag. In a further embodiment, the peptide is selected from the group consisting of a peptide tag, a peptide tag, and a FLAG tag. The tag includes, but is not limited to, avitag (biotinylation of the tag and streptavidin) (enabling isolation by ELISA), calmodulin, E-tag, hemagglutinin (HA), S- Tag, SBP-tag, softag1, streptavidin, tetra- or polycystaden Examples of tags include the V5, VSV, and Xpress tags. Additionally, polyhistidine tags are also available. In a further embodiment, covalently attached peptide tags, protein Covalent peptide tags can be used, including but not limited to isopeptides. (covalently binds to pyrin C protein), Spytag (spycatcher protein) covalently attached to the protein), and Snoop Tag (covalently attached to the Spycatcher protein) In still further embodiments, the nucleotides include, but are not limited to, biotin carboxylates. Carrier protein (BCCP), glutathione-s-transferase, green fluorescent protein Proteins (or other fluorophores), HaloTag, NusTag, thioredoxin, and Protein tags, including Fc tags, may optionally be used. Polymorphic tags may be used. In yet further embodiments, no tags are used. In some embodiments, the DDpp fusion protein comprises a removable tag. Therefore, any combination of extracellular, transmembrane and intracellular domains disclosed herein can be used. It can be used.

[0210] DDpp linker The terms "linker" and "spacer" are used interchangeably herein and are not intended to be limiting unless otherwise specified. otherwise, peptide bonds or other bonds that serve to link separate functional domains. In one embodiment, the linker in the DDpp is Located between DDpp and another polypeptide component containing an independent functional domain. Suitable linkers for connecting two or more conjugated DDpps will be apparent to those skilled in the art. and are typically used to bind peptides, proteins or other organic molecules. It may be any linker used in the art. In certain embodiments, such Suitable linkers are useful for constructing proteins or polypeptides intended for pharmaceutical use. Suitable.

[0211] DDpp and additional components of DDpp fusion proteins can be operated in a single amino acid sequence Suitable linkers for linking to include, but are not limited to, glycine linkers, serine linkers, -, mixed glycine / serine linker, glycine and serine rich linker or near polar Examples of polypeptide linkers include linkers composed of functional polypeptide fragments. It can be obtained.

[0212] In one embodiment, the linker is composed of mostly glycine, alanine, proline, asparagine, In one embodiment, the amino acids are selected from the group consisting of methylaminopropyl, ... , the linker is mostly glycine, alanine, proline, asparagine, The amino acids are selected from the group consisting of threonine, glutamine, and lysine. In embodiments, the DDpp fusion protein linker is selected from the group consisting of glycine, alanine, proline, and amino acids. consisting of one or more amino acids selected from the group consisting of paragine, glutamine, and lysine In one embodiment, the DDpp fusion protein linker is composed of glycine, alanine, From proline, asparagine, aspartic acid, threonine, glutamine, and lysine In another embodiment, the DDpp fusion is comprised of one or more amino acids selected from the group consisting of: The protein linker is composed of mostly sterically unhindered amino acids. In this embodiment, the linker is configured such that the amino acids therein are mostly glycine, serine, and / or is alanine. In some embodiments, the peptide linker is a polyglycine, e.g. , (Gly)5 (SEQ ID NO: 975), and (Gly)8 (SEQ ID NO: 976), poly(G In some embodiments, the peptide is selected from: Drinker is Gly-Gly-Gly-Gly-Thr-Gly-Gly-Gly-Gl y-Ser (SEQ ID NO: 4). In some embodiments, the peptide linker comprises Gly-Gly-Gly-Gly-Asp-Gly-Gly-Gly-Gly-Ser It contains the sequence (SEQ ID NO: 5).

[0213] In one embodiment, the DDpp fusion is directly linked ( In one embodiment, the DDpp fusion comprises: at least two, at least three, or at least two nucleotides directly linked to another component of the DDpp fusion Both contain four DDpp.

[0214] In another embodiment, the DDpp is linked via a linker to another component of the DDpp fusion protein. DDpp fusion proteins can be operably linked to a single linker, multiple linkers, In one embodiment, the DDpp fusion may comprise a linker peptide. DDpp fusion protein operably linked to another component of the DDpp fusion protein via a peptide. In one embodiment, the DDpp fusion protein is linked via a linker peptide. The protein comprises 2, 3, 4, or 5 DDs operably linked to another component of the protein.

[0215] The linker may be BCMA, CD123, CS1, HER2, AFP, or AFP p2 As long as the DDpp can be operably linked to the DDpp so as to bind the DDpp in a targeted manner such as 6. In some embodiments, phosphorus Carriers can be made of approximately 1 to 100 amino acids, approximately 1 to 50 amino acids, approximately 1 to 20 amino acids, or approximately 1 to 15 amino acids. about amino acids, about 1-10 about amino acids, about 1-5 about amino acids, about 2-20 about amino acids, about 2- The length of the linker is 15 amino acids, about 2 to 10 amino acids, or about 2 to 5 amino acids. The degree of flexibility and / or other characteristics may vary depending on, but not limited to, the target and The term "protein" includes affinity, specificity, or avidity for one or more other protein targets of interest. It must be clarified that the properties of the resulting DD-containing protein may be affected in some way. In the DDpp fusion protein, two or more linkers are used. If present, these linkers may be the same or different. In the context and disclosure, one skilled in the art would understand how to activate DDpp and other components of the DDpp fusion protein. For operable linkage, optimal linker composition and length can be routinely determined.

[0216] The linker may also be an alkyl linker or a non-peptide linker such as a PEG linker. For example, alkyl groups such as -NH-(CH2)sC(O)- (s=2 to 20) can be used. These alkyl linkers can also be used with lower alkyl (e.g., C1-C6), lower acyl, halogen (e.g., Cl, Br), CN, NH2, phenyl Exemplary non-peptide linkers include PEG-linkers, ... In certain embodiments, the PEG linker is about 100 to 5000 kDa, or has a molecular weight of approximately 100-500 kDa.

[0217] Suitable linkers for joining DDpp fusion protein components by chemical cross-linking include: Homobifunctional chemical cross-linking compounds such as, but not limited to, glutaraldehyde, adipimidine Dimethyl methyl methacrylate (DMA), dimethyl suberimidate (DMS) and dimethyl pimelimidate imidoesters such as dithiobis(succinimidyl propionate) or dithiobis(succinimidyl propionate) Dithiobis(sulfosuccinimidyl propionate) (DSP) and dithiobis(sulfosuccinimidyl propionate) (DTSS) N-hydroxysuccinimide (NHS) esters such as P) are used for crosslinking. a suitable linker for attaching the DDpp fusion protein component of the heterobifunctional reagent to Examples include, but are not limited to, amine-reactive amino acids having one amine-reactive end and a sulfhydryl-reactive end at the other end. Crosslinkers with a moiety, or an NHS ester and an SH-reactive group (e.g., maleimide) at one end Examples of crosslinkers include crosslinkers having a hydroxyl group (imide or pyridyl).

[0218] In a further embodiment, one or more linkers in the DDpp fusion protein are cleavable. Examples of cleavable linkers include, but are not limited to, those that can be cleaved by proteases (in vivo). Various types of peptide sequences recognized in vitro or in vivo, e.g., Tev, Tro plasmin, factor Xa, plasmin (blood protease), metalloproteases, cathepsins These include proteases found in the endothelial cells (e.g., GFLG, etc.), as well as in other body compartments. can be.

[0219] In some embodiments, the linker facilitates release of the DDpp or cytotoxic agent in the cell. For example, an acid-labile linker (e.g., a hydrazoline) may be used. protease-sensitive (e.g., peptidase-sensitive) linkers, photolabile linkers, Dimethyl linkers or disulfide-containing linkers (Chari, Can. Res. 52 :127-131 (1992); U.S. Patent No. 5,208,020; U.S. Patent Application Publication No. No. 20090110753, the contents of which are incorporated herein by reference in their entireties. Once the composition is internalized into the cell, it becomes fused to a DDpp or a cytotoxic agent. It is desirable that the covalent bond between the partners be cleaved intracellularly. "Intracellular cleavage" refers to the metabolic or cellular processes that affect the DDpp-drug complex. This means that the reaction occurs within the cytotoxic agent, i.e., the covalent bond between DDpp and the cytotoxic agent, DDpp. The bond between the fusion protein and the DDpp or the linker between the two DDpps is broken, resulting in the formation of free DDpp in the cell. Dpp and / or cytotoxic agents are produced.

[0220] Linker optimization can be performed using techniques described herein and / or other techniques of interest. The linker can be evaluated using techniques known in the art. Another DDp, such as an antibody domain or fragment that binds to a target molecule and / or antigen p does not interfere with the ability of DDpp to bind to the fusion protein component.

[0221] DDpp as a chemical complex DDpp fusion proteins that promote specific binding to target molecules include fluorescent dyes, radioactive dyes, and Isotopes, chromatographic compositions (e.g., beads, resins, gels, etc.) and chemical The DDpp conjugates can be chemically conjugated to a variety of compounds, including but not limited to, therapeutic agents. , has applications including diagnostic, analytical, manufacturing and therapeutic applications.

[0222] The inherent lack of cysteines in the DD sequence provides a unique cysteine ​​for site-specific conjugation. This provides an opportunity for the introduction of the technology.

[0223] In some embodiments, the DDpp (e.g., DDpp fusion protein) comprises at least The reactive residue is a functional group that can be used for the attachment of a complex, such as a chemotherapeutic agent. Reactive residues are useful as sites for, for example, cysteine, lysine, or other reactive Thus, cysteine ​​can be at the N- or C-terminus of DDpp, or A cysteine ​​can be added within the DDpp sequence. In addition, lysines can be added to either end of the DDpp or within the DDpp sequence. and / or the lysine can replace another amino acid in the sequence of DDpp. In embodiments, reactive residues (e.g., cysteine, lysine, etc.) are present in the loop sequence of DD. (e.g., amino acid residues 22-24 and 25 of SEQ ID NOs: 11-949 and 950) In one embodiment, the reactive residues are located between the components of the DDpp fusion, e.g., located in the linker located between DDpp and other components of the Dpp fusion protein Reactive residues (e.g., cysteine, lysine, etc.) may also be present within the sequence of DDpp, or can be located within other components of the DDpp fusion protein. or DDpp fusion protein, at least one, at least two, at least three In one embodiment, the DDpp, such as the DDpp fusion protein, comprises at least The amino acid sequence may contain one, at least two, or at least three cysteine ​​residues.

[0224] BCMA-bound DDpp In some embodiments, the DD of the DDpp specifically binds to BCMA. In embodiments, the DD of the DDpp is a BCMA having an amino acid sequence consisting of SEQ ID NO:7. In some embodiments, DDpp specifically binds to SEQ ID NOs: 11-305, and and 306. In another embodiment, the BCMA binding DDpp is an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 to 305, and 306. Includes variants of the sequence.

[0225] In some embodiments, the BCMA-binding DDpp binds to a single target, or In some embodiments, the DDpp domain comprises a dimer, a trimer, etc. has an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 to 305, and 306. The antibody comprises two, three, four, five, or more than five DDs that specifically bind to BCMA. In some embodiments, the DDpp comprises two, three, four, five, or more than five identical sequences. In some embodiments, the DDpp comprises a DD having a different epitope of BCMA. an antibody that specifically binds to the antibody and is selected from the group consisting of SEQ ID NOs: 11 to 305, and 306; Some embodiments contain 2, 3, 4, 5, or more than 5 DDs with the amino acid sequence In an embodiment, the DDpp comprises a DD that specifically binds to BCMA, and is capable of binding to BCMA or a different 2, 3, 4, 5, or more than 5 additional distinct Ds that specifically bind to the target antigen In some embodiments, the antibody further comprises a target-binding domain (e.g., scFv). DDpp is a DD (e.g., SEQ ID NOs: 11-305) that specifically binds to BCMA. and DD) having an amino acid sequence selected from the group consisting of 306 and 307, one or more additional DDs or In some embodiments, the DDpp further comprises another target binding domain. DD (e.g., selected from the group consisting of SEQ ID NOs: 11 to 305, and 306) that specifically binds to A a DD having an amino acid sequence selected from the group consisting of: Further comprising one or more additional DD or other target binding domains. In this embodiment, the DDpp is a DD that specifically binds to BCMA (e.g., SEQ ID NO: 11 DD) having an amino acid sequence selected from the group consisting of: Specifically bind to two, three, four, five, or more than five different targets. In this embodiment, the DDpp is a DD (e.g., SEQ ID NOs: 11 to 305) that specifically binds to BCMA. DD) having an amino acid sequence selected from the group consisting of 2, 3, In some embodiments, the antibody specifically binds to four, five, or more than five different cancer antigens. DDpp is a DD (e.g., SEQ ID NOs: 11-305) that specifically binds to BCMA. and 306), Specifically binds to five or more different cancer antigens expressed on the surface of cancer cells In some embodiments, the DDpp is a DD (e.g., a DD) that specifically binds to BCMA. DD having an amino acid sequence selected from the group consisting of sequence numbers 11 to 305 and 306 ) expressed on the surface of two, three, four, five, or more than five different cancer cells It specifically binds to the antigen.

[0226] In some embodiments, the DDpp is a DDpp-specific BCMA-binding protein disclosed herein. Some embodiments include variants of the BCMA-binding DD (reference DD) that retain the ability to bind BCMA. In this embodiment, the sequences of the BCMA-binding DD mutants consist of SEQ ID NOs: 11 to 305, and 306. compared to a reference BCMA-binding DD having an amino acid sequence selected from the group consisting of: 4, 5, 6, 7, 8, 9, or 10, 1-3, 1-5, or 1-10 conservative or or non-conservative substitutions. In some embodiments, the sequence of the BCMA-binding DD variant is A reference having an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 to 305, and 306. Compared to the illuminated BCMA-binding DD, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 In some embodiments, the BCM The sequence of the A-linked DD mutant is selected from the group consisting of SEQ ID NOs: 11 to 305, and 306. 1, 2, 3, 4, 5, 6, 7 compared to a reference BCMA-binding DD with an amino acid sequence , 8, 9, or 10, 1 to 3, 1 to 5, or 1 to 10 non-conservative substitutions.

[0227] In some embodiments, the sequence of the BCMA-binding DD mutant is SEQ ID NO: 11-305, and 306. A total of 1, 2, and 3 amino acid residues were found at positions corresponding to amino acid residues 1 to 22, 29 to 46, and 52 to 72. , 4, 5, 6, 7, 8, 9, or 10, 1-3, 1-5, or 1-10 conservative or non-conservative substitutions. In some embodiments, the sequence of the BCMA-binding DD variant is , having an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 to 305, and 306 Corresponding to amino acid residues 1–22, 29–46, and 52–72 of reference BCMA-DD A total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1-3, 1-5, or or 1 to 10 conservative substitutions. In some embodiments, the BCMA-binding DD variant The sequence is an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 to 305 and 306. Amino acid residues 1-22, 29-46, and 52-72 of the reference BCMA-binding DD A total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 pieces, 1 to 3, Contains 1 to 5, or 1 to 10 non-conservative substitutions.

[0228] In some embodiments, the sequence of the BCMA-binding DD mutant is SEQ ID NO: 11-305, and 306. Amino acid residues 2-6, 8-10, 12, 13, 15-17, 19, 20, 29, 30, and 32 ~34, 36, 37, 39~41, 43, 44, 52~55, 57~59, 61, 62, A total of 1, 2, 3, 4, 5, 6, 7, and 8 are placed in positions corresponding to 64-66 and 68-70. , 9, or 10, 1 to 3, 1 to 5, or 1 to 10 conservative or non-conservative substitutions In some embodiments, the sequence of the BCMA-binding DD mutant is SEQ ID NO: 11-30. 5, and 306, Groups 2~6, 8~10, 12, 13, 15~17, 19, 20, 29, 30, 32~34, 36, 37, 39-41, 43, 44, 52-55, 57-59, 61, 62, 64-6 A total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 68 to 70 are displayed in positions corresponding to the numbers 6, 68, 69, 70, and 69. or 10, 1 to 3, 1 to 5, or 1 to 10 conservative substitutions. In this embodiment, the sequences of the BCMA-binding DD mutants consist of SEQ ID NOs: 11 to 305, and 306. The amino acid residues 2-6, 8-10, 1 of the reference DD have an amino acid sequence selected from the group consisting of: 2, 13, 15-17, 19, 20, 29, 30, 32-34, 36, 37, 39-41 , 43, 44, 52–55, 57–59, 61, 62, 64–66, and 68–70 In the corresponding positions, a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1 to 3, 1 Contains up to 5, or 1 to 10 non-conservative substitutions.

[0229] In some embodiments, the present disclosure provides a method for detecting a nucleotide sequence that is completely or partially (e.g., overlapping with an epitope) providing a BCMA-binding DDpp that blocks binding of a reference DD to BCMA; DD has an amino acid sequence selected from SEQ ID NOs: 11 to 305, and 306. In embodiments, the present disclosure provides an amino acid sequence selected from SEQ ID NOs: 11-305, and 306. The present invention provides a BCMA-binding DDpp that binds to the same epitope on BCMA as a reference DD consisting of the sequence To provide.

[0230] In some embodiments, the DDpp is a fusion tag comprising a DD that specifically binds BCMA. In some embodiments, the DD comprises the amino acid sequence of SEQ ID NO:7. In some embodiments, the BCMA-binding The DDpp fusion protein is selected from the group consisting of SEQ ID NOs: 11 to 305, and 306. In another embodiment, DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 to 305, and and 306. The DDpp fusion protein may comprise a full-length antibody or a portion (fragment) of an antibody. In some embodiments, the DDpp fusion protein is a full-length IgG antibody (e.g., Ig In a further embodiment, the antibody comprises DDpp The fusion protein comprises a full-length antibody that specifically binds to a cancer antigen. , DDpp is a commercially approved therapeutic antibody (e.g., rituximab, ofatumumab, Ocrelizumab, veltuzumab, MEDI-551, epratuzumab, belimumab, tabacum lumab, AMG-557, MEDI-570, and NN882). In a further embodiment, the BCMA-binding DDpp is an Fc fusion protein. The protein contains a variable human Fc domain.

[0231] In some embodiments, the DDpp fusion protein is operably linked to a serum protein. In a further embodiment, the DDpp fusion protein comprises a BCMA-binding DD. It comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 to 305, and 306. In this embodiment, the BCMA-binding DDpp fusion protein is selected from the group consisting of SEQ ID NOs: 11-305, and and 306. In a further embodiment, the present invention includes variants of the amino acid sequence selected from the group consisting of: The DDpp fusion protein includes human serum albumin or a fragment thereof. In some embodiments, the DDpp fusion protein is AFP or AFP p26, or In some embodiments, the DDpp fusion protein comprises AF P (e.g., SEQ ID NO: 9), or a fragment thereof. The p-fusion protein comprises AFP p26 (SEQ ID NO: 10), or a fragment thereof. In some embodiments, the DDpp fusion protein is selected from the group consisting of SEQ ID NOs: 10, 968, 969 , 970, 971, 972, 973, or 974. In some embodiments, the DDpp fusion protein is a serum protein or Some antigen fragments of proteins (e.g., AFP and AFP p26) are also included. In embodiments, the DDpp fusion protein may be 5 to 500, 5 to 400, 5 to 300, 5 to 200, 5-100, 5-50, 10-500, 10-400, 10-300, 10-2 Fragments consisting of 00, 10-100, or 10-50 amino acids of serum proteins Includes ent.

[0232] In some embodiments, the BCMA-binding DDpp fusion protein binds to the extracellular domain of the receptor. In some embodiments, the DDpp fusion protein comprises a The material contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 to 305, and 306. In another embodiment, the BCMA-binding DDpp fusion protein comprises a DD comprising SEQ ID NO: 1. 1 to 305, and 306. In certain embodiments, the BCMA-binding DDpp fusion protein is a BCMA-binding DDpp fusion protein that binds CD123 (e.g., the sequence In some embodiments, the extracellular domain of BCMA-binding DDpp fusion proteins bind to CD19, CD20, CD22, HVEM, and B Selected from the group consisting of TLA, DR3, CD37, TSLPR, IL7R, and gp96 The antibody comprises the extracellular domain of the receptor to be treated or a fragment thereof.

[0233] In some embodiments, the BCMA-binding DDpp fusion protein binds to a cell surface receptor Extracellular domain: 5-500, 5-400, 5-300, 5-200, 5-100, 5- 50, 10-500, 10-400, 10-300, 10-200, 10-100, and In some embodiments, DD comprises a fragment of 10 to 50 amino acids. The pp fusion protein binds to BCMA (SEQ ID NO: 7) or CD123 (SEQ ID NO: 8) cells. In some embodiments, the DDpp fusion protein comprises a fragment of the ectodomain. , BCMA (SEQ ID NO: 7), CD123 (SEQ ID NO: 8), or CS1 (SEQ ID NO: 965 In some embodiments, DDpp comprises a fragment of the extracellular domain of CD 19, CD20, CD22, HVEM, BTLA, DR3, CD37; TSLPR, IL 7R, and a fragment of the extracellular domain of a receptor selected from the group consisting of gp96 Includes:

[0234] In a further embodiment, the BCMA-binding DDpp fusion protein is an intracellular protein ( In some embodiments, the DD The pp fusion protein is selected from the group consisting of SEQ ID NOs: 11 to 305, and 306. In another embodiment, the BCMA-binding DDpp fusion protein comprises a DD comprising the amino acid sequence The quality is a mutation of an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 to 305, and 306. In some embodiments, the BCMA-binding DDpp fusion protein comprises 5 to 5 00, 5~400, 5~300, 5~200, 5~100, 5~50, 10~500, 1 0-400, 10-300, 10-200, 10-100, or 10-50 cells It includes fragments consisting of amino acid residues of proteins (eg, nuclear proteins).

[0235] CD123-bound DDpp In some embodiments, the DD of the DDpp specifically binds to CD123. In one embodiment, the DD is specific for CD123 having an amino acid sequence consisting of SEQ ID NO:8. In some embodiments, DDpp binds to SEQ ID NOs: 307-739, and 7 40. In another embodiment, DDpp comprises an amino acid sequence selected from the group consisting of: A variant of an amino acid sequence selected from the group consisting of sequences 307 to 739 and 740. include.

[0236] In some embodiments, the CD123-binding DDpp is a multi-target binding DDpp that binds to a single target. In some embodiments, the DDp comprises a fusion domain (e.g., a dimer, a trimer, etc.). p specifically binds to CD123 and is selected from the group consisting of SEQ ID NOs: 307 to 739 and 740 and a 2, 3, 4, 5, or more than 5 DDs having an amino acid sequence selected from the group consisting of: In some embodiments, the DDpp comprises 2, 3, 4, 5, or more than 5 identical In some embodiments, the DDpp comprises a DD having the same sequence as a different DD of CD123. A polypeptide that specifically binds to an epitope and is selected from the group consisting of SEQ ID NOs: 307 to 739 and 740. The present invention relates to a method for producing a nucleotide sequence comprising the steps of: In some embodiments, the DDpp comprises a DD that specifically binds to CD123 and 2, 3, 4, 5, or more than 5 antibodies that specifically bind to 23 or different target antigens; It may further comprise an additional different DD or target binding domain (e.g., scFv). In some embodiments, the DDpp is a DD that specifically binds to CD123 (e.g., SEQ ID NO: DD) having an amino acid sequence selected from the group consisting of Nos. 307 to 739, and 740 and one or more additional molecules that bind to one or more antigens expressed on the surface of B cells. In some embodiments, the antibody further comprises an additional DD or other target binding domain. DDpp is a DD (e.g., SEQ ID NOs: 307 to 739) that specifically binds to CD123. and 740), and one or more additional DDs or other target-binding binding domains that bind to multiple cancer antigens In some embodiments, the DDpp further comprises a D that specifically binds to CD123. D (e.g., an amino acid selected from the group consisting of SEQ ID NOs: 307-739, and 740) DD) specific for two, three, four, five, or more than five different targets. In a further embodiment, the DDpp binds to DD( For example, an amino acid sequence selected from the group consisting of SEQ ID NOs: 307 to 739 and 740 and a DD having a specificity for two, three, four, five, or more than five different cancer antigens. In some embodiments, the DDpp binds to a DD( For example, an amino acid sequence selected from the group consisting of SEQ ID NOs: 307 to 739 and 740 DD) and expressed on the surface of cancer cells in 2, 3, 4, 5, or more than 5 In some embodiments, DDpp specifically binds to different cancer antigens. DD that specifically binds to 3 (e.g., the group consisting of SEQ ID NOs: 307 to 739, and 740) DD) having an amino acid sequence selected from the group consisting of 2, 3, 4, 5, or more than 5 It specifically binds to cancer antigens expressed on the surface of different cancer cells.

[0237] In some embodiments, the DDpp is a polypeptide that specifically binds to CD123, as disclosed herein. Some experiments have demonstrated that the CD123-binding DD retains its ability to bind CD123 (reference DD). In some embodiments, the sequences of the CD123-binding DD mutants are set forth in SEQ ID NOs: 307-739, and 74 1 compared to a reference CD123-binding DD having an amino acid sequence selected from the group consisting of: , 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1 to 3, 1 to 5, or 1 to 10 In some embodiments, the CD123-binding DD mutation The sequence of the target amino acid is selected from the group consisting of SEQ ID NOs: 307 to 739, and 740. Compared to the reference CD123-binding DD with the sequence: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1 to 3, 1 to 5, or 1 to 10 conservative substitutions. In this embodiment, the sequences of the CD123-binding DD mutants are SEQ ID NOs: 307 to 739, and 740. 1, compared to a reference CD123-binding DD having an amino acid sequence selected from the group consisting of: 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1 to 3, 1 to 5, or 1 to 10 Includes non-conservative substitutions.

[0238] In some embodiments, the sequence of the CD123-binding DD mutant is SEQ ID NO: 307-73 a reference CD123-binding D having an amino acid sequence selected from the group consisting of: A total of 1 at positions corresponding to amino acid residues 1 to 22, 29 to 46, and 52 to 72 of D 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1 to 3, 1 to 5, or 1 to 10 Containing conservative or non-conservative substitutions. In some embodiments, the CD123-binding DD variants The sequence is an amino acid sequence selected from the group consisting of SEQ ID NOs: 307 to 739 and 740. Amino acid residues 1-22, 29-46, and 52-7 of reference CD123-DD with In the position corresponding to 2, a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1 to 3 In some embodiments, the CD123 The sequence of the binding DD mutant is selected from the group consisting of SEQ ID NOs: 307 to 739, and 740. Amino acid residues 1-22, 29-46 of a reference CD123-binding DD having an amino acid sequence , and positions corresponding to 52 to 72, a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, and contains 10, 1 to 3, 1 to 5, or 1 to 10 non-conservative substitutions.

[0239] In some embodiments, the sequence of the CD123-binding DD mutant is SEQ ID NO: 307-73 a reference CD123-binding D having an amino acid sequence selected from the group consisting of: Amino acid residues 2-6, 8-10, 12, 13, 15-17, 19, 20, 29, and 30 of D , 32-34, 36, 37, 39-41, 43, 44, 52-55, 57-59, 61, A total of 1, 2, 3, 4, 5, 6, and 7 are located at positions corresponding to 62, 64-66, and 68-70. 7, 8, 9, or 10, 1-3, 1-5, or 1-10 conservative or non-conservative In some embodiments, the sequence of the CD123-binding DD mutant is SEQ ID NO:3 a reference DD having an amino acid sequence selected from the group consisting of 07 to 739 and 740; Amino acid residues 2-6, 8-10, 12, 13, 15-17, 19, 20, 29, 30, and 3 2-34, 36, 37, 39-41, 43, 44, 52-55, 57-59, 61, 62 , 64 to 66, and 68 to 70, a total of 1, 2, 3, 4, 5, 6, 7, Contains 8, 9, or 10, 1 to 3, 1 to 5, or 1 to 10 conservative substitutions. In some embodiments, the sequences of the CD123-binding DD mutants are SEQ ID NOs: 307-739, and and 740. , 8-10, 12, 13, 15-17, 19, 20, 29, 30, 32-34, 36, 3 7, 39-41, 43, 44, 52-55, 57-59, 61, 62, 64-66, and and 68 to 70, a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 1, 1 to 3, 1 to 5, or 1 to 10 non-conservative substitutions.

[0240] In some embodiments, the present disclosure provides a method for detecting a nucleotide sequence that is completely or partially (e.g., overlapping with an epitope) providing a CD123-binding DDpp that blocks the binding of a (comparable) reference DD to CD123; Reference DD has an amino acid sequence selected from SEQ ID NOs: 307-739, and 740 In other embodiments, the present disclosure provides a method for the preparation of a nucleic acid sequence selected from SEQ ID NOs: 307-739, and 740. CD123 binding that binds to the same epitope on CD123 as the reference DD consisting of amino acid sequence Provide DDpp.

[0241] In some embodiments, the DDpp is a fusion comprising a DD that specifically binds to CD123. In some embodiments, the DD of the DDpp fusion protein is Specifically binds to CD123 having an amino acid sequence consisting of SEQ ID NO:8. In the present invention, DDpp is an amino acid sequence selected from the group consisting of SEQ ID NOs: 307 to 739 and 740. In another embodiment, DDpp comprises the amino acid sequences of SEQ ID NOs: 307-739, and 7 In some embodiments, the amino acid sequence comprises a variant selected from the group consisting of: DDpp fusion proteins include full-length antibodies or portions (fragments) of antibodies. In some embodiments, the DDpp fusion protein is a full-length IgG antibody (e.g., IgG1 , IgG2, IgG2, or IgG4). In a further embodiment, the DDpp fusion The protein comprises a full-length antibody that specifically binds to a cancer antigen. Dpp is a therapeutic antibody that has been commercially approved (e.g., rituximab, ofatumumab, octanoic acid, ribosomal ... Relizumab, veltuzumab, MEDI-551, epratuzumab, belimumab, tabacum In another embodiment, the compounds include ribozymes (e.g., AMG-557, MEDI-570, and NN882). , CD123-binding DDpp is an Fc fusion protein.

[0242] In some embodiments, the DDpp is a CD1 In a further embodiment, the fusion protein comprises a CD123-binding DD. The pp fusion protein is selected from the group consisting of SEQ ID NOs: 307-739, and 740. In another embodiment, the CD123-binding DDpp fusion protein comprises an amino acid sequence A variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 307 to 739 and 740 In some embodiments, the CD123-binding DDpp fusion protein is In some embodiments, the DDpp fusion protein comprises all or a portion of albumin. comprises AFP (SEQ ID NO: 9), or a fragment thereof. The CD123-binding DDpp fusion protein may be AFP p26 (SEQ ID NO: 10), or In some embodiments, the CD123-binding DDpp fusion protein comprises a fragment of The quality is SEQ ID NO: 10, 968, 969, 970, 971, 972, 973, or 974 In some embodiments, the DDpp fusion protein comprises a polypeptide having the sequence: are serum proteins or antigenic fragments of serum proteins (e.g., AFP, and In some embodiments, the DDpp fusion protein comprises 5 to 5 00, 5~400, 5~300, 5~200, 5~100, 5~50, 10~500, 1 0-400, 10-300, 10-200, 10-100, or 10-50 serum samples It contains fragments of amino acids of proteins.

[0243] In some embodiments, the CD123-binding DDpp fusion protein binds to the extracellular domain of the receptor. In some embodiments, the DDpp fusion protein comprises a DDpp domain or a fragment thereof. The protein has an amino acid sequence selected from the group consisting of SEQ ID NOs: 307 to 739 and 740. In another embodiment, the CD123-binding DDpp fusion protein comprises a DD comprising the sequence and variants of the amino acid sequence selected from the group consisting of numbers 307 to 739 and 740. In a further embodiment, the CD123-binding DDpp fusion protein binds to CD123 (e.g., For example, the extracellular domain of SEQ ID NO: 8, or a fragment thereof. In this form, the CD123-binding DDpp fusion protein binds to CD19, CD20, CD22, Consists of HVEM, BTLA, DR3, CD37, TSLPR, IL7R, and gp96 The antibody comprises the extracellular domain of a receptor selected from the group consisting of:

[0244] In some embodiments, the CD123-binding DDpp fusion protein binds to a cell surface receptor of the extracellular domain of 5-500, 5-400, 5-300, 5-200, 5-100, and 5 ~50, 10~500, 10~400, 10~300, 10~200, 10~100, or a fragment of 10 to 50 amino acids. The Dpp fusion protein contains a fragment of the extracellular domain of BCMA (SEQ ID NO: 7). In some embodiments, the DDpp fusion protein is a fusion protein of CD123 (SEQ ID NO: 8). In some embodiments, the DDpp fusion protein comprises a fragment of the extracellular domain. The substance contains a fragment of the extracellular domain of CS1 (SEQ ID NO: 965). In an embodiment, the DDpp is selected from the group consisting of CD19, CD20, CD22, HVEM, BTLA, and DR3. , CD37; TSLPR, IL7R, and gp96. Contains a fragment of the extracellular domain.

[0245] In a further embodiment, the CD123-binding DDpp fusion protein is an intracellular protein (e.g., nucleoprotein) or fragments thereof. In some embodiments, D The Dpp fusion protein is selected from the group consisting of SEQ ID NOs: 307-739, and 740. In another embodiment, the CD123-binding DDpp fusion tag comprises a DD comprising an amino acid sequence The protein is selected from the group consisting of amino acids 307-739 and 740. In some embodiments, the CD123-binding DDpp fusion protein includes variants of the sequence. 5~500, 5~400, 5~300, 5~200, 5~100, 5~50, 10~ 500, 10-400, 10-300, 10-200, 10-100, or 10-50 A fragment consisting of amino acid residues of an intracellular protein (e.g., a nuclear protein) include.

[0246] CS1 combined DDpp In some embodiments, the DD of the DDpp specifically binds to CS1. In one embodiment, the DD specifically binds to CS1 having the amino acid sequence of SEQ ID NO: 965. In some embodiments, DDpp is selected from the group consisting of SEQ ID NOs: 896-909 and 910. In another embodiment, the DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NO: and variants of the amino acid sequence selected from the group consisting of Nos. 896 to 909 and 910. .

[0247] In some embodiments, the CS1-binding DDpp is a multi-target binding DDpp that binds to a single target. In some embodiments, the DDpp comprises a dimer, a trimer, etc. , specifically binds to CS1 and is selected from the group consisting of SEQ ID NOs: 896 to 909, and 910 The amino acid sequence of the DD may be any of the following: In some embodiments, the DDpp contains two, three, four, five, or more than five identical sequences. In some embodiments, the DDpp comprises a DD having a different epitope of CS1. and specifically binds to an amino acid sequence selected from the group consisting of SEQ ID NOs: 896 to 909, and 910. In some embodiments, the DDs may include 2, 3, 4, 5, or more than 5 DDs having the amino acid sequence. In this form, the DDpp comprises a DD that specifically binds to CS1 and can bind to either CS1 or a different target. 2, 3, 4, 5, or more than 5 additional distinct DDs or further comprises a target binding domain (e.g., an scFv). In some embodiments, DDpp is a DD (e.g., SEQ ID NOs: 896-909) that specifically binds to CS1. DD) having an amino acid sequence selected from the group consisting of: 910, One or more additional DDs or other targets that bind to the expressed antigen or antigens In some embodiments, the DDpp further comprises a specific binding domain. DD (e.g., selected from the group consisting of SEQ ID NOs: 896-909, and 910) that specifically binds to and one or more nucleotides that bind to one or more cancer antigens. The antibody further comprises a plurality of additional DD or other target binding domains. In this embodiment, DDpp is a DD (e.g., SEQ ID NOs: 896-90) that specifically binds to CS1. DD) having an amino acid sequence selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, and 10; In a further embodiment, the antibody specifically binds to four, five, or more than five different targets. DDpp is a DD (e.g., SEQ ID NOs: 896 to 909) that specifically binds to CS1. and 910), In some embodiments, D Dpp is a DD (e.g., SEQ ID NOs: 896-909, and 909) that specifically binds to CS1. 10), and or specifically binds to more than five different cancer antigens expressed on the surface of cancer cells. In some embodiments, the DDpp is a DD that specifically binds to CS1 (e.g., SEQ ID NO: 8). DD) having an amino acid sequence selected from the group consisting of 96 to 909, and 910 and two, three, four, five, or more than five cancer antigens expressed on the surface of different cancer cells. Specific binding.

[0248] In some embodiments, DDpp specifically binds to CS1, as disclosed herein. In some embodiments, the CS1-binding DD includes a variant of the DD that retains the ability to bind CS1 (reference DD). The sequence of the CS1-binding DD mutant is the group consisting of SEQ ID NOs: 896 to 909 and 910. 1, 2, 3, 4, 5 compared to a reference CS1-binding DD having an amino acid sequence selected from , 6, 7, 8, 9, or 10, 1-3, 1-5, or 1-10 conservative or non- In some embodiments, the sequence of the CS1-binding DD mutant is SEQ ID NO: A reference CS having an amino acid sequence selected from the group consisting of 896 to 909 and 910. Compared to 1 bond DD, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1 to 3, 1 In some embodiments, the CS1-binding DD The sequence of the variant is an amino acid sequence selected from the group consisting of SEQ ID NOs: 896-909, and 910. Compared to the reference CS1-binding DD with the amino acid sequence 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1 to 3, 1 to 5, or 1 to 10 non-conservative substitutions.

[0249] In some embodiments, the sequences of the CS1-binding DD mutants are SEQ ID NOs: 896-909, and 910. A total of 1, 2, 3, and 4 were present at positions corresponding to the amino acid residues 1 to 22, 29 to 46, and 52 to 72. 4, 5, 6, 7, 8, 9, or 10, 1-3, 1-5, or 1-10 conservative or In some embodiments, the sequence of the CS1-binding DD mutant comprises a sequence A reference having an amino acid sequence selected from the group consisting of SEQ ID NOs: 896 to 909, and 910. Positions corresponding to amino acid residues 1 to 22, 29 to 46, and 52 to 72 of CS1-DD , a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1 to 3, 1 to 5, or In some embodiments, the sequence of the CS1-binding DD mutant is The antibody has an amino acid sequence selected from the group consisting of SEQ ID NOs: 896 to 909 and 910. These correspond to amino acid residues 1-22, 29-46, and 52-72 of the reference CS1-binding DD. A total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1 to 3, 1 to 5, , or containing 1 to 10 non-conservative substitutions.

[0250] In some embodiments, the sequences of the CS1-binding DD mutants are SEQ ID NOs: 896-909, and 910. Acid residues 2-6, 8-10, 12, 13, 15-17, 19, 20, 29, 30, 32 34, 36, 37, 39-41, 43, 44, 52-55, 57-59, 61, 62, 6 A total of 1, 2, 3, 4, 5, 6, 7, 8, and 9 are placed in positions corresponding to 4 to 66 and 68 to 70. Contains 9, 10, 1-3, 1-5, or 1-10 conservative or non-conservative substitutions In some embodiments, the sequences of the CS1-binding DD mutants are set forth in SEQ ID NOs: 896-909. and 910 amino acid residues of reference DD having an amino acid sequence selected from the group consisting of 2-6, 8-10, 12, 13, 15-17, 19, 20, 29, 30, 32-34, 3 6, 37, 39-41, 43, 44, 52-55, 57-59, 61, 62, 64-66 , and positions corresponding to 68 to 70, a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, and contains 10, 1 to 3, 1 to 5, or 1 to 10 conservative substitutions. The sequence of the CS1-binding DD mutant is SEQ ID NOs: 896-909 and 910. Amino acid residues 2-6, 8-10, 12 of a reference DD having an amino acid sequence selected from the group , 13, 15-17, 19, 20, 29, 30, 32-34, 36, 37, 39-41, 43, 44, 52-55, 57-59, 61, 62, 64-66, and 68-70 A total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1 to 3, 1 to Contains 5, or 1 to 10 non-conservative substitutions.

[0251] In some embodiments, the present disclosure provides a method for detecting a nucleotide sequence that is completely or partially (e.g., overlapping with an epitope) providing a CS1-binding DDpp that blocks the binding of a reference DD (which overlaps with the reference DD) to CS1; The antibody has an amino acid sequence selected from SEQ ID NOs: 896 to 909, and 910. In embodiments, the present disclosure provides an amino acid sequence selected from SEQ ID NOs: 896-909, and 910. and providing a CS1-binding DDpp that binds to the same epitope of CS1 as a reference DD consisting of the sequence. do.

[0252] In some embodiments, the DDpp is a fusion protein comprising a DD that specifically binds to CS1. In some embodiments, the DD of the DDpp fusion protein is a protein selected from the group consisting of SEQ ID NO:9 In some embodiments, the CS1 polypeptide specifically binds to a CS1 having an amino acid sequence consisting of 65 amino acids. DDpp is an amino acid selected from the group consisting of SEQ ID NOs: 896 to 909, and 910 In another embodiment, DDpp comprises the sequences of SEQ ID NOs: 896-909, and 910. In some embodiments, the amino acid sequence of the DD The pp fusion protein may contain a full-length antibody or a portion (fragment) of an antibody. In this embodiment, the DDpp fusion protein is a full-length IgG antibody (e.g., IgG1, I In a further embodiment, the DDpp fusion protein comprises a fusion protein of the present invention. The protein comprises a full-length antibody that specifically binds to a cancer antigen. p is a commercially approved therapeutic antibody (e.g., rituximab, ofatumumab, ocreglitazone, uzumab, veltuzumab, MEDI-551, epratuzumab, belimumab, tabalumab, AMG-557, MEDI-570, and NN882). The S1-binding DDpp is an Fc fusion protein.

[0253] In some embodiments, the DDpp comprises a CS1 operably linked to a serum protein. In some embodiments, the CS1-binding DDpp fusion protein is a fusion protein comprising the CS1-binding DDpp. The fusion protein is an amino acid sequence selected from the group consisting of SEQ ID NOs: 896-909, and 910. In another embodiment, the CS1-binding DDpp fusion protein comprises the amino acid sequence of SEQ ID NO:8. 96 to 909, and 910. In some embodiments, the CS1-binding DDpp fusion protein is a fusion protein that binds the entirety of human serum albumin. In some embodiments, the DDpp fusion protein comprises an AFP (sequence 9), or a fragment thereof. The pp fusion protein contains AFP p26 (SEQ ID NO: 10), or a fragment thereof. In some embodiments, the CS1-binding DDpp fusion protein is selected from the group consisting of SEQ ID NOs: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 39, 38, 39, 4 Polypeptides having the sequences 68, 969, 970, 971, 972, 973, or 974 In some embodiments, the DDpp fusion protein comprises a serum protein or contains antigenic fragments of serum proteins (e.g., AFP, and AFP p26) In some embodiments, the DDpp fusion protein is 5 to 500, 5 to 400, 5 to 600, 5 to 800, 5 to 1000, 5 to 1500, 5 to 2000, 5 to 2000, 5 to 3000, 5 to 4000, 5 to 5 ...6000, 5 300, 5-200, 5-100, 5-50, 10-500, 10-400, 10-30 From 0, 10-200, 10-100, or 10-50 amino acids of serum proteins The fragment includes:

[0254] In some embodiments, the CS1-binding DDpp fusion protein binds to the extracellular domain of the receptor. In some embodiments, the DDpp fusion protein comprises a DDpp fusion protein or a fragment thereof. The present invention relates to a method for producing a human medicament comprising the steps of: In another embodiment, the CS1-binding DDpp fusion protein comprises a DD comprising SEQ ID NO: 89. 6 to 909, and 910. In another embodiment, the CS1-binding DDpp fusion protein is In some embodiments, the CS1-binding extracellular domain comprises a CS1-binding extracellular domain, or a fragment thereof. DDpp fusion proteins bind to CD19, CD20, CD22, HVEM, BTLA, and DR 3, a receptor selected from the group consisting of CD37, TSLPR, IL7R, and gp96 or a fragment thereof.

[0255] In some embodiments, the CS1-binding DDpp fusion protein binds to the cellular domain of a cell surface receptor. Extracellular domain: 5-500, 5-400, 5-300, 5-200, 5-100, 5-5 0, 10-500, 10-400, 10-300, 10-200, 10-100, or In some embodiments, the DDp fragment comprises a fragment of 10 to 50 amino acids. The p-fusion protein comprises a fragment of the extracellular domain of BCMA (SEQ ID NO: 7). In some embodiments, the DDpp fusion protein binds to the CD123 (SEQ ID NO: 8) In some embodiments, the DDpp fusion protein comprises a fragment of the ectodomain. , a fragment of the extracellular domain of CS1 (SEQ ID NO: 965). In this state, DDpp binds to CD19, CD20, CD22, HVEM, BTLA, DR3, and C. D37: Cells expressing a receptor selected from the group consisting of TSLPR, IL7R, and gp96 Contains a fragment of the ectodomain.

[0256] In a further embodiment, the CS1-binding DDpp fusion protein binds to an intracellular protein (e.g., In some embodiments, the DDp comprises a nucleoprotein (e.g., a nucleoprotein) or a fragment thereof. pFusion proteins are selected from the group consisting of SEQ ID NOs: 896-909, and 910 In another embodiment, the CS1-binding DDpp fusion protein comprises a DD comprising the amino acid sequence The amino acid sequence of the variant selected from the group consisting of SEQ ID NOs: 896 to 909 and 910. In some embodiments, the CS1-binding DDpp fusion protein comprises 5 to 50 0, 5-400, 5-300, 5-200, 5-100, 5-50, 10-500, 10 ~400, 10-300, 10-200, 10-100, or 10-50 intracellular tags This includes fragments consisting of amino acid residues of proteins (e.g., nucleoproteins).

[0257] HER2 binding DDpp In some embodiments, the DD of the DDpp specifically binds to HER2. In embodiments, the DD is specific for HER2 having the amino acid sequence of SEQ ID NO: 967. In some embodiments, DDpp binds to SEQ ID NOs: 911-949, and 9 50. In another embodiment, DDpp comprises an amino acid sequence selected from the group consisting of: A variant of an amino acid sequence selected from the group consisting of sequences 911 to 949 and 950. include.

[0258] In some embodiments, the HER2-binding DDpp is a multi-target binding DDpp that binds to a single target. In some embodiments, the DDpp domain comprises a dimer, a trimer, etc. specifically binds to HER2 and is selected from the group consisting of SEQ ID NOs: 911 to 949, and 950. It comprises 2, 3, 4, 5 or more than 5 DDs with selected amino acid sequences. In some embodiments, the DDpp comprises 2, 3, 4, 5, or more than 5 identical sequences. In some embodiments, the DDpp comprises a DD having a sequence of different epitopes of HER2. and a sequence selected from the group consisting of SEQ ID NOs: 911 to 949, and 950. It contains two, three, four, five or more DDs having an amino acid sequence corresponding to the In this embodiment, the DDpp comprises a DD that specifically binds to HER2, and Two, three, four, five, or more than five additional different antigens that specifically bind to different target antigens. In some embodiments, the antibody further comprises a target-binding domain (e.g., scFv) that binds the target. In this embodiment, the DDpp is a DD that specifically binds to HER2 (e.g., SEQ ID NOs: 911-919). 49, and 950), one or more additional DDs or In some embodiments, the DDpp further comprises other target binding domains. DDs that specifically bind to HER2 (e.g., SEQ ID NOs: 911-949, and 950) and a DD having an amino acid sequence selected from the group consisting of: The target further comprises one or more additional DD or other target binding domains that bind to the target. In some embodiments, the DDpp is a DD that specifically binds to HER2 (e.g., the sequence DD having an amino acid sequence selected from the group consisting of numbers 911 to 949 and 950 ) and specifically bind to two, three, four, five, or more than five different targets. In certain embodiments, the DDpp is a DD that specifically binds to HER2 (e.g., SEQ ID NO: 9). DD) having an amino acid sequence selected from the group consisting of 11 to 949, and 950. specifically bind to two, three, four, five, or more than five different cancer antigens. In this embodiment, the DDpp is a DD that specifically binds to HER2 (e.g., SEQ ID NO: 91 DD) having an amino acid sequence selected from the group consisting of 1 to 949, and 950 , 2, 3, 4, 5, or more than 5 different cancer antigens expressed on the surface of cancer cells. In some embodiments, DDpp specifically binds to HER2. D (e.g., an amino acid selected from the group consisting of SEQ ID NOs: 911-949, and 950) DD having a sequence) and a representation of 2, 3, 4, 5, or more than 5 different cancer cells. It specifically binds to cancer antigens expressed on the surface.

[0259] In some embodiments, the DDpp is a HER2-specific agonist as disclosed herein. In some embodiments, the HER2-binding DD may be a variant (reference DD) that retains the ability to bind HER2. In this embodiment, the sequences of the HER2-binding DD mutants are SEQ ID NOs: 911 to 949, and 950 to 1, 2, 3, or more than a reference HER2-binding DD having an amino acid sequence selected from the group consisting of: , 4, 5, 6, 7, 8, 9, or 10, 1-3, 1-5, or 1-10 conservative or non-conservative substitutions. In some embodiments, the sequence of the HER2-binding DD variant is , having an amino acid sequence selected from the group consisting of SEQ ID NOs: 911 to 949, and 950 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 HER2-binding DDs compared to the reference HER2-binding DD , 1 to 3, 1 to 5, or 1 to 10 conservative substitutions. The sequences of the ER2-binding DD mutants are selected from the group consisting of SEQ ID NOs: 911 to 949 and 950. 1, 2, 3, 4, 5, compared to a reference HER2-binding DD having a selected amino acid sequence Contains 6, 7, 8, 9, or 10, 1-3, 1-5, or 1-10 non-conservative substitutions nothing.

[0260] In some embodiments, the sequences of the HER2-binding DD mutants are SEQ ID NOs: 911-949 and a reference HER2-binding DD having an amino acid sequence selected from the group consisting of: A total of 1, 2, and 3 were located at positions corresponding to amino acid residues 1 to 22, 29 to 46, and 52 to 72. Save 3, 4, 5, 6, 7, 8, 9, or 10, 1-3, 1-5, or 1-10 In some embodiments, the sequence of the HER2-binding DD variant is The antibody has an amino acid sequence selected from the group consisting of SEQ ID NOs: 911 to 949 and 950. corresponding to amino acid residues 1-22, 29-46, and 52-72 of the reference HER2-DD A total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1 to 3, 1 to 5, In some embodiments, the HER2-binding DD variant comprises 1 to 10 conservative substitutions. The variant sequences are selected from the group consisting of SEQ ID NOs: 911 to 949, and 950. Amino acid residues 1-22, 29-46, and 52 of the reference HER2 binding DD with the amino acid sequence A total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 pieces are placed in positions corresponding to ~72. Contains up to 3, 1 to 5, or 1 to 10 non-conservative substitutions.

[0261] In some embodiments, the sequences of the HER2-binding DD mutants are SEQ ID NOs: 911-949 and a reference HER2-binding DD having an amino acid sequence selected from the group consisting of: Amino acid residues 2-6, 8-10, 12, 13, 15-17, 19, 20, 29, 30, and 3 2-34, 36, 37, 39-41, 43, 44, 52-55, 57-59, 61, 62 , 64 to 66, and 68 to 70, a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1-3, 1-5, or 1-10 conservative or non-conservative substitutions In some embodiments, the sequence of the HER2-binding DD mutant comprises SEQ ID NO: 911 to 918. 949, and 950 of the amino acid sequence of reference DD Acid residues 2~6, 8~10, 12, 13, 15~17, 19, 20, 29, 30, 32~3 4, 36, 37, 39-41, 43, 44, 52-55, 57-59, 61, 62, 64 A total of 1, 2, 3, 4, 5, 6, 7, 8, 9 are in positions corresponding to ~66 and 68~70. Containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1-3, 1-5, or 1-10 conservative substitutions. In embodiments, the sequences of the HER2-binding DD mutants are SEQ ID NOs: 911-949, and 950. Amino acid residues 2 to 6, 8 to 1 of reference DD having an amino acid sequence selected from the group consisting of: 0, 12, 13, 15-17, 19, 20, 29, 30, 32-34, 36, 37, 39 ~41, 43, 44, 52~55, 57~59, 61, 62, 64~66, and 68~ A total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 pieces are placed in the positions corresponding to 70. Contains 3, 1 to 5, or 1 to 10 non-conservative substitutions.

[0262] In some embodiments, the present disclosure provides a method for detecting a nucleotide sequence that is completely or partially (e.g., overlapping with an epitope) providing a HER2-binding DDpp that blocks the binding of a reference DD to HER2, DD has an amino acid sequence selected from SEQ ID NOs: 911 to 949, and 950. In one embodiment, the present disclosure provides an amino acid sequence selected from SEQ ID NOs: 911-949, and 950. HER2-binding DDp that binds to the same epitope as the reference DD consisting of the amino acid sequence Provide p.

[0263] In some embodiments, the DDpp is a fusion tag comprising a DD that specifically binds to HER2. In some embodiments, the DD of the DDpp fusion protein is a protein selected from the group consisting of SEQ ID NO: 967. In the present invention, DDpp is an amino acid sequence selected from the group consisting of SEQ ID NOs: 911 to 949 and 950. In another embodiment, DDpp comprises the amino acid sequences of SEQ ID NOs: 911-949, and 9 In some embodiments, the amino acid sequence comprises a variant selected from the group consisting of: DDpp fusion proteins include full-length antibodies or portions (fragments) of antibodies. In some embodiments, the DDpp fusion protein is a full-length IgG antibody (e.g., IgG1 , IgG2, IgG2, or IgG4). In a further embodiment, the DDpp fusion The protein comprises a full-length antibody that specifically binds to a cancer antigen. Dpp is a therapeutic antibody that has been commercially approved (e.g., rituximab, ofatumumab, octanoic acid, ribosomal ... Relizumab, veltuzumab, MEDI-551, epratuzumab, belimumab, tabacum In another embodiment, the compounds include ribozymes (e.g., AMG-557, MEDI-570, and NN882). , HER2-binding DDpp is an Fc fusion protein.

[0264] In some embodiments, the DDpp is a HER2 receptor operably linked to a serum protein. In some embodiments, the fusion protein comprises a HER2-binding DDp. pFusion proteins are selected from the group consisting of SEQ ID NOs: 911-949, and 950 In another embodiment, the HER2-binding DDpp fusion protein comprises the amino acid sequence and variants of the amino acid sequence selected from the group consisting of numbers 911 to 949 and 950. In some embodiments, the HER2-binding DDpp fusion protein is a human serum albumin (HSA)-binding DDpp fusion protein. In some embodiments, the DDpp fusion protein comprises all or part of A FP (SEQ ID NO: 9), or a fragment thereof. The 2-linked DDpp fusion protein is AFP p26 (SEQ ID NO: 10), or its flag In some embodiments, the HER2-binding DDpp fusion protein comprises the sequence Sequences having numbers 10, 968, 969, 970, 971, 972, 973, or 974 In some embodiments, the DDpp fusion protein comprises a polypeptide that binds to a serum tag. antigenic fragments of proteins or serum proteins (e.g., AFP, and AFP p In some embodiments, the DDpp fusion protein comprises 5 to 500, 5 to 26 400, 5-300, 5-200, 5-100, 5-50, 10-500, 10-400 , 10-300, 10-200, 10-100, or 10-50 serum proteins It includes fragments consisting of amino acids.

[0265] In some embodiments, the HER2-binding DDpp fusion protein binds to the extracellular domain of the receptor. In some embodiments, the DDpp fusion protein comprises a The quality is an amino acid sequence selected from the group consisting of SEQ ID NOs: 911 to 949, and 950. In another embodiment, the HER2-binding DDpp fusion protein comprises a DD comprising SEQ ID NO: and variants of the amino acid sequence selected from the group consisting of 911-949 and 950. In a further embodiment, the HER2-binding DDpp fusion protein is HER2 (SEQ ID NO: 9 67), or a fragment thereof. ER2-binding DDpp fusion proteins bind to CD19, CD20, CD22, HVEM, and BT. LA, DR3, CD37; TSLPR, IL7R and gp96 The antibody comprises the extracellular domain of a receptor or a fragment thereof.

[0266] In some embodiments, the HER2-binding DDpp fusion protein binds to a cell surface receptor Extracellular domain: 5-500, 5-400, 5-300, 5-200, 5-100, 5- 50, 10-500, 10-400, 10-300, 10-200, 10-100, and In some embodiments, DD comprises a fragment of 10 to 50 amino acids. The pp fusion protein contains a fragment of the extracellular domain of BCMA (SEQ ID NO: 7). In some embodiments, the DDpp fusion protein is a fusion protein of CD123 (SEQ ID NO: 8). In some embodiments, the DDpp fusion protein comprises a fragment of the extracellular domain. comprises a fragment of the extracellular domain of CS1 (SEQ ID NO: 965). In terms of morphology, DDpp is a member of the cytoplasm of CD19, CD20, CD22, HVEM, BTLA, DR3, CD37; cells of a receptor selected from the group consisting of TSLPR, IL7R, and gp96 Contains a fragment of the extracellular domain.

[0267] In a further embodiment, the HER2-binding DDpp fusion protein is an intracellular protein ( In some embodiments, the DD The pp fusion protein is selected from the group consisting of SEQ ID NOs: 911-949, and 950. In another embodiment, the HER2-binding DDpp fusion protein comprises a DD comprising an amino acid sequence The protein has an amino acid sequence selected from the group consisting of SEQ ID NOs: 911 to 949 and 950. In some embodiments, the HER2-binding DDpp fusion protein comprises a variant of 5 ~500, 5~400, 5~300, 5~200, 5~100, 5~50, 10~500 , 10-400, 10-300, 10-200, 10-100, or 10-50 fine particles It includes fragments consisting of amino acid residues of intracellular proteins (eg, nuclear proteins).

[0268] AFP binding DDpp In some embodiments, the DD of the DDpp is specific for AFP or a fragment thereof. In a further embodiment, DD of DDpp binds to the amino acid sequence consisting of SEQ ID NO: 9. In some embodiments, the nucleotide sequence specifically binds to AFP or a fragment thereof. DDpp is selected from the group consisting of SEQ ID NOs: 741 to 874, and 886 to 895. In a further embodiment, the AFP-binding DDpp comprises the amino acid sequence of SEQ ID NO: 741. -874, and 886-895. .

[0269] In some embodiments, the AFP-conjugated DDpp is a multi-target binding DDpp that binds to a single target. In some embodiments, the DDpp comprises a dimer, a trimer, etc. , specifically binds to AFP, and is a group consisting of SEQ ID NOs: 741 to 874, and 886 to 895 and a 2, 3, 4, 5, or more than 5 DDs having an amino acid sequence selected from the group consisting of: In some embodiments, the DDpp comprises 2, 3, 4, 5, or more than 5 identical In some embodiments, the DDpp comprises a DD having the same sequence as the AFP. and specifically binds to the tope, and is selected from the group consisting of SEQ ID NOs: 741 to 874, and 886 to 895. 2, 3, 4, 5, or more than 5 DDs having an amino acid sequence selected from the group consisting of: In some embodiments, the DDpp comprises a DD that specifically binds to AFP, or 2, 3, 4, 5, or more than 5 additional antibodies that specifically bind different target antigens. The antibody further comprises a different DD or target binding domain (e.g., scFv). In this embodiment, the DDpp is a DD that specifically binds to AFP (e.g., SEQ ID NO: 741 DD) having an amino acid sequence selected from the group consisting of: and one or more additional molecules that bind to one or more antigens expressed on the surface of B cells. In some embodiments, the antibody further comprises an additional DD or other target binding domain. DDpp is a DD that specifically binds to AFP (e.g., SEQ ID NOs: 741-874, and DD) having an amino acid sequence selected from the group consisting of 886 to 895, The antibody has one or more additional DDs or other target-binding domains that bind to multiple cancer antigens. In some embodiments, the DDpp further comprises a D that specifically binds to AFP. D (e.g., selected from the group consisting of SEQ ID NOs: 741 to 874, and 886 to 895) DD) with an amino acid sequence, and In a further embodiment, DDpp specifically binds to AFP. D (e.g., selected from the group consisting of SEQ ID NOs: 741 to 874, and 886 to 895) and 2, 3, 4, 5, or more than 5 different cancer anti-cancer drugs. In some embodiments, DDpp specifically binds to AFP. DD (e.g., selected from the group consisting of SEQ ID NOs: 741-874, and 886-895) 2, 3, 4, 5, or more than 5 cancer cells, each of which contains a DD having an amino acid sequence that is In some embodiments, the DD pp is a DD that specifically binds to AFP (e.g., SEQ ID NOs: 741-874, and 88 DD) having an amino acid sequence selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, Specifically binds to five or more cancer antigens expressed on the surface of different cancer cells .

[0270] In some embodiments, the DDpp specifically binds to AFP, as disclosed herein. In some embodiments, the present invention includes a variant of the DD that retains the ability to bind AFP (reference DD). The sequences of the AFP-binding DD mutants are SEQ ID NOs: 741 to 874 and 886 to 895. compared to a reference AFP-binding DD having an amino acid sequence selected from the group consisting of: , 4, 5, 6, 7, 8, 9, or 10, 1-3, 1-5, or 1-10 conservative or non-conservative substitutions. In some embodiments, the sequence of the AFP-binding DD variant is An amino acid sequence selected from the group consisting of SEQ ID NOs: 741 to 874, and 886 to 895 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1 compared to a reference AFP-binding DD having In some embodiments, the amino acid sequence contains 0, 1 to 3, 1 to 5, or 1 to 10 conservative substitutions. The sequences of the AFP-binding DD mutants are SEQ ID NOs: 741 to 874 and 886 to 895. compared to a reference AFP-binding DD having an amino acid sequence selected from the group consisting of: 4, 5, 6, 7, 8, 9, or 10, 1-3, 1-5, or 1-10 non-conservative Contains substitutions.

[0271] In some embodiments, the sequences of the AFP-binding DD mutants are SEQ ID NOs: 741-874, and a reference AFP-binding D having an amino acid sequence selected from the group consisting of 886 to 895 A total of 1 at positions corresponding to amino acid residues 1 to 22, 29 to 46, and 52 to 72 of D 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1 to 3, 1 to 5, or 1 to 10 Conservative or non-conservative substitutions are included. In some embodiments, the sequence of the AFP-binding DD mutant is The sequence is an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874, and 886-895. Amino acid residues 1-22, 29-46, and 52-7 of reference AFP-DD with the sequence In the position corresponding to 2, a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1 to 3 In some embodiments, the AFP-binding The sequence of the DD mutant is selected from the group consisting of SEQ ID NOs: 741 to 874 and 886 to 895. Amino acid residues 1-22, 29-46 of a reference AFP-binding DD having the amino acid sequence of interest , and positions corresponding to 52 to 72, a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, and contains 10, 1 to 3, 1 to 5, or 1 to 10 non-conservative substitutions.

[0272] In some embodiments, the sequences of the AFP-binding DD mutants are SEQ ID NOs: 741-874, and a reference AFP-binding D having an amino acid sequence selected from the group consisting of 886 to 895 Amino acid residues 2-6, 8-10, 12, 13, 15-17, 19, 20, 29, and 30 of D , 32-34, 36, 37, 39-41, 43, 44, 52-55, 57-59, 61, A total of 1, 2, 3, 4, 5, 6, and 7 are located at positions corresponding to 62, 64-66, and 68-70. 7, 8, 9, or 10, 1-3, 1-5, or 1-10 conservative or non-conservative In some embodiments, the sequence of the AFP-binding DD mutant is SEQ ID NO: 741 Reference D having an amino acid sequence selected from the group consisting of: Amino acid residues 2-6, 8-10, 12, 13, 15-17, 19, 20, 29, and 30 of D , 32-34, 36, 37, 39-41, 43, 44, 52-55, 57-59, 61, A total of 1, 2, 3, 4, 5, 6, and 7 are located at positions corresponding to 62, 64-66, and 68-70. Contains 7, 8, 9, or 10, 1 to 3, 1 to 5, or 1 to 10 conservative substitutions. In some embodiments, the sequences of the AFP-binding DD mutants are SEQ ID NOs: 741-874, and and amino acid residues 886 to 895 of reference DD having an amino acid sequence selected from the group consisting of Groups 2~6, 8~10, 12, 13, 15~17, 19, 20, 29, 30, 32~34, 36, 37, 39-41, 43, 44, 52-55, 57-59, 61, 62, 64-6 A total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 68 to 70 are displayed in positions corresponding to the numbers 6, 68, 69, 70, and 69. or 10, 1 to 3, 1 to 5, or 1 to 10 non-conservative substitutions.

[0273] In some embodiments, the present disclosure provides a method for detecting a nucleotide sequence that is completely or partially (e.g., overlapping with an epitope) providing an AFP-binding DDpp that blocks the binding of the reference DD to AFP; The antibody has an amino acid sequence selected from SEQ ID NOs: 741 to 873, and 874. In embodiments, the present disclosure provides an amino acid sequence selected from SEQ ID NOs: 741-873, and 874. and providing an AFP-binding DDpp that binds to the same epitope of AFP as a reference DD consisting of the sequence. do.

[0274] In some embodiments, the DDpp is a fusion protein comprising a DD that specifically binds to AFP. In some embodiments, DDpp is a protein consisting of the amino acid sequence SEQ ID NO:9. Further embodiments are fusion proteins comprising a DD that specifically binds to an AFP having the formula: DDpp is selected from the group consisting of SEQ ID NOs: 741 to 874 and 886 to 895. In another embodiment, DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874, and a variant of DD comprising an amino acid sequence selected from the group consisting of 886 to 895. It is a fusion protein containing an AFP-binding DD.

[0275] In some embodiments, the DDpp is a full-length antibody or a portion (fragment) of an antibody. A fusion protein comprising a DD that specifically binds to AFP operably linked to In some embodiments, the DDpp is an Fc fusion protein. The DDpp fusion protein is a full-length IgG antibody (e.g., IgG1, IgG2, IgG In a further embodiment, the DDpp fusion protein comprises a fusion protein selected from the group consisting of IgG, IgG2, and IgG4. In a further embodiment, the DDpp comprises a full-length antibody that specifically binds to the antigen. approved therapeutic antibodies (e.g., rituximab, ofatumumab, ocrelizumab, bell Tuzumab, MEDI-551, epratuzumab, belimumab, tabalumab, AMG-55 7, MEDI-570, and NN882). In other embodiments, AFP-conjugated DD pp is an Fc fusion protein.

[0276] In some embodiments, the DDpp comprises an AFP operably linked to a serum protein. In some embodiments, the DDpp fusion protein is a fusion protein comprising a conjugated DD. The substance is an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874, and 886-895. In another embodiment, the AFP-binding DDpp fusion protein comprises the sequence of SEQ ID NO: 74. 1 to 874, and 886 to 895. In a further embodiment, the DDpp fusion protein is human serum albumin or In some embodiments, the DDpp fusion protein comprises a fragment of a serum protein. Some embodiments include fragments of proteins or antigenic fragments of serum proteins. In this state, the DDpp fusion protein is , 5~100, 5~50, 10~500, 10~400, 10~300, 10~200, Fragments of serum proteins consisting of 10-100 or 10-50 amino acids include.

[0277] In some embodiments, the AFP-binding DDpp fusion protein binds to the extracellular domain of the receptor. In some embodiments, the DDpp fusion protein comprises a DDpp fusion protein or a fragment thereof. is an amino acid sequence selected from the group consisting of SEQ ID NOs: 741 to 874, and 886 to 895 In another embodiment, the AFP-binding DDpp fusion protein comprises a DD comprising the sequence Amino acid sequence variants selected...

Claims

1. 1. A cell engineered to express a first CAR and a second CAR, wherein the first CAR comprises a first D domain target binding domain (DD) that specifically binds to BCMA and comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 168, and the second CAR comprises a second DD that binds to a different target than the first DD.

2. The cell of claim 1, wherein the first DD comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

168.

3. The cell of claim 1, wherein the first DD comprises the amino acid sequence of any one of SEQ ID NOs: 157 or 164-169.

4. The cell of claim 1, wherein the first DD comprises the amino acid sequence of SEQ ID NO:

168.

5. 2. The cell of claim 1, wherein the first DD comprises SEQ ID NO: 168 or a sequence containing 1, 2, 3, 4, or 5 or fewer substitutions relative to SEQ ID NO:

168.

6. The cell of any one of claims 1 to 5, wherein the second DD specifically binds to AFP, AFP p26, CD19, CD20, CD22, CD123, CS1, HVEM, BTLA, DR3, HER2, or PDL1.

7. The cell of claim 6 , wherein the second DD specifically binds to CD19.

8. The cell of any one of claims 1 to 7, wherein the first CAR or the second CAR comprises a target-binding domain, a transmembrane domain, and an intracellular signaling domain.

9. 9. The cell of claim 8, (a) the transmembrane domain comprises a CD8, 41BB, or CD28 transmembrane domain; (b) the intracellular signaling domain is selected from the group consisting of a human T-cell receptor alpha, beta, or zeta chain domain; a human 41BB domain; a human CD28 domain; and any combination thereof; and / or (c) the intracellular signaling domain comprises an intracellular domain of a costimulatory molecule selected from the group consisting of CD27, CD28, 41BB, OX40, CD30, CD40, PD1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, NKG2D, B7-H3, a ligand that specifically binds to CD83, and any combination thereof; cell.

10. 10. The cell of any one of claims 1 to 9, wherein the cell comprises one or more isolated nucleic acids encoding the first CAR and the second CAR.

11. The cell of any one of claims 1 to 9, wherein the cell comprises one or more vectors encoding the first CAR and the second CAR.

12. The cell of claim 11 , wherein the one or more vectors are lentiviral vectors.

13. The cell according to any one of claims 1 to 12, which is a T cell or a natural killer (NK) cell.

14. A pharmaceutical composition comprising the cells according to any one of claims 1 to 13.

15. A kit comprising the cells according to any one of claims 1 to 13.

16. A pharmaceutical composition for use in treating blood cancer, comprising the cells of any one of claims 1 to 13.

17. 17. The pharmaceutical composition of claim 16, wherein the hematological cancer is multiple myeloma.

18. A pharmaceutical composition for use in the treatment of a B-cell related disorder, comprising a cell according to any one of claims 1 to 13.

19. A pharmaceutical composition for use in treating a disorder of the immune system, comprising a cell according to any one of claims 1 to 13.

20. 20. The pharmaceutical composition of claim 19, wherein the immune system disorder is an autoimmune disease.

21. 21. The pharmaceutical composition of claim 20, wherein the autoimmune disease is rheumatoid arthritis.

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