Modified Proteins or Polypeptides

Abstract

The present invention provides modified immunoglobulin single variable domains and proteins or polypeptides comprising said immunoglobulin single variable domains, which have reduced binding to preparative anti-drug antibodies. In particular, the C-terminus of said immunoglobulin single variable domains comprises an amino acid modification. The present invention also relates to pharmaceutical uses of said immunoglobulin single variable domains, proteins or polypeptides comprising said immunoglobulin single variable domains, pharmaceutical compositions, encoding nucleic acids and methods of preparation.

Description

[Technical field]

[0001] This disclosure claims priority to a Chinese patent application filed on November 29, 2021 (application number CN202111429892.3).

[0002] The present disclosure relates to the biopharmaceutical field, in particular to modified immunoglobulin single variable domains with reduced binding to preparative anti-drug antibodies and to proteins or polypeptides comprising said immunoglobulin single variable domains. The C-terminus of said immunoglobulin single variable domains comprises an amino acid modification. The present disclosure further relates to pharmaceutical uses, pharmaceutical compositions, encoding nucleic acids and preparation methods of said immunoglobulin single variable domains, proteins or polypeptides comprising said immunoglobulin single variable domains. [Background technology]

[0003] Immunogenicity of antibody drugs is a common phenomenon. Preclinical and clinical reports have shown that various antibody drugs, both commercially available and under development, induce anti-drug antibodies (ADAs) in animals, and the ADAs produced may affect the therapeutic efficacy and in vivo metabolism of the antibody drugs. Various techniques and methods have been developed over the years to avoid or mitigate this phenomenon, including the preparation of chimeric antibodies, humanization of antibodies, and development of fully human antibodies.

[0004] However, even fully human antibodies cannot completely avoid the production of immunogenicity, and one of the reasons for this is the presence in the human body of autoantibodies that can bind to natural immunoglobulins or fragments of immunoglobulins, i.e., pre-anti-drug antibodies (pre-ADAs). These pre-ADAs have epitope specificity for various immunoglobulins present throughout the human body, including epitopes in the Fc region, epitopes in the variable region / CDR region, epitopes in the hinge region of the heavy chain, etc. Functionally, current research has shown that these pre-ADAs are related to infectious diseases, homeostasis of the immune system of the body, etc., but many specific functions have not yet been fully recognized.

[0005] Pre-ADA can bind to endogenous immunoglobulins as well as to therapeutic proteins or polypeptides (including monoclonal antibodies) or polypeptide molecules. Pre-ADA binding to therapeutic proteins or polypeptide molecules can retain, eliminate, or neutralize the molecules, affecting their function and metabolism, leading to altered clinical responses and bioavailability, as well as the occurrence of side effects and special phenomena, such as hypersensitivity reactions.

[0006] Single-domain antibodies (sdAbs), including sdAbs of the heavy chain variable region (VH) of IgG molecules derived from humans and non-human primates and sdAbs of the heavy chain (VHH) derived from camel / alpaca, are generally subjected to immunogenicity-related detection and optimization so that ADA is reduced before entering clinical development. However, even after humanization, these molecules may still bind to pre-ADA. It has been reported that drug molecules based on the structure of sdAbs and their derivatives cause strong immune-related side effects during clinical development, and that the side effects are closely related to the binding of pre-ADA.

[0007] Therefore, providing a modification method capable of reducing the degree of binding of sdAbs and their derivatives to pre-ADA would be very beneficial for better development of corresponding drugs and exploiting their clinical value. In the present disclosure, a series of modification methods for reducing the binding of sdAbs to pre-ADA have been tested, and desirable candidate molecules have been successfully obtained. It is expected that these modification methods and the candidate molecules produced thereby will be widely used in the future to reduce the binding of sdAb-based therapeutic biopolymers to pre-ADA, and further optimize the clinical therapeutic effects of drugs. Summary of the Invention

[0008] The present disclosure provides immunoglobulin single variable domains with reduced binding to anti-drug antibodies (ADA), pre-drug antibodies (pre-ADA), and the disclosure also provides encoding nucleic acids, methods of preparation, pharmaceutical compositions, pharmaceutical uses, and detection uses for said immunoglobulin single variable domains, proteins or polypeptides comprising said immunoglobulin single variable domains, related products.

[0009] Immunoglobulin Single Variable Domains In a first aspect, some embodiments of the disclosure provide an immunoglobulin single variable domain, which comprises: X1X2(X3) n or VTVX1X2(X3) n wherein X1 is selected from S, F and R, X2 is selected from A, P and S, X3 is A or absent, and n is an integer of 0 to 5; When n is 0, X3 is absent; When n is 1 to 5, X3 is A; In some specific embodiments, the modified immunoglobulin single variable domain has any one of the following (a) to (e): (a) SAA or VTVSAA (SEQ ID NO: 37), (b) FP or VTVFP (SEQ ID NO: 38), (c) FPA or VTVFPA (SEQ ID NO: 39), (d) RS or VTVRS (SEQ ID NO: 45), and (e) RSA or VTVRSA (SEQ ID NO: 46) It contains a C-terminus consisting of any one of the following:

[0010] In some embodiments, immunoglobulin single variable domains are provided whose C-terminus comprises or terminates in an amino acid sequence designated X1X2X3 or VTVX1X2X3, where X1 is selected from S, F, R, X2 is selected from A, P, S or X2 is absent and X3 is selected from A or X3 is absent.

[0011] In some embodiments, the X1X2X3 or VTVX1X2X3 is any one of the following (1) to (10), i.e., (1) SAA or VTVSAA (SEQ ID NO: 37), (2) FP or VTVFP (SEQ ID NO: 38), (3) FPA or VTVFPA (SEQ ID NO: 39), (4) RP or VTVRP (SEQ ID NO: 40), (5) R or VTVR (SEQ ID NO: 41), (6) RA or VTVRA (SEQ ID NO: 42), (7) FS or VTVFS (SEQ ID NO: 43), (8) SP or VTVSP (SEQ ID NO: 44), (9) RS or VTVRS (SEQ ID NO: 45), and (10) RSA or VTVRSA (SEQ ID NO: 46) Any one of the following may be selected:

[0012] In some embodiments, immunoglobulin single variable domains are provided that have a C-terminus that comprises or terminates in any one of the following amino acid sequences: SA, SAS, SAST (SEQ ID NO: 47), SASTK (SEQ ID NO: 48), SASTKG (SEQ ID NO: 49), SASTKGP (SEQ ID NO: 50), SAT, SATV (SEQ ID NO: 51), SAAS (SEQ ID NO: 52), SAAST (SEQ ID NO: 53), SAASTK (SEQ ID NO: 54), SAASTKG (SEQ ID NO: 55), SAASTKGP (SEQ ID NO: 56), FS, FSA, FSAS (SEQ ID NO: 57), No. 57), FSAST (SEQ ID NO: 58), FSASTK (SEQ ID NO: 59), FSASTKG (SEQ ID NO: 60), FSASTKGP (SEQ ID NO: 61), FST, FSTV (SEQ ID NO: 62), FPAS (SEQ ID NO: 63), FPAST (SEQ ID NO: 64), FPASTK (SEQ ID NO: 65), FPASTKG (SEQ ID NO: 66), FPASTKGP (SEQ ID NO: 67), RPA, RPAA (SEQ ID NO: 68), RPAST (SEQ ID NO: 69), RPASTK (SEQ ID NO: 70), RPASTKG (SEQ ID NO: 71), RPASTKGP (SEQ ID NO: 72), RPT, RPTV ( SEQ ID NO:73), RPAAST (SEQ ID NO:74), RPAASTK (SEQ ID NO:75), RPAASTKG (SEQ ID NO:76), RPAASTKGP (SEQ ID NO:77), RAA, RAST (SEQ ID NO:78), RASTK (SEQ ID NO:79), RASTKG (SEQ ID NO:80), RASTKGP (SEQ ID NO:81), RAAST (SEQ ID NO:82), RAASTK (SEQ ID NO:83), RAASTKG (SEQ ID NO:84), RAASTKGP (SEQ ID NO:85), RAT, RATV (SEQ ID NO:86), SAAG (SEQ ID NO:87), SAAGG (SEQ ID NO:88), FPG, FP GG (SEQ ID NO: 89), FPAG (SEQ ID NO: 90), FPAGG (SEQ ID NO: 91), RAG, RAGG (SEQ ID NO: 92), RS, RSA, RSAA (SEQ ID NO: 93), RSAAA (SEQ ID NO: 94), RSAS (SEQ ID NO: 95), RSAST (SEQ ID NO: 96), RSASTK (SEQ ID NO: 97), RSASTKG (SEQ ID NO: 98), RSASTKGP (SEQ ID NO: 99), RSG or RSGG (SEQ ID NO: 100), VTVSA (SEQ ID NO: 101), VTVSAS (SEQ ID NO: 102), VTVSAST (SEQ ID NO: 103), VTVSASTK (SEQ ID NO: 104),VTVSASTKG (SEQ ID NO: 105), VTVSASTKGP (SEQ ID NO: 106), VTVSAT (SEQ ID NO: 107), VTVSATV (SEQ ID NO: 108), VTVSAAS (SEQ ID NO: 109), VTVSAAST (SEQ ID NO: 110), VTVSAASTK (SEQ ID NO: 111), VTVSAASTKG (SEQ ID NO: 112), VTVSAASTKGP (SEQ ID NO: 113), VTVSAT (SEQ ID NO: 114), VTVSATV (SEQ ID NO: 115), VTVFS (SEQ ID NO: 116), VTVFSA (SEQ ID NO: 117), VTVFSAS (SEQ ID NO: 118), VT VFSAST (SEQ ID NO: 119), VTVFSASTK (SEQ ID NO: 120), VTVFSASTKG (SEQ ID NO: 121), VTVFSASTKGP (SEQ ID NO: 122), VTVFST (SEQ ID NO: 123), VTVFSTV (SEQ ID NO: 124), VTVFPAS (SEQ ID NO: 125), VTVFPAST (SEQ ID NO: 126), VTVFPASTK (SEQ ID NO: 127), VTVFPASTKG (SEQ ID NO: 128), VTVFPASTKGP (SEQ ID NO: 129), VTVRPA (SEQ ID NO: 130), VTVRPAA (SEQ ID NO: 131), VTVRPAST (SEQ ID NO: 132), VTVRPASTK (SEQ ID NO: 133), VTVRPASTKG (SEQ ID NO: 134), VTVRPASTKGP (SEQ ID NO: 135), VTVRPAA (SEQ ID NO: 136), VTVRPT (SEQ ID NO: 137), VTVRPTV (SEQ ID NO: 138), VTVRPAAST (SEQ ID NO: 139), VTVRPAASTK (SEQ ID NO: 140), VTVRPAASTKG (SEQ ID NO: 141), VTVRPAASTKGP (SEQ ID NO: 142), VTVRAA (SEQ ID NO: 143), VTVRAST (SEQ ID NO: 144), VTVRASTK (SEQ ID NO: 145), VT VRASTKG (SEQ ID NO: 146), VTVRASTKGP (SEQ ID NO: 147), VTVRAAST (SEQ ID NO: 148), VTVRAASTK (SEQ ID NO: 149), VTVRAASTKG (SEQ ID NO: 150), VTVRAASTKGP (SEQ ID NO: 151), VTVRAT (SEQ ID NO: 152), VTVRATV (SEQ ID NO: 153), VTVSAAG (SEQ ID NO: 154), VTVSAAGG (SEQ ID NO: 155), VTVFPG (SEQ ID NO: 156), VTVFPGG (SEQ ID NO: 157), VTVFPAG (SEQ ID NO: 158), VTVFPAGG (SEQ ID NO: 159),VTVRAG (SEQ ID NO: 160), VTVRAGG (SEQ ID NO: 161), VTVRS (SEQ ID NO: 162), VTVRSA (SEQ ID NO: 163), VTVRSAA (SEQ ID NO: 164), VTVRSAAA (SEQ ID NO: 165), VTVRSAS (SEQ ID NO: 166), VTVRSAST (SEQ ID NO: 167), VTVRSASTK (SEQ ID NO: 168), VTVRSASTKG (SEQ ID NO: 169), VTVRSASTKGP (SEQ ID NO: 170), VTVRSG (SEQ ID NO: 171) or VTVRSGG (SEQ ID NO: 172).

[0013] In a second aspect, some embodiments of the disclosure provide an immunoglobulin single variable domain, the C-terminus of which comprises or is terminated by an amino acid sequence designated X4X5 or VTVX4X5, wherein X4 is selected from F or R, X5 may be present or absent, and when present, X5 represents an amino acid extension of 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or, for example, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9) amino acid residues, and the amino acid extension is, for example, P, PA, A, AA, AAA, AAAA (SEQ ID NO: 173), PAA, PAAA (SEQ ID NO: 174), AS, AST, ASTK (SEQ ID NO: 175), ASTKG (SEQ ID NO: 176), ASTK GP (SEQ ID NO: 177), PAS, PAST (SEQ ID NO: 178), PASTK (SEQ ID NO: 179), PASTKG (SEQ ID NO: 180), PASTKGP (SEQ ID NO: 181), PT, PTV, PG, PGG, PAG, S, SA, SAA, SAAA (SEQ ID NO: 182), SAS, SSAST (SEQ ID NO: 183), SSASTK (SEQ ID NO: 184), SSASTKG (SEQ ID NO: 185), SSASTKGP (SEQ ID NO: 186), SRSG (SEQ ID NO: 187) or SRSGG (SEQ ID NO: 188); or X4 is S and X5 represents an amino acid extension of 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, also e.g., 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9) amino acid residues, such as A, AA, AAA, AAAA, AS, AST, ASTK, ASTKG, ASTKGP, T, TV, G, GG.

[0014] In some embodiments, an immunoglobulin single variable domain is provided, the C-terminus of which comprises or is terminated with an amino acid sequence designated X4X5 or VTVX4X5, wherein X4 is selected from F or R and X5 represents an amino acid stretch of 1 to 5 amino acid residues, said amino acid stretch being preferably P, PA, S, SA, or X4 is S and X5 represents an amino acid stretch of 1 to 5 amino acid residues, said amino acid stretch being preferably AA, For example, the above X4X5 or VTVX4X5, SAA or VTVSAA, FP or VTVFP, FPA or VTVFPA, RS or VTVRS, or RSA or VTVRSA Any one of the following may be selected:

[0015] In a third aspect, some embodiments of the disclosure provide immunoglobulin single variable domains that comprise framework sequences derived from human germline.

[0016] In some embodiments, there is an amino acid modification at position 112 relative to the human germline framework sequence, for example selected from 112F, 112R, or 112A.

[0017] In some embodiments, there is an amino acid modification at position 113 relative to the human germline framework sequence, for example, 113P, 113A, or 113S.

[0018] In some embodiments, when an amino acid modification of 112F is present, an amino acid modification of 113P, 113A or 113S is simultaneously present at position 113, or when an amino acid modification of 112R is present and / or an amino acid modification of 113P or 113A is present at position 113.

[0019] In some embodiments, the immunoglobulin single variable domain has any one of the combinations of amino acid modifications at positions 112 and 113 selected from 112F and 113P, 112F and 113A, 112F and 113S, 112R and 113P, 112R and 113A, 112R and 113S, 112A and 113P, 112A and 113A, 112A and 113S, 112S and 113P.

[0020] In some embodiments, an immunoglobulin single variable domain is provided, which comprises an amino acid modification at position 112 relative to a human germline framework sequence of 112F or 112R; Optionally, there is further an amino acid modification at position 113 relative to the human germline framework sequence of 113P, or position 113 is S; In some specific embodiments, positions 112 and 113 relative to the human germline framework sequences include: 112F and 113P, 112R and 113S, and 112F and 113S Any one of the amino acid residue combinations is present.

[0021] In a fourth aspect, some embodiments of the present disclosure provide immunoglobulin single variable domains that comprise framework sequences derived from human germline.

[0022] In some embodiments, position 112 relative to the human germline heavy chain framework sequence is an S, including an amino acid modification at position 113 relative to the human germline heavy chain framework sequence, said modification being selected from 113A, 113P.

[0023] In some embodiments, after position 113 there is an amino acid extension of 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, also e.g., 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9) additional amino acid residues, such as A, AA, AAA, AAAA, AS, AST, ASTK, ASTKG, ASTKGP, TV, T, G, GG.

[0024] In some embodiments, the C-terminus can be produced by an amino acid modification (eg, substitution or replacement, deletion, addition) such that the immunoglobulin single variable domain comprises the amino acid modification.

[0025] In some embodiments, the immunoglobulin single variable domain may further comprise: 1) One or more amino acid modifications at positions 14, 41, 108 relative to the human germline framework sequence. In some specific embodiments, the modification at position 14 includes, but is not limited to, 14A, 14K, 14Q, 14T (e.g., P14A, P14K, P14Q, P14T), the modification at position 41 includes, but is not limited to, 41A (e.g., P41A), and the modification at position 108 includes, but is not limited to, 108A, 108Q (e.g., L108A, L108Q). The relevant mutations in WO2013024059A are incorporated herein in their entirety.

[0026] 2) one or more amino acid modifications at positions 11, 13, 14, 15, 82, 82a or 82b, 83, 84, 107, 108 relative to the human germline framework sequence, incorporating herein in their entirety the relevant mutations in WO2012175741A.

[0027] 3) Amino acid modifications at positions 11 and / or 88 relative to human germline framework sequences. In some specific embodiments, modifications at position 11 include, but are not limited to, 11K, 11R, 11D, 11E (e.g., L11K, L11R, L11D, L11E), and modifications at position 88 include, but are not limited to, 88E, 88D, 88R, A8K (e.g., A88E, A88D, A88R, A88K). In some specific embodiments, the immunoglobulin single variable domain is 11E / 88E, 11E / 88D, 11E / 88K, 11E / 88R, 11D / 88E, 11D / 88D, 11D / 88K, 11D / 88R, 11K / 88E, 11K / 88D, 11K / 88K, 11K / 88R, 11R / 88E, 11R / 88D, 11R / 88K, or 11R / 88R (e.g., L11E / A88E, L11E / A88D, L11E / A88K, L11E / A88R, L11D / A88E, L11D / A88D, L11D / A88K, L11D / A88R, L11K / A88E, L11K / A88D, L11K / A88K, L11K / A88R, L11R / A88E, L11R / A88D, L11R / A88K or L11R / A88R). The relevant mutations in WO2016118733 are incorporated herein in their entirety.

[0028] In some embodiments, the human germline framework sequences may be human germline heavy chain framework sequences.

[0029] In some embodiments, the modified immunoglobulin single variable domains provided herein above have reduced side effects and / or improved safety, e.g., reduced immunogenicity, compared to unmodified immunoglobulin single variable domains.

[0030] In some specific embodiments, the modified immunoglobulin single variable domains provided herein have reduced binding to, for example, pre-ADA or ADA present in serum, compared to an unmodified immunoglobulin single variable domain. Reduced binding ability means that the molecule binds to pre-ADA (or ADA) with reduced affinity or reduced avidity. For example, the K D is the K of unmodified sdAb to pre-ADA (or ADA). D 1.5-fold or more (e.g., 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 8-fold) binding of the modified immunoglobulin single variable domains provided herein above to pre-ADA (or ADA) is reduced by 10% or more (e.g., 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 90%) compared to binding of the unmodified sdAb to pre-ADA (or ADA), and detection methods that can be employed are conventional in the art, such as the ELISA shown in Example 1 of the present disclosure. D refers to the equilibrium dissociation constant, and those skilled in the art will recognize D It is understood that the smaller the value of , the stronger the binding.

[0031] In some embodiments, the immunoglobulin single variable domain provided in the present disclosure may be a single domain antibody (sdAb) or a nanobody, such as a VH sdAb or a VHH. The immunoglobulin single variable domain may be human or non-human, such as a human VH sdAb or a humanized camelid VHH. The immunoglobulin single variable domain (e.g. sdAb, nanobody) may be phage-displayed or chimeric, camelid, humanized. The immunoglobulin single variable domain (e.g. sdAb, nanobody) may be sequence-optimized, such as modified by humanization, affinity maturation, removal of T-cell epitopes, reduction of antibody deamidation and / or reduction of antibody isomerization. The immunoglobulin single variable domains (e.g., sdAbs) may be post-translationally modified, such as by cleavage of leader sequences, addition of various sugar moieties in various glycosylation and phosphorylation modes, deamidation, oxidation, disulfide bond scrambling, isomerization, truncation of C-terminal lysines, and cyclization of N-terminal glutamines.

[0032] In some embodiments, the binding (e.g., monovalent binding) of the immunoglobulin single variable domain provided herein to a target antigen comprises specific binding to a CDR region (e.g., including CDR1, CDR2, CDR3) of the target antigen, and the sdAb has a K D and binds to a target antigen with a K within the range of, for example, about 500 nM to about 10 pM, about 200 nM to about 10 pM, about 50 nM to about 10 pM, or about 10 nM to about 10 pM. D and binds to the target antigen.

[0033] In some embodiments, the immunoglobulin single variable domains provided in this disclosure include variants thereof, which have one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid mutations compared to the parent immunoglobulin single variable domain, which may be conservative replacements, substitutions or modifications, and / or deletions, additions that do not affect function, which may occur in the CDR and / or FR regions, and which have a similar or very similar biological function, binding affinity to pre-ADA or ADA compared to the parent immunoglobulin single variable domain. In some specific embodiments, the variants are, for example, amino acid conservative substitution variants of the immunoglobulin single variable domain.

[0034] In some embodiments, the immunoglobulin single variable domain of the disclosure is fused or conjugated to another molecule and is present in the form of a fusion or conjugate, including, but not limited to, another immunoglobulin single variable domain, a protein or polypeptide, a polypeptide, an antibody or an antigen-binding fragment thereof, an Fc region of an immunoglobulin, a PEG molecule. In some specific embodiments, the immunoglobulin single variable domain of the disclosure is located or exposed at the C-terminus of the fusion or conjugate, e.g., when the fusion or conjugate consists of one polypeptide chain, the immunoglobulin single variable domain of the disclosure is located or exposed at the C-terminus of the polypeptide chain, and when the fusion or conjugate is polymerized with two or more polypeptide chains, the immunoglobulin single variable domain of the disclosure is located or exposed at the C-terminus of at least one of the polypeptide chains, or at the C-terminus of two or more polypeptide chains.

[0035] Proteins or Polypeptides The present disclosure provides proteins or polypeptides comprising at least one (eg, 1, 2, 3, 4, 5, 6, 7, 8) of the immunoglobulin single variable domains of the present disclosure.

[0036] In some embodiments, the protein or polypeptide is a fusion protein or polypeptide of an immunoglobulin single variable domain of the disclosure and another polypeptide (eg, a therapeutic polypeptide).

[0037] In some embodiments, the at least one immunoglobulin single variable domain is located at the C-terminus of the protein or polypeptide.

[0038] In some embodiments, the protein or polypeptide is an antibody (including an antigen-binding fragment thereof).

[0039] In some embodiments, the antibody is a monoclonal antibody, a bispecific antibody, a multispecific antibody (eg, a triabody, a tetrabody).

[0040] In some embodiments, the antibody is monovalent, bivalent, trivalent, tetravalent, pentavalent, or hexavalent.

[0041] In some embodiments, the antibody comprises a heavy chain and a light chain and the at least one immunoglobulin single variable domain is located at the C-terminus of the heavy chain and / or the C-terminus of the light chain.

[0042] In some embodiments, when the protein or polypeptide (e.g., an antibody) is composed of one polypeptide chain, the immunoglobulin single variable domain of the disclosure is located at the C-terminus of the polypeptide chain, when the protein or polypeptide (e.g., an antibody) is composed / polymerized of two polypeptide chains, the immunoglobulin single variable domain of the disclosure may be located at the C-terminus of one or two of the two polypeptide chains, when the protein or polypeptide (e.g., an antibody) is composed / polymerized of three, four or more polypeptide chains, the immunoglobulin single variable domain of the disclosure may be located at the C-terminus of one, two, more or all of the polypeptide chains. The present disclosure does not exclude the linkage of other immunoglobulin variable domains, Fab, Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single domain antibodies (e.g., VH sdAb or VL sdAb or VHH), scFv, polypeptides to the N-terminus of the polypeptide chain.

[0043] In some embodiments, the protein or polypeptide (e.g., an antibody) comprises an immunoglobulin Fc region, e.g., the Fc region is a human IgG1, IgG2, IgG3, or IgG4 Fc region. In some specific embodiments, the Fc region has increased or decreased effector function, the effector function being antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC). In some specific embodiments, the Fc region has enhanced or diminished binding to FcγR. In some specific embodiments, the Fc region comprises a heavy chain mismatch prevention mutation, e.g., knob-into-hole (KIH). In some specific embodiments, an exemplary IgG1 with enhanced effector function is The Fc region may include the following substitutions or any combination thereof: S239D, S239E, S239K, F241A, V262A, V264D, V264L, V264A, V264S, D265A, D265S, D265V, F296A, Y296A, R301A, I332E, or any combination thereof, for example, S239D / I332E, S239D / A330S / I332E, S239D / A330L / I332E, S298A / D333A / K334A, P247I / A339D, P247I / A339Q, D280H / K290S, D280H / K290S / S298D, D280H / K290S / S298V, F243L / R292P / Y300L, F243L / R292P / Y300L / P396L, F243L / R292P / Y300L / V305I / P396L, G236A / S239D / I332E, K326A / E333A, K326W / E333S, K290E / S298G / T299A, K290N / S298G / T299A, K290E / S298G / T299A / K326E or K290N / S298G / T299A / K326E.In some embodiments, exemplary IgG Fc regions with reduced effector function include L234A / L235A, L234F / L235E / D265A, or L234A / L235A / G237A / P238S / H268A / A330S / P331S, K214T / E233P / L234V / L235A / G236 deletion / A327G / P331A / D365E / L358M on IgG1, V234A / G237A, V234A / G237A / P238S / H268A / V309L / A330S / P331S, H268Q / V309L / A330S / P331S, S IgG1, IgG2, IgG3 or N297A substitutions may also be used, such as 267E / L328F, F234A / L235A on IgG4, S228P / F234A / L235A, S228P / F234A / L235A / G236 deletion / G237A / P238S, S228P / F234A / L235A / G237A / P238S, N297A on IgG1, IgG2, IgG3 or IgG4, and further comprising a hybrid IgG2 / 4 Fc domain, e.g. an Fc having residues 117-260 from IgG2 and residues 261-447 from IgG4.

[0044] In some embodiments, the protein or polypeptide (e.g., an antibody) has the N-terminus of an immunoglobulin variable domain of the present disclosure linked directly or via a linker to the C-terminus of an Fc region (e.g., its CH3). In some embodiments, the antibody has the N-terminus of an immunoglobulin variable domain of the present disclosure linked directly or via a linker to the C-terminus of the antibody's light chain (e.g., its CL). The present disclosure does not exclude the linkage of other immunoglobulin variable domains, Fab, Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single domain antibodies (e.g., VH sdAb or VL sdAb or VHH), scFv, polypeptides to the N-terminus of the heavy and / or light chain. In some embodiments, the linker is conventional, e.g., (G m S n ) h or (GGNGT) h or (YGNGT) h or (EPKSS) hIn which m and n are each independently selected from integers of 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8), and h is independently selected from integers of 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). For example, the linker is G4S, GS, GAP, (G4S)2, (G4S)3, (G4S)4, (G4S)5, or ASGS.

[0045] In some embodiments, the protein or polypeptide (e.g., an antibody) comprises at least two (e.g., 2, 3, 4, 5, 6, 7, 8) antigen binding domains, and in some specific forms, the at least two (e.g., 2, 3, 4, 5, 6, 7, 8) antigen binding domains target two different antigens, or target multiple (e.g., 3, 4) different antigens, or target two or multiple (e.g., 3, 4, 5, 6) different domains or epitopes of the same antigen.

[0046] In some embodiments, the antigens include, but are not limited to, PD-1, PD-L1, CTLA4, TIGIT, CD40, TNFα, TNFR (including TNFR1), VEGF, IL-1R, IL-6R, IL-4, IL-5, IL-13, DC-SIGN, ASGPR, HSA, TGFβR2, and 4-1BB.

[0047] In some embodiments, the protein or polypeptide (e.g., an antibody) is therapeutic or prophylactic for treating or preventing a disease / condition, including, but not limited to, a cell proliferative disorder (e.g., a tumor), a metabolic disorder, an autoimmune disease, or an infectious disease.

[0048] In some embodiments, the protein or polypeptide (e.g., an antibody) further comprises a half-life prolonging domain, such as albumin or polypeptide (HSA), an HSA binding domain (e.g., an anti-HSA antibody, an anti-HSA single domain antibody), a PEG molecule, or an immunoglobulin Fc region.

[0049] The present disclosure further provides a complex or derivative comprising an sdAb according to the present disclosure, or comprising a protein or polypeptide according to the present disclosure. The complex may, for example, comprise any detectable label.

[0050] Exemplary Immunoglobulin Single Variable Domains As an example of an immunoglobulin single variable domain according to the present disclosure described above, the present disclosure further provides a 4-1BB protein or polypeptide comprising an immunoglobulin single variable domain that specifically binds to 4-1BB.

[0051] In some embodiments, the immunoglobulin single variable domain that specifically binds to 4-1BB comprises CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 are CDR1, CDR2, and CDR3 in the amino acid sequence set forth in any one of SEQ ID NOs: 6, 15 to 18, and the CDR1, CDR2, and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system.

[0052] In some embodiments, the immunoglobulin single variable domain that specifically binds to 4-1BB comprises any one or any combination of the CDR1, CDR2, and CDR3 described above.

[0053] In some embodiments, the amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain that specifically binds to 4-1BB are set forth in SEQ ID NOs: 7, 8, 9, or SEQ ID NOs: 7, 8, 18, respectively, which are the CDRs defined by the Kabat numbering system.

[0054] In some embodiments, the immunoglobulin single variable domain that specifically binds to 4-1BB has been modified by humanization, affinity maturation, removal / reduction of T cell epitopes (TCE), reduction of antibody deamidation and / or reduction of antibody isomerization. In some specific embodiments, the heavy chain framework regions (FR) of the human germline template used for the humanization are derived from IGHV3-64*04, IGHV3-23*03 and / or IGHV3-74*01. In some embodiments, FR1 is derived from IGHV3-64*04, FR2 is derived from IGHV3-23*03 and FR3 is derived from IGHV3-74*01.

[0055] In some embodiments, the amino acid sequence of the immunoglobulin single variable domain that specifically binds to 4-1BB is set forth in any one of SEQ ID NOs: 6, 15 to 18, and 20 to 35, respectively, or has at least 90%, at least 95%, identity thereto.

[0056] In this disclosure, "at least 80% (sequence) identity" refers to at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% (sequence) identity. "at least 90% (sequence) identity" covers at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% (sequence) identity, and "at least 95% (sequence) identity" covers at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% (sequence) identity.

[0057] In some embodiments, an immunoglobulin single variable domain that specifically binds to 4-1BB is provided, the immunoglobulin single variable domain having an amino acid sequence set forth in any one of SEQ ID NOs: 21, 30 to 35, and having reduced binding to pre-ADA (or ADA) compared to an sdAb that specifically binds to 4-1BB set forth in any one of SEQ ID NOs: 20, 22 to 29.

[0058] In some embodiments, a protein or polypeptide (including an antibody) is provided that comprises an immunoglobulin single variable domain that specifically binds to 4-1BB. The immunoglobulin single variable domain that specifically binds to 4-1BB may be one or more (e.g., 1, 2, 3, 4, 5, 6), and may be the same or different. The antibody may be, for example, a mono-, bi- or multispecific antibody.

[0059] In some embodiments, the protein or polypeptide (including an antibody) further comprises a human immunoglobulin Fc region, for example, the Fc region is an Fc region of human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc region is an Fc region with reduced effector function, for example, reduced ADCC, ADCP, or CDC. In some embodiments, the Fc region is represented by any one of SEQ ID NOs: 10 to 12, or has at least 80%, at least 90%, sequence identity thereto.

[0060] In some embodiments, the immunoglobulin single variable domain that specifically binds to 4-1BB has at least one activity selected from the following: (a)≦10 -7 K D binding to human 4-1BB or an epitope thereof at a value (b) When not cross-linked with FcγRIIb (i.e., CD32b), it weakly activates or does not activate the 4-1BB signaling pathway, e.g., at an antibody concentration of 100 n, the degree of activation when not cross-linked with FcγRIIb is 10% or less of the activity under saturating antibody concentration conditions when cross-linked with FcγRIIb; (c) When cross-linked with FcγRIIb, it relatively strongly activates or strongly activates the 4-1BB signaling pathway, e.g., EC 50 is less than 1 nM; (d) activating T cells and / or promoting the proliferation of T cells; and (e) inhibiting tumor growth.

[0061] Among these, for detection of activation of the 4-1BB signaling pathway in (b) and (c), for example, the 4-1BB / NF-κB luciferase reporter gene assay in Example 5 is referred to.

[0062] In some embodiments, the K of an immunoglobulin single variable domain binding 4-1BB that specifically binds 4-1BB is D The value is ≦1×10 -7 M, for example, ≦1×10 -8 M, or ≦1×10 -9 M, or ≦1×10 -10 It may be M.

[0063] In some embodiments, the immunoglobulin single variable domain that specifically binds to 4-1BB can inhibit tumor growth by at least about 10%, e.g., at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%.

[0064] In some embodiments, a protein or polypeptide (e.g., an antibody) is provided that binds to or competitively binds to the same epitope as an sdAb that specifically binds to 4-1BB of the present disclosure, or that blocks the binding to 4-1BB of an sdAb that specifically binds to 4-1BB of the present disclosure, or whose binding to 4-1BB is blocked by an sdAb that specifically binds to 4-1BB of the present disclosure.

[0065] Polynucleotides and Vectors There is provided a nucleic acid molecule encoding the immunoglobulin single variable domain of the present disclosure, a protein or polypeptide (including an antibody) comprising the immunoglobulin single variable domain. The nucleic acid may be RNA, DNA or cDNA. According to some embodiments of the present disclosure, the nucleic acid is essentially an isolated nucleic acid.

[0066] The nucleic acid according to the present disclosure may be in the form of, present in, and / or part of a vector, such as a plasmid, cosmid, YAC, or viral vector. The vector may in particular be an expression vector, i.e. a vector that allows for the expression of the PD-1 binding protein or polypeptide in vitro and / or in vivo (i.e. in a suitable host cell, host organism, and / or expression system). Such an expression vector generally comprises at least one nucleic acid according to the present disclosure, operably linked to one or more suitable expression control elements (e.g. promoters, enhancers, terminators, etc.). The selection of such elements and their sequences for expression in a particular host is within the knowledge of a person skilled in the art. Regulatory elements and other elements useful or necessary for the expression of the sdAb of the present disclosure, proteins or polypeptides (including antibodies) comprising said sdAb, are for example promoters, enhancers, terminators, integration factors, selection markers, leader sequences, reporter genes.

[0067] The nucleic acids according to the present disclosure may be prepared or obtained by known methods (e.g., automated DNA synthesis and / or recombinant DNA techniques) based on information about the amino acid sequence of the polypeptide according to the present disclosure, and / or may be isolated from a suitable natural source.

[0068] host cell Recombinant host cells are provided that express or are capable of expressing an immunoglobulin single variable domain according to the disclosure, a protein or polypeptide (including an antibody) comprising the immunoglobulin single variable domain, and / or that comprise a nucleic acid or vector of the disclosure. In some embodiments, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

[0069] Bacterial cells include, for example, cells of gram-negative strains (e.g., Escherichia coli, Proteus and Pseudomonas strains) and gram-positive strains (e.g., Bacillus, Streptomyces, Staphylococcus and Lactococcus strains).

[0070] Fungal cells include, for example, cells of species of Trichoderma, Neurospora, and Aspergillus, or cells of species of Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula.

[0071] Mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

[0072] However, the present disclosure may also be used with amphibian cells, insect cells, plant cells and any other cells known in the art for expressing heterologous proteins or polypeptides.

[0073] Preparation method The present invention provides a method for preparing the immunoglobulin single variable domain according to the present disclosure, a protein or polypeptide (including an antibody) comprising the immunoglobulin single variable domain, which comprises expressing the target protein or polypeptide in the host cell and isolating the target protein or polypeptide from the host cell. Optionally, a purification step may be included, for example, by purifying on an A or G Sepharose FF column with a conditioned buffer, washing off non-specifically bound components, and eluting the bound antibody by a pH gradient method, detecting it by SDS-PAGE, and collecting it. Optionally, the product is filtered and concentrated by conventional methods. Soluble compounds and polymers may be removed by conventional methods such as molecular sieving, ion exchange, etc. The resulting product must be immediately frozen, for example at -70°C, or lyophilized.

[0074] The engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified by conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into an expression vector. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. Mammalian expression systems lead to glycosylation of the antibody, especially at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded in serum-free medium in a bioreactor to produce the antibody. The culture medium into which the antibody is secreted can be purified and collected by conventional techniques. The antibody can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can be removed by conventional methods such as molecular sieving, ion exchange, etc.

[0075] composition Compositions are provided that include an immunoglobulin single variable domain according to the disclosure, a protein or polypeptide (including an antibody) that includes the immunoglobulin single variable domain, etc. For example, pharmaceutical compositions are provided that include a disease (e.g., cancer) treating, mitigating, or prophylactically effective amount of an immunoglobulin single variable domain according to the disclosure, a protein or polypeptide (including an antibody) that includes the immunoglobulin single variable domain, etc., and at least one pharma- ceutically acceptable excipient, diluent, or vector.

[0076] In some specific embodiments, a unit dose of the pharmaceutical composition may contain 0.01 to 99% by weight of an immunoglobulin single variable domain according to the present disclosure, or a protein or polypeptide (including an antibody) comprising said immunoglobulin single variable domain, or the content of an immunoglobulin single variable domain according to the present disclosure, or a protein or polypeptide (including an antibody) comprising said immunoglobulin single variable domain in a unit dose of the pharmaceutical composition is 0.1 to 2000 mg, and in some specific embodiments, 1 to 1000 mg.

[0077] In some embodiments, an article of manufacture or product is provided that includes an immunoglobulin single variable domain according to the present disclosure, or a protein or polypeptide (including an antibody) that includes the immunoglobulin single variable domain. Optionally, the article of manufacture includes a container and a label. The container is, for example, a vial, syringe, or test tube. The container holds a composition that is effective for treating a medical condition (e.g., cancer, autoimmune disease, metabolic disease). A label on or associated with the container indicates that the composition is used to treat a selected medical condition (e.g., cancer, autoimmune disease, metabolic disease).

[0078] In some embodiments, there is provided a pharmaceutical composition comprising an immunoglobulin single variable domain according to the present disclosure, a protein or polypeptide (including an antibody) comprising said immunoglobulin single variable domain, optionally in an amount effective to treat or alleviate a disease (e.g. cancer, an autoimmune disease, a metabolic disease), and optionally further comprising at least one pharma- ceutical acceptable excipient, diluent or vector.

[0079] When the immunoglobulin single variable domain is an immunoglobulin single variable domain that specifically binds 4-1BB, the disease state can be, for example, a cancer selected from lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, rectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, renal cancer, squamous cell carcinoma, hematological cancer, or any combination thereof.

[0080] Methods of Treatment and Pharmaceutical Use The present disclosure also provides methods of using the immunoglobulin single variable domains, proteins or polypeptides (including antibodies) comprising the immunoglobulin single variable domains, and pharmaceutical compositions thereof for the treatment, mitigation, prevention, or diagnosis of a disease or condition.

[0081] In some embodiments, methods are provided for ameliorating, alleviating, treating or preventing a disease, comprising administering to a subject an ameliorating, alleviating, treating or preventing effective amount of an immunoglobulin single variable domain according to the present disclosure, a protein or polypeptide (including an antibody) comprising said immunoglobulin single variable domain, and pharmaceutical compositions thereof.

[0082] In some embodiments, there is provided the use of an immunoglobulin single variable domain according to the present disclosure, a protein or polypeptide (including an antibody) comprising said immunoglobulin single variable domain, and a pharmaceutical composition thereof, for the preparation of a medicament for ameliorating, alleviating, treating or preventing a disease.

[0083] In some embodiments, the disease is a proliferative condition or any other disease or condition characterized by uncontrolled cell proliferation (e.g., cancer, although cancer and tumor may be used interchangeably in this disclosure). In some embodiments, the cancer is a solid tumor or a hematological tumor. In some embodiments, the cancer is advanced or metastatic. In some embodiments, the cancer is selected from lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, rectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, renal cancer, squamous cell carcinoma, hematological cancer, or any combination thereof.

[0084] In some embodiments, a method of treating or preventing an infectious disease or an autoimmune disease in a subject is provided, comprising administering to the subject a therapeutically or prophylactically effective amount of an immunoglobulin single variable domain according to the present disclosure, a protein or polypeptide (including an antibody) comprising said immunoglobulin single variable domain, and pharmaceutical compositions thereof. In some embodiments, the infectious disease is a pathogen infection, such as HIV, HCV, HBV, characterized by different degrees of dysfunction of virus-specific T cell responses. In some embodiments, the autoimmune disease is selected from systemic lupus erythematosus, myasthenia gravis, multiple sclerosis, insulin-dependent diabetes mellitus, Crohn's disease, rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, and psoriatic arthritis.

[0085] detection The present disclosure provides for the detection of immunoglobulin single variable domains, proteins or polypeptides (including antibodies) that contain said immunoglobulin single variable domains, which can be used in vivo or in vitro.

[0086] Further provided is a method, system or device for detecting side effects (including immunogenicity, binding to ADA or pre-ADA) caused by said immunoglobulin single variable domain, a protein or polypeptide (including an antibody) comprising said immunoglobulin single variable domain, which comprises treating a sample, e.g. a serum sample from a subject, with an immunoglobulin single variable domain according to the present disclosure, a protein or polypeptide (including an antibody) comprising said immunoglobulin single variable domain, wherein said subject may be administered an immunoglobulin single variable domain according to the present disclosure, a protein or polypeptide (including an antibody) comprising said immunoglobulin single variable domain for the treatment or prevention of a disease.

[0087] In some embodiments, an immunoglobulin single variable domain according to the present disclosure, a protein or polypeptide (including an antibody) comprising said immunoglobulin single variable domain, is used in a method to predict whether it will cause protein or polypeptide interference, for example to determine whether said immunoglobulin single variable domain, a protein or polypeptide (including an antibody) comprising said immunoglobulin single variable domain, has a high or increased risk of causing protein or polypeptide interference or binding to an interfering factor (e.g., pre-ADA) in human blood or serum.

[0088] In some embodiments, a method is provided for determining whether an immunoglobulin single variable domain, a protein or polypeptide (including an antibody) comprising said immunoglobulin single variable domain generates protein or polypeptide interference in an immunoassay (e.g., an ADA or pre-ADA assay), the method comprising: (i) contacting any of said immunoglobulin single variable domains, proteins or polypeptides (including antibodies) comprising said immunoglobulin single variable domains with an antibody obtained from a human subject and selected / isolated for its ability to recognise and / or bind to the C-terminus of an immunoglobulin single variable domain that is C-terminally terminated with the amino acid sequence VTVSS (an "unmodified immunoglobulin single variable domain" in the present disclosure); (ii) determining whether any of the immunoglobulin single variable domains, proteins or polypeptides (including antibodies) comprising the immunoglobulin single variable domains bind to the antibody in the immunoassay; Binding to said antibody in step (ii) means that said any said immunoglobulin single variable domain, protein or polypeptide (including antibody) comprising said immunoglobulin single variable domain is capable of (or has a high or increased risk of) causing such protein or polypeptide interference.

[0089] In some embodiments, a method is provided for predicting the extent to which an immunoglobulin single variable domain, a protein or polypeptide comprising an immunoglobulin single variable domain, or an antibody will generate protein or polypeptide interference in an immunoassay, the method comprising: (i) contacting any immunoglobulin single variable domain, protein or polypeptide (including antibody) comprising said immunoglobulin single variable domain according to the present disclosure with an antibody obtained from a human subject and selected / isolated for its ability to recognize and / or bind to the C-terminus of an immunoglobulin single variable domain, the C-terminus of which is terminated with the amino acid sequence VTVSS; (ii) determining whether said immunoglobulin single variable domain, a protein or polypeptide (including an antibody) comprising said immunoglobulin single variable domain binds to said antibody in said immunoassay, and the strength of said binding (in some embodiments, said strength of binding is compared to the binding of an immunoglobulin single variable domain terminated at its C-terminus with the amino acid sequence VTVSS to said antibody under the same experiment or comparable detection conditions); performing an immunoassay comprising at least Here, the occurrence of binding or a strong degree of binding in step (ii) means that the immunoglobulin single variable domain, the protein or polypeptide (including an antibody) comprising the immunoglobulin single variable domain may cause protein or polypeptide interference or has a high or increased risk of causing protein or polypeptide interference, and the absence of binding or a weak degree of binding in step (ii) means that the immunoglobulin single variable domain, the protein or polypeptide (including an antibody) comprising the immunoglobulin single variable domain does not cause protein or polypeptide interference or has a low risk of causing protein or polypeptide interference, for example, the immunoassay is an ELISA assay.

[0090] In some specific embodiments, the antibody is a polyclonal antibody. In some forms, the antibody is a polyclonal antibody obtainable by a method comprising at least one immunoaffinity chromatography step from a biological sample obtained from a subject and applied as a source material for obtaining a polyclonal antibody, and optionally one or more other steps for isolating and / or purifying the polyclonal antibody from said sample, in which an immunoglobulin single variable domain, a protein or polypeptide (including an antibody) comprising said immunoglobulin single variable domain is used as an affinity moiety or antigen, wherein the immunoglobulin single variable domain used as the affinity moiety or antigen is terminated at its C-terminus with the amino acid sequence VTVSS.

[0091] In some embodiments, a method is provided for reducing the tendency of an immunoglobulin single variable domain, the C-terminus of which is terminated with the sequence VTVSS (i.e., an "unmodified immunoglobulin single variable domain" in the context of this disclosure), a protein or polypeptide (including an antibody) comprising said sdAb, to generate protein or polypeptide interference in an anti-drug antibody assay, or the use of a C-terminal modification for reducing the tendency of an immunoglobulin single variable domain, the protein or polypeptide (including an antibody) comprising said sdAb, to generate protein or polypeptide interference comprising modifying the C-terminus of an immunoglobulin single variable domain by the method provided in this disclosure such that it has a reduced or essentially reduced tendency to generate protein or polypeptide interference compared to an immunoglobulin single variable domain with the sequence VTVSS at its terminus (i.e., an "unmodified immunoglobulin single variable domain" in the context of this disclosure).

[0092] In some embodiments, the in vitro detection method, system, or device includes, for example, (i) contacting a sample with an immunoglobulin single variable domain according to the disclosure, a protein or a polypeptide (including an antibody) comprising said immunoglobulin single variable domain; (ii) detecting a complex formed between any immunoglobulin single variable domain, a protein or polypeptide (including an antibody) comprising said immunoglobulin single variable domain and the sample; and / or (iii) contacting a reference sample (e.g., a control sample) with any immunoglobulin single variable domain, protein or polypeptide (including an antibody) comprising said immunoglobulin single variable domain, and (iv) determining the extent of complex formation by comparison with a reference sample, where a change (e.g., a statistically significant change) formed in the complex in the sample or subject compared to in the control sample or subject indicates the extent of binding of any of the immunoglobulin single variable domains in the sample, or a protein or polypeptide (including an antibody) comprising the immunoglobulin single variable domain, to pre-ADA (or ADA) in vivo in the subject from the sample; may include.

[0093] Detection may include determining the location or time of complex formation. The immunoglobulin single variable domain, the protein or polypeptide (including antibodies) comprising said immunoglobulin single variable domain may also be labeled with a detectable substance, and detection may be achieved by said label. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances and radioactive substances. Conventional detection assays such as, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or tissue immunohistochemistry may be used. For detection purposes, the sdAb of the present disclosure, the protein or polypeptide (including antibodies) comprising said immunoglobulin single variable domain may be labeled with a fluorophore chromophore.

[0094] In some embodiments, a reagent kit is further provided, which includes the binding protein or polypeptide, polynucleotide, and may further include a diagnostic manual. The reagent kit may further include at least one additional reagent, such as a marker or an additional diagnostic agent. For in vivo use, the antibody may be prepared as a pharmaceutical composition.

[0095] This disclosure incorporates the detection methods of WO2012175741A in their entirety.

[0096] Definition of Terms In order that this disclosure may be more readily understood, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined in this disclosure, all other technical and scientific terms used in this disclosure have the meanings commonly understood by those of ordinary skill in the art.

[0097] Unless the context clearly indicates otherwise, throughout the specification and claims, the words "comprise," "having," "containing," and the like, are to be understood to have an inclusive meaning, i.e., "including but not limited to," rather than an exclusive or exhaustive meaning.

[0098] The three-letter and one-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem, 243, p3558 (1968).

[0099] "4-1BB protein or polypeptide" or "4-1BB" refers to CD137, also known as tumor necrosis factor receptor superfamily 9, a member of the TNF receptor superfamily (TNFRSF) and CD8 + and CD4 + 4-1BB is a costimulatory molecule expressed on the cell surface of T cells, regulatory T cells (Tregs), NK cells and NKT cells, B cells, and neutrophils. 4-1BB belongs to costimulatory molecules and is expressed after immune cells are activated. Human 4-1BB protein or polypeptide has NCBI accession number NP_001552.2. In the present disclosure, "4-1BB" may optionally include any of the proteins or polypeptides or fragments or variants thereof, including (but not limited to) the known or wild-type 4-1BB described in the present disclosure, and any naturally produced splice variants, amino acid variants, or isoforms.

[0100] "Antibody" or "immunoglobulin" is used in the broadest sense and covers a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding portions), provided they exhibit the desired antigen-binding activity. "Antigen-binding fragments" include, but are not limited to, single chain antibodies (i.e., full length heavy and light chains), Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single domain antibodies (e.g., VH / VL or VHH), scFv, bivalent, trivalent or tetravalent antibodies, Bis-scFv, diabody, tribody, triabody, tetrabody and epitope-binding fragments of any one of the above (see, e.g., Holliger and Hudson, 2005, Nature Biotech. 23(9):1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217).

[0101] "Immunoglobulin single variable domain" is generally used to refer to an immunoglobulin variable domain (which may be a heavy or light chain domain and includes a VH, VHH or VL domain) that is capable of forming a functional antigen-binding site when it does not interact with other variable domains (e.g. in the absence of the necessary VH / VL interactions between the VH and VL domains of a conventional four-chain monoclonal antibody). Examples of "immunoglobulin single variable domains" are nanobodies (including VHH, humanized VHH, camelized VH (e.g. camelized human VH)), IgNARs, domains, (single domain) antibodies as or derived from a VH domain (e.g. dAbs TM ) and (single domain) antibodies as or derived from the VL domain (e.g., dAbs TM). Immunoglobulin single variable domains based on and / or derived from heavy chain variable domains (e.g. VH or VHH domains) are generally preferred. One specific example of an immunoglobulin single variable domain is a "VHH domain" (or abbreviated as "VHH") as defined below. In the present disclosure, domain antibody (dAb), single domain antibody, single domain antibody (sdAb) may be used interchangeably.

[0102] "VHH" is also called nanobody, VHH antibody, VHH antibody fragment, VHH domain, heavy chain single domain antibody (sometimes called single domain antibody), and refers to the variable region of a heavy chain antibody (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains", Nature 363, 446-448 (1993)). "VHH" is used to distinguish it from the heavy chain variable region (VH) in a conventional tetrapeptide chain antibody. Due to its domain specificity, VHH can bind to an epitope alone without the need for another antigen binding domain. In a conventional tetrapeptide chain antibody, both the VH domain and the VL domain are required to recognize the epitope together. The VHH domain is a small, stable and efficient antigen recognition unit formed by a single immunoglobulin domain. In some embodiments, for example with respect to 4-1BB, the terms "anti-4-1BB single domain antibody", "4-1BB VHH", "4-1BB VHH antibody" and "4-1BB nanobody" may be used interchangeably.

[0103] VHH generally consists of 110-120, e.g. 112-115 amino acids. However, it should be noted that longer and shorter sequences are also applicable for the purposes described in this disclosure. Other structural and functional properties of VHH and VHH-containing polypeptides can be summarized as follows: VHH functionally binds to antigen when VL is not present and does not interact with VL. The VH and VL domains of a conventional tetrapeptide chain antibody are not suitable for practical use as antigen-binding molecules or immunoglobulin single variable domains alone, and the VH and VL domains need to be in a form or combined (e.g. in the form of Fab or scFv) to provide a functional antigen-binding unit. VHH can be used as a single and relatively small functional antigen-binding unit, domain or polypeptide. Due to their unique properties, the use of VHH domains (either alone or as part of a larger polypeptide) offers a number of significant advantages over the use of conventional VH and VL domains, scFv or conventional antibody fragments (e.g. Fab or F(ab')2 fragments): - only a single domain needs to bind to the antigen with high affinity and high selectivity, so there is no need to have two separate domains and to ensure that the two domains are in the correct spatial conformation and arrangement (e.g. scFvs generally require the use of specifically designed linkers); - the VHH domain can be expressed by a single gene and does not require post-translational folding or modification; - VHH domains can be easily engineered into multivalent and multispecific formats; - VHH domains are highly soluble and have no tendency to aggregate; - VHH domains are highly stable against heat, pH, proteins or polypeptides and other denaturing agents or conditions and therefore do not require the use of refrigeration equipment during preparation, storage or transportation, thus saving costs, time and protecting the environment; - VHH domains are easy and relatively cheap to prepare, as well as on the scale required for production; - VHH domains are relatively small (approximately 15 kDa or 1 / 10 the size of a conventional IgG) compared to conventional tetrapeptide chain antibodies and their antigen-binding fragments, and therefore exhibit high tissue penetration and can be administered in high doses; - VHH domains can exhibit so-called cavity binding properties (compared to conventional VH domains, VHHs have an extended CDR3 loop and can therefore reach target epitopes that cannot be reached by conventional tetrapeptide chain antibodies and their antigen-binding fragments).

[0104] Methods for obtaining VHHs that bind to target antigens or epitopes have already been disclosed in the following documents: R. van der Linden et al., Journal of Immunological Methods, 240 (2000) 185-195; Li et al., J Biol Chem., 287 (2012) 13713-13721; Deffar et al., African Journal of Biotechnology Vol. 8 (12), pp. 2645-2652, 17 June, 2009; and WO94 / 04678.

[0105] A VHH domain can be "humanized" by replacing one or more amino acid residues in the amino acid sequence of the original VHH (e.g., a VHH derived from the Camelidae family) with one or more amino acid residues at the corresponding positions in the VH domain of a human conventional tetrapeptide chain antibody (also referred to as "sequence optimization" in the present disclosure, which may cover other modifications that confer improved properties to the VHH, such as removal of potential post-translational modification sites, in addition to humanization). The humanized VHH domain may comprise one or more fully human framework region sequences, and in one specific embodiment, may comprise human framework region sequences of IGHV3. Methods of humanization include, for example, resurfacing, CDR grafting to a universal framework, framework shuffling, etc. Human FR germline sequences can be obtained, for example, from the ImMunoGeneTics (IMGT) homepage.

[0106] A "domain" (of a protein or polypeptide) refers to a folded protein or polypeptide structure. Generally, a domain is responsible for a single function of a protein or polypeptide. In many cases, it may be added, removed, or transferred to other proteins without losing the function of other portions and / or domains of the protein or polypeptide. In some embodiments of the present disclosure, a "domain" may be used as an antibody-antigen binding site.

[0107] A "variable domain" refers to a domain that essentially consists of four "framework regions", referred to here and below as "framework region 1" (FR1), "framework region 2" (FR2), "framework region 3" (FR3), and "framework region 4" (FR4), respectively, which are separated by three "complementarity determining regions", referred to here and below as "complementarity determining region 1" (CDR1), "complementarity determining region 2" (CDR2), and "complementarity determining region 3" (CDR3). The general structure (or sequence) of a variable domain may therefore be depicted as FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The variable domain contains the antigen binding site and thus confers specificity for the antigen.

[0108] "Framework region (FR)" or "framework" refers to the portion of a variable domain that serves as a stent for the CDRs.

[0109] For example, as shown in FIG. 2 of Riechmann and Muyldermans, J. Immunol. Methods 231, 25-38 (1999), the amino acid residues used in VHH domains are numbered according to the general numbering scheme for VH domains by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91). However, it should be noted that, as known in the art for VH and VHH domains, the total number of amino acid residues in each CDR may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (i.e., one or more positions according to the Kabat numbering may not be occupied in the actual sequence or the actual sequence may contain more amino acid residues than the number allowed by the Kabat numbering). This means that, in general, the numbers according to Kabat may or may not correspond to the actual number of amino acid residues in the actual sequence.

[0110] One of skill in the art can determine the amino acid sequence boundaries of an antibody CDR according to any one of a number of known numbering schemes, including those described in Kabat et al., supra (the "Kabat" numbering scheme), Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 (the "Chothia" numbering scheme), MacCallum et al., 1996, J. Mol. Biol., 262:732-745 (the "Contact" numbering scheme), Lefran et al., Dev. Comp. Immunol., 2003, 27:55-77 (the "IMGT" numbering scheme), and Honegge & Pluckthun, J. Mol. Biol., 2001, 309:657-70 (the "AHo" numbering scheme), each of which is incorporated by reference in its entirety. Alternative methods for numbering the amino acid residues of VH domains are known in the art and can be used for VHH domains as well, however, unless otherwise indicated, the specification, claims and figures of this disclosure will follow the numbering appropriate for VHH domains according to Kabat, as set forth above.

[0111] A "humanized" antibody is an antibody that retains the reactivity of a non-human antibody while having reduced immunogenicity in humans, which may be achieved, for example, by retaining the non-human CDR regions and replacing the remainder of the antibody with its human counterpart (i.e., the constant and variable region framework region portions).

[0112] An "affinity matured" antibody, particularly a VHH or domain antibody, has one or more changes in one or more CDRs that increase its affinity for the target antigen compared to its parent antibody. Affinity matured antibodies can be prepared by methods known in the art.

[0113] "Fc region" or "fragment crystallizable region" is used to define the C-terminal region of an antibody heavy chain, including native Fc regions and modified Fc regions. In some embodiments, the Fc region comprises two subunits, which may be the same or different. In some embodiments, the Fc region of a human IgG heavy chain is defined to extend from the amino acid residue at position Cys226 or from Pro230 to its carboxy terminus. Suitable Fc regions for use in the antibodies described herein include the Fc regions of human IgG1, IgG2 (IgG2A, IgG2B), IgG3 and IgG4. In some embodiments, the boundaries of the Fc region may be altered, such as a deletion of the C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region, or a deletion of the C-terminal glycine and lysine (residues 446 and 447 according to the EU numbering system) of the Fc region. Unless otherwise stated, the numbering convention for the Fc region is the EU numbering system, also referred to as the EU index.

[0114] An "unmodified immunoglobulin single variable domain", e.g. an immunoglobulin single variable domain that binds a target antigen, comprises three complementarity determining regions (CDRs) in a framework structure. Considering that in the genetics of naturally occurring immunoglobulin chains, the V region is terminated at the beginning of the CDR3 and the remainder of the CDR3 is provided by the D and J regions (resulting in a VDJ fusion), an immunoglobulin single variable domain according to the present disclosure comprises all of the CDR3 and is terminated at its C-terminus with FR4 residues. A VH sdAb is terminated at its C-terminus with residues LVTVSS. A VHH sdAb is terminated at its C-terminus with residues VTVSS. A VL sdAb is terminated at its C-terminus with VEIKR. A "modified immunoglobulin single variable domain" is an immunoglobulin single variable domain as described in the present disclosure, further having a modification that alters the three-dimensional conformation of the C-terminus of the immunoglobulin single variable domain. Modified immunoglobulin single variable domains include immunoglobulin single variable domains that include C-terminal additions, extensions or tags and / or certain amino acid substitutions as disclosed herein.

[0115] "Pre-drug antibodies" or "pre-ADA" are ADAs that are already present in a subject or individual to whom an agent (e.g., a protein or polypeptide-based agent, further e.g., an antibody agent) is to be administered. Pre-drug antibodies can be in a subject or individual that is being used for the first time in an experiment (i.e., a subject or individual to whom the agent has not been administered before).

[0116] An "antigen" is a molecule with which an immunocompetent vertebrate is immunized so that antibodies that recognize the antigen are produced or an expression library (e.g., phage, yeast or ribosome display libraries, among others) is screened. In this disclosure, an antigen is defined more broadly and includes a target molecule that is specifically recognized by an antibody, and includes portions or mimetics of molecules used in the immunization process to produce antibodies or library screening to select antibodies.

[0117] "Epitope" or, interchangeably, "antigenic determinant" refers to any antigenic determinant on an antigen to which an antibody binds. Antigenic determinants generally comprise chemically active surface groupings of molecules, such as amino acids or sugar side chains, and generally have specific three-dimensional structural characteristics and / or specific charge characteristics. An epitope may be a "linear" epitope or a "conformational" epitope. In a linear epitope, all of the interaction sites between the antigen and the interacting molecule (e.g., an antibody) occur linearly along the primary amino acid sequence of the antigen. In a conformational epitope, the interaction sites occur across amino acid residues that are separated from one another.

[0118] "Specificity" refers to the number of different types of antigens or epitopes that a target antigen-binding molecule or antigen-binding molecule can bind. The specificity can be confirmed based on the affinity and / or avidity of the antigen-binding molecule. The dissociation equilibrium constant (K D Affinity, which is expressed as K, is a measure of the binding strength between an epitope and an antigen-binding site on an antigen-binding molecule. D The smaller the value, the stronger the binding strength between the epitope and the antigen-binding molecule (or affinity, 1 / KD Affinity may be expressed as an association constant (KA), which is: Affinity = 1 / K (K A ) ...

[0119] "Binding affinity" or "affinity" is used in this disclosure as a measure of the strength of a non-covalent interaction between two molecules (e.g., an antibody or portion thereof and an antigen). The binding affinity between two molecules is determined by the dissociation constant (K D ) can be quantified by measuring the kinetics of complex formation and dissociation using, for example, surface plasmon resonance (SPR) techniques (Biacore). D The rate constants corresponding to the association and dissociation of the monovalent complex can be determined as the association rate constant k a (or k on ) and the dissociation rate constant k d (or k off ) is called. D is K D =k d / k a By the equation k a and k d The value of the dissociation constant can be determined directly by well-known methods and can also be calculated for complex mixtures, for example, by methods such as those described in Caceci et al. (1984, Byte 9:340-362). For example, the K can be calculated by the double filtration nitrocellulose filter binding assay as disclosed in Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90:5428-5432). DOther standard assays to assess the binding ability of an antibody to a target antigen are known in the art, including, for example, ELISA, Western blot, RIA and flow cytometry analysis, and other assays exemplified elsewhere in this disclosure. The binding kinetics and binding affinity of an antibody can be determined by standard assays known in the art, such as surface plasmon resonance (SPR), e.g., Biacore. TM The K of each antibody / antigen complex may be evaluated by the KinExA system. D By comparing the K values, one can compare the binding affinities associated with interactions with different molecules, for example, comparing the binding affinities of different antibodies to a given antigen. Similarly, the specificity of an interaction can be determined by comparing the K of an interaction of interest (e.g., a specific interaction between an antibody and an antigen). D value and the K of a non-target interaction (e.g., a known control antibody that does not bind to the target antigen). D It can be evaluated by determining and comparing values.

[0120] "Homology" and "identity" are the sequence similarity between two polynucleotide sequences or two polypeptides. If a position in two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if each position in two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared, multiplied by 100. For example, when optimally aligning sequences, if 6 out of 10 positions in two sequences are matched or homologous, then the two sequences are 60% homologous, and if 95 out of 100 positions in two sequences are matched or homologous, then the two sequences are 95% homologous.

[0121] "Variants" refer to nucleotide and amino acid sequences having different percentage levels of sequence identity to the nucleotide and amino acid sequences of the present disclosure, obtained by making appropriate mutational modifications, such as substitutions, insertions, and deletions, to the nucleotide and amino acid sequences of the present disclosure. The sequence identity may be at least 85%, 90% or 95%, including, as non-limiting examples, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%. Sequence comparison between two sequences and percentage identity determination can be performed using the default settings of the BLASTN / BLASTP algorithm available from the homepage of the National Center For Biotechnology Institute.

[0122] "Conservative substitution" refers to a substitution of an original amino acid residue with another amino acid residue having similar properties. It has little or no effect on the function, activity or other biological properties of a polypeptide. The above-mentioned conservative amino acid substitution is known in the art. For example, the conservative amino acid substitution is preferably a substitution of one amino acid residue in the following groups (i) to (v) with another amino acid residue in the same group: (i) small aliphatic non-polar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (ii) polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln, (iii) polar positively charged residues: His, Arg and Lys, (iv) Large aliphatic non-polar residues: Met, Leu, Ile, Val and Cys, and (v) Aromatic residues: Phe, Tyr and Trp.

[0123] Particularly preferred conservative amino acid substitutions are: Ala is replaced by Gly or Ser, Arg is replaced by Lys, Asn is replaced by Gln or His, Asp is replaced by Glu, Cys is replaced by Ser, Gln is replaced by Asn, Glu is replaced by Asp, Gly is replaced by Ala or Pro, His is replaced by Asn or Gln, Ile is replaced by Leu or Val, Leu is replaced by Ile or Val, Lys is replaced by Arg, Gln or Glu, Met is replaced by Leu, Tyr or Ile, Phe is replaced by Met, Leu or Tyr, Ser is replaced by Thr, Thr is replaced by Ser, Trp is replaced by Tyr, Tyr is replaced by Trp or Phe, and Val is replaced by Ile or Leu.

[0124] "Back mutation" refers to the mutation of amino acid residues in the framework region derived from a human antibody to amino acid residues at the corresponding positions in the original antibody. In general, to avoid the reduction in activity caused by the reduced immunogenicity of a humanized antibody, the activity of the antibody can be maintained by performing the least amount of back mutations on the variable region of the humanized antibody. In the present disclosure, "back mutation" also includes mutations in CDRs.

[0125] "Polynucleotide", "nucleic acid molecule" covers DNA and RNA and may be single-stranded or double-stranded, although double-stranded DNA is preferred. A nucleic acid is "operatively linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operatively linked to a coding sequence if it affects the transcription of the coding sequence.

[0126] A "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid linked to it. In one embodiment, the vector is a "plasmid," which refers to a circular double stranded DNA circle to which other DNA segments can be ligated. In another embodiment, the vector is a viral vector, in which another DNA segment can be ligated to the viral genome. The vectors of this disclosure can either autonomously replicate in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors), or can integrate into the genome of the host cell after introduction into the host cell, thereby replicating along with the host genome (e.g., non-episomal mammalian vectors).

[0127] "Cells," "cell lines," and "cell cultures" may be used interchangeably and all such designations include progeny. Thus, "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It should also be understood that all progeny may not be precisely identical in DNA content, due to intentional or unintentional mutations. Mutant progeny that have the same function or biological activity as screened for from the originally transformed cell are included. When different designations are referred to, they will be clear from the context.

[0128] "Host cell" refers to a cell into which an expression vector has been introduced. Host cells may include microbial (e.g., bacterial), plant or animal cells. Bacteria amenable to transformation include members of the enterobacteriaceae family, such as strains of Escherichia coli and Salmonella, Bacillaceae, such as Bacillus subtilis, Pneumococcus, Streptococcus, and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese hamster ovary cell line), NS0 cells, 293 cells.

[0129] "Pharmaceutical composition" refers to a mixture containing one or more compounds described in the present disclosure or their physiologically / pharmaceutical acceptable salts or prodrugs, and other chemical components such as physiologically / pharmaceutical acceptable vectors and excipients. The pharmaceutical composition is intended to facilitate administration to a living body and contribute to the absorption of the active ingredient to further exert biological activity.

[0130] A "pharmaceutically acceptable vector" or "pharmaceutically acceptable excipient" includes any material that, when combined with an active ingredient, allows the ingredient to retain its biological activity and does not react with the subject's immune system. Examples include, but are not limited to, any standard pharmaceutical vector, such as phosphate buffered saline solution, water, emulsions such as oil / water emulsions, and various wetting agents. In some embodiments, the diluent used for aerosol or parenteral administration is phosphate buffered saline (PBS) or normal (0.9%) saline. Compositions containing such vectors are prepared by conventional methods well known in the art (see, for example, Remington's Pharmaceutical Sciences, 18th edition, edited by A. Gennaro, Mack Publishing Co., Easton, PA, 1990, and R Remington, The Science and Practice of Pharmacy, 20th edition, Mack Publishing, 2000).

[0131] "Giving", "administration" and "treatment" refer to the contact of an exogenous agent, therapeutic agent, diagnostic agent or composition with an animal, human, subject, cell, tissue, organ or biological fluid when used with an animal, human, subject, cell, tissue, organ or biological fluid. "Giving", "administration" and "treatment" may refer to, for example, therapeutic, pharmacokinetic, diagnostic, research and experimental methods. Treatment of cells includes contact of a reagent with a cell and contact of a reagent with a fluid, where the fluid contacts the cell. "Giving", "administration" and "treatment" also refer to treating cells ex vivo and in vitro with a reagent, diagnostic, binding composition, or through another cell. "Treatment" refers to therapeutic treatment, preventative or prophylactic measures, research and diagnostic uses when used with a human, veterinary or research subject.

[0132] An "effective amount" includes an amount sufficient to ameliorate or prevent the symptoms or pathology of a medical condition. Effective amount also refers to an amount sufficient to permit or facilitate diagnosis. The effective amount used in a subject can vary depending on factors such as the condition being treated, the overall health of the subject, the route and dose of administration, and the severity of side effects. An effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects. The subject of the present disclosure may be an animal or human subject.

[0133] By "subject" or "patient" is meant mammals, particularly primates, especially humans.

[0134] "Treatment" refers to administering to a subject an oral or topical therapeutic agent, such as a composition comprising any one of the antibodies or antigen-binding fragments thereof according to the present disclosure, or a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof, wherein the patient has one or more disease symptoms and the therapeutic agent is known to have a therapeutic effect on those symptoms. Typically, the patient or population being treated is provided with a therapeutic agent in an amount that effectively relieves one or more disease symptoms, thereby inducing the resolution of those symptoms or inhibiting those symptoms from progressing to any clinically measurable extent. The amount of therapeutic agent that effectively relieves any particular disease symptom (also referred to as a "therapeutically effective amount") can vary depending on a variety of factors, including the disease state, age and weight of the patient, and the ability of the drug to produce the required therapeutic effect in the patient. Whether the disease symptom has been alleviated can be assessed by any clinical detection method commonly used by a physician or other professional health care provider to assess the severity or progression of the condition. Although embodiments of the present disclosure (e.g., methods of treatment or products) may be ineffective in alleviating the respective target disease symptoms, they should alleviate the target disease symptoms in a statistically significant number of patients, as determined by any statistical testing method known in the art, such as Student's t-test, chi-square test, Mann and Whitney U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0135] A "sequence" (e.g. in "immunoglobulin sequence", "antibody sequence", "single variable domain sequence", "VHH sequence" or "protein or polypeptide sequence") should generally be understood to include not only the related amino acid sequence but also the nucleic acid or nucleotide sequence encoding said sequence, unless a more restrictive interpretation is required in this disclosure.

[0136] "Optionally" or "optionally" means that the subsequently described event or circumstance may, but need not, occur, and the description includes cases where the event or circumstance occurs and cases where it does not occur. [Brief description of the drawings]

[0137] [Figure 1] 1 shows the results of detecting the frequency of pre-ADA in the serum of healthy individuals. [Diagram 2] This shows the results of detecting the effects of different amino acid modification methods on the binding of sdAb to pre-ADA. [Diagram 3] This shows the frequency of detection of pre-ADA against amino acid-modified sdAb in the serum of healthy individuals. [Figure 4] This shows the results of a FACS binding test of antibodies to human 4-1BB antigen, awaiting measurement. [Diagram 5] This shows the results of 4-1BB / NF-κB luciferase reporter gene detection before (FIG. 5A) and after (FIG. 5B) FcγRIIb cross-linking of the antibody awaiting measurement. [Figure 6] This is the result of FACS detection of the binding of anti-4-1BB single domain antibody C5 and its humanized antibodies C5_V1, C5_V2, and C5_V3 to human 4-1BB on the surface of HEK293 cells, with urelumab used as a control. [Figure 7] These are the results of detecting the activation effect of the anti-4-1BB single domain antibody C5 and its humanized antibodies C5_V1, C5_V2, and C5_V3 on the NF-κB signaling pathway. Urelumab and isotype IgG1 antibody were used as controls. [Figure 8] FACS detection of binding of C5_V2 and C5_V2-YTI to human 4-1BB on the surface of HEK293 cells, with Urelumab, an isotype IgG1 antibody, used as a control. [Figure 9] This shows the results of detection of binding of different C5_V2-YTI amino acid modified mutants to pre-ADA. [Figure 10] This shows the results of detecting the frequency of pre-ADA against C5_V2-YTI amino acid modification variants in the serum of healthy individuals. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0138] The present disclosure will be further described below with reference to examples, but these examples are not intended to limit the scope of the present disclosure.

[0139] Experimental methods for which no specific conditions are specified in the Examples or Test Examples of this disclosure generally follow conventional conditions or conditions recommended by the manufacturers of materials or products. Reference is made to Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, and Modern Methods in Molecular Biology, Ausubel et al., Greene Publishing Company, Wiley Interscience, NY. Reagents for which no specific source is specified are conventional reagents that are commercially available.

[0140] Example 1. Detection of pre-ADA against single domain antibodies (sdAbs) This disclosure uses enzyme-linked immunosorbent assay (ELISA) to measure pre-ADA for single domain antibodies, which is a commonly used detection method to measure the effect of pre-ADA antibodies. The serum used in this experiment is a single sample of healthy volunteer serum (Shanghai Xinfan Biotechnology Co., Ltd.), and the secondary antibody is an anti-human antibody λ chain HRP-labeled antibody (Southern Biotech, 2070-05).

[0141] According to previous studies, antibody sequences P2-1 and P2-2 derived from sdAb (sequence 1 and sequence 16) in GSK patent WO2013024059A were synthesized, of which P2-1 is the N-terminal fused His6 tag of sequence 1 in WO2013024059A, and P2-2 is the N-terminal fused His6 tag of sequence 16 in WO2013024059A. The corresponding sequence numbers of P2-1 and P2-2 in the present disclosure are SEQ ID NO: 1 and SEQ ID NO: 2. Also, an IgG1 molecule was synthesized with the same amino acid sequence as Cetuximab, and the heavy and light chain amino acid sequences of the IgG1 molecule are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

[0142] The ELISA assay is as follows: 1) Antigen coating: Dilute the antibody samples to be measured, including P2-1 and P2-2, with PBS to an effective final concentration of 2ng / μL each, add to a 96-well plate, 50μL / well, and incubate at room temperature for 1h. 2) Blocking: Discard the supernatant, wash once with PBST, add 4% M-PBST blocking solution, 150 μL / well, and incubate at room temperature for 1 h. 3) Incubation with serum: Wash once with PBST, add 30 μL / well of one person's serum (diluted 1:5 with PBS), and incubate at room temperature for 1 h. 4) Washing: Discard the supernatant and wash 8 times with PBST. 5) Secondary antibody incubation: Diluted secondary antibody (diluted with 4% M-PBST, see corresponding specifications for dilution ratio) was added at 50 μL / well and incubated at room temperature for 1 h. 6) Washing: Discard the supernatant and wash 8 times with PBST. 7) Color development: 50 μL of the substrate ABTS was added and incubated in the dark for about 20 minutes, after which OD415 was detected using SpectraMax iD3 (Molecular Devices).

[0143] In this experiment, a total of 40 healthy donors' serum samples were subjected to background reactivity detection for each antibody sample, and the samples were divided into negative control PBS group, P2-1 group, P2-2 group, and IgG1 group, and the results are shown in FIG. 1. The results showed that, compared with the negative control PBS group, about 57.5% of serum samples in the P2-1 group had detectable sdAb-reactive pre-ADA, while about 12.5% ​​of serum samples in the P2-2 group, which was modified to reduce immunogenicity, had detectable sdAb-reactive pre-ADA, which is basically consistent with the data reported in the GSK literature. In this disclosure, pre-ADA positivity means that a sample with a difference of +50% or more in the single-point signal corresponding to the IgG1 group of the sample group waiting to be measured is considered a positive sample, and vice versa.

[0144] >P2-1 (SEQ ID NO: 1) [ka] >P2-2 (SEQ ID NO:2) [ka] >The heavy chain of the IgG1 molecule (SEQ ID NO: 3) [ka] >Light chain of IgG1 molecule (SEQ ID NO: 4) [ka]

[0145] Example 2. Amino acid modification of the VH framework (FR) of a single domain antibody To study whether the binding of pre-ADA to the framework of a single domain antibody can be reduced, the single domain antibody P2-42 was selected as the parent clone, and amino acid modifications (including addition, substitution, and deletion) were made to the framework region of P2-42 by site-directed mutagenesis technology to obtain various modified mutants, the structures of which are shown in Table 1. The parent clone of the clones in Tables 1 and 2 is both P2-42.

[0146] The above P2-42 is a VHH antibody terminated with VTVSS at the C-terminus, and clones P2-43 to P2-58 are C-terminal modified mutants of P2-42 only, and no other sequences were changed except for the amino acid residues of the C-terminal modifications (including additions and substitutions) made in Table 1. All of the above positions were numbered based on the human germline framework sequences.

[0147] The above modified variants were measured and verified by the ELISA method in Example 1, and the sera used were eight individual serum samples that were subjected to the detection in Example 1 and showed pre-ADA positivity as a result. See FIG. 2 and Table 1 for the results. The results in Table 1 show the average signal reduction percentage (%) and the number of positive individuals for each modified variant compared to the parent clone. The greater the average signal reduction (%), the lower the modified variant's binding ability to pre-ADA and the lower the possibility of being neutralized by reserve pre-ADA in the human body.

[0148] The formula for calculating the mean % signal reduction of the present disclosure is as follows: Mean signal reduction % = (arithmetic mean signal value of corresponding sample group - arithmetic mean signal value of P2-42 group) / arithmetic mean signal value of P2-42 group x 100% As a result, the pre-ADA binding signals of six molecules, P2-53, P2-54, P2-55, P2-56, P2-57 and P2-58, were clearly reduced.

[0149] [Table 1]

[0150] Here, VTVSS, VTVSSA, VTVSF, VTVSG, VTVSL, VTVSR, VTVST, and VTVSV are represented by sequence numbers 189 to 196, respectively.

[0151] Example 3. Detection of pre-ADA for modified variants of single domain antibodies For six molecules, P2-53, P2-54, P2-55, P2-56, P2-57 and P2-58, this example further validates the effect of the mutations by detecting the frequency of pre-ADA for each modified variant in serum samples of 40 healthy donors, and the serum used is a single serum from a healthy donor (similar to the 40 samples in Example 1).

[0152] The above modified mutants were measured and verified by the ELISA method in Example 1. FIG. 3 shows the signal inhibition level of each mutant, and Table 2 shows the average signal inhibition (%) and pre-ADA positivity frequency of each mutant.

[0153] As a result, both modifications are clearly capable of reducing the binding of single domain antibodies to pre-ADA, since all clones showed an average signal reduction close to or exceeding 40%.

[0154] [Table 2]

[0155] Example 4. Screening and preparation of anti-4-1BB single domain antibodies The proteins or polypeptides used in the present disclosure are human 4-1BB protein or polypeptide (Human 4-1BB / TNFSF9 Protein, His Tag, purchased from Acrobiosystems, product number 41B-H5227), human 4-1BB protein or polypeptide (biotin / His tag) (Biotinylated Human 4-1BB / TNFRSF9 Protein, Avitag™, His Tag, purchased from Acrobiosystems, product number 41B-H82E3), and monkey 4-1BB protein or polypeptide (Cynomolgus / Rhesus macaque 4-1BB / TNFRSF9 Protein, His Tag, purchased from Acrobiosystems, product number 41B-C52H4), and the amino acid sequence begins and ends with Leu24-Gln186.

[0156] 1. Alpaca Immunization, Titer Detection, and Affinity Panning of the Phage Library Alpacas were immunized with His-tagged human 4-1BB recombinant protein or polypeptide (Acrobiosystems, 41B-H5227), once every 2 weeks, for a total of four immunizations. At the first immunization, 0.5 mg of antigen was mixed with 1 mL of complete Freund's adjuvant (CFA) and injected subcutaneously, and at the three subsequent immunizations, 0.25 mg of antigen was mixed with 1 mL of incomplete Freund's adjuvant (IFA) and injected subcutaneously. Blank serum was collected before immunization, and 50 mL of peripheral blood was collected one week after the third immunization and one week after the fourth immunization, respectively. PBMCs were isolated, total RNA was extracted, purity was detected, reverse transcribed to DNA, and the target fragments of the nanobody were ligated into a phage display vector after two rounds of nested PCR. The phage library was obtained by electroporation.

[0157] >Human 4-1BB protein or polypeptide sequence (SEQ ID NO:5) [ka]

[0158] To obtain anti-4-1BB nanobodies that simultaneously recognize humans and monkeys, a two-round strategy of cross-screening of human and monkey antigens was used. The antigens used in the first and second rounds of screening were human 4-1BB and monkey 4-1BB, or monkey 4-1BB and human 4-1BB, respectively. In each round of screening, the Gly-HCl acid elution method was used to elute phages that specifically bind to 4-1BB. 96 clones (192 clones in total) were randomly selected from the titration plates of the first and second rounds, respectively, and positive clones were screened using phage ELISA to detect the optical density at 450 nm. The positive clones were sequenced. Based on the sequencing results, sequence alignment and phylogenetic tree analysis were performed to screen 14 unique sequences, including H27, H170, C3, C5, C145, etc., of which the sequence of C5 is as follows, and the sequences of H27, H170, C3, C145, etc. are not shown.

[0159] >C5 (SEQ ID NO:6) [ka] [Table 3]

[0160] 2. Expression and purification of VHH-Fc fusion proteins or polypeptides The sequence of C5 was linked to human IgG1-Fc (SEQ ID NO: 10, where the underlined parts are mutations) with C220A, S267E and L328F mutations (numbered by the Eu system). The sequence of the linked VHH-Fc fusion protein or polypeptide is as follows. Also, by introducing mutations such as L234A, L235A and N297A (according to the Eu numbering system) into the Fc of human IgG1, the antibody FcγR-mediated effector function (e.g., as shown in SEQ ID NO: 10) is completely eliminated, and by introducing S228P (according to the Eu numbering system) into the Fc of human IgG4, the antibody molecule is stabilized to prevent the formation of half molecules (e.g., as shown in SEQ ID NO: 11), both of which are selectable IgG Fc. In SEQ ID NOs: 10 to 12, the underlined parts are Fc mutations.

[0161] >Human IgG1-Fc (containing C220A, S267E, L328F mutations) (SEQ ID NO: 10) [ka] (SEQ ID NO:10) > Human IgG1-Fc (containing C220A, L234A, L235A, N297A mutations) (SEQ ID NO: 11) [ka] >Human IgG4-Fc (including S228P mutation) (SEQ ID NO: 12) [ka] >Human IgG1 Fc (SEQ ID NO: 13) [ka] Take as an example the antibody sequence shown in SEQ ID NO:14, in which C5 is linked to SEQ ID NO:10. >C5-Fc (SEQ ID NO: 14) [ka] The plasmid was constructed and transiently transfected into cells, and the antibody was expressed and purified. The target antibody was obtained by detection.

[0162] Example 5. Detection of antigen-binding activity of anti-4-1BB single domain antibody and its agonistic activity 1. Detection of binding ability to 4-1BB antigen The binding activity of the anti-4-1BB single domain antibody to the human 4-1BB protein or polypeptide was detected by flow cytometry.

[0163] HEK293-Hu4-1BB cells were obtained by transiently transfecting HEK293 cells (ATCC CRL-1573) with a gene expressing human 4-1BB protein or polypeptide (CD137 cDNA ORF Clone, Human, C-OFPSpark tag, purchased from Sino Biological, Cat#HG10041-ACR), and the cell culture medium was DMEM (Gibco, Cat#11995065) containing 10% fetal bovine serum. The experimental medium was sterile PBS (phosphate buffer, pH7.40) containing 2% fetal bovine serum. The HEK293-Hu4-1BB cells were washed twice with the experimental medium and cultured at 1 × 10 5 HEK293-Hu4-1BB cells were seeded at 1 / well in a 96-well U-bottom plate, different concentrations of anti-4-1BB single domain antibody VHH-Fc samples to be measured were added, the cells were incubated at 4°C for 1h, washed twice with experimental medium, and sheep anti-human IgG (H+L) Alexa Fluor 488 antibody (Thermo, Cat#A11013) was added and washed twice, and the fluorescence signal value was read by flow cytometer. Urelumab monoclonal antibody was used as a positive control, and the MFI value of each antibody is shown in Figure 4.

[0164] As a result, such anti-4-1BB single domain antibodies have different degrees of binding ability to 4-1BB on the surface of HEK293-Hu4-1BB cells, among which C5 has good cell membrane surface antigen binding activity.

[0165] Detection of agonist activity against the 2,4-1BB signaling pathway The agonistic activity of anti-4-1BB single domain antibodies was assessed using a 4-1BB / NF-κB reporter gene.

[0166] HEK293 cells (ATCC CRL-1573) were transiently transfected with a gene expressing human 4-1BB (CD137 cDNA ORF Clone, Human, C-OFPSpark tag, purchased from Sino Biological, Cat#HG10041-ACR) and an NF-κB reporter gene (pGL4.32[luc2P / NF-κB-RE / Hygro]Vector, purchased from Promega, Cat#E849A) to obtain HEK293-Hu4-1BB / NF-κB double-transfected cells, and the activation of 4-1BB can be characterized at the activation level of the NF-κB signaling pathway. HEK293 cells were transiently transfected with FcγRIIb plasmid (CD32B / Fcgr2b cDNA ORF Clone, Human, N-His tag, purchased from Sino Biological, Cat#HG10259-NH) to obtain FcγRIIb-highly expressing HEK293 cells. The cell culture medium was DMEM (Gibco, Cat#11995065) containing 10% fetal bovine serum. HEK293-Hu4-1BB / NF-κB cells (2 × 10 6 50 μL of 100 mM NaCl / mL was seeded into a 96-well cell culture plate and 40 μL of medium or FcγRIIb-expressing HEK293 cells (2.5 × 10 6 10 μL / well of 10x gradient diluted anti-4-1BB single domain antibody was added and incubated at 37°C for 6 h. The cells were removed and an equal volume of Bio-Glo Luciferase Assay System reagent (Promega, Cat#G7940) was added to each well, incubated in the dark for 5 min, and the fluorescent signal was measured using an Envision plate reader (PerkinElmer, 2150). EC 50 The EC value and Emax value (fluorescence intensity of the control group without antibody) were calculated. 50The in vitro cellular agonist activity of the anti-4-1BB single domain antibody was evaluated based on the values. The results are shown in FIG.

[0167] The results showed that without FcγRIIb (i.e., CD32b) positive cross-linking, the anti-4-1BB single domain antibodies all showed weaker activation of the 4-1BB / NF-κB luciferase reporter gene signaling pathway than the Urelumab control, suggesting that the anti-4-1BB single domain antibodies of the present disclosure are safer, and after FcγRIIb cross-linking, the anti-4-1BB single domain antibodies showed significant activation of the 4-1BB / NF-κB luciferase reporter gene signaling pathway, with C5 having the strongest activation ability and comparable to the Urelumab control. Summarizing the activity results, the sequence C5, which has low background activation before FcγRIIb cross-linking and has even stronger activation activity after FcγRIIb cross-linking, was screened and humanized.

[0168] [Table 4]

[0169] Example 6. Modification of anti-4-1BB single domain antibody 1. Humanization The amino acid numbers of the CDR and FR regions (human framework regions, framework) of the C5 sequence were labeled according to the Kabat numbering system, and the FR1+CDR1 sequence, FR2+CDR2 sequence, and FR3+CDR3 sequence were aligned with the antibody germline database to obtain highly homologous FR human germline templates. FR1 is derived from IGHV3-64*04, FR2 is derived from IGHV3-23*03, and FR3 is derived from IGHV3-74*01. Each of the above human germline FR regions was replaced and inserted into the original sequence to reduce immunogenicity in the human body. The binding strength and activity were restored by performing back mutations on important amino acids that affect the structure and function of the antibody. The humanized sequences are as follows:

[0170] >C5_V1 (sequence number 15) [ka] >C5_V2 (sequence number 16) [ka] >C5_V3 (sequence number 17) [ka] The above three humanized sequences were linked to human IgG1-Fc (SEQ ID NO: 10) respectively. The plasmid was constructed, transiently transfected, expressed and purified. The specific process is as follows: the culture solution was diluted with 60μL of transfection reagent, then mixed evenly with 15μg of plasmid, incubated at 37℃ for 15min, and the mixed transfection solution was added dropwise to 30mL of cell solution, placed on a shaker and cultured for one week to express, and the supernatant was collected. It was then subjected to Protein A affinity purification, eluted with citrate buffer (pH 3.4), and finally dialyzed with 1×PBS buffer and frozen for storage.

[0171] 2. Removal of TCE sites Then, T-cell epitope prediction (TCE) was performed on the C5_V2 sequence, and the CDR3 sequence of C5_V2 was modified based on the prediction results so that the number of TCEs was reduced. The F mutation in the CDR3 region was mutated to Y (F99Y, according to the Kabat numbering system) to obtain a TCE-optimized version of the C5_V2 sequence, named C5_V2-YTI, whose sequence is as follows: >C5_V2-YTI (sequence number 18) [ka] That is, according to the Kabat numbering system, the amino acid sequence of CDR1 in C5_V2-YTI is shown in SEQ ID NO: 7, the amino acid sequence of CDR2 is shown in SEQ ID NO: 8, and the amino acid sequence of CDR3 is shown in HPLTYTIATMNDYDY (SEQ ID NO: 19).

[0172] The above sequences of C5_V2-YTI, C5, C5_V1, C5_V2, and C5_V3 were linked to human IgG1-Fc (SEQ ID NO: 10). Plasmids were constructed, transiently transfected, expressed, purified, and used for subsequent detection.

[0173] Example 7. Detection of antigen-binding activity and agonistic activity of modified anti-4-1BB single domain antibodies 1. Detection of binding ability to 4-1BB antigen The antigen-binding activity of the humanized antibodies was detected by the FACS detection method as described in Example 2. The results are shown in FIG.

[0174] As a result, the humanized anti-4-1BB single domain antibodies had different degrees of binding ability to 4-1BB on the surface of HEK293-Hu4-1BB cells, and C5_V1, C5_V2, and C5_V3 all maintained good binding activity.

[0175] [Table 5]

[0176] Detection of agonist activity against the 2,4-1BB signaling pathway The activation of the humanized antibody to the 4-1BB / NF-κB signal was detected by the NF-κB luciferase reporter gene experiment as described in Example 2, and the results are shown in FIG.

[0177] As a result, after the addition of FcγRIIb cross-linking, such humanized anti-4-1BB single domain antibodies showed activation to the 4-1BB / NF-κB luciferase reporter gene signaling pathway, and the activation ability of C5, C5_V1, C5_V2, C5_V3 was comparable to that of the Urelumab (according to WO2005035584A) control.

[0178] [Table 6]

[0179] The antigen-binding activities of C5_V2 and C5_V2-YTI were compared by the FACS detection method described in Example 5, and the results are shown in Figure 8 and Table 7. As a result, such C5_V2-YTI maintained good binding activity to 4-1BB on the surface of HEK293-Hu4-1BB cells, and was equivalent to C5_V2.

[0180] [Table 7]

[0181] Example 8. Amino acid modifications to the VH framework (FR) of anti-4-1BB single domain antibody and detection of pre-ADA The anti-4-1BB single domain antibody C5_V2-YTI according to the present disclosure was subjected to C-terminal modifications as shown in Table 8.

[0182] [Table 8] >C5_V2-YTI-42 (SEQ ID NO: 20) [ka] >C5_V2-YTI-43 (SEQ ID NO: 21) [ka] >C5_V2-YTI-44 (SEQ ID NO: 22) [ka] >C5_V2-YTI-46 (SEQ ID NO: 23) [ka] >C5_V2-YTI-47 (SEQ ID NO: 24) [ka] >C5_V2-YTI-48 (SEQ ID NO: 25) [ka] >C5_V2-YTI-49 (SEQ ID NO: 26) [ka] >C5_V2-YTI-50 (SEQ ID NO: 27) [ka] >C5_V2-YTI-51 (SEQ ID NO: 28) [ka] >C5_V2-YTI-52 (SEQ ID NO: 29) [ka] >C5_V2-YTI-53 (SEQ ID NO: 30) [ka] >C5_V2-YTI-54 (SEQ ID NO: 31) [ka] >C5_V2-YTI-55 (SEQ ID NO: 32) [ka] >C5_V2-YTI-56 (SEQ ID NO: 33) [ka] >C5_V2-YTI-57 (SEQ ID NO: 34) [ka] >C5_V2-YTI-58 (SEQ ID NO: 35) [ka] The N-terminus of the sequences shown in SEQ ID NOs: 20 to 35 and the C-terminus of IgG1 Fc (SEQ ID NO: 13) were linked with a GGGG (SEQ ID NO: 197) linker, and detection in Tables 9 and 10 was performed. Exemplary sequences are as follows, where the italicized part is IgG1 Fc and the underlined part is the linker.

[0183] >C5_V2-YTI-42 (SEQ ID NO: 36) [ka] According to the experimental method of Example 2, each C-terminal modified mutant of C5_V2-YTI was subjected to ELISA detection, and the results are shown in Table 9. As a result, six molecules, C5_V2-YTI-53, C5_V2-YTI-54, C5_V2-YTI-55, C5_V2-YTI-56, C5_V2-YTI-57 and C5_V2-YTI-58, showed clearly reduced pre-ADA binding signals.

[0184] [Table 9] According to the experimental method of Example 3, ELISA detection was performed for each of the C-terminal modified mutants of C5_V2-YTI, and the results are shown in Table 10. As a result, the average signal reduction for all clones was close to or exceeded 40%, indicating that all modifications can clearly reduce the binding of single domain antibodies to pre-ADA.

[0185] [Table 10]

[0186] Although specific embodiments of the present disclosure have been described above, those skilled in the art should understand that these are merely illustrative and that various changes and modifications can be made to these embodiments without departing from the principles and spirit of the present disclosure. Accordingly, the scope of the present disclosure is limited by the appended claims.

Claims

1. A modified immunoglobulin single variable domain, X １ X ２ (X ３ ) ｎ or VTVX １ X ２ (X ３ ) ｎ comprising the C-terminus of the amino acid sequence represented by, wherein X １ is selected from S, F and R, X ２ is selected from A, P, S, X ３ is A or does not exist, and n is an integer from 0 to 5, If n is 0, X ３ It does not exist. If n is between 1 and 5, then X ３ is A, Preferably, the modified immunoglobulin single variable domain is (a) to (e) below, i.e., (a) SAA or VTVSAA, (b) FP or VTVFP, (c) FPA or VTVFPA, (d) RS or VTVRS, and (e) RSA or VTVRSA A modified immunoglobulin monovariable domain containing a C-terminus consisting of one of the following.

2. A modified immunoglobulin monovariable domain comprising a heavy chain framework sequence derived from human germline, wherein the human germline heavy chain framework sequence has an amino acid modification of 112F or 112R at position 112, Selectively, the immunoglobulin monovariate domain further has an amino acid modification of 113P at position 113 relative to the human germline heavy chain framework sequence, or position 113 is S. Preferably, the immunoglobulin single variable domain has the following (f) to (h) at positions 112 and 113 relative to the human germline framework sequence, i.e., (f) 112F and 113P, (g) 112R and 113S, and (h) 112F and 113S A modified immunoglobulin monovariate domain containing any one of the following amino acid residue combinations.

3. A modified immunoglobulin monovariable domain comprising a heavy chain framework sequence derived from human germline cells, wherein the 112th position relative to the human germline heavy chain framework sequence is S, the 113th position relative to the human germline heavy chain framework sequence has amino acid modifications 113A and 113P, and there is an amino acid elongation of 1 to 5 amino acid residues after the 113th position, wherein the amino acid elongation is preferably A, AA, or AAA. Modified immunoglobulin single variable domain.

4. A VH single-domain antibody or a nanobody is selected, wherein the VH single-domain antibody is preferably of human origin, and the nanobody is preferably of camel origin or humanized. The modified immunoglobulin monovariate domain according to claim 1.

5. Compared to an unmodified immunoglobulin monovariate domain, the modified immunoglobulin monovariate domain has reduced binding to pre-ADA or anti-ADA, and the unmodified immunoglobulin monovariate domain has the C-terminus of the amino acid sequence VTVSS (SEQ ID NO: 189). The modified immunoglobulin monovariate domain according to claim 1.

6. It specifically binds to the target antigen, and a) Three complementarity determination (CDR) regions specific to the target antigen, b) Four framework (FR) areas, A modified immunoglobulin monovariate domain according to claim 1, comprising:

7. a) One or more amino acid modifications at positions 14, 41, and 108 of the human germline heavy chain framework sequence, preferably 14A, 14K, 14Q, or 14T, and / or 41A, and / or 108A, or 108Q, and / or b) Amino acid modifications at the 11th and / or 88th positions of the human germline heavy chain framework sequence, preferably 11K, 11R, 11D or 11E, and / or 88E, 88D, 88R or 88K. A modified immunoglobulin monovariate domain according to claim 1, comprising:

8. The aforementioned immunoglobulin monovariate domain specifically binds to 4-1BB, Preferably, the immunoglobulin monovariate domain comprises CDR1, CDR2, and CDR3 in any one of the amino acid sequences of SEQ ID NOs. 6 and 15-18, wherein CDR1, CDR2, and CDR3 are defined by the Kabat, IMGT, Chothia, AbM, or Contact numbering system. More preferably, the immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3, respectively, as indicated by SEQ ID NOs: 7, 8, and 9, or CDR1, CDR2, and CDR3, respectively, as indicated by SEQ ID NOs: 7, 8, and 19. Most preferably, the amino acid sequence of the immunoglobulin single variable domain that specifically binds to 4-1BB is represented by any one of SEQ ID NOs: 21, 30-35, or has at least 90%, and optionally at least 95%, identity thereto. The modified immunoglobulin monovariate domain according to claim 1.

9. A protein or polypeptide comprising a modified immunoglobulin monovariable domain as described in claim 1 at its C-terminus.

10. The protein or polypeptide according to claim 9, which is an antibody, preferably a monovalent, bivalent, polyvalent, single-specific, bispecific, or multispecific antibody.

11. Targeting at least two different antigens, or targeting at least two different domains or epitopes of the same antigen, Preferably, at least one of the at least two antigens or the same antigen is 4-1BB. The protein or polypeptide according to claim 9.

12. The protein or polypeptide according to claim 9, further comprising an immunoglobulin Fc region, preferably the Fc region of human IgG1, human IgG2, human IgG3, or human IgG4.

13. The protein or polypeptide according to claim 12, wherein the N-terminus of the modified immunoglobulin single variable domain is ligated directly or via a linker to the C-terminus of the Fc region.

14. A polynucleotide encoding a modified immunoglobulin monovariable domain according to any one of claims 1 to 8, or a protein or polypeptide according to any one of claims 9 to 13.

15. A vector that contains or expresses the polynucleotide described in claim 14.

16. A pharmaceutical composition comprising a modified immunoglobulin monovariate domain according to any one of claims 1 to 8, a protein or polypeptide according to any one of claims 9 to 13, a polynucleotide encoding the modified immunoglobulin monovariate domain according to any one of claims 1 to 8 or the protein or polypeptide according to any one of claims 9 to 13, or a vector containing or expressing a polynucleotide encoding the modified immunoglobulin monovariate domain according to any one of claims 1 to 8 or the protein or polypeptide according to any one of claims 9 to 13, and at least one pharmaceutically acceptable excipient, diluent, or vector.

17. A pharmaceutical composition comprising a modified immunoglobulin monovariate domain according to any one of claims 1 to 8, wherein binding to pre-ADA or ADA in the body of the subject is reduced, and a protein or polypeptide according to any one of claims 9 to 13.

18. A pharmaceutical composition for preventing or treating a disease, comprising a modified immunoglobulin monovariate domain according to any one of claims 1 to 8, and a protein or polypeptide according to any one of claims 9 to 13, wherein the disease is preferably a proliferative disorder, a metabolic disorder, an autoimmune disorder or an infectious disorder, and more preferably the proliferative disorder is cancer.

19. A method for reducing the binding of a single immunoglobulin variable domain to pre-ADA or ADA, wherein the C-terminus of the single immunoglobulin variable domain is X １ X ２ (X ３ ) ｎ Or VTVX １ X ２ (X ３ ) ｎ This includes including the C-terminus of the amino acid sequence shown by, of which X １ is selected from S, F, and R, X ２ It is selected from A, P, and S, X ３ is either A or does not exist, and n is an integer from 0 to 5. If n is 0, X ３ It does not exist. If n is between 1 and 5, then X ３ is A, Preferably, the modified immunoglobulin single variable domain is (a) to (e) below, i.e., (a) SAA or VTVSAA, (b) FP or VTVFP, (c) FPA or VTVFPA, (d) RS or VTVRS, and (e) RSA or VTVRSA A method comprising a C-terminus consisting of any one of the following.

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