Antibodies against chelated radionuclides

Abstract

This provides antibodies usable in pre-targeted radioimmunotherapy (PRIT) and radioimmunoimaging. [Solution] A multispecific antibody is provided for use in therapy, comprising an antigen-binding site specific to DOTAM-lead (Pb) chelate and an antigen-binding site specific to a target antigen.

Description

[Technical Field]

[0001] This application relates to antibodies, including bispecific antibodies, that specifically bind to chelated radionuclides. This application further relates to the use of such bispecific antibodies in applications such as radioimmunoimaging and radioimmunotherapy. This application further relates to scavenging agents and compositions useful in such methods. [Background technology]

[0002] Monoclonal antibodies have been developed to target drugs to cancer cells. By conjugating toxic drugs with antibodies that bind to tumor-associated antigens, it may be possible to kill tumors more specifically with less damage to surrounding tissue.

[0003] In pretargeted radioimmunotherapy (PRIT), an antibody construct is used that has affinity for tumor-associated antigens on the one hand and affinity for a radiolabeled compound on the other. In the first step, the antibody is administered and localizes within the tumor. Subsequently, the radiolabeled compound is administered. Because the radiolabeled compound is small, it can be rapidly delivered to the tumor and quickly removed. This reduces radiation exposure to areas other than the tumor (Goldenberg et al., Theranostics 2012, 2(5), pp. 523-540). In addition to its direct cytotoxic effect, PRIT can also act as an inducer of cell death in immunogenic cells and as a potential combination partner for cancer immunotherapy and endogenous vaccination techniques. A similar procedure can also be used for imaging. Pretargeting can use a bispecific antibody or a system using avidin-biotin, although the latter has the disadvantage that avidin / streptavidin is immunogenic.

[0004] For use in PRIT, radionuclides are generally in the form of chelates containing the desired radionuclide.

[0005] Su et al. (Nucl Med Biol 2005, 32:741-747) evaluated antibody pretargeting systems including mAb-streptavidin and DOTA-biotin. 212 Pb-DOTA-biotin is not stable, with over 30% being free. 212 Bi( 212 Pb decay products) 212 It was found that it is emitted from Pb-DOTA.

[0006] WO2010 / 099536 describes a bispecific antibody capable of binding to DOTA complexes of yttrium, ruthenium, and gadolinium. However, DOTA does not stably bind to all radionuclides and may exhibit slow complex formation rates (Yong and Brechbiel, Dalton Trans. June 21, 2001; 40(23), pp. 6068-6076). Chelating agents that do not stably bind to radionuclides risk reducing radiation delivery to tumors while increasing toxicity. [Overview of the project]

[0007] The present invention provides antibodies that bind to DOTAM and metal chelates containing lead (Pb). DOTAM can chelate Pb in a stable manner to form a Pb[DOTAM] complex.

[0008] The antibody of the present invention is bound to a chelate containing DOTAM and Pb, where Pb may be a stable (non-radioactive) isotope or a radioactive isotope. Radioactive isotopes of lead are useful in applications such as radiation imaging and radioimmunotherapy.

[0009] Preferably, the antibody of the present invention has a very high affinity for Pb-DOTAM chelates in the pM to fM range.

[0010] The antibody further binds to bismuth (Bi) chelated by DOTAM. 212 Pb is, 212It is a radiophilic nuclide of Bi, 212 It can act as an in vivo source of Bi. The ability of antibodies to bind to chelated Bi and chelated Pb increases their usefulness in applications such as radioimmunotherapy, where Bi isotopes are produced as decay products from Pb isotopes. In some embodiments, antibodies can bind to both Bi-DOTAM chelates and Pb-DOTAM with very high affinity in the pM-fM range.

[0011] Furthermore, the antibodies of the present invention are selective for Bi-DOTAM and Pb-DOTAM, as needed or preferably, compared to other chelating agent-metal complexes such as Cu-DOTAM chelates.

[0012] In one embodiment, the present invention relates to an antibody comprising an antigen-binding site specific to a Pb-DOTAM chelate, wherein the antigen-binding site comprises a heavy chain containing at least one, two, or three heavy chain CDR sequences: a) Heavy chain CDR1 contains the amino acid sequence GFSLSTYSMS (SEQ ID NO: 1); b) The heavy chain CDR2 contains the amino acid sequence FIGSRGDTYYASWAKG (SEQ ID NO: 2); c) Heavy chain CDR3 contains the amino acid sequence ERDPYGGGAYPPHL (SEQ ID NO: 3); Furthermore / or the antigen-binding site comprises a light chain containing at least one, two, or three light chain CDR sequences: d) The light chain CDR1 contains the amino acid sequence QSSHSVYSDNDLA (SEQ ID NO: 4); e) The light chain CDR2 contains the amino acid sequence QASKLAS (SEQ ID NO: 5); f) Provide an antibody in which the light chain CDR3 contains the amino acid sequence LGGYDDESDTYG (SEQ ID NO: 6).

[0013] In some embodiments, the antigen-binding site includes both the light chain and the heavy chain as defined above.

[0014] In another embodiment, the present invention relates to an antibody comprising an antigen-binding site specific to a Pb-DOTAM chelate, wherein the antigen-binding site comprises at least: a) Heavy chain CDR2 containing the amino acid sequence FIGSRGDTYYASWAKG (SEQ ID NO: 2), or variants thereof having one, two, or up to three substitutions in SEQ ID NO: 2, where these substitutions do not include Phe50, Asp56, and / Tyr58, and optionally also do not include Gly52 and / or Arg54; b) Heavy chain CDR3 containing the amino acid sequence ERDPYGGGAYPPHL (SEQ ID NO: 3), or variants thereof having one, two, or up to three substitutions in SEQ ID NO: 3, wherein these substitutions do not include Glu95, Arg96, Asp97, Pro98, and optionally also omit Ala100C, Tyr100D, and / or Pro100E, and / or optionally also omit Tyr99; c) Light chain CDR1 containing the amino acid sequence QSSHSVYSDNDLA (SEQ ID NO: 4), or a variant thereof having one, two, or up to three substitutions in SEQ ID NO: 4, wherein these substitutions do not include Tyr28 and Asp32; d) Light chain CDR3 containing the amino acid sequence LGGYDDESDTYG (SEQ ID NO: 6), or a variant thereof having one, two, or up to three substitutions in SEQ ID NO: 6, where these substitutions do not include Gly91, Tyr92, Asp93, Thr95c, and Tyr96. We provide antibodies that include [the specified substance].

[0015] The residue numbering was done by Kabat.

[0016] In some embodiments, the antibody may be further used as needed: i) Heavy chain CDR1 containing the amino acid sequence GFSLSTYSMS (SEQ ID NO: 1) or its variants having one, two, or three substitutions in SEQ ID NO: 1; ii) A variant thereof having at least one, two, or three substitutions in the light chain CDR2 or SEQ ID NO: 5 containing the amino acid sequence QASKLAS (SEQ ID NO: 5), and optionally lacking Gln50. It includes a heavy chain CDR1 and a light chain CDR2.

[0017] In any embodiment of the present invention relating to a variant of the sequence containing the above CDR, the protein may have one or more of the above residues as an invariant.

[0018] In some embodiments, the antibody according to the present invention binds to the same or overlapping epitopes of the chelated radionuclide to which the antibody disclosed herein binds.

[0019] In some embodiments, the antibody binds to the same epitope as Fab PRIT-0213 or PRIT-0214, or to an overlapping epitope. For example, the antibody, i) An antibody having a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 7 and a light chain variable domain containing the amino acid sequence of SEQ ID NO: 8; or i) An antibody having a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 9 and a light chain variable domain containing the amino acid sequence of SEQ ID NO: 10 It may be bound to the same epitope, or to an overlapping epitope.

[0020] In one embodiment, the antigen-binding site is a) Heavy chain CDR1 containing the amino acid sequence GFSLSTYSMS (SEQ ID NO: 1); b) Heavy chain CDR2 containing the amino acid sequence FIGSRGDTYYASWAKG (SEQ ID NO: 2); c) Heavy chain CDR3 containing the amino acid sequence ERDPYGGGAYPPHL (SEQ ID NO: 3); d) Light chain CDR1 containing the amino acid sequence QSSHSVYSDNDLA (SEQ ID NO: 4); e) Light chain CDR2 containing the amino acid sequence QASKLAS (SEQ ID NO: 5); f) Light chain CDR3 containing the amino acid sequence LGGYDDESDTYG (SEQ ID NO: 6) Includes at least one, two, three, four, five, or six CDRs selected from the following.

[0021] If necessary, the antibody in any of the above embodiments may be human, chimeric, or humanized.

[0022] If necessary, the antigen-binding site may include a heavy chain variable domain containing an amino acid sequence selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 9, or a variant thereof containing an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO: 7 or SEQ ID NO: 9.

[0023] If necessary, the antigen-binding site may include a light chain variable domain containing an amino acid sequence selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 10, or a variant thereof containing an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO: 8 or SEQ ID NO: 10.

[0024] If necessary, the antigen-binding site specific to the Pb-DOTAM chelate may include a heavy chain variable domain containing an amino acid sequence selected from the group consisting of SEQ ID NO: 7 or SEQ ID NO: 9, or a variant thereof as defined above, and a light chain variable domain containing an amino acid sequence selected from the group consisting of SEQ ID NO: 8 or SEQ ID NO: 10, or a variant thereof as defined above. For example, the antigen-binding site specific to the Pb-DOTAM chelate may include a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 7 or a variant thereof, and a light chain variable domain containing the amino acid sequence of SEQ ID NO: 8 or a variant thereof. In another embodiment, it may include a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 9 or a variant thereof, and a light chain variable domain containing the amino acid sequence of SEQ ID NO: 10 or a variant thereof.

[0025] Antibodies may be in any form, including whole antibodies and antibody fragments. Antibodies may also be monospecific. In this form, antibodies are useful, for example, in sorting and purification schemes to effectively separate the radiolabeled portion.

[0026] In some embodiments, an antibody that specifically binds to a Pb-DOTAM chelate is coupled to a cell-binding / targeting moiety to produce a targeted agent. Such agents are useful, for example, for pre-targeted radioimmunotherapy or pre-targeted radioimmunoimaging.

[0027] Coupling may preferably occur by expression as a fusion polypeptide or protein. Fusion may be direct or via a linker. The fusion polypeptide or protein can be produced recombinantly, avoiding the need for conjugation chemical reactions.

[0028] In some embodiments, the targeting portion (including the antigen-binding site for the target) is an antibody or a fragment thereof. That is, in some embodiments, the antibody may be in the form of a multispecific (e.g., bispecific) antibody, as will be further discussed below.

[0029] In another embodiment, the present invention further relates to a multispecific antibody / antibody conjugate suitable for targeting Pb-DOTAM chelates to target cells.

[0030] Therefore, in another embodiment, the present invention relates to a bispecific or multispecific antibody that specifically binds to both Pb-DOTAM chelate and a target antigen, for example, an antigen expressed on the surface of a target cell. The bispecific antibody comprises at least one antigen-binding site specific to DOTAM-chelated lead and at least one antigen-binding site for the target antigen.

[0031] In some embodiments, the antigen-binding site specific to the Pb-DOTAM chelate may be one of the embodiments described above.

[0032] The target antigen may be any antigen as further discussed herein, for example, any tumor-specific antigen. In some embodiments, it may be a protein or polypeptide expressed by a pathogen such as a prokaryote or virus.

[0033] In some embodiments, the tumor-associated antigen may be CEA (carcinoembryonic antigen). That is, in some embodiments, the bispecific antibody may include at least one antigen-binding site specific to Pb-DOTAM chelate and at least one antigen-binding site specific to CEA. CEA is advantageous in the context of the present invention because it is internalized relatively slowly, and thus a high percentage of the bispecific antibody remains available on the cell surface after initial treatment for binding to radionuclides. Other low-internalization target / tumor-associated antigens are also preferred and are described herein. Other examples of tumor-associated antigens that may be useful in the present invention include CD20 or HER2.

[0034] In some embodiments, when the target antigen is CEA, the antigen-binding site specific to CEA is: d) The heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 11; e) The heavy chain CDR2 contains the amino acid sequence of SEQ ID NO: 12; f) Heavy chain CDR3 contains the amino acid sequence of SEQ ID NO: 13, It may contain at least one, two, or three heavy chain CDRs; and / or antigen-binding sites specific to CEA are, a) Light chain CDR1 contains the amino acid sequence of SEQ ID NO: 14; b) Light chain CDR2 contains the amino acid sequence of SEQ ID NO: 15; c) Light chain CDR3 contains the amino acid sequence of SEQ ID NO: 16, It may contain at least one, two, or three light chain CDRs.

[0035] In some embodiments, the antigen-binding site for CEA is a) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 11; b) Heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 12; c) Heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 13; d) Light chain CDR1 containing the amino acid sequence of SEQ ID NO: 14; e) Light chain CDR2 containing the amino acid sequence of SEQ ID NO: 15; f) Light chain CDR3 containing the amino acid sequence of SEQ ID NO: 16 It may include at least one, two, three, four, five, or six (i.e., all) CDRs selected from the above.

[0036] In some embodiments, the antigen-binding site for CEA may include a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 17, or a variant thereof containing an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO: 17.

[0037] If necessary, the antigen-binding site may include a light chain variable domain containing the amino acid sequence of SEQ ID NO: 18, or a variant thereof containing an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO: 18.

[0038] If necessary, the antigen-binding site specific to CEA may include a heavy chain variable domain or a variant thereof containing the amino acid sequence of SEQ ID NO: 17, and a light chain variable domain or a variant thereof containing the amino acid sequence of SEQ ID NO: 18.

[0039] Various possible forms for bispecific or multispecific antibodies, including those further described herein, are known in the art. The antibodies of the present invention may employ any of these forms. For example, in some embodiments, the bispecific antibody may be bivalent, trivalent, or tetravalent.

[0040] The antibodies of the present invention may preferably contain an Fc region. The presence of the Fc region is beneficial in the context of radioimmunotherapy and radioimaging, for example, by extending the circulating half-life of the protein and / or resulting in higher tumor uptake than can be observed with respect to smaller fragments.

[0041] In some embodiments, if an Fc region is present, it may be preferable that the Fc region be manipulated to reduce its effector function. This may include substitutions of one or more of the Fc region residues 234, 235, 238, 265, 269, 270, 297, 327, and / or 329, for example, one or more of 234, 235, and / or 329. In some embodiments, the Fc region may be manipulated to include substitutions of Pro329 to Gly, Leu234 to Ala, and / or Leu235 to Ala (numbered by EU index).

[0042] Various forms of multispecific antibodies containing an Fc domain are known.

[0043] In one embodiment, a bispecific or multispecific antibody may include i) at least one Fab, cross-Fab, Fv, scFab, or scFv fragment or single-domain antibody (VHH) containing an Fc domain, ii) an antigen-binding site specific to Pb-DOTAM chelate, and iii) at least one Fab, cross-Fab, Fv, scFab, or scFv fragment or single-domain antibody (VHH) containing an antigen-binding site specific to the target antigen.

[0044] In some embodiments, bispecific or multispecific antibodies may be preferably polyvalent, e.g., bivalent, with respect to the target antigen (e.g., tumor-associated antigen). This has the advantage of increasing avidity.

[0045] In some embodiments, the bispecific or multispecific antibody is monovalent with respect to Pb-DOTAM. This reduces the risk of high molecular weight complex formation when a removal agent is used (see further discussion below).

[0046] Thus, in some embodiments, the antibody may be trivalent: that is, bivalent with respect to the target antigen and monovalent with respect to Pb-DOTAM.

[0047] In one exemplary form, a bispecific or multispecific antibody may include a full-length antibody (e.g., IgG) comprising first and second antibody heavy chains and first and second antibody light chains, wherein the first heavy chain and first light chain assemble to form an antigen-binding site for a first antigen, and the second heavy chain and second light chain assemble to form an antigen-binding site for a second antigen. If necessary, additional antigen-binding moieties can be fused to the N-terminuses or C-terminuses of the first and / or second heavy chains, for example, via a polypeptide linker, to increase the valence for one or both antigens. For example, additional antigen-binding moieties for the first antigen can be fused to the N-terminuses of one or both of the heavy chain molecules.

[0048] In another exemplary form, a bispecific or multispecific antibody may comprise a full-length antibody (e.g., IgG) containing an antigen-binding site for a first antigen (which may be bivalent for the first antigen), and further comprising at least one antigen-binding moiety specific to a second antigen. In various embodiments, the antigen-binding moiety may be a Fab fragment, a crossover Fab molecule, scFab, an Fv molecule, scFv, or a single-domain antibody (VHH), or a portion of a second full-length antibody. For example, the antibody may comprise a full-length antibody containing an antigen-binding site for a first antigen, and further comprising at least a second heavy-chain variable domain and a second light-chain variable domain that together form an antigen-binding site for a second antigen. Either the first or second antigen is a Pb-DOTAM chelate, and the other antigen is a target antigen.

[0049] In some embodiments of the format of this specification, the second antigen is a Pb - DOTAM chelate, and the first antigen is a target, for example, a tumor - associated antigen (in some embodiments, CEA, CD20, or ERBB2).

[0050] In another exemplary format, the bispecific or multispecific antibody is a full - length antibody that includes an antigen - binding site for a first antigen (which may be bivalent for the first antigen), wherein one of the N - or C - termini of the heavy chain is linked via a polypeptide linker to a first polypeptide, and the first polypeptide associates with a second polypeptide to form a Fab or crossed Fab that includes a binding site for a second antigen. For example, this format may be such that the first and second polypeptides together form an antigen - binding site for the second antigen, i) a first polypeptide consisting of a VH domain and a CH1 domain that associates with a second polypeptide consisting of a VL and a CL domain; or ii) a first polypeptide consisting of a VL domain and a CH1 domain that associates with a second polypeptide consisting of a VH and a CL domain; or iii) a first polypeptide consisting of a VH domain and a CL domain that associates with a second polypeptide consisting of a VL and a CH1 domain and may be included.

[0051] In some embodiments, the fusion may be at the N - terminus of one of the heavy chains of the full - length antibody.

[0052] In another specific embodiment, the antibody is a) a full - length antibody that specifically binds to a first antigen and consists of two antibody heavy chains and two antibody light chains; b) i) an antibody heavy - chain variable domain (VH); or ii) an antibody heavy - chain variable domain (VH) and an antibody heavy - chain constant domain (CH1); or iii) an antibody heavy - chain variable domain (VH) and an antibody light - chain constant domain (CL) A polypeptide consisting of, A polypeptide in which the N-terminus of the VH domain is fused to the C-terminus of one of the two heavy chains of the full-length antibody via a peptide linker; c) i) Antibody light chain variable domain (VL); or ii) Antibody light chain variable domain (VL) and antibody light chain constant domain (CL); or iii) Variable domain (VL) and constant domain (CH1) of the antibody light chain A polypeptide consisting of, A polypeptide in which the N-terminus of the VL domain is fused to the other C-terminus of the two heavy chains of the full-length antibody via a peptide linker. Includes; The antibody may also be a bispecific antibody in which the antibody heavy chain variable domain of the peptide under (b) and the antibody light chain variable domain of the peptide under (c) combine to form an antigen-binding site for a second antigen.

[0053] In this form, either the first or second antigen may be a Pb-DOTAM chelate. The other may be a target antigen, such as a tumor-associated antigen.

[0054] In some embodiments, the second antigen is a Pb-DOTAM chelate, and the first antigen is a target, such as a tumor-associated antigen (in some embodiments, CEA, CD20, or ERBB2).

[0055] The antibodies described above may be trivalent. In another possible embodiment, further antigen-binding moieties may be fused to increase the valence with respect to one or both antigens, as further discussed herein.

[0056] If necessary, the linker (and any linker considered herein) may be a peptide of at least 5 amino acids, preferably between 25 and 50 amino acids. The linker may be a rigid linker or a flexible linker. In some embodiments, it is flexible and contains or consists of Thr, Ser, Gly and / or Ala residues. For example, it may contain or consist of Gly and Ser residues. In some embodiments, it may be (Gly-Gly-Gly-Gly-Ser) n The linker may have a repeating motif such as [wherein n is, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10]. In some embodiments, the linker may be or include the sequence GGGGSGGGGSGGGGSGGGGS (Sequence ID 26). Other linkers may be used and can be identified by those skilled in the art.

[0057] Further details of this antibody format are provided in WO2010 / 115589A1 (Roche Glycart AG), which is incorporated herein by reference in its entirety.

[0058] as needed, i) In the constant domain CL of the first light chain of the full-length antibody under a), the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (numbered by Kabat) (in one preferred embodiment, independently by lysine (K) or arginine (R)); and in the constant domain CH1 of the first heavy chain under a), the amino acid at position 147 or the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbered by Kabat's EU index); or ii) In the constant domain CL of the second light chain under b), the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (numbered by Kabat) (in one preferred embodiment, independently by lysine (K) or arginine (R)), and in the constant domain CH1 of the second heavy chain under b), the amino acid at position 147 or the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbered by Kabat's EU index).

[0059] In one embodiment, the bispecific antibody of the present invention may have the above-described trivalent structure. a) A full-length antibody that specifically binds to CEA and consists of two antibody heavy chains and two antibody light chains, The heavy chain has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with respect to the heavy chain of amino acids 1-450 (including the values ​​at both ends, based on consecutive numbering) of sequence number 22 or 23 (i.e., with respect to the portion of the sequence preceding the linker); A full-length antibody whose light chain has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the light chain of SEQ ID NO: 21; and / or b) i) An antibody heavy chain variable domain (VH) having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the heavy chain variable domain of SEQ ID NO: 7; or ii) The antibody heavy chain variable domain (VH) and antibody heavy chain constant domain (CH1) having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the heavy chain variable domain of SEQ ID NO: 7; or iii) The antibody heavy chain variable domain (VH) and antibody light chain constant domain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with the heavy chain variable domain of SEQ ID NO: 7 A polypeptide consisting of, A polypeptide in which the N-terminus of the VH domain is fused to the C-terminus of one of the two heavy chains of the full-length antibody via a peptide linker; and / or c) i) An antibody light chain variable domain (VL) having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the light chain variable domain of SEQ ID NO: 8; or ii) The antibody light chain variable domain (VL) and antibody light chain constant domain (CL) having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with the light chain variable domain of Sequence ID No. 8; or iii) The antibody light chain variable domain (VL) and antibody heavy chain constant domain (CH1) having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with respect to the light chain variable domain of Sequence ID No. 8. A polypeptide consisting of, The polypeptide may also include a polypeptide in which the N-terminus of the VL domain is fused to the other C-terminus of the two heavy chains of the full-length antibody via a peptide linker; The antibody heavy chain variable domain of the peptide under (b) and the antibody light chain variable domain of the peptide under (c) together form an antigen-binding site for the Pb-DOTAM chelate.

[0060] In one example, one heavy chain of a full-length antibody contains a so-called "knob mutation" (T366W and, if necessary, one of S354C or Y349C, preferably S354C) according to EU index numbering, and the other contains a so-called "hole mutation" (T366S, L368A and Y407V, and, if necessary, Y349C or S354C, preferably Y349C) (see, for example, Carter, P et al., Immunotechnol. 2 (1996) 73).

[0061] In some embodiments, the two antibody heavy chains under (a) are: i) a first antibody heavy chain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the heavy chain of amino acids 1 to 450 (including both end values and based on consecutive numbering) of SEQ ID NO: 23 (i.e., for the portion of the sequence before the linker), having C at position 349, S at position 366, A at position 368 and V at position 407 (EU numbering); and ii) a second antibody heavy chain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the heavy chain of amino acids 1 to 450 (including both end values and based on consecutive numbering) of SEQ ID NO: 22, having C at position 354 and W at position 366 (EU numbering).

[0062] The "fixed" residues described herein are either "knob-into-hole" mutations in CH3 or other residues such as disulfide bridge-forming residues that pair with corresponding residues on the CH3 of the other heavy chain to favor the formation of the desired molecule. Possible residues that may be present on the other heavy chain may be derived from the sequences of Table 2 (e.g., sequences 19 and 20 or 22 and 23). For example, in one embodiment, if the first antibody heavy chain has C at position 349, the second antibody heavy chain has C at position 354.

[0063] If necessary, the linker is as described above.

[0064] In another embodiment, the bispecific antibody of the invention may have the above-described trivalent structure, a) a full-length antibody that specifically binds to CEA and consists of two antibody heavy chains and two antibody light chains, where the heavy chain has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the heavy chain of amino acids 1 to 450 (including both end values and based on consecutive numbering: i.e., the sequence before the linker) of SEQ ID NO: 19 or 20; A full-length antibody whose light chain has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the light chain of SEQ ID NO: 21; b) i) An antibody heavy chain variable domain (VH) having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the heavy chain variable domain of SEQ ID NO: 9; or ii) The antibody heavy chain variable domain (VH) and antibody heavy chain constant domain (CH1) having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the heavy chain variable domain of Sequence ID No. 9; or iii) The antibody heavy chain variable domain (VH) and antibody light chain constant domain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with the heavy chain variable domain of SEQ ID NO: 9 A polypeptide consisting of, A polypeptide in which the N-terminus of the VH domain is fused to the C-terminus of one of the two heavy chains of the full-length antibody via a peptide linker; c) i) An antibody light chain variable domain (VL) having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the light chain variable domain of SEQ ID NO: 10; or ii) The antibody light chain variable domain (VL) and antibody light chain constant domain (CL) having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with the light chain variable domain of Sequence ID No. 10; or iii) The antibody light chain variable domain (VL) and antibody heavy chain constant domain (CH1) having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with respect to the light chain variable domain of Sequence ID No. 10. A polypeptide consisting of, A polypeptide in which the N-terminus of the VL domain is fused to the other C-terminus of the two heavy chains of the full-length antibody via a peptide linker. It may include; The antibody heavy chain variable domain of the peptide under (b) and the antibody light chain variable domain of the peptide under (c) together form an antigen-binding site for the Pb-DOTAM chelate.

[0065] As described above, in any of the embodiments described above, one of the heavy chains of the full-length antibody contains a so-called "knob mutation" (T366W and, if necessary, one of S354C or Y349C, preferably S354C) according to EU index numbering, and the other contains a so-called "hole mutation" (T366S, L368A and Y407V, and, if necessary, Y349C or S354C, preferably Y349C) (see, for example, Carter, P et al., Immunotechnol. 2 (1996) 73).

[0066] In one embodiment, the two antibody heavy chains under (a) may include: i) a first antibody heavy chain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the heavy chain of amino acids 1-450 of SEQ ID NO: 22, with C at position 354 and W (EU numbering) at position 366; and ii) a second antibody heavy chain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the heavy chain of amino acids 1-450 of SEQ ID NO: 23, with C at position 349, S at position 366, A at position 368 and V (EU numbering) at position 407.

[0067] If necessary, the linker is as described above.

[0068] In another embodiment, the bispecific antibody is i) A first heavy chain having an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the heavy chain of SEQ ID NO: 22, ii) A second heavy chain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with the heavy chain of Sequence ID No. 23, iii) Two antibody light chains having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the light chain of SEQ ID NO: 21 Includes.

[0069] In yet another embodiment, the bispecific antibody is i) A first heavy chain having the amino acid sequence of SEQ ID NO: 22; ii) A second heavy chain having the amino acid sequence of SEQ ID NO: 23; and iii) Two antibody light chains having the amino acid sequence of SEQ ID NO: 21 This molecule, which includes [the specified substance], is referred to herein as PRIT-0213.

[0070] In another embodiment, the bispecific antibody is i) A first heavy chain having an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the heavy chain of SEQ ID NO: 19, ii) A second heavy chain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with the heavy chain of Sequence ID No. 20, iii) Two antibody light chains having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the light chain of SEQ ID NO: 21 Includes.

[0071] In yet another embodiment, the bispecific antibody is i) The first heavy chain having the amino acid sequence of SEQ ID NO: 19; ii) A second heavy chain having the amino acid sequence of SEQ ID NO: 20; and iii) Two antibody light chains having the amino acid sequence of SEQ ID NO: 21 This molecule, which includes [the specified substance], is referred to herein as PRIT-0214.

[0072] In one embodiment, the bispecific antibody of the present invention may have the above-described trivalent structure. a) A full-length antibody that specifically binds to ERBB2 and consists of two antibody heavy chains and two antibody light chains, The heavy chain has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with respect to the heavy chain of amino acids 1-449 (including the values ​​at both ends and based on consecutive numbering) of sequence number 36 or 37 (i.e., with respect to the portion of the sequence preceding the linker); A full-length antibody whose light chain has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the light chain of SEQ ID NO: 38; and / or b) i) An antibody heavy chain variable domain (VH) having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the heavy chain variable domain of SEQ ID NO: 7 or SEQ ID NO: 9 (preferably SEQ ID NO: 7 in some embodiments); or ii) The antibody heavy chain variable domain (VH) and antibody heavy chain constant domain (CH1) having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with the heavy chain variable domain of SEQ ID NO: 7 or SEQ ID NO: 9 (preferably SEQ ID NO: 7 in some embodiments); or iii) The antibody heavy chain variable domain (VH) and antibody light chain constant domain (CL) having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with the heavy chain variable domain of SEQ ID NO: 7 or SEQ ID NO: 9 (preferably SEQ ID NO: 7 in some embodiments). A polypeptide consisting of, A polypeptide in which the N-terminus of the VH domain is fused to the C-terminus of one of the two heavy chains of the full-length antibody via a peptide linker; and / or c) i) An antibody light chain variable domain (VL) having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the light chain variable domain of SEQ ID NO: 8 or SEQ ID NO: 10 (preferably SEQ ID NO: 8 in some embodiments); or ii) The antibody light chain variable domain (VL) and antibody light chain constant domain (CL) having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with the light chain variable domain of SEQ ID NO: 8 or SEQ ID NO: 10 (preferably SEQ ID NO: 8 in some embodiments); or iii) The antibody light chain variable domain (VL) and antibody heavy chain constant domain (CH1) having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with the light chain variable domain of SEQ ID NO: 8 or SEQ ID NO: 10 (preferably SEQ ID NO: 8 in some embodiments). A polypeptide consisting of, The polypeptide may also include a polypeptide in which the N-terminus of the VL domain is fused to the other C-terminus of the two heavy chains of the full-length antibody via a peptide linker; The antibody heavy chain variable domain of the peptide under (b) and the antibody light chain variable domain of the peptide under (c) together form an antigen-binding site for the Pb-DOTAM chelate.

[0073] In one example, one heavy chain of a full-length antibody contains a so-called "knob mutation" (T366W and, if necessary, one of S354C or Y349C, preferably S354C) according to EU index numbering, and the other contains a so-called "hole mutation" (T366S, L368A and Y407V, and, if necessary, S354C or Y349C, preferably Y349C) (see, for example, Carter, P et al., Immunotechnol. 2 (1996) 73).

[0074] In some embodiments, the two antibody heavy chains under (a) include i) a first antibody heavy chain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the heavy chain of SEQ ID NO: 37 (i.e., relative to the portion of the sequence preceding the linker) from amino acids 1 to 449 (including the values ​​at both ends and based on consecutive numbering), and having C at position 349, S at position 366, A at position 368 and V at position 407 (EU numbering); and ii) a second antibody heavy chain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the heavy chain of SEQ ID NO: 36 (including the values ​​at both ends and based on consecutive numbering), and having C at position 354 and W at position 366 (EU numbering).

[0075] If necessary, the linker is as described above.

[0076] In another embodiment, the bispecific antibody is i) A first heavy chain having an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the heavy chain of SEQ ID NO: 36, ii) A second heavy chain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with the heavy chain of Sequence ID No. 37, iii) Two antibody light chains having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the light chain of Sequence ID No. 38. Includes.

[0077] In yet another embodiment, the bispecific antibody is i) A first heavy chain having the amino acid sequence of SEQ ID NO: 36; ii) A second heavy chain having the amino acid sequence of SEQ ID NO: 37; and iii) Two antibody light chains having the amino acid sequence of SEQ ID NO: 38 This molecule, which includes [the specified substance], is referred to herein as P1AD9827.

[0078] In another embodiment, the bispecific antibody of the present invention may have the above-described trivalent structure. a) A full-length antibody that specifically binds to CD20 and consists of two antibody heavy chains and two antibody light chains, The heavy chain has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with respect to the heavy chain of amino acids 1-448 of sequence number 47 or 48 (including the values ​​at both ends and based on consecutive numbering) (i.e., with respect to the portion of the sequence preceding the linker); A full-length antibody whose light chain has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the light chain of SEQ ID NO: 49; and / or b) i) An antibody heavy chain variable domain (VH) having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the heavy chain variable domain of SEQ ID NO: 7 or SEQ ID NO: 9 (preferably SEQ ID NO: 7 in some embodiments); or ii) The antibody heavy chain variable domain (VH) and antibody heavy chain constant domain (CH1) having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with the heavy chain variable domain of SEQ ID NO: 7 or SEQ ID NO: 9 (preferably SEQ ID NO: 7 in some embodiments), iii) The antibody heavy chain variable domain (VH) and antibody light chain constant domain (CL) having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with the heavy chain variable domain of SEQ ID NO: 7 or SEQ ID NO: 9 (preferably SEQ ID NO: 7 in some embodiments). A polypeptide consisting of, A polypeptide in which the N-terminus of the VH domain is fused to the C-terminus of one of the two heavy chains of the full-length antibody via a peptide linker; and / or c) i) An antibody light chain variable domain (VL) having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the light chain variable domain of SEQ ID NO: 8 or SEQ ID NO: 10 (preferably SEQ ID NO: 8 in some embodiments); or ii) The antibody light chain variable domain (VL) and antibody light chain constant domain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with the light chain variable domain of SEQ ID NO: 8 or SEQ ID NO: 10 (preferably SEQ ID NO: 8 in some embodiments); iii) The antibody light chain variable domain (VL) and antibody heavy chain constant domain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with the light chain variable domain of SEQ ID NO: 8 or SEQ ID NO: 10 (preferably SEQ ID NO: 8 in some embodiments). A polypeptide consisting of, A polypeptide in which the N-terminus of the VL domain is fused to the other C-terminus of the two heavy chains of the full-length antibody via a peptide linker. It may include; The antibody heavy chain variable domain of the peptide under (b) and the antibody light chain variable domain of the peptide under (c) together form an antigen-binding site for the Pb-DOTAM chelate.

[0079] In one example, one heavy chain of a full-length antibody contains a so-called "knob mutation" (T366W and, if necessary, one of S354C or Y349C, preferably S354C) according to EU index numbering, and the other contains a so-called "hole mutation" (T366S, L368A and Y407V, and, if necessary, Y349C or S354C, preferably Y349C) (see, for example, Carter, P et al., Immunotechnol. 2 (1996) 73).

[0080] In some embodiments, the two antibody heavy chains under (a) include: i) a first antibody heavy chain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the heavy chain of SEQ ID NO: 48 from amino acids 1 to 448 (including the values ​​at both ends and based on consecutive numbering) (i.e., the portion of the sequence prior to the linker), with C at position 349, S at position 366, A at position 368 and V at position 407 (EU numbering); and ii) a second antibody heavy chain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the heavy chain of SEQ ID NO: 47 from amino acids 1 to 448 (including the values ​​at both ends and based on consecutive numbering), with C at position 354 and W at position 366 (EU numbering).

[0081] If necessary, the linker is as described above.

[0082] In another embodiment, the bispecific antibody is i) A first heavy chain having an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the heavy chain of SEQ ID NO: 47, ii) A second heavy chain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with the heavy chain of Sequence ID No. 48, iii) Two antibody light chains having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the light chain of SEQ ID NO: 49 Includes.

[0083] In yet another embodiment, the bispecific antibody is i) The first heavy chain having the amino acid sequence of SEQ ID NO: 47; ii) A second heavy chain having the amino acid sequence of SEQ ID NO: 48; and iii) Two antibody light chains having the amino acid sequence of SEQ ID NO: 49 This molecule, which includes [the specified substance], is referred to herein as P1AD9826.

[0084] In a further aspect, the invention relates to a polynucleotide or set of polynucleotides encoding any of the antibodies described herein. In a further embodiment, the invention relates to a vector or set of expression vectors comprising the polynucleotide or polynucleotides, and optionally, an expression vector. For a further object, the invention relates to a prokaryotic or eukaryotic host cell comprising the vector of the invention. Further provided is a method of producing an antibody, comprising culturing the host cell such that the antibody is produced.

[0085] The bispecific or multispecific antibodies described herein may be useful in a variety of applications, including therapeutic and diagnostic applications, such as pretargeted radioimmunotherapy and pretargeted radioimmunimaging.

[0086] Thus, in a further aspect, the invention relates to any bispecific or multispecific antibody described herein for use in pretargeted radioimaging. In such embodiments, the chelated Pb is preferably 203 Pb.

[0087] A method of targeting a radioisotope to a tissue or organ for imaging may i) administering to a subject the multispecific or bispecific antibody described herein, which binds to a target antigen and localizes on the surface of cells expressing the target antigen; and ii) subsequently administering to the individual a Pb radionuclide chelated with DOTAM or a functional variant thereof, which binds to the antibody localized on the surface of the target cells may comprise.

[0088] If necessary, between steps (i) and (ii), a scavenger is administered that binds to an antigen-binding site specific to the Pb-DOTAM chelate. The scavenger blocks the antigen-binding site for Pb-DOTAM, preventing circulating antibodies from binding to the chelated Pb radionuclide. Alternatively, the scavenger can further increase the rate of antibody clearance from the body. The “scavenger” may also be called a “blocking agent”: these terms can be substituted for each other in the following discussion.

[0089] The removal agent may include a complex of a metal ion with DOTAM or a functional variant thereof, the complex being recognized by an antigen-binding site for Pb-DOTAM. Preferably, the metal ion is a stable isotope or an intrinsically stable isotope. "Stable isotope" means an isotope that does not undergo radioactive decay. "Intrinsically stable isotope" means an isotope that undergoes radioactive decay with a very long half-life, making it safe for use. Preferably, the metal ion is selected from Pb, Ca, and Bi ions. For example, the removal agent may be Pb complexed with DOTAM or a functional variant thereof, Ca complexed with DOTAM or a functional variant thereof, or a complexed metal ion with DOTAM or a functional variant thereof 209 Bi(1.9x10 19 It may also contain stable isotopes of Pb (an intrinsically stable isotope with a half-life of 1 year). 204 Pb, 206 Pb, 207 Pb and 208 It may be a mixture of Pb, or naturally occurring lead.

[0090] DOTAM or a functional variant thereof is conjugated to the removal portion. This portion provides a removal agent with low uptake into the tumor, for example, due to its size and / or high hydrodynamic radius. Preferred removal portions are discussed further below. In some embodiments, the removal agent may include DOTAM or a functional variant thereof conjugated to dextran or a derivative thereof.

[0091] In another embodiment of the imaging method, a multispecific or bispecific antibody can be conjugated to a chelated Pb radionuclide at the time of administration.

[0092] If necessary, in any embodiment, the method is iii) Imaging tissues or organs in which Pb radionuclides chelated with DOTAM or its functional variants are localized. It may also include the following.

[0093] In some embodiments, the target antigen may be a tumor-specific antigen, and the imaging may be a method for imaging a tumor or multiple tumors.

[0094] In further embodiments, the present invention relates to antibodies described herein for use in a method of pretargeted radioimmunotherapy. In such embodiments, chelated Pb is preferably 212 It is Pb.

[0095] Methods for targeting radioactive isotopes to tissues or organs for therapeutic purposes are: i) administering to the subject a multispecific or bispecific antibody as described herein, which binds to a target antigen and localizes to the surface of cells expressing the target antigen; and ii) Next, administer a Pb radionuclide chelated with DOTAM or a functional variant thereof, which binds to an antibody localized on the cell surface. It may include.

[0096] If necessary, administer the removal agent between steps (i) and (ii) as described above.

[0097] In some embodiments, the target antigen is a tumor-associated antigen, and the method is a method for treating cancer. In other embodiments, the target antigen may be an infection-associated antigen, such as a protein expressed by a prokaryotic or virus-infected cell.

[0098] In some embodiments, the antibodies described herein may be administered as part of a combination therapy. For example, they may be administered in combination with one or more radiosensitizers and / or chemotherapeutic agents: the radiosensitizer or chemotherapeutic agent and the antibody may be administered simultaneously or sequentially in either order.

[0099] The methods of radioimaging and radioimmunotherapy described herein may, if necessary, be used with, for example, antibodies and 203 Pb-DOTAM and 212 Both Pb-DOTAM can be combined, for example, by administering them as a mixture.

[0100] The present invention also relates to a pharmaceutical composition comprising the antibody of the present invention and a pharmaceutically acceptable additive.

[0101] In further embodiments, the present invention relates to the antibody of the present invention, i) Pharmaceutically acceptable additives; ii) Pb radionuclides chelated with DOTAM or its functional variants; iii) The removal agents described herein; iv) One or more additional chemotherapeutic agents; and / or v) One or more radiosensitizers This pertains to kits that include one, two, three, four, or all of the following.

[0102] In a further aspect of the present invention, the inventors have developed novel decontaminants. Such decontaminants can be used in any of the diagnostic, imaging, or therapeutic methods described herein.

[0103] In one embodiment, the present invention relates to a scavenger comprising dextran or a derivative thereof conjugated with a chelating agent selected from DOTAM and functional variants of DOTAM, wherein the chelating agent forms a complex with Pb, Zn, Ca, or Bi. The scavenger typically comprises a chelated metal ion, such as a Pb, Zn, Ca, or Bi ion.

[0104] The removal agent may include aminodextran coupled to DOTAM or a functional variant of DOTAM. For example, the removal agent may include DOTAM coupled to aminodextran using isothiocyanate coupling (e.g., a compound obtained by reacting aminodextran with p-SCN-Bn-TCMC).

[0105] One potential difficulty associated with the use of removal agents is the possibility that they may enter the tumor and negatively affect the subsequent binding of radioactive ligands.

[0106] The inventors have further found that when using a dextran-based scavenger that i) has a high average molecular weight and ii) is subjected to a molecular weight cutoff so that fragments below a certain size are removed, good clearance from the blood can be achieved along with low penetration of the scavenger into the tumor.

[0107] Thus, a preferred removal agent may be one in which: i) the average molecular weight of dextran or its derivative in the removal agent is 200 to 800 kDa, optionally higher than 300, 350, 400 or 450 kDa, optionally lower than 700, 650, 600 or 550 kDa, and optionally about 500 kDa; and ii) dextran, dextran derivatives or removal agents below a specific molecular weight cutoff are removed, and the molecular weight cutoff is 50 kDa or higher, 100 kDa or higher or 200 kDa or higher, optionally in the range of 50 kDa to 250 kDa or 50 kDa to 200 kDa, optionally in the range of 100 kDa to 200 kDa, and optionally about 100 kDa, 150 kDa or 200 kDa.

[0108] In a further embodiment, the present invention relates to a method for preparing a scavenger and to the use of a scavenger in a radioimmunotherapy or radioimmunoimaging method.

[0109] These and other embodiments of the present invention are further discussed below. [Brief explanation of the drawing]

[0110] [Figure 1] Figure 1 shows a schematic diagram of a possible bispecific antibody format. This format includes two antigen-binding sites for one target (A) and one antigen-binding site for a second target (B) (2:1 format). [Figure 2] Figure 2 shows the structure of PRIT-0213 after complex formation with Pb-DOTAM. [Figure 3] Figure 3 shows the interaction sites of PRIT-0213 complexed with Pb-DOTAM, numbered according to Kabat. [Figure 4]Figure 4 shows the distribution of 212Pb 24 hours after injection of radiolabeled DOTAM (%ID / g±SD, n=3). PRIT-0206, -0207, -0208, and -0165 target T84.66, while PRIT-0186, -0187, and -0156 target CH1A1A. PRIT-0175 is a non-CEA binding control. [Figure 5] Figure 5 shows the distribution of 203Pb, expressed as counts per minute (CPM) 96 hours after injection of PRIT antibody pre-conjugated with radiolabeled DOTAM (CPM ± SD, n=3). PRIT-0205, -0206, -0207, -0208, and -0209 are fully humanized constructs, while PRIT-0165 and -0175 are the positive and negative controls, respectively. [Figure 6] Figure 6 shows the accumulation / clearance of 203Pb-DOTAM-bsAb in BxPC3 tumors and blood, expressed as CPM±SD (n=3), at various time points after injection of radiolabeled DOTAM and pre-conjugated PRIT antibody. PRIT-0206 is a fully humanized version of PRIT-0165; PRIT-0175 is a non-CEA-binding control. [Figure 7] Figure 7 shows the radioactivity distribution in selected tissues 2 hours after injection of a 212Pb-labeled removal agent in mice with MKN45 tumors (%ID / g±SD, n=3). [Figure 8] Figure 8 shows the radioactivity distribution in selected tissues and urine 24 hours after injection of a 212Pb-labeled removal agent in mice with MKN45 tumors (%ID / g±SD, n=3). [Figure 9] Figure 9 shows the organ-wise radioactivity distribution in selected tissues and urine 24 hours after injection of a 212Pb-labeled removal agent in mice with MKN45 tumors (%ID±SD, n=3). [Figure 10]Figure 10 shows the radioactivity distribution in selected tissues one week after injection of 203Pb-labeled removal agent in tumor-free mice (%ID / g±SD, n=3). [Figure 11] Figure 11 shows the organ-specific radioactivity distribution in selected tissues one week after injection of 203Pb-labeled removal agent in tumor-free mice (%ID±SD, n=3). [Figure 12] Figure 12 shows the radioactivity content in blood 4 hours after injection of 212Pb-DOTAM (%ID / g±SD, n=3). The striped bars represent the CA-free control (no removal agent) compared to all candidate reagents. Asterisks mark the level of statistical significance from low (*) to high (***). [Figure 13] Figure 13 shows the average radioactivity content in blood 24 hours after injection of 212Pb-DOTAM (%ID / g±SD, n=3). The striped bars represent the CA-free control (no removal agent) compared to all candidate reagents. [Figure 14] Figure 14 shows the radioactivity content in blood and tumors 24 hours after injection of 212Pb-DOTAM (%ID / g±SD, n=3). [Figure 15] Figure 15 shows the distribution of 212Pb 24 hours after injection of radiolabeled DOTAM using 30 or 100 μg of bispecific antibody and 10–100 μg of decontamination agent with a 100 or 30 kDa filtration cutoff, or completely decontamination-free (PBS) (%ID / g±SD, n=3). [Figure 16] Figure 16 shows the effect of gradually increasing doses of scavenging agents (0–100 μg) on ​​the activity concentration of 212Pb in blood and tumors. Tumors were pre-targeted with 100 μg of PRIT-0165, followed 4 days later by Dex500 diafiltration with a 100 kDa cutoff, or by using PBS. 212Pb-DOTAM was administered 2 hours after scavenging. The symbols represent %ID / g 24 hours after radioactivity injection, and the lines represent linear regression of tumor data. [Figure 17]Figure 17 shows the radioactivity distribution in selected tissues 24 hours after injection of 212Pb-DOTAM (%ID / g±SD, n=3). Dark gray and black bars represent the CA-free positive control (no removal agent) compared to the candidate reagent. [Figure 18] Figure 18 shows the 212Pb content (%ID / g±SD, n=3) in blood and tumors 24 hours after injection of 212Pb-DOTAM, as well as the corresponding tumor-to-blood ratio. Dark gray and black bars represent the CA-free positive control (no removal agent) compared to the candidate reagent. [Figure 19] Figure 19 shows the tumor-to-blood ratio 24 hours after injection of 212Pb-DOTAM as a function of the amount of scavenging agent (CA) (PJRD08-46) and TCMC saturation (9:20:39:00 or 84:1). The dashed lines represent the linear regression (R²=0.82) and nonlinear curve fitting (R²=0.74) of each data point. [Figure 20] Figure 20 shows the distribution of 212Pb 24 hours after injection of radiolabeled DOTAM (%ID / g±SD, n=3). Bars with white and gray backgrounds represent targeting of T84.66 and CH1A1A, respectively; black bars represent non-CEA binding controls. [Figure 21] Figure 21 shows the radioactivity distribution in selected tissues 24 hours after injection of 212Pb-DOTAM for treatment cycles 1 and 2 in the BxPC3 model (%ID / g ± SEM, n=3). [Figure 22] Figure 22 shows the mean body weight in groups A–G (n=8) after CEA-PRIT in the BxPC3 model. The curves were truncated at the first death in each group. Vertical dotted lines indicate 212Pb-DOTAM administration for some or all groups, as per the study design. [Figure 23] Figure 23 shows the mean weight change in groups A–G (n=8) after CEA-PRIT in the BxPC3 model, expressed as a percentage of initial body weight. Curves were truncated at the first death in each group. Vertical dotted lines indicate 212Pb-DOTAM administration for some or all groups, as per the study design. [Figure 24] Figure 24 shows the mean and standard error of tumor growth for groups A-G in the BxPC3 model (n=8). Curves were truncated at the first death in each group. Vertical dotted lines indicate 212Pb-DOTAM administration for some or all groups, as per the study design. [Figure 25] Figure 25 shows the individual tumor growth curves for groups A through G in the BxPC3 model. The vertical dotted line indicates administration of 212Pb-DOTAM. [Figure 26] Figure 26 shows Kaplan-Meier curves illustrating survival in groups A-G in the BxPC3 model (n=8). The vertical dotted line indicates administration of 212Pb-DOTAM. [Figure 27] Figure 27 shows the radioactivity distribution in selected tissues 24 hours after injection of 212Pb-DOTAM for treatment cycles 1 and 2 in the LS174T model (%ID / g±SD, n=3). [Figure 28] Figure 28 shows the mean body weight in groups A–G (n=8) after CEA-PRIT in the LS174T model. Curves were truncated at the first death in each group. Vertical dotted lines indicate 212Pb-DOTAM administration for some or all groups, as per the study design. [Figure 29] Figure 29 shows the mean weight change in groups A–G (n=8) after CEA-PRIT in the LS174T model, expressed as a percentage of initial body weight. Curves were truncated at the first death in each group. Vertical dotted lines indicate 212Pb-DOTAM administration for some or all groups, as per the study design. [Figure 30] Figure 30 shows the mean and standard error of tumor growth for groups A-G in the LS174T model (n=8). Curves were truncated at the first death in each group. Vertical dotted lines indicate 212Pb-DOTAM administration for some or all groups, according to the study design. [Figure 31] Figure 31 shows the individual tumor growth curves for groups A through G in the LS174T model. The vertical dotted line indicates administration of 212Pb-DOTAM. [Figure 32]Figure 32 shows Kaplan-Meier curves illustrating survival in groups A-G in the LS174T model (n=8). The vertical dotted line indicates administration of 212Pb-DOTAM. [Figure 33] Figure 33 shows the radioactivity distribution in selected tissues 24 hours after injection of 212Pb-DOTAM (%ID / g±SD, n=3). Gray bars represent tissue accumulation after injection of varying amounts of Dex500-(50%) scavenger (CA); black bars represent CA-free controls (no scavenger). [Figure 34] Figure 34 shows the 212Pb content in blood and tumors 24 hours after injection of 212Pb-DOTAM, as well as the corresponding tumor-to-blood ratio (%ID / g±SD, n=3). Gray bars represent tissue accumulation after injection of varying amounts of Dex500-(50%) scavenger (CA); black bars represent CA-free controls (no scavenger). [Figure 35] Figure 35 shows the radioactivity content in the blood 4 hours after injection of 212Pb-DOTAM (%ID / g±SD, n=3). [Figure 36] Figure 36 shows the binding of one antibody (PRIT-0165) to MKN-45 cells, which can be detected using either secondary detection (right panel, Alexa 488) or DOTAM FITC (left panel, FITC-A). [Figure 37] Figure 37 shows possible forms of bispecific antibodies using CEA as an exemplary target antigen. Other target antigens can also be used. [Figure 38] Figure 38 shows the binding of P1AD8927 to KPL-4 cells to demonstrate Her2 binding ability: antibody detection using a human IgG-specific secondary antibody. [Figure 39] Figure 39 shows the binding of P1AD8927 to KPL-4 cells to demonstrate DOTAM binding ability: isotype-corrected detection using Pb-DOTAM-FITC. [Figure 40] Figure 40 shows the binding of P1AD8926 to Raji cells to demonstrate CD20 binding ability: antibody detection using a human IgG-specific secondary antibody. [Figure 41] Figure 41 shows the binding of P1AD8926 to Raji cells to demonstrate DOTAM binding ability: isotype-corrected detection using Pb-DOTAM-FITC. [Figure 42] Figure 42 shows a summary of the Protocol 103 study evaluating CEA-PRIT in scBxPC3 tumors in SCID mice (h = hours, d = days, w = weeks). [Figure 43] Figure 43: Panel A shows the mean accumulation of 212Pb in collected tissue after both treatment cycles, expressed as %ID / g±SD (n=3). Panel B shows the individual tumor uptake of 212Pb per mouse, along with the corresponding tumor volume (mm3) at euthanasia. [Figure 44] Figure 44 shows the mean tumor growth curves and standard errors for groups A-G in the BxPC3 model (n=10). Curves were truncated for n<5. Vertical dotted lines indicate 212Pb-DOTAM administration (30 or 10 μCi) for some or all groups, according to the study design. [Figure 45] Figure 45 shows the individual tumor growth curves for groups A-G in the BxPC3 model (n=10). The vertical dotted line indicates administration of 212Pb-DOTAM (30 or 10 μCi). [Figure 46] Figure 46 shows Kaplan-Meier curves illustrating survival in groups A-G in the BxPC3 model (n=10). The vertical dotted line indicates administration of 212Pb-DOTAM (30 or 10 μCi). [Figure 47] Figure 47 shows the mean weight loss in groups A-G after CEA PRIT in the BxPC3 model (n=10). Curves were truncated for n<5. Vertical dotted lines indicate 212Pb-DOTAM administration for some or all groups, according to the study design. [Figure 48]Figure 48 shows the distribution of 203Pb-BsAb (20 μCi, 100 μg) in SCID mice with scBxPC3 tumors. Mice were injected with 203Pb-DOTAM-CEA-DOTAM or 203Pb-DOTAM-DIG-DOTAM (negative control) pre-bound with 20 μCi, and organs were subsequently collected 1, 4, 7, or 10 days post-injection. The accumulated radioactivity in the collected tissues was evaluated (%ID / g ± SD, n=5). [Figure 49] Figure 49 shows the accumulation of 203Pb-BsAb in scBxPC3 tumors 1 to 10 days after injection of pre-bound 203Pb-DOTAM-CEA-DOTAM or 203Pb-DOTAM-DIG-DOTAM (negative control) at a dose of 20 μCi / 100 μg (%ID / g ± SD, n=5). [Figure 50] Figure 50 shows the distribution of 212Pb in SCID mice with scBxPC3 tumors. Mice were injected with CEA-DOTAM BsAb and CA 5 minutes to 48 hours after radioactive injection, prior to 212Pb-DOTAM administration (%ID / g ± SD, n=5). *Cumulative 212Pb content in urine and feces over time, i.e., at each time point including previous values. Estimated %ID / g in urine was based on 1 / 5 (10 mL) of pooled urine / wash solution (50 mL) from 5 mice. [Figure 51] Figure 51 shows a summary of the Protocol 131 study, which evaluated the in vivo distribution of 212Pb after PRIT using CEA-DOTAM BsAb, Pb-DOTAM-dextran-500 CA, and 212Pb-DOTAM quenched with one of five different metals (Zn, Gd, Cu, Ca, or Pb) in SCID mice carrying scBxPC3 tumors (d=days, h=hours). [Figure 52] Figure 52 shows the distribution of 212Pb in tumor-bearing SCID mice 2 hours after injection of CEA-DOTAM-pretargeted 212Pb-DOTAM (%ID / g±SD, n=4). [Figure 53]Figure 53 shows the distribution of 212Pb in selected normal tissues of SCID mice with tumors 2 hours after injection of 212Pb-DOTAM quenched with different metals (%ID / g). [Figure 54] Figure 54 shows the distribution of 212Pb in tumor-bearing SCID mice 24 hours after injection of 212Pb-DOTAM pretargeted with CD20-DOTAM BsAb or negative control DIG-DOTAM (%ID / g±SD, n=3). [Figure 55] Figure 55 shows the distribution of 212Pb in tumor-bearing SCID mice 24 hours after injection of 212Pb-DOTAM pretargeted with HER2-DOTAM BsAb or negative control DIG-DOTAM (%ID / g±SD, n=3). [Figure 56] Figure 56 shows a summary of Protocol 154, which evaluates the in vivo distribution of 212Pb-DOTAM after CEA-PRIT using various BsAb constructs in SCID mice carrying scHPAF-II tumors (h = hours, d = days). [Figure 57] Figure 57 shows the distribution of 212Pb in tumor-bearing SCID mice 6 hours after injection of 212Pb-DOTAM pretargeted with either a negative control DIG-DOTAM, a standard CEA-DOTAM BsAb, or one of the alternative BsAb constructs (%ID / g±SD, n=3). [Figure 58] Figure 58 shows the blood content and tumor accumulation of 212Pb 6 hours after injection of 212Pb-DOTAM pretargeted with either a negative control DIG-DOTAM, a standard CEA-DOTAM BsAb, or an alternative BsAb construct (%ID / g±SD, n=3). [Figure 59]Figure 59 shows the experimental schedule for protocol 162. CD20-PRIT was performed in SCID mice carrying scWSU-DLCL2 tumors using CD20-DOTAM BsAb, Ca-DOTAM-dextran-500 CA, and 212Pb-DOTAM; a one-step RIT was performed in SCID mice carrying scWSU-DLCL2 tumors using CD20-DOTAM BsAb pre-bound with 212Pb-DOTAM (212Pb-DOTAM-CD20-DOTAM). [Figure 60] Figure 60 shows the distribution of 212Pb in tumor-bearing SCID mice 24 hours after injection of CD20-DOTAM-pretargeted 212Pb-DOTAM or pre-conjugated 212Pb-DOTAM-CD20-DOTAM. Radioactivity content in organs and tissues is expressed as mean %ID / g and standard deviation (SD; n=3). [Figure 61] Figure 61 shows the mean WSU-DLCL2 sc tumor growth for groups A to G, expressed as mm3±SEM (n=10). [Figure 62] Figure 62 shows the mean change in mouse body weight after various treatments, expressed as initial body weight ± % of SEM. The dotted line indicates 212Pb or antibody injection depending on the treatment scheme. [Modes for carrying out the invention]

[0111] definition For the purposes of this specification, “acceptor human framework” is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or human consensus framework, as defined below. An acceptor human framework “derived from” a human immunoglobulin framework or human consensus framework may contain the same amino acid sequence, or it may contain amino acid sequence variations. In some embodiments, the number of amino acid variations is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is sequence-identical to the VL human immunoglobulin framework sequence or human consensus framework sequence.

[0112] "Affinity" refers to the combined strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can generally be expressed by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific demonstrative and exemplary embodiments for measuring binding affinity are described below.

[0113] An "affinity-mature" antibody refers to an antibody that has one or more modifications in its hypervariable regions (HVRs) compared to a parent antibody that does not have one or more such modifications, and such modifications result in improved affinity of the antibody to the antigen.

[0114] The terms “anti-Pb-DOTAM antibody,” “antibody that binds to Pb-DOTAM,” “antibody that binds to Pb-DOTAM chelate,” and equivalent terms refer to an antibody that can bind to a Pb-DOTAM chelate with sufficient affinity to be useful in sorting and / or purification schemes for separating the Pb-DOTAM-labeled portion, and / or to allow the antibody to localize Pb-DOTAM to the site of the antibody, for example, to target Pb-DOTAM to cells. The terms “anti-target antibody” and “target-binding antibody” refer to an antibody that can bind to a target with sufficient affinity to be useful in therapeutic and / or diagnostic applications, including localization of the antibody to a target, for example, when the antibody is expressed on the surface of a cell. In one embodiment, the degree of binding of the antibody to unrelated portions and / or unrelated target proteins is less than about 10% of the binding of the antibody to Pb-DOTAM or the target, as measured, for example, by radioimmunoassay (RIA). In a particular embodiment, the antibody may have a concentration of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (for example, 10 -8 M or less, for example, 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 M) has a dissociation constant (Kd) for Pb-DOTAM and / or the target.

[0115] The term "antibody" as used herein is used in its broadest sense and is not limited to any particular form, but encompasses a variety of antibody structures, including monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0116] An "antibody fragment" refers to a molecule other than an intact antibody, which contains a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments, though not limited to them, include Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0117] The reference antibody and "antibody that binds to the same epitope" may refer to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competitive assay, or conversely, a reference antibody that blocks the binding of the antibody to its antigen by 50% or more in a competitive assay. Exemplary competitive assays are provided herein.

[0118] The term "chimeric" antibody refers to an antibody in which part of the heavy chain and / or light chain originates from a specific source or species, but the rest of the heavy chain and / or light chain originates from a different source or species.

[0119] The "class" of an antibody refers to the type of constant domain or constant region in its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further classified into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0120] As used herein, the term “cytotoxic agent” means a substance that inhibits or prevents cellular function and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., 225 Ac, 211 At, 131 I, 125 I, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi,213 Bi, 32 P, 212 Radioisotopes of Pb and Lu; chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other inserts); growth inhibitors; enzymes and their fragments, such as diffusion-degrading enzymes; antibiotics; toxins such as low molecular weight toxins or enzyme-active toxins of bacterial, fungal, plant or animal origin (including their fragments and / or variants); and various antitumor or anticancer agents disclosed below.

[0121] "Effector function" refers to the biological activity resulting from the Fc region of an antibody, which changes with the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cell-mediated cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0122] The “effective dose” of a drug, such as a pharmaceutical preparation, refers to the amount required to achieve the desired therapeutic or preventive outcome, and the amount that remains effective over a period of time.

[0123] In this specification, the term “Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. This term includes the Fc region of the natural sequence and Fc region variants. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 toward the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0124] The term "framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The variable domain FR generally consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, HVR and FR sequences generally appear in VH (or VL) in the following sequence: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0125] The terms “full-length antibody,” “intact antibody,” and “whole antibody” are used interchangeably herein and refer to antibodies having a structure substantially similar to that of a natural antibody, or having a heavy chain containing an Fc region as defined herein. A full-length antibody may be, for example, IgG.

[0126] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including the offspring of such cells. Host cells include “transformers” and “transformed cells,” which include primary transformed cells and their offspring, regardless of the number of passages. Offspring do not have to be completely identical to the parent cells in terms of nucleic acid content and may contain mutations. Mutant offspring having the same function or biological activity as those screened or selected in the originally transformed cells are also included herein.

[0127] A "human antibody" is one that is produced by a human or human cell, or has an amino acid sequence corresponding to an antibody of non-human origin that utilizes the human antibody repertoire or other human antibody coding sequences. Specifically, this definition of a human antibody excludes humanized antibodies that contain non-human antigen-binding residues.

[0128] The "Human Consensus Framework" is a framework representing the most commonly present amino acid residues in selected human immunoglobulin VL or VH framework sequences. Generally, selected human immunoglobulin VL or VH sequences are derived from subgroups of variable domain sequences. Generally, the sequence subgroups are those described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, NIH Publication 91-3242, Bethesda MD (1991), Vol. 1-3. In one embodiment, for VL, the subgroup is subgroup Kappa I as described above by Kabat et al. In one embodiment, for VH, the subgroup is subgroup III as described above by Kabat et al.

[0129] A “humanized” antibody refers to a chimeric antibody containing amino acid residues derived from a non-human HVR and amino acid residues derived from a human FR. In certain embodiments, the humanized antibody may contain at least one, typically two, substantially all of the variable domains, with all or substantially all of the HVR (e.g., CDR) corresponding to that of a non-human antibody and all or substantially all of the FR corresponding to that of a human antibody. The humanized antibody may optionally contain at least a portion of the antibody constant region derived from the human antibody. A “humanized” antibody, e.g., a non-human antibody, refers to an antibody that has been humanized.

[0130] As used herein, the terms “hypervariable region” or “HVR” refer to each region of an antibody variable domain that is hypervariable in sequence (“complementarity-determining region” or “CDR”), and / or forms a structurally defined loop (“hypervariable loop”), and / or contains an antigen contact residue (“antigen contact”). Generally, an antibody contains six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). An example of an HVR is: (a) Hypervariable loops present at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196: pp. 901-917 (1987)); (b) CDRs present at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda MD (1991)); (c) Antigen contact present at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al., J.Mol.Biol.262:732-745 (1996)); and (d) HVR amino acid residues 46-56(L2), 47-56(L2), 48-56(L2), 49-56(L2), 26-35(H1), 26-35b(H1), 49-65(H2), 93-102(H3), and 94-102(H3) This includes combinations of (a), (b), and / or (c).

[0131] Alternatively, the sequence of CDR-H1 described herein may extend from Kabat26 to Kabat35.

[0132] In one embodiment, the HVR or CDR residues include those identified in Table 2 or elsewhere in this specification.

[0133] Unless otherwise specified, HVR / CDR residues and other residues in the variable domain (e.g., FR residues) are numbered herein by Kabat et al., cited above.

[0134] An "immunoconjugate" is, but is not limited to, an antibody conjugated to one or more heterologous molecules, including cytotoxic agents.

[0135] The “individual” or “subject” is a mammal. Mammals include, but are not limited to, livestock (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0136] The molecules described herein may be “isolated.” “Isolated” antibodies are those separated from components of their natural environment. In some embodiments, antibodies are purified to a purity higher than 95% or 99%, as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For an overview of methods for evaluating antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848: pp. 79-87 (2007).

[0137] The term “nucleic acid molecule” or “polynucleotide” includes any compound and / or substance containing a polymer of nucleotides. Each nucleotide consists of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Nucleic acid molecules are often described by a sequence of bases that represents the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically represented from 5' to 3'. In this specification, the term nucleic acid molecule includes, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and deoxyribonucleic acid (DNA) containing mixed polymers of two or more of these molecules. Nucleic acid molecules may be linear or cyclic. Furthermore, the term nucleic acid molecule includes both sense and antisense strands, as well as both single-stranded and double-stranded forms. Additionally, nucleic acid molecules described herein may contain natural or non-natural nucleotides. Examples of non-natural nucleotides include modified nucleotide bases having derivatized sugar or phosphate backbone links or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules that are suitable as vectors for the direct expression of the antibodies of the present invention in vitro and / or in vivo, for example, in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors may be unmodified or modified. For example, mRNA may be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule, and the mRNA may be injected into a target to generate antibodies in vivo (see, e.g., Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi:10.1038 / nm.4356 or EP2101823B1).

[0138] "Isolated" nucleic acids refer to nucleic acid molecules that have been separated from the components of their natural environment. Isolated nucleic acids typically contain nucleic acid molecules found in cells that contain nucleic acid molecules, but the nucleic acid molecules are located outside of chromosomes or in chromosomal locations different from their natural chromosomal locations.

[0139] "Antibody-encoding isolated nucleic acid" means one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecules in a single vector or in separate vectors, where such nucleic acid molecules are located at one or more positions in a host cell.

[0140] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., possible antibody variants, including, for example, naturally occurring mutations or those arising during the production of a monoclonal antibody preparation, where, generally, the individual antibodies comprising the population are identical and / or bind to the same epitope, except for such variants present in small amounts. Typically, in contrast to a polyclonal antibody preparation, which contains different antibodies against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the characteristic of an antibody obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring the production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, and such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0141] A "naked antibody" refers to an antibody that is not conjugated with a heterogeneous portion (e.g., a cytotoxic portion) or a radiolabel. Naked antibodies may be present in pharmaceutical formulations.

[0142] "Natural antibodies" refer to naturally occurring immunoglobulin molecules with various structures. For example, a natural IgG antibody is a heterotetrameric glycoprotein with approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each heavy chain has a variable region (VL), also called a variable light chain domain or light chain variable domain, followed by a constant light chain (CL) domain. The light chains of an antibody can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains.

[0143] The term “package insert” is used to refer to the instruction manual that is customarily included in the market packaging of such therapeutic drugs, containing information regarding indications, use, dosage, administration, combination therapy, contraindications, and / or warnings regarding the use of the therapeutic drug.

[0144] The "amino acid sequence identity percentage (%)" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence, after the sequences have been aligned and gaps introduced as necessary to achieve the maximum sequence identity percentage, without considering any conservative substitutions as part of the sequence identity. Alignment for determining the amino acid sequence identity percentage can be achieved in various ways within the scope of the art of the art, such as using publicly available computer software, for example, BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning the sequences, including any algorithm required to achieve the maximum alignment over the full length of the sequences being compared. However, for the purposes of this specification, the amino acid sequence identity % value is generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program is authored by Genentech, Inc., and its source code, along with user documentation, is filed with the U.S. Copyright Office, Washington DC, 20559, and is registered under U.S. Copyright Office registration number TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from the source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change.

[0145] In situations where ALIGN-2 is used for amino acid sequence comparison, the percentage of amino acid sequence identity of a given amino acid sequence A with respect to a given amino acid sequence B (or, to put it another way, a given amino acid sequence A that has or contains a certain percentage of amino acid sequence identity with respect to a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y [In the formula, X is the number of amino acid residues scored as identical by the ALIGN-2 sequence alignment program for A and B, and Y is the total number of amino acid residues in B.] If the length of amino acid sequence A is not equal to the length of amino acid sequence B, it will be understood that the amino acid sequence identity % of A to B is not equal to the amino acid sequence identity % of B to A. Unless otherwise specifically stated, all amino acid sequence identity % values ​​used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0146] The term "pharmaceutical preparation" refers to a preparation that is in a form that allows the biological activity of the active ingredient it contains to be effective, and that does not contain any further ingredients that are unacceptably toxic to the person to whom the preparation is administered.

[0147] A "pharmaceutically acceptable carrier" refers to a component in a pharmaceutical preparation other than the active ingredient that is non-toxic to the target substance. Examples of pharmaceutically acceptable carriers, though not limited to them, include buffers, additives, stabilizers, or preservatives.

[0148] As used herein, “treatment” (and its grammatical variations such as “to treat” or “to treat”) refers to a clinical intervention in an attempt to alter the natural course of the individual being treated, which may be carried out for preventive purposes or during the course of a clinicopathological condition. Desired effects of treatment include, but are not limited to, prevention of the onset or recurrence of the disease, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, slowing of the rate of disease progression, improvement or alleviation of the disease state, and remission or improved prognosis. In some embodiments, the antibodies of the present invention are used to delay the onset of the disease or to slow the progression of the disease.

[0149] The term "variable region" or "variable domain" refers to a domain in the antibody heavy or light chain involved in antibody binding to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, and each domain contains four conserved framework regions (FRs) and three hypervariable regions (HVRs) (see, e.g., Kindt et al., Kuby Immunology, 6th edition, WHFreeman and Co., p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen can be isolated using the VH or VL domain derived from the antibody binding to that antigen, and complementary libraries of VL or VH domains can be screened, respectively. For example, see Portolano et al., J.Immunol. 150: pp. 880-887 (1993); Clarkson et al., Nature 352: pp. 624-628 (1991).

[0150] As used herein, the term “vector” refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is ligated. This term includes vectors as self-replicating nucleic acid structures, as well as vectors incorporated into the genome of a host cell into which they are introduced. Certain vectors can instruct the expression of the nucleic acid to which they are operably ligated. Such vectors are referred herein to as “expression vectors.”

[0151] As used herein, the term "Pb" or "lead" includes its ion, for example, Pb(II). Thus, a knowledgeable reader will know, for example, lead, Pb, 212 Pb or 203 It can be understood that the term Pb is intended to encompass the ionic form of the element, in particular Pb(II). In various aspects of the present invention, Pb may be a radioactive isotope (for example, when used in radioimmunotherapy or radioimmunoimaging methods) or a stable, non-radioactive isotope (for example, as may be preferred in the context of a decontamination agent).

[0152] "DOTAM" has the chemical name: 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane. The following formula: This is the compound TIFF2026062683000001.tif64170.

[0153] 212 Pb-DOTAM has the following structure: It has TIFF2026062683000002.tif56170.

[0154] In certain aspects and embodiments, the present invention also utilizes functional mutants or derivatives of DOTAM containing metal ions. Preferred mutants / derivatives of DOTAM differ from the structure of DOTAM to a certain limited extent and retain the ability to function (i.e., retain sufficient activity for use for one or more purposes described herein). In such aspects and embodiments, DOTAM and the functional mutants / derivatives of DOTAM may be one of the active mutants disclosed in WO2010 / 099536.

[0155] A suitable functional variant / derivative is given by the following formula: Compounds of TIFF2026062683000003.tif66170, or their pharmaceutically acceptable salts. [In the formula, R N H, C 1~6 Alkyl, C 1~6 Haloalkyl, C 2~6 Alkenil, C 2~6 Alkinyl, C 3~7 Cycloalkyl, C 3~7 Cycloalkyl-C 1~4 Alkyl, C 2~7 Heterocycloalkyl, C 2~7 Heterocycloalkyl-C 1~4 Alkyl, phenyl, phenyl-C 1~4 -alkyl, C 1~7 Heteroaryls, and C 1~7 Heteroaryl-C 1~4 -It is alkyl; C 1~6 Alkyl, C 1~6 Haloalkyl, C 2~6 Alkenyl and C 2~6 Alkinyl is selected from 1, 2, 3, or 4 independently chosen R W Each of the bases may be substituted; the above C 3~7 Cycloalkyl, C 3~7 Cycloalkyl-C 1~4 Alkyl, C 2~7 Heterocycloalkyl, C 2~7 Heterocycloalkyl-C 1~4 Alkyl, phenyl, phenyl-C1~4 -alkyl, C 1~7 heteroaryl, and C 1~7 heteroaryl-C 1~4 -alkyl may each be substituted with 1, 2, 3, or 4 independently selected R X groups; L 1 is independently C 1~6 alkylene, C 1~6 alkenylene, or C 1~6 alkynylene, and each may be substituted with 1, 2, or 3 independently selected R 1 groups; L 2 is C 2~4 linear alkylene and may be substituted with independently selected R 1 groups; C 1~4 alkyl and / or C 1~4 haloalkyl and may be substituted with 1, 2, 3, or 4 groups independently selected therefrom; R 1 is D 1 -D 2 -D 3 , halogen, cyano, nitro, hydroxyl, C 1~6 alkoxy, C 1~6 haloalkoxy, C 1~6 alkylthio, C 1~6 alkylsulfinyl, C 1~6 alkylsulfonyl, amino, C 1~6 alkylamino, di-C 1~6 alkylamino, C 1~4 alkylcarbonyl, carboxy, C 1~6 alkoxycarbonyl, C 1~6 alkylcarbonylamino, di-C 1~6 alkylcarbonylamino, C 1~6 alkoxycarbonylamino, C 1~6 alkoxycarbonyl-(C 1~6 alkyl)amino, carbamyl, C 1~6 alkylcarbamyl, and di-C 1~6 alkylcarbamyl, and is independently selected from, D 1These are C 6~10 Aryl-C 1~4 Alkyl, C 1~9 Heteroaryl-C 1~4 Alkyl, C 3~10 Cycloalkyl-C 1~4 Alkyl, C 2~9 Heterocycloalkyl-C 1~4 Alkyl, C 1~8 Alkylene, C 1~8 Alkenylene and C 1~8 Selected independently from alkynylene; the C 1~8 Alkylene, C 1~8 Alkenylene and C 1~8 Alkynylene is selected from 1, 2, 3, or 4 independently selected R 4 It may be substituted with the base; the above C 6~10 Aryl-C 1~4 Alkyl, C 1~9 Heteroaryl-C 1~4 Alkyl, C 3~10 Cycloalkyl-C 1~4 Alkyl, C 2~9 Heterocycloalkyl-C 1~4 Alkyl is 1, 2, 3, or 4 independently selected R 5 The bases may be substituted in each case; D 2 Each of them either does not exist independently, or C 1~20 It is a linear alkylene, and the C 1~20 One to six non-adjacent methylene groups of a linear alkylene are independently selected -D 4 -The parts may be replaced, however, C 1~20 At least one methylene unit in a linear alkylene is -D as needed. 4 - Provided that it is not replaced in part; the C 1~20 Linear alkylenes include halogen, cyano, nitro, hydroxyl, and C. 1~4 Alkyl, C 1~4 Haloalkyl, C 1~4 Alkoxy, C 1~4 Haloalkoxy, amino, C 1~4 Alkylamino, di-C 1~4 Alkylamino, C1~4 Alkylcarbonyl, Carboxylate, C 1~4 Alkoxycarbonyl, C 1~4 Alkylcarbonylamino, di-C 1~4 Alkylcarbonylamino, C 1~4 Alkoxycarbonylamino, C 1~4 Alkoxycarbonyl-(C 1~4 Alkyl)amino, carbamyl, C 1~4 Alkylcarbamyl and di-C 1~4 It may be substituted with one or more groups independently selected from alkylcarbamyl; D 3 These are H, halogen, cyano, nitro, hydroxyl, and C, respectively. 1~6 Alkyl, C 1~6 Haloalkyl, C 2~6 Alkenil, C 2~6 Alkinyl, C 3~14 Cycloalkyl, C 3~14 Cycloalkyl-C 1~4 Alkyl, C 2~14 Heterocycloalkyl, C 2~14 Heterocycloalkyl-C 1~4 Alkyl, C 6~14 Ariel, C 6~14 Aryl-C 1~4 Alkyl, C 1~13 Heteroaryl, C 1~13 Heteroaryl-C 1~4 Selected independently of alkyl; the C 1~6 Alkyl, C 1~6 Haloalkyl, C 2~6 Alkenil, C 2~6 Alkinyl is selected from 1, 2, 3, or 4 independently chosen R 6 Each of the bases may be substituted; the above C 3~14 Cycloalkyl, C 3~14 Cycloalkyl-C 1~4 Alkyl, C 2~14 Heterocycloalkyl, C 2~14 Heterocycloalkyl-C 1~4 Alkyl, C 6~14 Ariel, C 6~14 Aryl-C 1~4 Alkyl, C1~13 Heteroaryl, C 1~13 Heteroaryl-C 1~4 Alkyl is 1, 2, 3, or 4 independently selected R 7 The bases may be substituted in each case; D 4 These are -O-, -S-, and -NR respectively. a C(=O)-, -NR a C(=S)-, -NR b C(=O)NR c NR b C(=S)NR c -, -S(=O)-, -S(=O)2-, -S(=O)NR a -, -C(=O)-, -C(=S)-, -C(=O)O-, -OC(=O)NR a -, -OC(=S)NR a -, -NR a -, -NR b S(=O)NR c -, and NR b S(=O)2NR O -Selected independently of; R 4 and R 6 These are halogen, cyano, nitro, hydroxyl, and C, respectively. 1~4 Alkoxy, C 1~4 Haloalkoxy, C 1~4 Alkylthio, C 1~4 Alkyl sulfinyl, C 1~4 Alkylsulfonyl, amino, C 1~4 Alkylamino, di-C 1~4 Alkylamino, C 1~4 Alkylcarbonyl, Carboxylate, C 1~4 Alkoxycarbonyl, C 1~4 Alkylcarbonylamino, di-C 1~4 Alkylcarbonylamino, C 1~4 Alkoxycarbonylamino, C 1~4 Alkoxycarbonyl-(C 1~4 Alkyl)amino, carbamyl, C 1~4 Alkylcarbamyl and di-C 1~4 Independently selected from alkylcarbamyls; R5 These are halogen, cyano, cyanate, isothiocyanate, nitro, hydroxyl, and C, respectively. 1~4 Alkyl, C 2~4 Alkenil, C 2~4 Alkinyl, C 1~4 Alkoxy, C 1~4 Haloalkoxy, C 1~4 Alkylthio, C 1~4 Alkyl sulfinyl, C 1~4 Alkylsulfonyl, amino, C 1~4 Alkylamino, di-C 1~4 Alkylamino, C 1~4 Alkylcarbonyl, Carboxylate, C 1~4 Alkoxycarbonyl, C 1~4 Alkylcarbonylamino, di-C 1~4 Alkylcarbonylamino, C 1~4 Alkoxycarbonylamino, C 1~4 Alkoxycarbonyl-(C 1~4 Alkyl)amino, carbamyl, C 1~4 Alkylcarbamyl and di-C 1~4 Independently selected from alkylcarbamyls; R 7 These are halogen, cyano, nitro, hydroxyl, and C, respectively. 1~6 Alkyl, C 2~6 Alkenil, C 2~6 Alkinyl, C 3~7 Cycloalkyl, C 3~7 Cycloalkyl-C 1~4 Alkyl, C 2~7 Heterocycloalkyl, C 2~7 Heterocycloalkyl-C 1~4 Alkyl, phenyl, phenyl-C 1~4 Alkyl, C 1~7 Heteroaryl, C 1~7 Heteroaryl-C 1~4 Alkyl, -OR O , -SR O -S(=O)R P -S(=O)2R P -S(=O)NR s R t -C(=O)R P, -C(=O)OR P -C(=O)NR s R t -OC(=O)R P -OC(=O)NR s R t , -NR s R t , -NR q C(=O)R r , -NR q C(=O)OR r , -NR q C(=O)NR r , -NR q S(=O)2R r , and NR P S(=O)2NR s R t Selected independently from; the C 1~6 Alkyl, C 2~6 Alkenil, C 2~6 The alkynyl may be substituted with 1, 2, 3, or 4 independently selected R' groups; the C 3~7 Cycloalkyl, C 3~7 Cycloalkyl-C 1~4 Alkyl, C 2~7 Heterocycloalkyl, C 2~7 Heterocycloalkyl-C 1~4 Alkyl, phenyl, phenyl-C 1~4 Alkyl, C 1~7 Heteroaryl, C 1~7 Heteroaryl-C 1~4 The alkyl group may be substituted with one, two, three, or four independently selected R'' groups; R a , R b , and R c H and C are respectively 1~6 Alkyl, C 1~6 Haloalkyl, C 2~6 Alkenil, C 2~6 Alkinyl, C 3~7 Cycloalkyl, C 3~7 Cycloalkyl-C 1~4 Alkyl, C 2~7 Heterocycloalkyl, C 2~7 Heterocycloalkyl-C1~4 Alkyl, phenyl, phenyl-C 1~4 Alkyl, C 1~7 Heteroaryl, C 1~7 Heteroaryl-C 1~4 Selected independently of alkyl; the C 1~6 Alkyl, C 1~6 Haloalkyl, C 2~6 Alkenil, C 2~6 Alkinyl is selected from 1, 2, 3, or 4 independently chosen R W Each of the bases may be substituted; the above C 3~7 Cycloalkyl, C 3~7 Cycloalkyl-C 1~4 Alkyl, C 2~7 Heterocycloalkyl, C 2~7 Heterocycloalkyl-C 1~4 Alkyl, phenyl, phenyl-C 1~4 Alkyl, C 1~7 Heteroaryl, C 1~7 Heteroaryl-C 1~4 Alkyl is 1, 2, 3, or 4 independently selected R X The bases may be substituted in each case; R O , R p , R q , R r , R s and R t H and C are respectively 1~6 Alkyl, C 1~6 Haloalkyl, C 2~6 Alkenil, C 2~6 Alkinyl, C 3~7 Cycloalkyl, C 3~7 Cycloalkyl-C 1~4 Alkyl, C 2~7 Heterocycloalkyl, C 2~7 Heterocycloalkyl-C 1~4 Alkyl, phenyl, phenyl-C 1~4 Alkyl, C 1~7 Heteroaryl, C 1~7 Heteroaryl-C 1~4 Selected independently of alkyl; the C 1~6 Alkyl, C 1~6 Haloalkyl, C2~6 Alkenil, C 2~6 Alkinyl is selected from 1, 2, 3, or 4 independently chosen R y Each of the bases may be substituted; the above C 3~7 Cycloalkyl, C 3~7 Cycloalkyl-C 1~4 Alkyl, C 2~7 Heterocycloalkyl, C 2~7 Heterocycloalkyl-C 1~4 Alkyl, phenyl, phenyl-C 1~4 Alkyl, C 1~7 Heteroaryl, C 1~7 Heteroaryl-C 1~4 Alkyl is 1, 2, 3, or 4 independently selected R z The bases may be substituted in each case; R', R w and R y These are hydroxyl, cyano, nitro, and C, respectively. 1~4 Alkoxy, C 1~4 Haloalkoxy, amino, C 1~4 Alkylamino and di-C 1~4 Selected independently from alkylaminos; R''oR x , and R z These are hydroxyl, halogen, cyano, nitro, and C, respectively. 1~4 Alkyl, C 1~4 Haloalkyl, C 1~4 Alkoxy, C 1~4 Haloalkoxy, amino, C 1~4 Alkylamino and di-C 1~4 Selected independently from alkylaminos; However, this is subject to the condition that the valence does not exceed that of each atom in the part that may be substituted. That's fine.

[0156] Preferably, the functional variant / derivative of the above formula has an affinity for the antibody of the present invention equivalent to or higher than that of DOTAM, and a binding strength to Pb equivalent to or higher than that of DOTAM ("affinity" is measured by the dissociation constant, as described above). For example, the dissociation constant of the functional variant / derivative of the antibody of the present invention with Pb may be 1.1 times or less, 1.2 times or less, 1.3 times or less, 1.4 times or less, 1.5 times or less, or 2 times or less than the dissociation constant of DOTAM with the same antibody / Pb.

[0157] R N H and C are respectively 1~6 Alkyl, or C 1~6 Haloalkyl; preferably H, C 1~4 Alkyl, or C 1~4 It may be a haloalkyl. Most preferably, R N These are H.

[0158] Regarding the DOTAM mutant, 1, 2, 3, or most preferably, each L 2 This is a C2 alkylene. Advantageously, the C2 alkylene variant of DOTAM may have a particularly high affinity for Pb. 2 Any substituent for R 1 , C 1~4 Alkyl, or C 1~4 It may also be a haloalkyl group. Preferably, L 2 Any substituent for C 1~4 Alkyl or C 1~4 Haloalkyl groups are also acceptable.

[0159] If necessary, L 2 Each of these may be an unsubstituted C2 alkylene-CH2CH2-.

[0160] L 1 Each is preferably C 1~4 Alkylenes, more preferably C1 alkylenes such as -CH2-.

[0161] Functional variants / derivatives may also include DOTAM or the above compounds conjugated to one or more further parts, e.g., small molecules, polypeptides, or carbohydrates. This conjugation may occur via a single carbon in the macrocyclic backbone. The small molecules may be, for example, dyes (Alexa 647 or Alexa 488), biotin, or biotin moieties. The polypeptides may be, for example, oligopeptides, therapeutic peptides such as antibodies, or polypeptides. Exemplary carbohydrates include dextran, linear or branched polymers or copolymers (e.g., polyalkylenes, poly(ethylene-lysine), polymethacrylates, polyamino acids, polysaccharides or oligosaccharides, dendrimers).

[0162] Functional variants / derivatives of DOTAM are given by the following formula: TIFF2026062683000004.tif63170[In the formula, Z is independently defined as R as defined above] 1 [and p, q, r, and s are 0, 1, or 2; and p+q+r+s is 1 or greater] The compound may be a compound of the form p, q, r, and s, preferably p, q, r, and s are 0 or 1, and / or p+q+r+s = 1. For example, the compound may have p+q+r+s=1, where Z is the p-SCN-benzyl moiety, and such compounds are commercially available from Macrocyclics, Inc. (Plano, Texas).

[0163] Detailed explanation Compositions and methods antibody In one embodiment, the present invention is based in part on providing an antibody that specifically binds to Pb-DOTAM (i.e., a chelate containing DOTAM complexed with Pb, also referred herein as "Pb-SOTAM chelate").

[0164] In a particular embodiment, the antibody that specifically binds to Pb-DOTAM may have one or more of the following properties: • Specifically binds to Pb-DOTAM and Bi-DOTAM; • It is selective for Pb-DOTAM compared to other chelated metals such as Cu-DOTAM; • Binds to Pb-DOTAM with very high affinity; The antibodies described herein, for example, PRIT-0213 or PRIT-0214, bind to the same epitope on Pb-DOTAM and / or have the same contact residue as the antibodies.

[0165] Radioactive isotopes of lead (Pb) are useful in diagnostic and therapeutic methods. Specific radioactive isotopes of lead that may be useful in this invention include: 212 Pb and 203 Pb is one example. Stable isotopes of lead, for example, 204 Pb, 206 Pb, 207 Pb or 208 Pb can also be used in the removal agent. Pb is a stable (non-radioactive) isotope. 204 Pb, 206 Pb, 207 Pb and 208 It may be a mixture of Pb, or naturally occurring lead.

[0166] Due to their combination of short path length and high linear energy delivery, alpha-particle radionuclides cause less damage to surrounding tissues and possess a more specific ability to kill tumor cells compared to beta-particle radionuclides. 212 Bi is an alpha particle emitter, but its short half-life prevents its direct use. 212 Pb is, 212 It is a radiophilic nuclide of Bi, 212 It acts as a source of Bi in vivo, thereby 212 This method efficiently overcomes the short half-life of Bi (Yong and Brechbiel, Dalton Trans. June 21, 2001; 40(23) pp. 6068-6076).

[0167] 203 Pb is useful as an imaging isotope. Thus,203 Antibodies bound to Pb-DOTAM may be useful in radioimmunoimaging (RII).

[0168] Generally, radioactive metals are used in chelated form. In aspects of the present invention, DOTAM is used as a chelating agent. DOTAM is a stable chelating agent for Pb(II) (Yong and Brechbiel, Dalton Trans. June 21, 2001; 40(23) pp. 6068-6076; Chappell et al., Nuclear Medicine and Biology, Vol. 27, pp. 93-100, 2000). Thus, DOTAM is used, 212 Pb and 203 It is particularly useful in conjugations with lead isotopes such as Pb, as discussed above.

[0169] As discussed above, the antibody according to the present invention binds to Pb-DOTAM. In some embodiments, it is preferable that the antibody binds to Pb-DOTAM with a binding affinity Kd value of 100 pM, 50 pM, 20 pM, 10 pM, 5 pM, 1 pM or less, for example, 0.9 pM or less, 0.8 pM or less, 0.7 pM or less, 0.6 pM or less, or 0.5 pM or less.

[0170] The antibody further binds to Bi chelated with DOTAM. In some embodiments, the antibody preferably binds to Bi-DOTAM (i.e., a chelate containing DOTAM complexed with bismuth, also referred to herein as "Bi-DOTAM chelate") with binding affinity Kd values ​​of 1 nM, 500 pM, 200 pM, 100 pM, 50 pM, 10 pM or less, for example, 9 pM, 8 pM, 7 pM, 6 pM, 5 pM or less.

[0171] In some embodiments, the antibody may bind to Bi-DOTAM and Pb-DOTAM with similar affinity. For example, the affinity ratio for Bi-DOTAM / Pb-DOTAM, e.g., the ratio of Kd values, is preferably in the range of 0.1 to 10, e.g., 1 to 10.

[0172] The sample affinity values ​​for the exemplary antibody (PRIT-0213) according to the present invention are provided below: TIFF2026062683000005.tif55170

[0173] Affinity was determined by KinExA equilibrium measurement.

[0174] Furthermore, the antibodies of the present invention are preferably selective for Bi-DOTAM and / or Pb-DOTAM compared to other chelated metals such as Cu-DOTAM. For example, the affinity ratio, e.g., the Kd value ratio, for Pb-DOTAM / Cu-DOTAM may be at least 100,000.

[0175] In some embodiments, the antibody is i) A first heavy chain having the amino acid sequence of SEQ ID NO: 22; ii) A second heavy chain having the amino acid sequence of SEQ ID NO: 23; and iii) Two antibody light chains having the amino acid sequence of SEQ ID NO: 21 It is preferable that the antibody binds to Pb-DOTAM and / or Bi-DOTAM with an affinity (e.g., affinity Kd value) equal to or higher than that of a bispecific antibody (referred to herein as PRIT-0213) having the same properties.

[0176] In some embodiments, the antibody is i) The first heavy chain having the amino acid sequence of SEQ ID NO: 19; ii) A second heavy chain having the amino acid sequence of SEQ ID NO: 20; and iii) Two antibody light chains having the amino acid sequence of SEQ ID NO: 21 It is preferable that the DOTAM-chelated Pb and / or DOTAM-chelated Bi bind to a bispecific antibody (referred to herein as PRIT-0214) with an affinity (e.g., affinity KD value) equal to or higher than that of a bispecific antibody (referred to herein as PRIT-0214).

[0177] In some embodiments, the antibody according to the present invention binds to the same or overlapping chelated radionuclide epitopes as the antibodies disclosed herein.

[0178] In some embodiments, the antibody is i) A first heavy chain having the amino acid sequence of SEQ ID NO: 22; ii) A second heavy chain having the amino acid sequence of SEQ ID NO: 23; and iii) Two antibody light chains having the amino acid sequence of SEQ ID NO: 21 It binds to the same or overlapping Pb-DOTAM chelate (Pb-DOTAM) epitope as Fab PRIT-0213.

[0179] The epitope of a chelated radionuclide (e.g., Pb-DOTAM) to which a given antibody binds can be determined and compared with the epitope of a chelated radionuclide to which an antibody disclosed herein (e.g., Fab PRIT-0213) binds.

[0180] This disclosure describes the determination of the crystal structure of Fab PRIT-0213 complexed with Pb-DOTAM at a resolution of 1.40 Å in Example 14, and the characterization of the binding interaction between Fab PRIT-0213 and Pb-DOTAM based on the analysis of this structure using the Protein Interface Surface and Assembly (PISA) program (Krissinel and Henrick, J Mol Biol (2007) 372(3):774-797).

[0181] In some embodiments, the antibody according to the present invention may, for example, exhibit interaction with one or more of the following sites with respect to Pb-DOTAM: edge-to-face, N6, N7, N8, N5 and / or C12 with respect to the azacyclododecane ring region below the azacyclododecane ring (e.g., the tetracyclododecane ring), as determined by PISA analysis of the structure of the antibody complexed with Pb-DOTAM. In some embodiments, the antibody may exhibit interaction with one or more of the following sites with respect to Pb-DOTAM: N7, N8, edge-to-face, tetracyclododecane ring and / or N6 with respect to the azacyclododecane ring.

[0182] In some embodiments, the antibody may exhibit one or more of the following interactions with respect to Pb-DOTAM, as determined, for example by PISA analysis of the structure of the antibody complexed with Pb-DOTAM: edge-to-face nonpolar interaction with the azacyclododecane ring, polar interaction with N8, hydrogen bonding with N7, hydrogen bonding with N8, polar interaction with N5, nonpolar interaction with C12, polar interaction with N7, polar (hydrogen) bonding with N6, and / or nonpolar interaction with the tetracyclododecane ring.

[0183] In some embodiments, the antibody may exhibit one or more of the following interactions with respect to Pb-DOTAM, as determined, for example, by PISA analysis of the structure of the antibody complexed with Pb-DOTAM: hydrogen bonding between one or more residues of antibody heavy chain CDR3 and N7, hydrogen bonding between one or more residues of antibody heavy chain CDR3 and N8, nonpolar interactions between one or more residues of antibody heavy chain CDR2 that are edge-to-face with the azacyclododecane ring, nonpolar interactions between antibody light chain CDR3 and the tetracyclododecane ring, and / or nonpolar interactions between antibody light chain CDR1 and N6.

[0184] In other embodiments, antibodies may share the same contact residues as described herein: for example, these residues may be invariant. These residues are: a) In the heavy chain CDR2: Phe50, Asp56 and / or Tyr58, and Gly52 and / or Arg54 as needed; b) In the heavy chain CDR3: Glu95, Arg96, Asp97, Pro98, Tyr99, Ala100C and / or Tyr100D, and Pro100E as needed; c) Light chain CDR1 contains: Tyr28 and / or Asp32; d) Light chain CDR3 contains: Gly91, Tyr92, Asp93, Thr95c and / or Tyr96; e) During light chain CDR2: Gln50 as needed It may include.

[0185] Certain aspects and embodiments of the antibody according to the present invention are discussed above. More preferred aspects and embodiments of the present invention are discussed below. In all embodiments, the antibody retains the ability to bind to Pb-DOTAM, preferably to Bi-DOTAM, and more preferably to the affinity and / or selectivity discussed above.

[0186] In one embodiment, the present invention can provide an anti-Pb-DOTAM antibody comprising at least one, two, three, four, five, or six CDRs selected from (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

[0187] In one embodiment, the present invention provides an antibody comprising at least one, at least two, or all three VH CDR sequences selected from (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3. In one embodiment, the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3. In another embodiment, the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3 and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6. In a further embodiment, the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6, and CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2. In a further embodiment, the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4. In further embodiments, the antibody comprises (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 1; (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 2; and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 3.

[0188] In another embodiment, the present invention provides an antibody comprising at least one, at least two, or all three VL CDR sequences selected from (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6. In one embodiment, the antibody comprises (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

[0189] In another embodiment, the antibody of the present invention comprises (a) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) CDR-H3 comprising the amino acid sequence selected from SEQ ID NO: 3; and (b) a VL domain comprising at least one, at least two, or all three VL CDRs selected from (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, and (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

[0190] In another embodiment, the present invention provides antibodies comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

[0191] In some embodiments, the antibody may include one or more of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and / or CDR-L3, each having substitutions, for example, one, two, or three substitutions, compared to the amino acid sequences of SEQ ID NOs. Preferably, these substitutions are not present in the invariant positions described above.

[0192] For example, in some embodiments, CDR-H2 may include variants having the amino acid sequence FIGSRGDTYYASWAKG (SEQ ID NO: 2), or having up to one, two, or three substitutions in SEQ ID NO: 2, wherein these substitutions do not include Phe50, Asp56, and / or Tyr58, and optionally also do not include Gly52 and / or Arg54 (all numbered by Kabat).

[0193] In some embodiments, CDR-H2 may be substituted at one or more positions shown below. Here, and in the substitution table below, substitutions are based on germline residues (underlined) or on amino acids that are theoretically sterically compatible and present in the repertoire crystallized at that site. In some embodiments, the above residues may be fixed and other residues substituted according to the table below; in other embodiments, substitutions of any residue may be made according to the table below. TIFF2026062683000006.tif127170

[0194] If necessary, CDR-H3 may include variants of the amino acid sequence ERDPYGGGAYPPHL (SEQ ID NO: 3), or SEQ ID NO: 3, having one, two, or three substitutions, wherein these substitutions do not include Glu95, Arg96, Asp97, Pro98, and optionally also do not include Ala100C, Tyr100D, and / or Pro100E, and / or optionally also do not include Tyr99. For example, in some embodiments, the substitutions do not include Glu95, Arg96, Asp97, Pro98, Tyr99, Ala100C, and Tyr100D.

[0195] In certain embodiments, CDR-H3 may be substituted at one or more of the positions shown below. In some embodiments, the above residue may be fixed and other residues may be substituted according to the following table; in other embodiments, substitution of any residue may be made according to the following table. TIFF2026062683000007.tif112170

[0196] If necessary, CDR-L1 may also include the amino acid sequence QSSHSVYSDNDLA (SEQ ID NO: 4), or its variants having up to one, two, or three substitutions in SEQ ID NO: 4, in which these substitutions do not include Tyr28 and Asp32 (Kavat numbering).

[0197] In certain embodiments, CDR-L1 may be substituted at one or more of the positions shown below. Again, in some embodiments, the above residue may be fixed and other residues may be substituted according to the table below; in other embodiments, substitution of any residue may be made according to the table below. TIFF2026062683000008.tif105170

[0198] If necessary, CDR-L3 may include the amino acid sequence LGGYDDESDTYG (SEQ ID NO: 6), or variants thereof having one, two, or three substitutions in SEQ ID NO: 6, wherein these substitutions do not include Gly91, Tyr92, Asp93, Thr95c, and / or Tyr96 (kabat).

[0199] In certain embodiments, CDR-L3 may be substituted at the following positions shown below (many substitutions are expected as many residues are solvent-exposed and do not have antigen contact). Again, in some embodiments, the above residue may be fixed and other residues may be substituted according to the following table; in other embodiments, substitution of any residue may be made according to the following table. TIFF2026062683000009.tif149170

[0200] The antibodies may further include, as needed, CDR-H1 and CDR-L2 having the sequence of SEQ ID NO: 1 or SEQ ID NO: 5, respectively, or variants thereof having at least one, two, or three substitutions, and optionally, conservative substitutions.

[0201] In any of the above embodiments, the anti-Pb-DOTAM antibody may be humanized. In one embodiment, the anti-Pb-DOTAM antibody comprises a CDR as in any of the above embodiments, and further comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework. In another embodiment, the anti-Pb-DOTAM antibody comprises a CDR as in any of the above embodiments, and further comprises framework regions derived from vk 1 39 and / or vh 2 26. For vk 1 39, in some embodiments, a reverse mutation may not be present. For vh 2 26, the germline Ala49 residue may not be reverse-mutated to Gly49.

[0202] If necessary, the antigen-binding site may include a heavy chain variable domain containing an amino acid sequence selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 9, or a variant thereof containing an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO: 7 or SEQ ID NO: 9. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the antibody containing the sequence preferably retains the ability to bind to Pb-DOTAM with the affinity described herein. The VH sequence may contain invariant residues as described above. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 7 or SEQ ID NO: 9. In certain embodiments, the substitutions, insertions, or deletions are located in a region outside the CDR (i.e., FR). If necessary, the antibody includes the VH sequence in SEQ ID NO: 7 or SEQ ID NO: 9, and includes post-translational modifications of that sequence. In certain embodiments, the VH includes one, two, or three CDRs selected from (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 1; (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 2; and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 3.

[0203] In another embodiment, an anti-Pb-DOTAM antibody is provided, comprising a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 10. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the anti-Pb-DOTAM antibody containing the sequence preferably retains the ability to bind to Pb-DOTAM with affinity as described herein. The VL sequence may contain invariant residues as described above. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 8 or SEQ ID NO: 10. In certain embodiments, substitutions, insertions, or deletions are located in regions outside the CDR (i.e., FR). Optionally, the anti-Pb-DOTAM antibody includes the VL sequence in SEQ ID NO: 8 or SEQ ID NO: 10, and includes post-translational modifications of that sequence. In certain embodiments, the VL includes one, two, or three CDRs selected from (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 4; (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 5; and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 6.

[0204] In another embodiment, an anti-Pb-DOTAM antibody is provided, wherein the antibody comprises a VH sequence as described in any of the embodiments provided above and a VL sequence as described in any of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 7 and SEQ ID NO: 8, respectively, and includes post-translational modifications of those sequences. In another embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 9 and SEQ ID NO: 10, respectively, and includes post-translational modifications of those sequences.

[0205] In further embodiments of the present invention, the antibody described in any of the above embodiments is a monoclonal antibody, including a chimeric, humanized, or human antibody. In one embodiment, the antibody is an antibody fragment, e.g., Fv, Fab, Fab', scFab, scFv, diabody, or F(ab')2 fragment. Examples of antibody fragments, but not limited to, include Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, as well as other fragments described below. For an overview of a particular antibody fragment, see Hudson et al., Nat. Med. 9: pp. 129-134 (2003). For an overview of the scFv fragment, see, for example, Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore (eds.), (Springer-Verlag, New York), pp. 269–315 (1994); also see WO93 / 16185; and U.S. patents 5,571,894 and 5,587,458. For a discussion of the Fab and F(ab')2 fragments, which contain salvage receptor-binding epitope residues and have increased in vivo half-lives, see U.S. patent 5,869,046.

[0206] A diabody is an antibody fragment having two antigen-binding sites, which may be bivalent or bispecific. See, for example, EP404,097;WO1993 / 01161;Hudson et al., Nat.Med.9:129-134 (2003); and Hollinger et al., Proc.Natl.Acad.Sci.USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat.Med.9:129-134 (2003).

[0207] A single-domain antibody is an antibody fragment containing all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, the single-domain antibody is a human single-domain antibody (see, for example, Domantis, Inc., Waltham, MA; U.S. Patent No. 6,248,516B1).

[0208] Antibody fragments can be produced by a variety of techniques, including, but not limited to, the proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or phages), as described herein.

[0209] In another embodiment, the antibody is a full-length antibody, for example, an intact IgG antibody or other antibody class or isotype as defined herein.

[0210] Targeted agent In some embodiments, an antibody that specifically binds to DOTAM-chelated Pb is coupled to a cell-binding / targeting moiety to produce a target agent. If necessary, the antibody that specifically binds to DOTAM-chelated Pb may be the antibody described in any of the embodiments above.

[0211] Coupling may preferably occur by expression as a fusion polypeptide or protein. Fusion may be direct or via a linker. The fusion polypeptide or protein can be produced recombinantly, avoiding the need for conjugation chemical reactions. Optionally, the linker may be a peptide of at least 5 amino acids, preferably between 25 and 30 amino acids. The linker may be a rigid or flexible linker. In some embodiments, it is flexible and contains or consists of Thr, Ser, Gly, and / or Ala residues. For example, it may contain or consist of Gly and Ser residues. In some embodiments, it is (Gly-Gly-Gly-Gly-Ser)n The linker may have a repeating motif such as [wherein n is, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10]. In some embodiments, the linker may be or include the sequence GGGGSGGGGSGGGGSGGGGS (Sequence ID 26). Other linkers may be used and can be identified by those skilled in the art.

[0212] Thus, a multispecific (e.g., bispecific) antibody complex is provided that specifically binds to both Pb-DOTAM chelate and another target antigen, such as an antigen present on the surface of a target cell.

[0213] With respect to the present invention relating to therapeutic methods and products for use in therapeutic methods, it is applicable to any condition that can be treated by cytotoxic activity targeted at diseased cells of a patient. The treatment is preferably of a tumor or cancer (e.g., pancreatic cancer, breast cancer, or prostate cancer). However, the applicability of the present invention is not limited to tumors and cancers. For example, the treatment may also be of a viral infection. Immunotoxins against viral antigens expressed on the surface of infected cells have been investigated for various viral infections such as HIV, rabies, and EBV. Cai and Berger 2011 Antiviral Research 90(3):143-50 used an immunotoxin containing PE38 for targeted killing of cells infected with Kaposi's sarcoma-associated herpesvirus. Furthermore, Resimmune® (A-dmDT390-bisFv(UCHT1)) is thought to selectively kill human malignant T cells and transiently deplete normal T cells, and is considered to have potential for the treatment of T cell-induced autoimmune diseases such as multiple sclerosis and graft-versus-host disease, as well as T cell hematological malignancies currently undergoing clinical trials.

[0214] Thus, suitable target antigens may include cancer cell antigens, particularly human cancer cell antigens, viral antigens, or microbial antigens.

[0215] The target antigens described herein are designed to bind to diseased cells, such as tumor cells, via their cell surface antigens. Antigens are typically normal cell surface antigens that are overexpressed or expressed at abnormal times. Ideally, target antigens are expressed only on diseased cells (such as tumor cells), but this is rarely observed in practice. Consequently, target antigens are usually selected based on differential expression between diseased and healthy tissues.

[0216] Thus, the target antibody can specifically bind to any suitable cell surface marker. The selection of a specific targeting moiety and / or cell surface marker can be chosen depending on the specific cell population to be targeted. Cell surface markers are known in the art (see, for example, Mufson et al., Front. Biosci., 11:337-43 (2006); Frankel et al., Clin. Cancer Res., 6:326-334 (2000); and Kreitman et al., AAPS Journal, 8(3):E532-E551 (2006)), and may be, for example, proteins or carbohydrates. In embodiments of the present invention, the targeting moiety (cell binding agent) is a ligand that specifically binds to a receptor on the cell surface. Examples of ligands include, but are not limited to, vascular endothelial growth factor (VEGF), Fas, TNF-related apoptosis-inducing ligands (TRAIL), cytokines (e.g., IL-2, IL-15, IL-4, IL-13), lymphokines, hormones, and growth factors (e.g., transforming growth factor (TGFa), nerve growth factor, epidermal growth factor).

[0217] The cell surface marker may be, for example, a tumor-associated antigen.

[0218] As used herein, the terms “tumor-associated antigen” or “tumor-specific antigen” refer to any molecule (e.g., protein, peptide, lipid, carbohydrate, etc.) that is exclusively, primarily, or overexpressed by tumor cells and / or cancer cells, such that the antigen is associated with tumors and / or cancer. Tumor-associated antigens may also be expressed by normal, non-tumor, or non-cancerous cells. However, in such cases, the expression of tumor-associated antigens by normal, non-tumor, or non-cancerous cells is not as robust as the expression by tumor or cancer cells. In this regard, tumor or cancer cells may overexpress the antigen or express it at significantly higher levels compared to the expression of the antigen by normal, non-tumor, or non-cancerous cells. Furthermore, tumor-associated antigens may also be expressed by cells at different stages of development or maturation. For example, tumor-associated antigens may also be expressed by embryonic or fetal cells that are not typically found in adult hosts. Alternatively, tumor-associated antigens may also be expressed by stem cells or progenitor cells that are not typically found in adult hosts.

[0219] Tumor-associated antigens may be antigens expressed by any cells of any cancer or tumor, including the cancers and tumors described herein. Tumor-associated antigens may be tumor-associated antigens of only one type of cancer or tumor, such that the tumor-associated antigen is associated with or characteristic of only one type of cancer or tumor. Alternatively, tumor-associated antigens may be tumor-associated antigens of more than one type of cancer or tumor (for example, characteristic of more than one type). For example, tumor-associated antigens may be expressed by both breast cancer cells and prostate cancer cells, and not expressed at all by normal, non-tumor, or non-cancerous cells.

[0220] Exemplary tumor-associated antigens to which cell-binding agents can specifically bind include, but are not limited to, mucin 1 (MUC1; tumor-associated epithelial mucin), preferentially expressed melanoma antigen (PRAME), carcinoembryonic antigen (CEA), prostate-specific membrane antigen (PSMA), PSCA, EpCAM, Trop2, granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR), CD56, human epidermal growth factor receptor 2 (HER2 / neu) (also known as erbB-2), CDS, CD7, tyrosinase-associated protein (TRP) I, and TRP2. In preferred embodiments, the cell surface markers to which the targeting portion (cell binding agent) specifically binds are: differentiation antigens (CD) 19, CD20, CD21, CD22, CD25, CD30, CD33 (sialic acid-bound Ig-like lectin 3, myeloid cell surface antigen), CD79b, CD123 (interleukin 3 receptor alpha), transferrin receptor, EGF receptor, mesothelin, cadherin, Lewis Y, glypican-3, FAP (fibroblast-activating protein alpha), PSMA (prostate-specific membrane antigen), CA9=CAIX (carbonic anhydrase IX), L1 The group is selected from CAM (neuronal cell adhesion molecule L1), endothialin, HER3 (activation conformation of epidermal growth factor receptor family member 3), Alk1 / BMP9 complex (anaplastic lymphoma kinase 1 / osteogenesis imperfecta 9), TPBG=5T4 (trophoblast glycoprotein), ROR1 (receptor tyrosine kinase-like surface antigen), HER1 (activation conformation of epidermal growth factor receptor), and CLL1 (type C lectin domain family 12, member A). Mesothelin is expressed in, for example, ovarian cancer, mesothelioma, non-small cell lung cancer, lung adenocarcinoma, fallopian duct carcinoma, head and neck cancer, cervical cancer, and pancreatic cancer. CD22 is expressed in, for example, pilocytic cell leukemia, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), non-Hodgkin lymphoma, small lymphocytic lymphoma (SLL), and acute lymphoblastic leukemia (ALL). CD25 is expressed in leukemias and lymphomas, including, for example, pilocytic cell leukemia and Hodgkin lymphoma.Lewis Y antigen is expressed in cancers such as bladder cancer, breast cancer, ovarian cancer, colorectal cancer, esophageal cancer, gastric cancer, lung cancer, and pancreatic cancer. CD33 is expressed in cancers such as acute myeloid leukemia (AML), chronic myelomonocytic leukemia (CML), and myeloproliferative disorders.

[0221] In embodiments of the present invention, the targeting portion is an antibody (including an antibody fragment) that specifically binds to a target, such as a tumor-associated antigen. In such embodiments, the drug may be called a bispecific or multispecific antibody.

[0222] Examples of antibodies that specifically bind to tumor-associated antigens include, but are not limited to, antibodies against the transferrin receptor (e.g., HB21 and its variants), antibodies against CD22 (e.g., RFB4 and its variants), antibodies against CD25 (e.g., anti-Tac and its variants), antibodies against mesothelin (e.g., SS1, MORAb-009, SS, HN1, HN2, MN, MB, and their variants), and antibodies against Lewis Y antigen (e.g., B3 and its variants). In this regard, the targeting portion (cell binding agent) may be an antibody selected from the group consisting of ofB3, RFB4, SS, SS1, MN, MB, HN1, HN2, HB21, and MORAb-009, as well as its antigen-binding portion. Further exemplary targeting moieties suitable for use in the chimeric molecule of the present invention include, for example, U.S. Patent No. 5,242,824 (anti-transferrin receptor); No. 5,846,535 (anti-CD25 antibody); No. 5,889,157 (anti-Lewis Y antibody); No. 5,981,726 (anti-Lewis Y antibody); No. 5,990,296 (anti-Lewis Y antibody); disclosed in Patent No. 7,081,518 (anti-mesoterin antibody); No. 7,355,012 (anti-CD22 antibody and anti-CD25 antibody); No. 7,368,110 (anti-mesoterin antibody); No. 7,470,775 (anti-CD30 antibody); No. 7,521,054 (anti-CD25 antibody); and No. 7,541,034 (anti-CD22 antibody); U.S. Patent Application Publication No. 2007 / 0189962 (anti-CD22 antibody); Frankel et al., Clin. Cancer Res., 6:326-334 (2000), and Kreitman et al., AAPS Journal, 8(3):E532-E551 (2006) (each incorporated herein by reference).

[0223] We have developed antibodies to target specific tumor-associated antigens such as Cripto, CD30, CD19, CD33, glycoprotein NMB, CanAg, Her2 (ErbB2 / Neu), CD56 (NCAM), CD22 (Siglec2), CD33 (Siglec3), CD79, CD138, PSCA, PSMA (prostate-specific membrane antigen), BCMA, CD20, CD70, E-selectin, EphB2, melanotransferrin, Muc16, and TMEEF2.

[0224] In some embodiments of the present invention, the tumor-associated antigen may preferably be a carcinoembryonic antigen (CEA). The CEA may be a human CEA, in particular having the amino acid sequence of carcinoembryonic antigen-associated cell adhesion molecule 5 (CEACAM5), as shown in UniProt (www.uniprot.org) accession number P06731 (version 119) or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_004354.2. Antibodies produced against CEA include T84.66 and its humanized and chimeric versions, such as T84.66-LCHA, CH1A1a, described in WO2016 / 075278A1 and / or WO2017 / 055389, anti-CEA antibodies described in WO2011 / 034660, and CEA hMN-14 (see also U.S. Patent Nos. 6,676,924 and 5,874,540) as described in Table 2 below.

[0225] CEA is advantageous in the context of the present invention because it is internalized relatively slowly, and thus a high percentage of the antibody remains available on the cell surface after initial treatment for binding to the radionuclide. Other low-internalization target / tumor-associated antigens are also preferred. For example, in some embodiments, the tumor-associated antigen may be CD20 or HER2. GenBank accessions NP_001005862, NP_004439, XP_005257196, and XP_005257197 disclose Her2 protein sequences, provided by GenBank on October 4, 2013, and SwissProt database accession P11836 discloses the CD20 sequence. In further embodiments, the target may be EGP-1 (also known as trophoblast-2, epithelial glycoprotein-1), colon-specific antigen-p (CSAp), or pancreatic mucin MUC1. For example, see Goldenberg et al., 2012 (Theranostics 2(5)), incorporated herein by reference. This reference also describes antibodies such as Mu-9, which binds to CSAp (see also Sharkey et al., Cancer Res. 2003; 63: pp. 354-63), hPAM4, which binds to MUC1 (see also Gold et al., Cancer Res. 2008: 68: pp. 4819-26), valtuzumab, which binds to CD20 (see also Sharkey et al., Cancer Res. 2008; 68: pp. 5282-90), and hRS7, which binds to EGP-1 (see also Cubas et al., Biochim Biophys Acta 2009; 1796: pp. 309-14). Any of these antibodies, or their antigen-binding moieties, may be useful in the present invention, that is, they can be incorporated into the antibodies described herein.

[0226] multispecific antibodies As discussed above, in certain embodiments, the antibodies provided herein are multispecific antibodies, for example, bispecific antibodies. A multispecific antibody is a monoclonal antibody having binding specificity to at least two different sites. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0227] In recent years, various recombinant antibody forms have been developed, such as IgG antibody forms and tetravalent bispecific antibodies formed by the fusion of single-chain domains (see, for example, Coloma, MJ et al., Nature Biotech 15 (1997) pp. 159-163; WO2001 / 077342; and Morrison, SL, Nature Biotech 25 (2007) pp. 1233-1234).

[0228] Furthermore, other novel antibody forms have been developed that can bind to two or more antigens and no longer retain the antibody core structure (IgA, IgD, IgE, IgG, or IgM), such as diabodies, triabodies or tetrabodies, minibodies, and several single-chain forms (scFv, bis-scFv) (Holliger, P. et al., Nature Biotech 23 (2005) pp. 1126-1136; Fischer, N., Leger, O., Pathobiology 74 (2007) pp. 3-14; Shen, J. et al., Journal of Immunological Methods 318 (2007) pp. 65-74; Wu, C. et al., Nature Biotech 25 (2007) pp. 1290-1297).

[0229] All such forms use a linker that fuses an antibody core (IgA, IgD, IgE, IgG, or IgM) with a further binding protein (e.g., scFv), or fuses two Fab fragments or scFvs, for example (Fischer, N., Leger, O., Pathobiology 74 (2007), pp. 3-14). Those skilled in the art should note that it is desirable to retain effector functions, such as complement-dependent cell-mediated cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC), mediated by Fc receptor binding, by maintaining a high degree of similarity to naturally occurring antibodies.

[0230] WO2007 / 024715 reports a bivariable-domain immunoglobulin as an engineered multivalent and multispecific binding protein. A process for the preparation of bioactive antibody dimers is reported in U.S. Patent No. 6,897,044. A multivalent Fv antibody construct having at least four variable domains linked to each other via a peptide linker is reported in U.S. Patent No. 7,129,330. Dimeric and multimeric antigen-binding structures are reported in U.S. Patent No. 2005 / 0079170. A trivalent or tetravalent monospecific antigen-binding protein containing three or four Fab fragments covalently bound to each other by a non-native immunoglobulin linkage structure is reported in U.S. Patent No. 6,511,663. WO2006 / 020258 reports a tetravalent bispecific antibody that can be efficiently expressed in prokaryotic and eukaryotic cells and is useful in therapeutic and diagnostic methods. A method for separating or preferentially synthesizing dimers linked by at least one interchain disulfide bond from dimers not linked by at least one interchain disulfide bond, derived from a mixture containing two types of polypeptide dimers, is reported in US2005 / 0163782. A bispecific tetravalent receptor is reported in U.S. Patent No. 5,959,083. An engineered antibody having three or more functional antigen-binding sites is reported in WO2001 / 077342.

[0231] Multispecific and multivalent antigen-binding polypeptides are reported in WO1997 / 001580. WO1992 / 004053 reports homoconjugates prepared from monoclonal antibodies of the IgG class that typically bind to the same antigenic determinant covalently linked by a synthetic crosslinking agent. Oligomer monoclonal antibodies with high avidity against antigens are reported in WO1991 / 06305, which typically secrete oligomers of the IgG class that have two or more immunoglobulin monomers that bind together to form a tetravalent or hexavalent IgG molecule. Sheep-derived antibodies and engineered antibody constructs are reported in U.S. Patent No. 6,350,860, which can be used to treat diseases in which interferon-gamma activity is pathogenic. US2005 / 0100543 reports a targetable construct that is a multivalent carrier of bispecific antibodies, i.e., a targetable construct in which each molecule of the targetable construct can act as a carrier for two or more bispecific antibodies. Genetically modified bispecific tetravalent antibodies are reported in WO1995 / 009917. WO2007 / 109254 reports a stabilized binding molecule consisting of or containing stabilized scFv.

[0232] Furthermore, multispecific antibodies can also be provided in an asymmetric form having domain crossovers in one or more binding arms of the same antigen specificity, i.e., by exchanging VH / VL domains (see, e.g., WO2009 / 080252 and WO2015 / 150447), CH1 / CL domains (see, e.g., WO2009 / 080253), or complete Fab arms (see, e.g., WO2009 / 080251, WO2016 / 016299, and also Schaefer et al., PNAS, 108(2011) pp. 1187-1191, and Klein et al., MAbs 8(2016) pp. 1010-20). In one embodiment, the multispecific antibody contains a cross-Fab fragment. The term “crossover Fab fragment,” “xFab fragment,” or “crossover Fab fragment” refers to a Fab fragment in which either the variable or constant regions of the heavy chain and light chain are exchanged. Crossover Fab fragments include polypeptide chains consisting of a light chain variable region (VL) and a heavy chain constant region 1 (CH1), and polypeptide chains consisting of a heavy chain variable region (VH) and a light chain constant region (CL). Asymmetric Fab arms can also be manipulated by introducing charged or uncharged amino acid mutations into the domain contact surfaces to direct precise Fab pairings. See, for example, WO2016 / 172485.

[0233] All of the above formats can be used for multispecific antibodies according to the present invention.

[0234] In one exemplary form, a bispecific antibody is a “trimerizer,” such as described in WO214 / 180754. This refers to a trimer antigen-binding molecule comprising three fusion polypeptides, each containing at least one antigen-binding moiety fused to a trimerizing domain derived from human cartilage matrix protein (CMP), the trimerizing domain being able to mediate the stable binding of a trimer antigen-binding molecule. The antigen-binding moieties may be, for example, Fab molecules, crossover Fab molecules, scFab, Fv molecules, scFv, or single-domain antibodies (VHH). In some embodiments, each fusion protein contains two (first and second) antigen-binding moieties, for example, both optionally via a peptide linker, where the first antigen-binding moiety is fused to the N-terminal amino acid of the trimerizing domain and the second antigen-binding moiety is fused to the C-terminal amino acid of the trimerizing domain. In this form, either the first or second antigen-binding moiety can bind to a Pb-DOTAM chelate. The other end will bind to a target antigen, such as a tumor-associated antigen. Each of the three antigen-binding molecules fused to the C-terminus may be specific to the same antigen; each of the three antigen-binding molecules fused to the N-terminus may be specific to a different antigen.

[0235] The CMP trimerization domains useful herein are derived from the following human cartilage proteins, and in one embodiment, include a sequence having at least 95% identity, and most preferably at least 98% identity, to the sequence of the trimerization domain shown below. In one embodiment, the trimerization domain includes the sequence of the trimerization domain.

[0236] Human cartilage protein sequence (496aa) MRVLSGTSLM LCSLLLLLQA LCSPGLAPQS RGHLCRTRPT DLVFVVDSSR SVRPVEFEKV KVFLSQVIES LDVGPNATRV GMVNYASTVK QEFSLRAHVS KAALLQAVRR IQPLSTGTMT GLAIQFAITK AFGDAEGGRS RSPDISKVVI VVTDGRPQDS VQDVSARARA SGVELFAIGV GSVDKATLRQ IASEPQDEHV DYVESYSVIE KLSRKFQEAF CVVSDLCATG DHDCEQVCIS SPGSYTCACH EGFTLNSDGK TCNVCSGGGG SSATDLVFLI DGSKSVRPEN FELVKKFISQ IVDTLDVSDK LAQVGLVQYS SSVRQEFPLG RFHTKKDIKA AVRNMSYMEK GTMTGAALKY LIDNSFTVSS GARPGAQKVG IVFTDGRSQD YINDAAKKAK DLGFKMFAVG VGNAVEDELR EIASEPVAEH YFYTADFKTI NQIGKKLQKK ICVEEDPCAC ESLVKFQAKV EGLLQALTRK LEAVSKRLAI LENTVV Exemplary sequence of the trimerization domain (39aa) CACESLVKFQ AKVEGLLQAL TRKLEAVSKR LAILENTVV

[0237] Another exemplary form is a bispecific or multispecific antibody comprising a full-length antibody (e.g., IgG) having first and second antibody heavy chains and first and second antibody light chains, wherein the first heavy chain and the first light chain assemble to form an antigen-binding site for a first antigen, and the second heavy chain and the second light chain assemble to form an antigen-binding site for a second antigen.

[0238] The precise set of heterodimeric weight chains can be supported, for example, by the use of knob-into-hole mutations and / or other modifications, which are further discussed below.

[0239] The precise assembly of light chains with their corresponding heavy chains can be supported by the cross-MAB technique. In this technique, a first heavy chain and a first light chain, or a second heavy chain and a second light chain, can assemble to form a cross-Fab fragment (while others assemble to form a conventional Fab). Thus, in one embodiment, the first heavy chain may contain a VL domain instead of a VH domain (e.g., VL-CH1-hinge-CH2-CH3), the first light chain may contain a VH domain replaced by a VL domain (e.g., VH-CL), or the first heavy chain may contain a CL domain instead of an HC1 domain (e.g., VH-CL-hinge-CH2-CH3), or the first light chain may contain a CH1 domain instead of a CL domain (e.g., VL-CH1). In this embodiment, the second heavy chain and the second light chain have a conventional domain structure (e.g., VH-CH1-hinge-CH2-CH3 and VL-CL, respectively). In an alternative embodiment, the second heavy chain may contain a VL domain instead of a VH domain (e.g., VL-CH1-hinge-CH2-CH3), the second light chain may contain a VH domain replaced by a VL domain (e.g., VH-CL), or the second heavy chain may contain a CL domain instead of an HC1 domain (e.g., VH-CL-hinge-CH2-CH3), and the second light chain may contain a CH1 domain instead of a CL domain (e.g., VL-CH1). In this embodiment, the first heavy chain and the first light chain have a conventional domain structure.

[0240] In some embodiments, the precise assembly of light chains and their corresponding heavy chains can be further or otherwise aided by the use of charge modifications, as discussed below.

[0241] One such antibody is shown in Figure 37 as P1AE1768, where the second heavy chain contains a CL domain instead of an HC1 domain (e.g., VH-CL-hinge-CH2-CH3), and the second light chain contains a CH1 domain instead of a CL domain (e.g., VL-CH1); the first heavy chain and the first light chain have a conventional domain structure. Fab having a conventional structure includes charge modification. Thus, in one embodiment, the antibody of the present invention comprises the first and second heavy chains of SEQ ID NO: 59 and SEQ ID NO: 58, respectively, and the first and second light chains of SEQ ID NO: 57 and SEQ ID NO: 60, respectively.

[0242] In some embodiments of the above form, the form may be bivalent. In another possible embodiment, additional antigen-binding moieties may be fused, for example, to the first and / or second heavy chain to increase the valence of one or both antigens. For example, additional antigen-binding moieties for the first antigen may be fused to one or both N-terminuses of the heavy chain molecule. The antibody may be polyvalent, e.g., bivalent, with respect to the first antigen (e.g., tumor-associated antigen) and monovalent with respect to the second antigen (e.g., DOTAM-chelated Pb).

[0243] Further antigen-binding sites may include, for example, an antigen-binding site for a first antigen (e.g., a tumor-associated antigen), such as an scFab. The scFab includes VH and CH1 domains, with the VL and CL domains substituted by a polypeptide linker to be expressed as a single chain. In other words, the scFab includes a polypeptide linker between the Fd and the light chain.

[0244] In another embodiment, the further antigen-binding moiety is a Fab or a cross-Fab. For example, the N or C terminus of one heavy chain can be linked by a polypeptide linker to a first polypeptide consisting of a VH domain and a CH1 domain, which associates with a second polypeptide consisting of VL and CL domains to form a Fab. In another embodiment, the N or C terminus of one heavy chain can be linked by a polypeptide linker to a first polypeptide consisting of a VL domain and a CH1 domain, which associates with a second polypeptide consisting of VH and CL domains. In another embodiment, the N or C terminus of one heavy chain can be linked by a polypeptide linker to a first polypeptide consisting of a VH domain and a CL domain, which associates with a second polypeptide consisting of VL and CH1 domains.

[0245] In this configuration, it is preferable that the same antigen-specific binding arms are formed by binding to the same light chain. Thus, the antigen-binding portion / arm for the first antigen may be a cross-Fab, and the antigen-binding portion / arm for the second antigen may be a conventional Fab. Alternatively, the antigen-binding portion / arm for the first antigen may be a conventional Fab, and the antigen-binding portion / arm for the second antigen may be a cross-Fab.

[0246] This form may also include charge modification, as will be further discussed below.

[0247] In one embodiment of this form, A full-length antibody comprising first and second antibody heavy chains and first and second antibody light chains, wherein the first heavy chain and the first light chain assemble to form a Fab containing an antigen-binding site for a first antigen (e.g., tumor-specific antigen, e.g., CEA), and the second heavy chain and the second light chain assemble to form a cross-Fab containing an antigen-binding site for a second antigen (e.g., DOTAM-chelated Pb) (e.g., the second heavy chain has a VL domain instead of a VH domain, and the second light chain has a VH domain instead of a VL domain); A full-length antibody in which either a first or second antibody heavy chain is fused via a linker to a polypeptide containing CH1 and VH domains, and the first polypeptide assembles with the second polypeptide containing CL and VL such that the first and second polypeptides assemble to form a Fab containing an antigen-binding site for a first antigen. A multivalent antibody containing [the specified substance] is provided.

[0248] The fusion may occur on one of the heavy chains of the full-length antibody, or optionally at the N-terminus of a second heavy chain.

[0249] Charge modifications may also be used as needed. For example, a Fab containing an antigen-binding site for the first antigen may include charge modification substitutions, as discussed below.

[0250] An example of such a form is P1AE1769, shown in Figure 37. Thus, in one embodiment, the antibody of the present invention comprises first and second heavy chains of SEQ ID NO: 64 and SEQ ID NO: 63, and first and second light chains of SEQ ID NO: 62 and SEQ ID NO: 61, respectively.

[0251] Another exemplary form includes a full-length antibody, such as IgG, which includes an antigen-binding site for a first antigen (which may be bivalent with respect to the first antigen) linked to an antigen-binding site for a second antigen.

[0252] For example, the antigen-binding portion for a second antigen may be an scFab containing an antigen-binding site for the second antigen (e.g., Pb-DOTAL chelate). In some embodiments, the scFab can be fused to the C-terminus of one of the two heavy chains of a full-length antibody, for example, to the C-terminus of its CH3 domain. The precise assembly of the heterodimeric heavy chain may be aided by, for example, the use of knob-into-hole mutations and / or other modifications further discussed below. One such antibody is exemplified in Figure 37 as P1AE1770. Thus, in one embodiment, the antibody of the present invention comprises the heavy chains of SEQ ID NOs. 66 and 67 and the light chain of SEQ ID NOs. 65.

[0253] Another exemplary form includes a full-length antibody comprising an antigen-binding site for a first antigen (which may be bivalent with respect to the first antigen), wherein one N-terminus or C-terminus of the heavy chain is linked to the first polypeptide via a polypeptide linker, and the first polypeptide associates with a second polypeptide to form a Fab or cross-Fab comprising a binding site for the second antigen. For example, this form includes such that the first and second polypeptides together form an antigen-binding site for the second antigen. i) A first polypeptide consisting of a VH domain and a CH1 domain, associated with a second polypeptide consisting of VL and CL domains; or ii) A first polypeptide consisting of a VL domain and a CH1 domain, associated with a second polypeptide consisting of VH and CL domains; or iii) The first polypeptide, consisting of a VH domain and a CL domain, is associated with a second polypeptide, consisting of a VL and a CH1 domain. It may include.

[0254] The precise assembly of heterodimeric weight chains may be supported, for example, by the use of knob-into-hole mutations and / or other modifications, which are further discussed below. For example, the Fab domain of a full-length antibody may include charge modifications.

[0255] In one embodiment, the first polypeptide is linked to one C-terminus of the heavy chain, for example, to the C-terminus of its CH3 domain, via a polypeptide linker. The first polypeptide may include an N-terminal VL domain and a C-terminal CH1 domain. Thus, the heavy chain having the fusion may include VH-CH1-hinge-CH2-CH3-linker-VL-CH1 from the N-terminus to the C-terminus. The light chain may include VH-CL. The Fab of the full-length antibody may include charge modification substitutions. One such antibody is shown in Figure 37 as P1AE1767. Thus, in one embodiment, the antibody of the present invention includes the heavy chain of SEQ ID NOs. 63 and 64 and the light chain of SEQ ID NOs. 61 and 62.

[0256] In another embodiment, the first polypeptide is linked to the N-terminus of the VH domain of the heavy chain via a polypeptide linker. The first polypeptide may include an N-terminal VL domain and a C-terminal CH1 domain. Thus, the heavy chain having the fusion may include VL-CH1-linker-VH-CH1-hinge-CH2-CH3 from the N-terminus to the C-terminus. The light chain may include VH-CL.

[0257] In another exemplary form, the antibody may include a full-length antibody that specifically binds to a first antigen and consists of two antibody heavy chains and two antibody light chains, with the C-terminus of each heavy chain fused to an antigen-binding portion that specifically binds to a second antigen.

[0258] In one embodiment, the first antigen is a target, such as a tumor-specific antigen, and the second antigen is a Pb-DOTAM chelate, although these may be reversed.

[0259] In another exemplary form, antibodies are a) A full-length antibody that specifically binds to the first antigen and consists of two antibody heavy chains and two antibody light chains; b) i) Antibody heavy chain variable domain (VH); or ii) Antibody heavy chain variable domain (VH) and antibody heavy chain constant domain (CH1); or iii) Variable domain (VH) of antibody heavy chain and constant domain (CL) of antibody light chain A polypeptide consisting of, A polypeptide in which the N-terminus of the VH domain is fused to the C-terminus of one of the two heavy chains of the full-length antibody via a peptide linker; c) i) Antibody light chain variable domain (VL); or ii) Antibody light chain variable domain (VL) and antibody light chain constant domain (CL); or iii) Variable domain (VL) and constant domain (CH1) of the antibody light chain A polypeptide consisting of, A polypeptide in which the N-terminus of the VL domain is fused to the other C-terminus of the two heavy chains of the full-length antibody via a peptide linker. Includes; The antibody may also be a bispecific antibody in which the antibody heavy chain variable domain of the peptide under (b) and the antibody light chain variable domain of the peptide under (c) combine to form an antigen-binding site for a second antigen.

[0260] In this format, if the first polypeptide is as described in b(i), the second polypeptide is as described in c(i); if the first polypeptide is as described in b(ii), the second polypeptide is as described in c(ii); and if the first polypeptide is as described in b(iii), the second polypeptide is as described in c(iii). Charge modification substitution can also be used, for example, in the Fab of a full-length antibody.

[0261] In this form, the first or second antigen may be DOTAM-chelated Pb. The other may be a target, for example, a tumor-associated antigen, such as CEA, CD20, or ERBB2. In some embodiments, the second antigen is DOTAM-chelated Pb and the first antigen is the target.

[0262] The antibody described above may be trivalent. In another possible embodiment, additional antigen-binding moieties can be fused to increase the valence for one or both antigens. For example, an additional antigen-binding moiety for the first antigen may be fused to one or both carboxyl ends of the heavy chain of a full-length antibody (e.g., a tumor-associated antigen), such that the antibody has a valence of 4 for the first antigen (if it is fused to the carboxyl ends of both heavy chains) and a valence of 1 for the second antigen.

[0263] Examples of the above form in which antibody (b) consists of a VH domain and antibody (c) consists of a VL domain are PRIT-213 and PRIT214. An example of the above form in which (b) consists of a VH domain and a CL domain and (c) consists of a VL domain and a CH1 domain is P1AE1766, as shown in Figure 37. Thus, in one embodiment, the antibody of the present invention comprises the first and second heavy chains of SEQ ID NO: 51 and SEQ ID NO: 52, and the light chain of SEQ ID NO: 50, respectively.

[0264] If necessary, the form used for the multispecific antibody of the present invention may be a trivalent form, such as that described in WO2010 / 115589A1 (Roche Glycart AG), which is incorporated herein by reference in whole.

[0265] WO2010 / 115589 describes arbitrary stabilization of the structure, thereby linking the antibody heavy chain variable region (VH) of the polypeptide below (b) and the antibody light chain variable domain (VL) of the polypeptide below (c) by interchain disulfide crosslinking, for example, at the following positions: i) From position 44 of the heavy chain variable domain to position 100 of the light chain variable domain, ii) From position 105 of the heavy chain variable domain to position 43 of the light chain variable domain, iii) From the 101st heavy chain variable domain to the 100th light chain variable domain (always numbered according to Kabat's EU index) It is stabilized by the introduction of a disulfide bond between them.

[0266] WO2010 / 115589 also states that the CH3 domain of the full-length antibody according to the present invention can be modified by the “knob-into-hole” technique, which is described in detail with several examples in, for example, WO96 / 027011, Ridgway, JB et al., Protein Eng 9 (1996) pp. 617-621; and Merchan, AM et al., Nat Biotechnol 16 (1998) pp. 677-681.

[0267] Thus, in some embodiments, the trivalent bispecific antibody is further characterized in that the CH3 domain of one heavy chain of the full-length antibody and the CH3 domain of the other heavy chain of the full-length antibody face each other at a contact surface including the original contact surface between the antibody CH3 domains, and the contact surface is modified to promote the formation of the trivalent bispecific antibody, and this modification is a) Within the original contact surface of the CH3 domain of one heavy chain in a trivalent bispecific antibody, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby modifying the CH3 domain of one heavy chain to generate a projection that can be located in the cavity within the contact surface of the CH3 domain of the other heavy chain. and b) The CH3 domain of the other heavy chain is modified such that, within the original contact surface of the second CH3 domain facing the original contact surface of the first CH3 domain in the trivalent bispecific antibody, an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby creating a cavity within the contact surface of the second CH3 domain where a projection within the contact surface of the first CH3 domain can be located. It is characterized by the following.

[0268] The amino acid residue having a larger side chain volume may, if necessary, be selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). The amino acid residue having a smaller side chain volume may, if necessary, be selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V).

[0269] If necessary, in some embodiments, both CH3 domains are further modified by introducing cysteine ​​(C) as an amino acid at the corresponding position in each CH3 domain so that they can form disulfide bridges between the two CH3 domains.

[0270] These and other details of the bispecific trivalent antibody format described in WO2010 / 115589A1 can be utilized in the present invention.

[0271] As used herein, the term “full-length antibody” refers to an antibody comprising two “full-length antibody heavy chains” and two “full-length antibody light chains.” A “full-length antibody heavy chain” may be a polypeptide comprising, in the direction from the N-terminus to the C-terminus of the antibody heavy chain variable domain (VH), an antibody constant heavy chain domain 1 (CH1), an antibody hinge region (HR), an antibody heavy chain constant domain 2 (CH2), and an antibody heavy chain constant domain 3 (CH3), abbreviated as VH-CH1-HR-CH2-CH3; and optionally, an antibody heavy chain constant domain 4 (CH4) in the case of an antibody of subclass IgE. Preferably, a “full-length antibody heavy chain” is a polypeptide comprising VH, CH1, HR, CH2, and CH3 in the direction from the N-terminus to the C-terminus. The possibility of cross-Mab formation is not intended to be excluded by the reference to “full-length,” thus the heavy chain may have a VH domain replaced with a VL domain, or a CH1 main replaced with a CL domain. A "full-length antibody light chain" may be a polypeptide consisting of an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL), abbreviated as VL-CL, from the N-terminus to the C-terminus. Alternatively, in the case of cross-Mab, the VL domain may be replaced with a VH domain, or the CL domain may be replaced with a CH1 domain. The antibody light chain constant domain (CL) may be κ (kappa) or λ (lambda). Two full-length antibody chains are linked together via inter-polypeptide disulfide bonds between the CL domain and the CH1 domain and between the hinge regions of the full-length antibody heavy chains. Typical examples of full-length antibodies are natural antibodies such as IgG (e.g., IgG1 and IgG2), IgM, IgA, IgD, and IgE). The full-length antibodies according to the present invention may be derived from a single species, e.g., human, or they may be chimeric or humanized antibodies. Each full-length antibody described herein contains two antigen-binding sites formed by a pair of VH and VL. The C-terminus of the heavy or light chain of the full-length antibody represents the last amino acid of the C-terminus of the heavy or light chain.

[0272] The N-terminus of the antibody heavy chain variable domain (VH) of the polypeptide under b) and the N-terminus of the antibody light chain variable domain (VL) of the polypeptide under c) indicates the last amino acid at the N-terminus of the VH or VL domain.

[0273] In any of the above forms, the first antigen may be a tumor-associated antigen, and the second antigen may be Pb-DOTAM (however, in some embodiments, these may be reversed).

[0274] In any of the above forms, the precise assembly of heavy chain heterodimers can be supported by modifications to the heavy chain sequence. In one embodiment, a knob-into-hole technique is used. The interaction surface of two CH3 domains can be modified to increase heterodimerization of both heavy chains containing these two CH3 domains. Each of the two CH3 domains (of the two heavy chains) may be a "knob" while the other is a "hole". For example, one may include a so-called "knob mutation" (T366W and optionally one of S354C or Y349C, preferably S354C) according to EU index numbering, and the other may include a so-called "hole mutation" (T366S, L368A, and Y407V, optionally Y349C or S354C, preferably Y349C) (see, e.g., Carter, P et al., Immunotechnol. 2 (1996) 73).

[0275] Furthermore, or alternatively, by introducing disulfide crosslinks, heterodimers can be stabilized (Merchant, AM et al., Nature Biotech 16 (1998) pp. 677-681; Atwell, S. et al., J. Mol. Biol. 270 (1997) pp. 26-35), and yields can be increased. For example, at the following locations: i) From position 44 of the heavy chain variable domain to position 100 of the light chain variable domain, ii) From position 105 of the heavy chain variable domain to position 43 of the light chain variable domain, iii) From the 101st heavy chain variable domain to the 100th light chain variable domain (always numbered according to Kabat's EU index) One example is the introduction of a disulfide bond between them.

[0276] charge modification The multispecific antibodies of the present invention are particularly efficient in reducing mispairing between light chains and mismatched heavy chains (Bence-Jones type byproducts) that can occur in the production of Fab-based bi- / multispecific antigen-binding molecules having VH / VL exchange in one (or more (in the case of molecules containing two or more antigen-binding Fab molecules)) of their binding arms, and may include amino acid substitutions in the Fab molecules contained herein (the whole is incorporated herein by reference, PCT publication no. WO2015 / 150447, see in particular the examples therein). The ratio of the desired multispecific antibody to undesirable byproducts, in particular Bence-Jones type byproducts that occur in one of its binding arms, can be improved by introducing charged amino acids with opposite charges at specific amino acid positions in the CH1 and CL domains of the Fab molecule (sometimes referred to herein as "charge modification").

[0277] Therefore, in some embodiments, the antibody of the present invention comprising a Fab molecule comprises at least one Fab having a heavy chain constant domain CH1 domain comprising the charge modification described herein and a light chain constant domain CL domain comprising the charge modification described herein.

[0278] Charge modification is generated in either (but not both) a conventional Fab molecule included in the antibody of the present invention (e.g., those shown in Figure 37: P1AE1766, P1AE1767, P1AE1768, P1AE1769) or a crossover Fab molecule included in the antibody of the present invention. In certain embodiments, charge modification is generated in a conventional Fab molecule included in the antibody of the present invention (which, in certain embodiments, specifically binds to a target cell antigen).

[0279] Accordingly, in some embodiments, the antibody of the present invention comprises a) a first antigen-binding moiety that binds to a first antigen (e.g., tumor-associated antigen) and b) a second binding moiety that binds to a second antigen (e.g., Dotam-Pb), wherein both the first and second antigen-binding moieties of the bispecific antigen-binding molecule are Fab molecules, one of the antigen-binding moieties (in some embodiments, particularly the second antigen-binding moiety) is a cross-Fab fragment, and one of the Fab molecules comprises a CH1 domain containing the charge modification described herein and a CL domain containing the charge modification described herein. The Fab containing the charge modification may preferably be a conventional (non-cross-binding) Fab, for example, in some embodiments, the antigen-binding moiety that binds to the first antigen.

[0280] The antibody according to the present invention may further include a third Fab molecule that specifically binds to a first antigen. In certain embodiments, the third Fab molecule is identical to the first Fab molecule under a). In these embodiments, the amino acid substitutions (charge modifications) according to the following embodiments can be made in the constant domains CL and CH1 of the first Fab molecule and the third Fa molecule, respectively. Alternatively, the amino acid substitutions according to the following embodiments can be made in the constant domains CL and CH1 of the second Fab molecule under b), rather than in the constant domains CL and CH1 of the first and third Fab molecules.

[0281] In some embodiments, in a Fab molecule comprising a charge-modified light chain constant domain CL and a charge-modified heavy chain constant domain CH1, the charge modification in the light chain constant domain CL is located at position 124 and optionally at position 123 (numbered by Kabat), and the charge modification in the heavy chain constant domain CH1 is located at position 147 and / or 213 (numbered by Kabat).

[0282] In some embodiments, in the light chain constant domain CL, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (as numbered by Kabat) (in one preferred embodiment, it is independently substituted with lysine (K)), and in the heavy chain constant domain CH1, the amino acid at position 147 and / or the amino acid at position 213 are independently substituted with glutamic acid (E) or aspartic acid (D) (as numbered by the Kabat EU index).

[0283] In another embodiment, in the light chain constant domain CL, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (numbered by Kabat) (in one preferred embodiment, independently by lysine (K) or arginine (R)), and in the heavy chain constant domain CH1, the amino acid at position 147 or the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbered by Kabat's EU index).

[0284] In further embodiments, in the light chain constant domain CL, the amino acid at position 124 is independently substituted with lysine (K) or arginine (R) (numbered by Kabat) (in one preferred embodiment, independently substituted with lysine (K) or arginine (R)), and in the heavy chain constant domain CH1, the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbered by Kabat's EU index).

[0285] In further embodiments, in the light chain constant domain CL, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (in one preferred embodiment, independently lysine (K) or arginine (R)) (numbered by Kabat), and in the heavy chain constant domain CH1, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbered by Kabat's EU index).

[0286] In further embodiments, in the light chain constant domain, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (numbered by Kabat) (in one preferred embodiment, independently substituted with lysine (K) or arginine (R)), the amino acid at position 123 is independently substituted with lysine (K), arginine (R), or histidine (H) (numbered by Kabat) (in one preferred embodiment, independently substituted with lysine (K) or arginine (R)), in the heavy chain constant domain CH1, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbered by Kabat's EU index), and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbered by Kabat's EU index).

[0287] In further embodiments, in the light chain constant domain CL, the amino acid at position 124 is substituted with lysine (K) (numbered by Kabat), the amino acid at position 123 is substituted with arginine (R) (numbered by Kabat), and in the heavy chain constant domain CH1, the amino acid at position 147 is substituted with glutamic acid (E) (numbered by Kabat's EU index), and the amino acid at position 213 is substituted with glutamic acid (E) (numbered by Kabat's EU index).

[0288] In further embodiments, in the light chain constant domain CL, the amino acid at position 124 is substituted with lysine (K) (numbered by Kabat), the amino acid at position 123 is substituted with lysine (K) (numbered by Kabat), and in the heavy chain constant domain CH1, the amino acid at position 147 is substituted with glutamic acid (E) (numbered by Kabat's EU index), and the amino acid at position 213 is substituted with glutamic acid (E) (numbered by Kabat's EU index).

[0289] In further embodiments, in the light chain constant domain CL, the amino acid at position 124 is substituted with lysine (K) (numbered by Kabat), the amino acid at position 123 is substituted with arginine (R) (numbered by Kabat), and in the heavy chain constant domain CH1, the amino acid at position 147 is substituted with glutamic acid (E) (numbered by Kabat's EU index), and the amino acid at position 213 is substituted with aspartic acid (D) (numbered by Kabat's EU index).

[0290] In further embodiments, in the light chain constant domain, the amino acid at position 124 is substituted with lysine (K) (numbered by Kabat), the amino acid at position 123 is substituted with lysine (K) (numbered by Kabat), and in the heavy chain constant domain CH1, the amino acid at position 147 is substituted with glutamic acid (E) (numbered by Kabat's EU index), and the amino acid at position 213 is substituted with aspartic acid (D) (numbered by Kabat's EU index).

[0291] In one embodiment, the antibody comprises a first heavy chain and a first light chain that specifically bind to a first antigen, and a second heavy chain and a second light chain that specifically bind to a second antigen. a) The constant domain CL of the first light chain and the constant domain CH1 of the first heavy chain include the charge mutation substitutions described herein; b) The light chain steady domain CL and heavy chain steady domain CH1 of the second light chain and the second heavy chain are interchangeable (thus forming tolerance Fab). An example of such an arrangement is shown for P1AE1768.

[0292] In another embodiment, the antibody is A full-length antibody that specifically binds to a first antigen and comprises two heavy chains and two light chains, wherein the two heavy chain constant domains CH1 and the two light chain constant domains CL of the full-length antibody include the charge modifications described herein; and The scFab comprises VH and CH1 domains (VH-CH1-linker-VL-CL) linked to VL and CL domains via a polypeptide linker, wherein the scFab is fused to one N-terminus of the heavy chain, and the scFab forms an antigen-binding site for a second antigen. An example of such an arrangement is P1AE1770.

[0293] In another embodiment, the multispecific antibody is a full-length antibody (for example, it may be bivalent with respect to the first antigen) containing an antigen-binding site for a first antigen, wherein two heavy chain constant domains CH1 and two light chain constant domains CL of the full-length antibody contain the charge modifications described herein. The antibody comprises a full-length antibody in which one C-terminus of the heavy chain (e.g., the C-terminus of its CH3 domain) is linked to a first polypeptide via a polypeptide linker, and the first polypeptide associates with a second polypeptide to form a cross-Fab containing a binding site for the second antigen.

[0294] The first polypeptide may contain an N-terminal VL domain and a C-terminal CH1 domain. Thus, the heavy chain having the fusion may contain VH-CH1-hinge-CH2-CH3-linker-VL-CH1 from the N-terminus to the C-terminus. The light chain may contain VH-CL. An example of such a configuration is P1AE1767.

[0295] In further embodiments, the antibody is a) A full-length antibody that specifically binds to a first antigen and comprises two antibody heavy chains and two antibody light chains, wherein the CH1 domain of the heavy chain and the CL domain of the light chain include the charge modifications described herein; b) i) Antibody heavy chain variable domain (VH); or ii) Antibody heavy chain variable domain (VH) and antibody heavy chain constant domain (CH1); or iii) Variable domain (VH) of antibody heavy chain and constant domain (CL) of antibody light chain A polypeptide consisting of, A polypeptide in which the N-terminus of the VH domain is fused to the C-terminus of one of the two heavy chains of the full-length antibody via a peptide linker; c) i) Antibody light chain variable domain (VL); or ii) Antibody light chain variable domain (VL) and antibody light chain constant domain (CL); or iii) Variable domain (VL) and constant domain (CH1) of the antibody light chain A polypeptide consisting of, A polypeptide in which the N-terminus of the VL domain is fused to the other C-terminus of the two heavy chains of the full-length antibody via a peptide linker. Includes; A bispecific antibody may be one in which the antibody heavy chain variable domain of the peptide under (b) and the antibody light chain variable domain of the peptide under (c) combine to form an antigen-binding site for a second antigen. Examples of such configurations are PRIT-213 and p1AE1766.

[0296] In some embodiments, the antibody of the present invention comprises a) a first antigen-binding moiety that binds to a first antigen, b) a second antigen-binding moiety that binds to a second antigen, and c) a third antigen-binding moiety that binds to a first antigen, wherein the first, second, and third antigen-binding moieties of the antibody are all Fab molecules, and in one of the antigen-binding moieties (particularly the second antigen-binding moiety), the variable domains VL and VH of the Fab light chain and Fab heavy chain are respectively substituted for each other. i) The amino acid substitutions according to the above embodiment are made in the constant domains CL and CH1 of the first and third Fab molecules, respectively, rather than in the constant domains CL and CH1 of the second Fab molecule under b); or ii) The amino acid substitutions according to the above embodiments are made in the constant domains CL and CH1 of the second Fab molecule under b) rather than in the constant domains CL and CH1 of the first and third Fab molecules. In some embodiments, the charge modification is present in the conventional (non-exchangeable) Fab: Thus, for example, if the second antigen-binding moiety is a cross-Fab, option (i) is preferred.

[0297] In one particular embodiment, the polyvalent antibody of the present invention is A full-length antibody comprising first and second antibody heavy chains and first and second antibody light chains, wherein the first heavy chain and the first light chain assemble to form a Fab containing an antigen-binding site for a first antigen (e.g., tumor-specific antigen, e.g., CEA), and the second heavy chain and the second light chain assemble to form a cross-Fab containing an antigen-binding site for a second antigen (e.g., DOTAM-chelated Pb) (e.g., the second heavy chain has a VL domain instead of a VH domain, and the second light chain has a VH domain instead of a VL domain); Either the first or second antibody heavy chain is fused via a linker to a polypeptide containing CH1 and VH domains, and the first polypeptide assembles with the second polypeptide containing CL and VL such that the first and second polypeptides assemble to form a Fab containing an antigen-binding site for the first antigen. The antibody comprises a full-length antibody in which the CH1 domain of the first heavy chain and the CL domain of the first light chain contain the charge modifications described herein.

[0298] Furthermore, the CH1 domain of the first polypeptide and the CL domain of the second polypeptide may include the charge modifications described herein.

[0299] The fusion may be located on one of the heavy chains of the full-length antibody, or optionally at the N-terminus of the second heavy chain. An example of such a configuration is P1AE1769.

[0300] Multispecific antibodies that bind to Pb-DOTAM and CEA In some embodiments, the antibodies of the present invention may preferably be multispecific, for example, bispecific antibodies that bind to both Pb-DOTAM and CEA. Thus, they include an antigen-binding site for Pb-DOTAM chelate and an antigen-binding site for CEA. In such embodiments, the antigen-binding site specific to Pb-DOTAM chelate may be according to any of the embodiments described herein. This form may be any of the forms described herein.

[0301] If necessary, the antigen-binding site that binds to CEA may bind with a Kd value of 1 nM or less, 500 pM or less, 200 pM or less, or 100 pM or less for monovalent binding.

[0302] If necessary, the antigen-binding site that binds to CEA may include at least one, two, three, four, five, or six CDRs selected from (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 11; (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 12; (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 13; (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 14; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 15; and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 16.

[0303] If necessary, the antigen-binding site that binds to CEA may include at least one, at least two, or all three VH CDR sequences selected from (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 11; (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 12; and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 13. In one embodiment, the antibody includes CDR-H3 containing the amino acid sequence of SEQ ID NO: 13. In another embodiment, the antibody includes CDR-H3 containing the amino acid sequence of SEQ ID NO: 13 and CDR-L3 containing the amino acid sequence of SEQ ID NO: 16. In a further embodiment, the antibody includes CDR-H3 containing the amino acid sequence of SEQ ID NO: 13, CDR-L3 containing the amino acid sequence of SEQ ID NO: 16, and CDR-H2 containing the amino acid sequence of SEQ ID NO: 12. In a further embodiment, the antibody includes (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 11; (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 12; and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 13.

[0304] If necessary, the antigen-binding site that binds to CEA includes at least one, at least two, or all three VL CDR sequences selected from (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 14; (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 15; and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 16. In one embodiment, the antibody includes (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 13; (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 14; and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 15.

[0305] If necessary, the antigen-binding site that binds to CEA includes (a) a VH domain containing at least one, at least two, or all three VH CDR sequences selected from (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 11, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 12, and (iii) CDR-H3 containing the amino acid sequence selected from SEQ ID NO: 13; and (b) a VL domain containing at least one, at least two, or all three VL CDRs selected from (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 14, and (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 15, and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 16.

[0306] In another embodiment, the antigen-binding sites that bind to CEA include (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 11; (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 12; (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 13; (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 14; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 15; and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 16.

[0307] In any of the above embodiments, the multispecific antibody may be humanized. In one embodiment, the anti-CEA antigen binding site comprises a CDR as in any of the above embodiments, and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0308] In another embodiment, the antigen-binding site that binds to CEA includes a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 17. In a particular embodiment, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the antigen-binding site containing that sequence preferably retains the ability to bind to CEA with the affinity described above. In a particular embodiment, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 17. In a particular embodiment, the substitutions, insertions, or deletions are located in a region outside the HVR (i.e., FR). If necessary, the antigen-binding site that binds to CEA includes the VH sequence in SEQ ID NO: 17, and includes post-translational modifications of that sequence. In certain embodiments, VH includes one, two, or three CDRs selected from (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 11; (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 12; and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 13.

[0309] In another embodiment, the antigen-binding site that binds to CEA includes a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 18. In a particular embodiment, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the antigen-binding site containing that sequence preferably retains the ability to bind to CEA with the affinity described above. In a particular embodiment, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 18. In a particular embodiment, the substitutions, insertions, or deletions are located in a region outside the HVR (i.e., FR). If necessary, the antigen-binding site for CEA includes the VL sequence in SEQ ID NO: 18, including post-translational modifications of that sequence. In certain embodiments, the VL includes one, two, or three CDRs selected from (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 14; (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 15; and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 16.

[0310] In another embodiment, the antigen-binding site that binds to CEA includes the VH sequence described in any of the embodiments provided above and the VL sequence described in any of the embodiments provided above. In one embodiment, the antibody includes the VH and VL sequences of SEQ ID NO: 17 and SEQ ID NO: 18, respectively, and includes post-translational modifications of those sequences.

[0311] In some embodiments, the multispecific antibody can bind to the same CEA epitope as PRIT-0213 or PRIT-0214 provided herein.

[0312] Multispecific antibodies that bind to Pb-DOTAM and ERBB2 In some embodiments, the antibodies of the present invention may preferably be multispecific, for example, bispecific antibodies that bind to both Pb-DOTAM and ERBB2. Thus, they include an antigen-binding site for Pb-DOTAM chelate and an antigen-binding site for ERBB2. In such embodiments, the antigen-binding site specific to Pb-DOTAM chelate may be according to any embodiment described herein. This form may be any form described herein.

[0313] If necessary, the antigen-binding site that binds to ERBB2 may bind with a Kd value of 1 nM or less, 500 pM or less, 200 pM or less, or 100 pM or less for monovalent binding.

[0314] If necessary, the antigen-binding site that binds to ERBB2 may include at least one, two, three, four, five, or six CDRs selected from (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 28; (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 29; (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 30; (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 31; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 32; and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 33.

[0315] If necessary, the antigen-binding site that binds to ERBB2 may include at least one, at least two, or all three VH CDR sequences selected from (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 28; (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 29; and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 30. In one embodiment, the antibody includes CDR-H3 containing the amino acid sequence of SEQ ID NO: 30. In another embodiment, the antibody includes CDR-H3 containing the amino acid sequence of SEQ ID NO: 30 and CDR-L3 containing the amino acid sequence of SEQ ID NO: 33. In a further embodiment, the antibody includes CDR-H3 containing the amino acid sequence of SEQ ID NO: 30, CDR-L3 containing the amino acid sequence of SEQ ID NO: 33, and CDR-H2 containing the amino acid sequence of SEQ ID NO: 29. In a further embodiment, the antibody includes (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 28; (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 29; and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 30.

[0316] If necessary, the antigen-binding site that binds to ERBB2 includes at least one, at least two, or all three VL CDR sequences selected from (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 31; (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 32; and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 33. In one embodiment, the antibody includes (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 31; (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 32; and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 33.

[0317] If necessary, the antigen-binding site that binds to ERBB2 includes (a) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 28, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29, and (iii) CDR-H3 comprising the amino acid sequence selected from SEQ ID NO: 30; and (b) a VL domain comprising at least one, at least two, or all three VL CDRs selected from (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 31, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 32, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 33.

[0318] In another embodiment, the antigen-binding site that binds to ERBB2 includes (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 28; (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 29; (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 30; (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 31; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 32; and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 33.

[0319] In any of the above embodiments, the multispecific antibody may be humanized. In one embodiment, the anti-ERBB2 antigen binding site comprises a CDR as in any of the above embodiments, and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0320] In another embodiment, the antigen-binding site that binds to ERBB2 includes a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 34. In a particular embodiment, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the antigen-binding site containing that sequence preferably retains the ability to bind to ERBB2 with the affinity described above. In a particular embodiment, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 34. In a particular embodiment, the substitutions, insertions, or deletions are located in a region outside the HVR (i.e., FR). If necessary, the antigen-binding site that binds to ERBB2 includes the VH sequence in SEQ ID NO: 34, and includes post-translational modifications of that sequence. In certain embodiments, VH includes one, two, or three CDRs selected from (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 28; (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 29; and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 30.

[0321] In another embodiment, the antigen-binding site that binds to ERBB2 includes a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 35. In a particular embodiment, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the antigen-binding site containing that sequence preferably retains the ability to bind to ERBB2 with the affinity described above. In a particular embodiment, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 35. In a particular embodiment, the substitutions, insertions, or deletions are located in a region outside the HVR (i.e., FR). If necessary, the antigen-binding site for CEA includes the VL sequence in SEQ ID NO: 35, including post-translational modifications of that sequence. In certain embodiments, the VL includes one, two, or three CDRs selected from (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 31; (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 32; and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 33.

[0322] In another embodiment, the antigen-binding site that binds to ERBB2 includes the VH sequence described in any of the embodiments provided above and the VL sequence described in any of the embodiments provided above. In one embodiment, the antibody includes the VH and VL sequences of SEQ ID NO: 34 and SEQ ID NO: 35, respectively, and includes post-translational modifications of those sequences.

[0323] In some embodiments, the multispecific antibody can bind to the same ERBB2 epitope as the P1AD9827 antibody provided herein.

[0324] Multispecific antibodies that bind to Pb-DOTAM and CD20 In some embodiments, the antibodies of the present invention may preferably be multispecific, for example, bispecific antibodies that bind to both Pb-DOTAM and CD20. Thus, they include an antigen-binding site for Pb-DOTAM chelate and an antigen-binding site for CD20. In such embodiments, the antigen-binding site specific to Pb-DOTAM chelate may be according to any of the embodiments described herein. This form may be any of the forms described herein.

[0325] If necessary, the antigen-binding site that binds to CD20 may bind with a Kd value of 1 nM or less, 500 pM or less, 200 pM or less, or 100 pM or less for monovalent binding.

[0326] If necessary, the antigen-binding site that binds to CD20 may include at least one, two, three, four, five, or six CDRs selected from (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 39; (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 40; (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 41; (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 42; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 43; and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 44.

[0327] If necessary, the antigen-binding site that binds to CD20 may include at least one, at least two, or all three VH CDR sequences selected from (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 39; (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 40; and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 41. In one embodiment, the antibody includes CDR-H3 containing the amino acid sequence of SEQ ID NO: 41. In another embodiment, the antibody includes CDR-H3 containing the amino acid sequence of SEQ ID NO: 41 and CDR-L3 containing the amino acid sequence of SEQ ID NO: 44. In a further embodiment, the antibody includes CDR-H3 containing the amino acid sequence of SEQ ID NO: 41, CDR-L3 containing the amino acid sequence of SEQ ID NO: 44, and CDR-H2 containing the amino acid sequence of SEQ ID NO: 40. In a further embodiment, the antibody includes (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 39; (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 40; and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 41.

[0328] If necessary, the antigen-binding site that binds to CD20 includes at least one, at least two, or all three VL CDR sequences selected from (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 42; (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 43; and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 44. In one embodiment, the antibody includes (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 42; (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 43; and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 44.

[0329] If necessary, the antigen-binding site that binds to CD20 includes (a) a VH domain containing at least one, at least two, or all three VH CDR sequences selected from (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 39, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 40, and (iii) CDR-H3 containing the amino acid sequence selected from SEQ ID NO: 41; and (b) a VL domain containing at least one, at least two, or all three VL CDRs selected from (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 42, (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 43, and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 44.

[0330] In another embodiment, the antigen-binding sites that bind to CD20 include (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 39; (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 40; (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 41; (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 42; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 43; and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 44.

[0331] In any of the above embodiments, the multispecific antibody may be humanized. In one embodiment, the anti-CD20 antigen binding site comprises a CDR as in any of the above embodiments, and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0332] In another embodiment, the antigen-binding site that binds to CD20 includes a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 45. In a particular embodiment, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the antigen-binding site containing that sequence preferably retains the ability to bind to CD20 with the affinity described above. In a particular embodiment, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 45. In a particular embodiment, the substitutions, insertions, or deletions are located in a region outside the HVR (i.e., FR). If necessary, the antigen-binding site that binds to CD20 includes the VH sequence in SEQ ID NO: 45, and includes post-translational modifications of that sequence. In certain embodiments, VH includes one, two, or three CDRs selected from (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 39; (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 40; and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 41.

[0333] In another embodiment, the antigen-binding site that binds to CD20 includes a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 46. In a particular embodiment, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the antigen-binding site containing that sequence preferably retains the ability to bind to CD20 with the affinity described above. In a particular embodiment, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 46. In a particular embodiment, the substitutions, insertions, or deletions are located in a region outside the HVR (i.e., FR). If necessary, the antigen-binding site for CD20 includes the VL sequence in SEQ ID NO: 46, including post-translational modifications of that sequence. In certain embodiments, the VL includes one, two, or three CDRs selected from (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 42; (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 43; and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 44.

[0334] In another embodiment, the antigen-binding site that binds to CD20 includes the VH sequence described in any of the embodiments provided above and the VL sequence described in any of the embodiments provided above. In one embodiment, the antibody includes the VH and VL sequences of SEQ ID NO: 45 and SEQ ID NO: 46, respectively, and includes post-translational modifications of those sequences.

[0335] In some embodiments, the multispecific antibody can bind to the same CD20 epitope as the P1AD9826 antibody provided herein.

[0336] Antibody variants In certain embodiments, amino acid sequence variants of antibodies provided herein are intended. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from and / or insertions of residues in the amino acid sequence of the antibody, and / or substitutions thereof. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct has the desired properties, such as antigen binding.

[0337] Substitution, insertion, and deletion variants In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Target sites for substitutional mutagenesis include HVR(CDR) and FR. Conservative substitutions are shown in Table 1 under the heading "Preferred Substitutions." More substantial substitutions are provided in Table 1 under the heading "Exemplary Substitutions" and are further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest, and the product can be screened for desired activity, such as retention / improvement of antigen binding, decreased immunogenicity, or improvement of ADCC or CDC. TIFF2026062683000010.tif167170

[0338] Amino acids can be classified according to their common side-chain characteristics: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basicity: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0339] Non-conservative substitutions would require replacing one member of these classes with one of another.

[0340] One type of substitution mutant involves the substitution of one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting mutants selected for further testing will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, decreased immunogenicity) compared to the parent antibody, and / or will substantially retain certain biological properties of the parent antibody. Exemplary substitution mutants are affinity-mature antibodies that can be readily generated using phage display-based affinity maturation techniques, such as those described herein. Briefly, one or more HVR residues are mutated, the mutant antibody is displayed on a phage, and it is screened for specific biological activity (e.g., binding affinity).

[0341] Modifications (e.g., substitutions) can be made in HVR, for example, to improve antibody affinity. Such modifications can be made in HVR "hot spots," i.e., residues encoded by codons that frequently undergo mutations during the somatic cell maturation process (see, e.g., Chowdfury, Methods Mol. Biol. 207: pp. 179-196 (2008)), and / or residues that come into contact with antigens having the resulting mutant VH or VL, which are tested for binding affinity. Affinity maturation by constructing a secondary library and then re-selecting from it is described, for example, in Hoogenboom et al., Methods in Molecular Biology 178: pp. 1-37 (O'Brien et al. (eds.), Human Press, Totowa, NJ (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by one of various methods (e.g., mutagenic PCR, chain shuffling, or oligonucleotide-targeted mutation). A secondary library is then constructed. Next, the library is screened to identify any antibody variant with the desired affinity. Another method for introducing diversity involves HVR designation techniques that randomize several HVR residues (e.g., 4-6 residues at a time). HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 are particularly often targeted.

[0342] In certain embodiments, substitutions, insertions, or deletions may be made within one or more HVRs, provided that such modifications do not substantially reduce the antibody's ability to bind to the antigen. For example, conservative modifications that do not substantially reduce binding affinity (e.g., conservative substitutions provided herein) can be made within an HVR. Such modifications may, for example, be located outside the antigen-contact residue in the HVR. In certain embodiments of the mutant VH and VL sequences provided above, each HVR is either unmodified or contains one, two, or three or fewer amino acid substitutions.

[0343] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, pp. 244:1081-1085. This method involves identifying residues or target residue groups (e.g., charged residues such as arg, asp, his, lys, and glu) and replacing them with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the antibody-antigen interaction is affected. Further substitutions may be introduced at amino acid positions that exhibit functional sensitivity to the initial substitutions. Alternatively, or even further, the crystal structure of the antigen-antibody complex may be used to identify the antibody-antigen contact sites. Such contact residues and adjacent residues can be targeted or removed as candidates for substitution. Mutants can be screened to determine whether they possess the desired properties.

[0344] Amino acid sequence insertions include amino-terminus fusions and / or carboxyl-terminus fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusions of antibodies to the N or C terminus of enzymes (e.g., those for ADEPT) or polypeptides that increase the serum half-life of the antibody.

[0345] Glycosylated mutants In certain embodiments, the antibodies provided herein are modified to increase or decrease the degree to which they are glycosylated. The addition or deletion of glycosylation sites to an antibody can be easily achieved by altering the amino acid sequence so that one or more glycosylation sites are created or removed.

[0346] If an antibody contains an Fc region, the carbohydrate bound to it can be modified. Natural antibodies produced by mammalian cells typically contain branched oligosaccharides that are commonly bound by an N-bond to Asn297 in the CH2 domain of the Fc region. See, for example, Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose bound to GlcNAc in the "stem" of the branched oligosaccharide structure. In some embodiments, modification of the oligosaccharide in the antibodies of the present invention can be performed to create antibody variants with improved specific properties.

[0347] In one embodiment, an antibody variant is provided having a glycan structure lacking fucose (directly or indirectly) bound to the Fc region. For example, the amount of fucose in such an antibody may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 compared to all glycan structures bound to Asn297 (e.g., complex hybrid and high-mannose structures) when measured by MALDI-TOF mass spectrometry, as described in WO2008 / 077546. Asn297 refers to the asparagine residue located near position 297 in the Fc region (EU numbering of the Fc region residue), although Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to slight sequence differences in the antibody. Such fucosylated variants may have improved ADCC function. For example, see U.S. Patent Application Publication No. 2003 / 0157108 (Presta, L.); No. 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include US2003 / 0157108; WO2000 / 61739; WO2001 / 29246; US2003 / 0115614; US2002 / 0164328; US2004 / 0093621; US2004 / 0132140; US2004 / 0110704; US2004 / 0110282; US2004 / 010 References include 9865;WO2003 / 085119;WO2003 / 084570;WO2005 / 035586;WO2005 / 035778;WO2005 / 053742;WO2002 / 031140;Okazaki et al., J.Mol.Biol.336:pp.1239~1249 (2004);Yamane-Ohnuki et al., Biotech.Bioeng.87:p.614 (2004).Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells lacking protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249: pp. 533-545 (1986); U.S. Patent Application No. 2003 / 0157108A1, Presta, L.; and WO2004 / 056312A1, Adams et al., particularly Example 11), and knockout cell lines such as those containing the alpha-1,6-fucosyltransferase gene, FUT8, or knockout CHO cells (see, for example, Yamane-Ohnuki et al., Biotech. Bioeng. 87: pp. 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng. 94(4): pp. 680-688 (2006); and WO2003 / 085107).

[0348] For example, antibody variants having a bifurcated oligosaccharide are provided, in which a bifurcated oligosaccharide bound to the Fc region of the antibody is bifurcated by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and U.S.2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide bound to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO1997 / 30087 (Patel et al.); WO1998 / 58964 (Raju, S.); and WO1999 / 22764 (Raju, S.).

[0349] It may be preferable to modify the antibody to reduce the degree of glycosylation. In some embodiments, the antibody may be unglycosylated or deglycosylated. The antibody may contain N297 substitutions, for example, N297D / A.

[0350] Fc region mutant In certain embodiments, Fc region variants can be generated by introducing one or more amino acid modifications into the Fc region of an antibody provided herein. The Fc region variant may include a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that includes amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0351] In certain embodiments, the present invention envisions antibody variants with reduced effector function, such as reduced or eliminated CDC, ADCC, and / or FcγR binding. In certain embodiments, the present invention envisions antibody variants having some, but not all, effector function, making them desirable candidates for applications where the in vivo half-life of the antibody is important, but certain effector function (complement-dependent cell-mediated cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC)) is unnecessary or detrimental.

[0352] In vitro and / or in vivo cytotoxicity assays can be performed to confirm decreased / depleted CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks FcγR binding (and therefore likely lacks ADCC activity). Primary cells for mediating ADCC, NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9: pp. 457-492 (1991). Non-limiting examples of in vitro assays for evaluating the ADCC activity of a target molecule are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985)); and U.S. Patent No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays may be used (see, for example, the ACTI® non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA); and the CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, the ADCC activity of the molecule of interest can be evaluated in vivo in animal models, such as those disclosed by Clynes et al. in Proc. Nat'l Acad. Sci. USA 95: pp. 652-656. Furthermore, a C1q binding assay can be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity.For example, see the C1q and C3c binding ELISAs described in WO2006 / 029879 and WO2005 / 100402. A CDC assay may also be performed to evaluate complement activation (see, for example, Gazzano-Santoro et al., J.Immunol.Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). Furthermore, FcRn binding and in vivo clearance / half-life can also be determined using methods known in the industry (see, for example, Petkova, SB et al., Int'l.Immunol.18(12):1759-1769 (2006); WO2013 / 120929A1).

[0353] Antibodies with reduced effector function include those having one or more substitutions among Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056), such as P329G. Such Fc variants include the so-called "DANA" Fc variant (U.S. Patent No. 7,332,581), which has alanine substitutions at residues 265 and 297, and Fc variants having two or more substitutions among amino acid positions 265, 269, 270, 297, and 327.

[0354] In certain embodiments, the antibody variant includes an Fc region having one or more amino acid substitutions that attenuate FcγR binding, e.g., substitutions at positions 234 and 235 (EU numbering of residues) of the Fc region. In one embodiment, the substitutions are L234A and L235A (LALA). In certain embodiments, the antibody variant further includes D265A and / or P329G in the Fc region derived from the human IgG1 Fc region. In one embodiment, the substitutions are L234A, L235A and P329G (LALA-PG) in the Fc region derived from the human IgG1 Fc region (see, for example, WO2012 / 130831). In another embodiment, the substitutions are L234A, L235A and D265A (LALA-DA) in the Fc region derived from the human IgG1 Fc region.

[0355] In other embodiments, it may be possible to use IgG subtypes with reduced effector functionality, such as IgG4 or IgG2.

[0356] Certain antibody variants with improved or reduced binding to FcR have been described (see, for example, U.S. Patent No. 6,737,056; WO2004 / 056312, and Shields et al., J. Biol. Chem. 9(2): pp. 6591-6604 (2001)).

[0357] In some embodiments, modifications resulting in alterations to C1q binding and / or complement-dependent cell injury (CDC) (i.e., improvement or attenuation, preferably attenuation) are made in the Fc region, such as those described in, for example, U.S. Patent No. 6,194,551, WO99 / 51642, and Idusogie et al., J.Immunol. 164: pp. 4178-4184 (2000).

[0358] In some embodiments, the number of FcRn bonds may be reduced, for example, to shorten the half-life. In other embodiments, the FcRn bonds may be normal. For example, in some embodiments, normal FcRn bonds can be used in a method that includes a removal agent.

[0359] In certain embodiments, the antibody variant includes an Fc region having one or more amino acid substitutions that reduce FcRn binding, for example, substitutions at positions 253 and / or 310 and / or 435 (EU numbering of residues) of the Fc region. In certain embodiments, the antibody variant includes an Fc region having amino acid substitutions at positions 253, 310 and 435. In one embodiment, the substitutions are I253A, H310A and H435A in the Fc region derived from the human IgG1 Fc region. See, for example, Grevys, A. et al., J.Immunol. 194 (2015), pp. 5497-5508.

[0360] In certain embodiments, the antibody variant includes an Fc region having one or more amino acid substitutions that reduce FcRn binding, e.g., substitutions at positions 310 and / or 433 and / or 436 (EU numbering of residues) of the Fc region. In certain embodiments, the antibody variant includes an Fc region having amino acid substitutions at positions 310, 433 and 436. In one embodiment, the substitutions are H310A, H433A and Y436A in the Fc region derived from the human IgG1 Fc region (see, for example, WO2014 / 177460A1). For example, in some embodiments, normal FcRn binding can be used.

[0361] For other examples of Fc region variants, see also Duncan & Winter, Nature 322:738-740 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO94 / 29351.

[0362] The C-terminus of the heavy chain of the antibodies reported herein may be a complete C-terminus having the amino acid residue PGK. The C-terminus of the heavy chain may be a shortened C-terminus from which one or two C-terminal amino acid residues have been removed. In one preferred embodiment, the C-terminus of the heavy chain is a shortened C-terminal PG.

[0363] In one aspect of all embodiments reported herein, an antibody comprising a heavy chain containing a C-terminal CH3 domain as identified herein includes a C-terminal glycine residue (G446, numbered by the EU index of amino acid position). This is still expressly encompassed with the terms “full-length antibody” or “full-length heavy chain” as used herein.

[0364] antibody derivative In certain embodiments, the antibodies provided herein may be further modified to include additional non-proteinoid moieties that are known in the art and readily available. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limited examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in production due to its stability in water. The polymers may have any molecular weight and may be branched or unbranched. The number of polymers bound to the antibody may vary, and if more than one polymer is bound, they may be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations such as, but are not limited to, the specific properties or functions of the antibody to be improved, and whether the antibody derivative will be used in a therapeutic setting under specified conditions.

[0365] In another embodiment, a conjugate is provided of an antibody and a non-proteinaceous moiety that can be selectively heated by exposure to radiation. In one embodiment, the non-proteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: pp. 11600-11605 (2005)). The radiation may be of any wavelength, but is not limited to any wavelength that does not harm normal cells, but heats the non-proteinaceous moiety to a temperature that kills cells close to the antibody-non-proteinaceous moiety.

[0366] Recombination method and composition For example, antibodies can be produced using recombinant methods and compositions, such as those described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding the antibody described herein is provided. Such nucleic acid may encode an amino acid sequence containing the VL of the antibody and / or an amino acid sequence containing the VH of the antibody (e.g., the light chain and / or heavy chain of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) containing such nucleic acid are provided. In a further embodiment, a host cell containing such nucleic acid is provided. In one such embodiment, the host cell comprises (1) a vector containing nucleic acid encoding an amino acid sequence containing the VL of the antibody and an amino acid sequence containing the VH of the antibody, or (2) a first vector containing nucleic acid encoding an amino acid sequence containing the VL of the antibody and a second vector containing nucleic acid encoding an amino acid sequence containing the VH of the antibody (e.g., it is transformed).

[0367] In the case of multispecific antibodies, nucleic acids encoding the heavy and light chain components of a particular antibody form can be provided. Vectors or sets of vectors containing such nucleic acids are also provided.

[0368] In one embodiment, the host cells are eukaryotic cells, such as Chinese hamster ovary (CHO) cells or lymphoid cells (e.g., Y0, NS0, Sp20 cells). In one embodiment, a method for producing an antibody according to the present invention is provided, comprising culturing host cells containing nucleic acids encoding the antibody provided above under conditions suitable for antibody expression, and, if necessary, recovering the antibody from the host cells (or host cell culture medium).

[0369] For recombinant antibody production, for example, nucleic acids encoding the antibody are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (for example, by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibody).

[0370] Suitable host cells for cloning or expressing antibody-encoding vectors include the prokaryotic or eukaryotic cells described herein. For example, antibodies may be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patents 5,648,237, 5,789,199, and 5,840,523 (see also Charlton, Methods in Molecular Biology, Vol. 248 (BKCLo (ed.), Humana Press, Totowa, NJ, 2003, pp. 245-254), which describes the expression of antibody fragments in Escherichia coli (E. coli)). After expression, antibodies can be isolated from bacterial cell paste in the soluble fraction and further purified.

[0371] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast, including fungal and yeast strains whose glycosylation pathways are "humanized," resulting in the production of antibodies with a partially or completely human glycosylation pattern, are suitable cloning or expression hosts for antibody-encoding vectors. See Gerngross, Nat. Biotech. 22: pp. 1409-1414 (2004), and Li et al., Nat. Biotech. 24: pp. 210-215 (2006).

[0372] Suitable host cells for the expression of glycosylated antibodies also originate from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. In particular, several baculovirus strains have been identified that can be used in conjunction with insect cells for transfection of armyworm (Spodoptera frugiperda) cells.

[0373] Plant cell cultures can also be used as hosts. See, for example, U.S. Patents 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLANTIBODIES® technology for producing antibodies in transgenic plants).

[0374] Vertebrate cells may be used as hosts. For example, mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include the SV40-transformed monkey kidney CV1 cell line (COS-7); human embryonic kidney cell lines (e.g., 293 or 293 cells described in Graham et al., J. Gen Virol. 36: pp. 59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells described in Mather, Biol. Reprod. 23: pp. 243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor cells (MMT060562); e.g., Mather et al., Annals These include TRI cells; MRC5 cells; and FS4 cells, as described in NYAcad.Sci.383:pp. 44-68 (1982). Other useful mammalian host cell lines include DHFR - Examples include Chinese hamster ovary (CHO) cells, including CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For an overview of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo (ed.), Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0375] Assay The antibodies provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activity by various assays known in the industry.

[0376] Binding assays and other assays In one embodiment, the antibody of the present invention is tested for its antigen-binding activity by known methods such as ELISA and Western blotting.

[0377] In another embodiment, a competitive assay can be used to identify antibodies that compete with, for example, PRIT-0213 or PRIT-0214 for binding to Pb-DOTAM or CEA. In certain embodiments, such competitive antibodies bind to the same epitopes (e.g., linear or stereoepitopes) to which PRIT-0213 or PRIT-0214 binds. Detailed exemplary methods for mapping the epitopes to which antibodies bind are provided in Morris (1996), "Epitope Mapping Protocols," Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

[0378] In an exemplary competition assay, an immobilized antigen is incubated in a solution containing a first labeled antibody that binds to the antigen (e.g., PRIT-0213 and PRIT-0214) and a second unlabeled antibody to be tested for its ability to compete with the first antibody for binding to the antigen. The second antibody may be present in the hybridoma supernatant. As a control, the immobilized antigen is incubated in a solution containing the first labeled antibody, rather than the second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to the antigen, excess unbound antibody is removed and the amount of label bound to the immobilized antigen is measured. If the amount of label bound to the immobilized antigen is substantially reduced in the test sample compared to the control sample, it indicates that the second antibody is competing with the first antibody for binding to the antigen. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0379] antibody affinity In certain embodiments, the antibodies provided herein have a dissociation constant (Kd) of 1 nM or less, 500 pM or less, 200 pM or less, 100 pM or less, 50 pM or less, 20 pM or less, 10 pM or less, 5 pM or less, or 1 pM or less, or otherwise as described herein.

[0380] In one embodiment, Kd is measured by radiolabeled antigen-binding assay (RIA). In one embodiment, the RIA is performed using the Fab version of the antibody of interest and its antigen. For example, the lysis-binding affinity of Fab to the antigen is measured in the presence of a titration series of unlabeled antigens with Fab and a minimum concentration of ( 125 I) After equilibrating with the labeled antigen, the bound antigen is captured using a plate coated with anti-Fab antibody (see, for example, Chen et al., J.Mol.Biol.293:865-881 (1999)). To establish the conditions for the assay, MICROTITER® multiwell plates (Thermo Scientific) are coated overnight with 5 μg / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and then blocked with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23°C). In a non-adsorbent plate (Nunc#269620), 100 pM or 26 pM [ 125Mix the [I]-antigen with serial dilutions of the Fab of interest (e.g., consistent with the evaluation of anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57: pp. 4593-4599 (1997)). Then incubate the Fab of interest overnight; however, incubation may be continued for a longer period (e.g., about 65 hours) to ensure equilibrium is reached. Subsequently, transfer the mixture to a capture plate for incubation at room temperature (e.g., over 1 hour). Then remove the solution and wash the plate eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. When the plate is dry, add 150 μL / well of flashing material (MICROSCINT-20®; Packard) and weigh the plate on a TOPCOUNT® gamma counter (Packard) for 10 minutes. The concentration of each Fab that gives a maximum binding of less than or equal to 20% is selected for use in competitive binding assays.

[0381] According to another embodiment, Kd is measured using a BIACORE® surface plasmon resonance assay. For example, the assay using BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) is performed at 25°C using a CM5 chip with approximately 10 reaction units (RUs) of immobilized antigen. In one embodiment, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. To achieve approximately 10 reaction units (RUs) of coupled protein, the antigen is diluted to 5 μg / ml (approximately 0.2 μM) before injection at a flow rate of 5 μl / min using 10 mM sodium acetate, pH 4.8. After antigen injection, 1 M ethanolamine is injected to block unreacted groups. For dynamic measurements, Fab's two-fold serial dilutions (0.78 nM to 500 nM) are injected into PBS containing 0.05% polysorbate 20 (TWEEN-20®) surfactant (PBST) at a flow rate of approximately 25 μl / min at 25°C. The binding rate (k) is calculated using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2) by simultaneously fitting binding and dissociation sensorgrams. on ) and dissociation rate (k off ) is calculated. The equilibrium dissociation constant (Kd) is k off / k on It is calculated as a ratio. For example, see Chen et al., J.Mol.Biol.293:865-881 (1999). The surface plasmon resonance assay above showed an association rate of 10 6 M -1 s -1If the value exceeds this, the association rate can be determined by using fluorescence quenching techniques to measure the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nx m, 16 nm band-passing) of a 20 nM anti-antigen antibody (Fab type) in PBS, pH 7.2 at 25°C in the presence of gradually increasing concentrations of antigen, when measured with a spectrophotometer such as a spectrophotometer with flow stop (Aviv Instruments) or an 8000 series SLM-AMINCO (Trademark) spectrophotometer (ThermoSpectronic) with a stirring cuvette.

[0382] In another embodiment, Kd is measured using a SET (solution equilibrium titration) assay. According to this assay, the test antibody is applied, typically at a constant concentration, and mixed with serial dilutions of the test antigen. After incubation to establish equilibrium, a portion of the free antibody is captured on an antigen-coated surface and detected with labeled / tagged anti-species antibodies, typically using electrochemiluminescence (e.g., as described by Haenel et al., Analytical Biochemistry 339 (2005), pp. 182-184).

[0383] For example, in one embodiment, a 384-well streptavidin plate (Nunc, Microcoat #11974998001) is incubated overnight at 4°C with 25 μl / well of an antigen-biotin-isomer mixture in PBS buffer at a concentration of 20 ng / ml. To equilibrate the antibody sample with the free antigen, 0.01 nM to 1 nM of antibody is titrated with the relevant antigen in dilution steps of 1:3, 1:2, or 1:1.7, starting with antigen concentrations of 2500 nM, 500 nM, or 100 nM. The sample is incubated overnight at 4°C in a sealed REMP-stored polypropylene microplate (Brooks). After overnight incubation, the streptavidin plate is washed three times with 90 μl of PBST per well. 15 μl of each sample from the equilibrated plate is transferred to an assay plate, incubated at RT for 15 minutes, and then washed three times with 90 μl of PBST buffer. Detection is performed by adding 25 μl of goat anti-human IgG antibody-POD conjugate (Jackson, 109-036-088, 1:4000 in OSEP) and then washing six times with 90 μl of PBST buffer. 25 μl of TMB substrate (Roche Diagnostics GmbH, catalog number 11835033001) is added to each well. Measurement is performed at 370 / 492 nm on a Safire2 reader (Tecan).

[0384] In another embodiment, Kd is measured using the KinExA (binding equilibrium exclusion) assay. According to this assay, typically, the antigen is titrated in a constant concentration of antibody binding sites to equilibrate the sample, and then the sample is passed through a flow cell where the free antibody binding sites are captured on antigen-coated beads, while washing away the antigen-saturated antibody complex. The antibodies captured on the beads are then detected with labeled, for example, fluorescently labeled, anti-species antibodies (Bee et al., PloS One, 2012;7(4):e36261). For example, in one embodiment, the KinExA experiment is performed at room temperature (RT) using PBS pH 7.4 as the running buffer. The sample is prepared in a running buffer ("sample buffer") to which 1 mg / ml BSA has been added. A flow rate of 0.25 ml / min is used. A constant amount of antibody with a binding site concentration of 5 pM is titrated with the antigen by 2-fold serial dilutions starting from 100 pM (concentration range of 0.049 pM to 100 pM). One sample of antibody without antigen acts as a 100% signal (i.e., no inhibition). The antigen-antibody complex is incubated at RT for at least 24 hours to reach equilibrium. The equilibrated mixture is then passed through a column of antigen-binding beads in a KinExA system in a volume of 5 ml to capture the unbound antibody by the beads without disturbing the equilibrium of the solution. The captured antibody is detected using an anti-human Fc fragment-specific secondary antibody conjugated to 250 ng / ml Dylight 650(C) in sample buffer. Each sample is measured twice for all equilibrium experiments. KD is obtained from nonlinear regression analysis of the data using the one-site homogeneous binding model included in the KinExA software (version 4.0.11) using the "Standard Analysis" method.

[0385] Treatment method and composition As discussed above, the multispecific antibodies according to the present invention are suitable for any treatment in which it is desirable to deliver a radionuclide as a target. Accordingly, the present invention provides targeted antibodies, such as the multispecific or bispecific antibodies described herein, for use in therapeutic methods. More specifically, targeted antibodies (e.g., multispecific or bispecific antibodies) described herein for use in pretargeted radioimmunotherapy methods are provided. In such embodiments, chelated Pb is preferably, 212 It is Pb.

[0386] As described above, the treatment may be for any condition that can be treated by cytotoxic activity targeted at diseased cells in the patient. The treatment is preferably for tumors or cancer. However, the applicability of the present invention is not limited to tumors and cancer. For example, the treatment may be for viral infection or infection by another pathogenic organism, such as a prokaryote. If necessary, the targeting may be T cells for the treatment of T cell-induced autoimmune diseases or T cell hematological malignancies. Thus, the conditions to be treated may include viral infections such as HIV, rabies, EBV and Kaposi's sarcoma-associated herpesvirus, as well as autoimmune diseases such as multiple sclerosis and graft-versus-host disease.

[0387] As used herein, the term "cancer" includes lymphoma, lymphocytic leukemia, lung cancer, non-small cell lung (NSCL) cancer, bronchioloalveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, gastric cancer, colon cancer, breast cancer, uterine cancer, Fallot-Pierce's canal cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, and thyroid cancer. This includes both solid tumors and hematological malignancies, such as cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, cancer of the kidney or ureter, renal cell carcinoma, renal pelvis cancer, mesothelioma, hepatocellular carcinoma, cholangiocarcinoma, neoplasms of the central nervous system (CNS), spinal tumors, brainstem glioma, glioblastoma multiforme, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, and Ewing's sarcoma, and includes refractory forms of any of the above cancers, or combinations of one or more of the above cancers.

[0388] Methods for targeting radioactive isotopes to tissues or organs for therapeutic purposes are: i) administering to the subject a multispecific or bispecific antibody as described herein, which binds to a target antigen and localizes to the surface of cells expressing the target antigen; and ii) Next, administering to an individual a Pb radionuclide chelated with DOTAM or a functional variant thereof, which binds to an antibody localized on the cell surface. It may include.

[0389] If necessary, a removal agent / blocking agent is administered between steps (i) and (ii). The removal agent / blocking agent can bind to an antigen-binding site specific to Pb-DOTAM and block subsequent binding by a chelated radionuclide. The removal agent may contain DOTAM or a functional variant thereof, chelated with a metal ion and conjugated to the removal portion.

[0390] Examples of suitable removal portions may include portions that increase the size and / or hydrodynamic radius of molecules, thereby inhibiting the ability of molecules to approach tumors without interfering with the ability of molecules to bind to antibodies in circulation. Exemplary portions include hydrophilic polymers. The portions may be polymers or copolymers of, for example, dextran, dextrin, PEG, polysialic acid (PSA), hyaluronic acid, hydroxyethyl starch (HES), or poly(2-ethyl 2-oxazoline) (PEOZ). In other embodiments, the portions may be unstructured peptides or proteins such as XTEN polypeptides (unstructured hydrophilic protein polymers), homoamino acid polymers (HAP), proline-alanine-serine polymers (PAS), elastin-like peptides (ELP), or gelatin-like proteins (GLK). Suitable molecular weights for polymers may be, for example, at least 50 kDa, for example, in the range of 50 kDa to 2000 kDa. For example, the molecular weight may be 200-800 kDa, preferably greater than 300, 350, 400 or 450 kDa, preferably less than 700, 650, 600 or 550 kDa, and preferably about 500 kDa.

[0391] In some embodiments, the scavenger may be DOTAM or a functional variant thereof (chelated with a metal ion), conjugated with dextran or a derivative thereof, as further described below.

[0392] In some embodiments, the weight ratio of antibody to scavenging agent may range from 1:1, 2:1, 3:1, or 4:1 to 20:1, 15:1, 10:1, 8:1, 6:1, or 5:1, for example, in the range of 1:1 to 20:1, 1:1 to 10:1, 2:1 to 8:1, or 2:1 to 6:1.

[0393] In some embodiments, the scavenger may be administered several hours or days after treatment with the multispecific antibody. In some embodiments, it may be preferable to administer the scavenger at least 2, 4, 6, 8, 10, 12, 16, 18, 22, or 24 hours after the multispecific antibody, or at least 1, 2, 3, 4, 5, 6, or 7 days after the antibody. In some embodiments, it may be preferable to administer the scavenger within 14 days after antibody administration, for example, within 10, 9, 8, 7, 6, 5, 4, 3, or 2 days.

[0394] If necessary, administer the elimination agent after the administration of the multispecific antibody at intervals of 4-10 days, 4-7 days, 2-7 days, or 2-4 days.

[0395] In some embodiments, the Pb radionuclide is administered within minutes, hours, or days after the removal agent. In some embodiments, it may be preferable to administer the Pb radionuclide at least 30 minutes after the removal agent, and, if necessary, within 48, 24, 8, or 4 hours after the administration of the removal agent. In some embodiments, the Pb radionuclide may be administered the day after the administration of the removal agent.

[0396] In some embodiments, the antibodies described herein can be administered as part of a combination therapy. For example, they can be administered together with one or more chemotherapeutic agents: the chemotherapeutic agents and antibodies can be administered simultaneously or sequentially in either order.

[0397] In some embodiments, the antibodies described herein may be administered further or together with a radiosensitizer. The radiosensitizer and the antibodies may be administered simultaneously or sequentially in either order.

[0398] Pharmaceutical preparations The anti-Pb-DOTAM antibodies described herein, for example, multispecific or bispecific antibodies, are prepared by mixing such antibodies of desired purity with one or more optionally selected pharmaceutically acceptable carriers, in the form of lyophilized formulations or aqueous solutions (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed.) (1980)). Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dose and concentration used, and are not limited to, buffers such as phosphoric acid, citrate, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; phenol, butyl, or benzyl alcohol; parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); and low molecular weight (less than approximately 10 residues) polypeptides. Examples of pharmaceutically acceptable carriers herein include: butylates; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersants such as soluble neutral active hyaluronidase glycoproteins (sHASEGP), e.g., human soluble PH-20 hyaluronidase glycoprotein such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGP and methods of use, including rHuPH20, are described in U.S. Patent Application Publications 2005 / 0260186 and 2006 / 0104968.In one embodiment, sHASEGP is combined with one or more further glycosaminoglycans, such as chondroitinase.

[0399] An example of a lyophilized antibody preparation is described in U.S. Patent No. 6,267,958. Examples of aqueous antibody preparations are described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter of which contains a histidine-acetate buffer.

[0400] The formulations described herein may also contain more than one active ingredient necessary for the specific indication being treated, preferably those having complementary activities that do not adversely affect each other. For example, it may be desirable to further provide the chemotherapeutic agents and / or radiosensitizers discussed above. Such active ingredients are preferably present together in amounts effective for the intended purpose.

[0401] The active ingredient can be captured in microcapsules prepared, for example, by droplet formation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or macroemulsions, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed.) (1980).

[0402] Sustained-release preparations can be prepared. A suitable example of a sustained-release preparation is a semipermeable matrix of a solid hydrophobic polymer containing an antibody, which may be in the form of a fabricated product, such as a film or microcapsule.

[0403] Formulations used for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration through a sterile filtration membrane.

[0404] Methods and compositions for diagnosis and detection The present invention further provides targeted antibodies, such as the multispecific antibodies described herein, for use in diagnostic methods performed on a subject. The diagnostic method may be, for example, a pre-targeted radioimmunoimaging method for diagnosing a subject suspected of having a proliferative disorder or infection. In such embodiments, chelated Pb is preferably 203 It is Pb.

[0405] Methods for targeting radioisotopes to tissues or organs for imaging include: i) administering to the subject a multispecific or bispecific antibody as described herein, which binds to a target antigen and localizes to the surface of cells expressing the target antigen; and ii) Next, administering to an individual a Pb radionuclide chelated with DOTAM or a functional variant thereof, which binds to an antibody localized on the surface of a cell. It may include.

[0406] In another embodiment, the multispecific or bispecific antibodies described herein may be conjugated to a chelated Pb radionuclide at the time of administration.

[0407] If necessary, the method is iii) Imaging tissues or organs where Pb radionuclides chelated with DOTAM or its functional variants are localized or are expected to be localized. It may also include the following.

[0408] In another embodiment, the method of the present invention applies to the subject, i) multispecific or bispecific antibodies as described herein that bind to a target antigen and localize to the surface of cells expressing the target antigen; and ii) Pb radionuclides chelated with DOTAM or its functional variants, wherein the Pb radionuclides chelated with DOTAM or its functional variants bind to antibodies localized on the surface of cells. This may include imaging the target tissue or organ to which the individual has been previously administered the substance.

[0409] If necessary, administer a removal agent / blocking agent between steps (i) and (ii). The removal agent, the administration regimen for the removal agent, and the weight ratio of the antibody to the removal agent may be as described above.

[0410] The target antigen may be any target antigen considered herein. In some embodiments, the target antigen may be a tumor-specific antigen considered above, and imaging may be a method for imaging a tumor or multiple tumors. The individual may be known to have a tumor or suspected to have one.

[0411] For example, the methods include: lung cancer, non-small cell lung (NSCL) cancer, bronchioloalveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, gastric cancer, colon cancer, breast cancer, uterine cancer, Fallot's canal cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, and urethral cancer. This may also be a method for imaging tumors in individuals who have or are suspected of having penile cancer, prostate cancer, bladder cancer, kidney or ureteral cancer, renal cell carcinoma, renal pelvis cancer, mesothelioma, hepatocellular carcinoma, cholangiocarcinoma, neoplasms of the central nervous system (CNS), spinal tumors, brainstem glioma, glioblastoma multiforme, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, and Ewing's sarcoma (including refractory forms of any of the above cancers, or combinations of one or more of the above cancers).

[0412] Removal agent In a further aspect of the present invention, the inventors have developed a novel decontamination agent that can be used in any of the diagnostic, imaging, or therapeutic methods described herein.

[0413] In one embodiment, the present invention relates to a dextran-based scavenger comprising dextran or a derivative thereof conjugated to M-DOTAM or a functional variant thereof.

[0414] In some embodiments, the removal agent is given by the following formula: Dextran-(linker-(M-DOTAM)) x [In the formula, Dextran is dextran or its derivatives; A linker is a connecting part; M-DOTAM is a functional variant of DOTAM in which a metal ion is incorporated; and x ≥ 1] It may also be a compound of the following.

[0415] In some embodiments, the linking portion consists of a urea group (-NH-C(O)-NH-) and a substituted urea group (-NR x -C(O)-NR x -[In the formula, one or both R x The group is not H), thiourea group (-NH-C(S)-NH-), substituted thiourea group (-NR x -C(S)-NR x -[In the formula, one or both R x The group is not H), amide group (-C(O)-NH-), substituted amide group (-C(O)-NR x -[wherein, R x (is not H), thioamide group (-C(S)-NH-), substituted amide group (-C(S)-NR x -[wherein, R xSubstituent R is not H), may be one or more divalent functional groups selected from triazole groups or substituted triazoles, or may include them. In these embodiments, the linking portion may optionally include one or more further divalent functional groups, such as alkylene groups, arylene groups, heteroarylene groups, alkylene groups and heteroaralkylene groups. x This is not particularly limited. In certain embodiments, R x If present, the group is selected from C1-C6 alkyl, C5-C12 aryl, C5-C12 heteroaryl, and halo groups.

[0416] In certain embodiments, the linking portion may be one or more divalent functional groups selected from urea groups, thiourea groups, amide groups, thioamide groups, or triazole groups, or may contain such groups.

[0417] In a preferred embodiment, the linking portion includes a divalent thiourea functional group or a divalent thioamide functional group.

[0418] In some embodiments, the linking portion comprises a divalent thiourea functional group and an optionally substituted arylene group. In some embodiments, the linking portion comprises a divalent thiourea functional group, an optionally substituted arylene group and an optionally substituted alkylene group. In a particular embodiment, the linking portion comprises a divalent thiourea functional group covalently bonded to an optionally substituted arylene group via one of its nitrogen atoms. In a further embodiment, the linking portion comprises a divalent thiourea functional group covalently bonded to an optionally substituted arylene group via one of its nitrogen atoms, and the optionally substituted arylene group covalently bonded to an optionally substituted alkylene group. In a preferred embodiment, the arylene group is unsubstituted. In a particular embodiment, the arylene group is a phenylene group. In a preferred embodiment, the alkylene group is unsubstituted. In a particular embodiment, the alkylene group is a C1-C6 alkyene group. In a particularly preferred embodiment, the alkylene group is selected from methylene and ethylene. When present in the linking portion, the arylene group and alkylene group may be unsubstituted. In certain embodiments, the linking portion consists of a divalent thiourea functional group covalently bonded to the arylene group via one of its nitrogen atoms, and the arylene group is covalently bonded to the alkylene group.

[0419] In some embodiments, the linking portion comprises a divalent thioamide functional group and an optionally substituted arylene group. In some embodiments, the linking portion comprises a divalent thioamide functional group, an optionally substituted arylene group and an optionally substituted alkylene group. In a particular embodiment, the linking portion comprises a divalent thioamide functional group covalently bonded to an optionally substituted arylene group via one of its nitrogen atoms. In a further embodiment, the linking portion comprises a divalent thioaide functional group covalently bonded to an optionally substituted arylene group via one of its nitrogen atoms, and the optionally substituted arylene group covalently bonded to an optionally substituted alkylene group. In a preferred embodiment, the arylene group is unsubstituted. In a particular embodiment, the arylene group is a phenylene group. In a preferred embodiment, the alkylene group is unsubstituted. In a particular embodiment, the alkylene group is a C1-C6 alkylene group. In a particularly preferred embodiment, the alkylene group is selected from methylene and ethylene. When present in the linking portion, the arylene group and alkylene group may be unsubstituted. In certain embodiments, the linking portion consists of a divalent thioamide functional group covalently bonded to the arylene group via one of its nitrogen atoms, and the arylene group is covalently bonded to the alkylene group.

[0420] In some embodiments, the connecting portion is given by the following formula: TIFF2026062683000011.tif38170[In the formula, y is 1 to 6 (preferably 1 or 2), * represents a binding site to dextran or its derivatives, ** [This represents a bond site to the ring atom of DOTAM or its functional variant.] It may be based on, or may include.

[0421] In some embodiments, the linking portion can be formed from a conjugation of an amine (preferably a primary amine) and an isocyanate or isothiocyanate. Such conjugations form a divalent urea functional group and a thiourea functional group, respectively. In such embodiments, if an isocyanate is one of the reactants, the linking portion may be considered to include a divalent urea functional group or a divalent amide functional group as appropriate. In such embodiments, if an isothiocyanate is one of the reactants, the linking portion may be considered to include a divalent thiourea functional group or a divalent thioamide functional group as appropriate.

[0422] Preferably, when x is greater than 1, each dextran has an average number of M-DOTAMs or functional variants greater than 1 per molecule. For example, x may be 2 or greater, 5 or greater, 10 or greater, 15 or greater, 20 or greater, 25 or greater, 30 or greater, 35 or greater, 40 or greater, or preferably 50 or greater. The inventors have found that improved removal can be achieved using dextrans labeled with multiple M-DOTAM groups.

[0423] DOTAM or its functional variants contain only one linker and can prevent dextran crosslinking.

[0424] Dextran derivatives that may be useful in scavenging agents include aminodextrans, in which dextran is substituted with one or more amines. Particularly useful are aminodextrans in which one or more hydroxyl groups of dextran are substituted with amino-substituted carboxymethylamide groups. Such compounds can be produced by modifying dextran with a carboxymethyl group (for example, by reacting it with chloroacetic acid) and then further reacting it with an optionally substituted diamine (preferably an α,ω-alkylenediamine, such as an alkyldiamine like ethylenediamine).

[0425] The amino groups provide linking sites for the linker. At least 30%, preferably at least 40%, and more preferably at least 50% of the available amino groups can be substituted with DOTAM or its functional variants.

[0426] Preferably, the "dextran" in the above formula is an aminodextran, which corresponds to a dextran substituted with one or more carboxymethyl groups that are themselves substituted with ethylenediamine.

[0427] Dextran is given by the formula -CH2C(=O)NH(CH2) f NHR F [In the formula, R F [where f represents a linker for hydrogen or DOTAM, and f is 1 to 6, most preferably 2] may be substituted with one or more groups of the following formula: TIFF2026062683000012.tif31170 [In the formula, the dashed line indicates the binding site to oxygen on dextran.] It may have.

[0428] The aminodextran may have a backbone primarily of α(1,6)-linked glucopyranosyl repeat units, which may have branching of other glucopyranosyl units linked by α(1,2), α(1,3), or α(1,4) glycosidic bonds, for example. At least some of the hydroxyl groups may be amino-substituted carboxymethylamide groups (in particular, those of the formula -CH2C(=O)NHCH2CH2NHR) as discussed above. F It is substituted with the group of . In other words, aminodextran is given by the following formula: TIFF2026062683000013.tif108170[R G These are H and an amino-substituted carboxymethylamide group (-CH2C(=O)NHCH2CH2NHR F (etc.), or primarily, binding to further glucopyranosyl units via α(1,6) bonds, with dashed lines indicating binding to adjacent units. It may include units of .

[0429] The removal agent is given by the following formula: TIFF2026062683000014.tif69170[In the formula, ** [where represents a binding site to DOTAM or a functional variant, and y is as defined above] may include one or more units of .

[0430] Dextran derivatives may include dextran or aminodextran modified with one or more groups selected from amino acids or sugars other than glucose. For example, dextran may be modified (e.g., capped) with one or more glutamic acid or polyglutamic acid units, including Glu, (Glu)2, (Glu)3, or (Glu)4. Furthermore, or alternatively, dextran may be modified (e.g., capped) with sugars other than glucose, such as N-acetylgalactosamine (GalNAc), or polysaccharides formed from such sugars, such as tri-GalNAc.

[0431] In some embodiments, the molecular weight of the dextran component may be at least 50 kDa, for example, between 50 kDa and 2000 kDa. For example, the molecular weight may be 200 to 800 kDa, optionally greater than 300, 350, 400 or 450 kDa, optionally less than 700, 650, 600 or 550 kDa, and optionally about 500 kDa.

[0432] The number of amino groups as a percentage of the number of glucose units in dextran or a dextran derivative ("saturation" of glucose units by amino groups) may be at least 0.5%, at least 1%, at least 2%, at least 5%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or preferably 100%. In some embodiments, the saturation of dextran by amino groups may preferably be at least, or about 1%, or 10%, for example, 1% to 10%.

[0433] The number of DOTAM groups as a percentage of the number of amino units in the dextran derivative ("saturation" of the aminodextran component by DOTAM) may be at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or preferably 100%. In some embodiments, the saturation of available amino groups in the dextran derivative by DOTAM may preferably be at least, or about 40% or 50%, for example, 40-60%.

[0434] One potential difficulty associated with the use of removal agents is the possibility that they may enter the tumor, bind to tumor-associated antigens, and negatively affect the subsequent binding of radioactive ligands.

[0435] The inventors have further found that when using a dextran-based scavenger that i) has a high average molecular weight and ii) is subjected to a molecular weight cutoff such that fragments below a certain size are removed, good clearance from the blood can be achieved along with low scavenger penetration into the tumor. The cutoff can be applied to dextran or a dextran derivative before the conjugation process; and / or to the scavenger after conjugation; and / or to the scavenger after complexation with a metal.

[0436] Thus, a useful removal agent in the present invention is a dextran-based removal agent comprising dextran or a derivative thereof (e.g., those defined above, preferably aminodextran) conjugated to a metal chelate, wherein i) the average molecular weight of dextran or its derivative is preferably 200 to 800 kDa, optionally greater than 300, 350, 400 or 450 kDa, optionally less than 700, 650, 600 or 550 kDa, and optionally about 500 kDa, and ii) dextran, dextran derivatives or removal agents below the molecular weight cutoff are removed, and the molecular weight cutoff The dextran-based scavenger may be 50 kDa or greater, 100 kDa or greater, or 200 kDa or greater, optionally in the range of 50 kDa to 250 kDa or 50 kDa to 200 kDa, optionally in the range of 100 kDa to 200 kDa, and optionally about 100 kDa or 150 kDa or 200 kDa (to avoid doubt, the inventors hereby state that when the cutoff is described as 50 kDa or greater, this means that the cutoff may be 50 kDa or any value greater than 50 kDa, but it still means that the dextran, dextran derivative or scavenger is below the cutoff that will be removed).

[0437] The amount of species having a molecular weight below the cutoff may be, for example, 5 wt% or less, 4 wt% or less, 3 wt% or less, 2 wt% or less, 1 wt% or less, 0.5 wt% or less, 0.4 wt% or less, 0.3 wt% or less, 0.2 wt% or less, 0.1 wt% or less, or 0.01 wt% or less, as a weight percentage of the removal agent. Preferably, the removal agent does not contain species having a molecular weight below the cutoff in essence.

[0438] Molecular weight cutoff can be achieved by filtration, such as diafiltration, ultrafiltration, tangent flow filtration, or cross-flow filtration. Preferably, at least two filtration steps are performed, and if necessary, at least three. "Average molecular weight" means, as the inventors, the weight-average molecular weight determined by SEC-MALS analysis.

[0439] When incorporated into DOTAM or its functional variants, it will be understood that the metals exist as metal ions and their oxidation state changes depending on the specific element. Thus, a knowledgeable reader will understand that, for example, lead, Pb, or 206 It can be understood that the term Pb is intended to encompass the ionic form of the element, particularly Pb(II).

[0440] The metal present in the removal agent may be a stable (non-radioactive) isotope of lead, or a stable or intrinsically stable isotope of another metal ion, provided that the metal ion-DOTAM complex is recognized with high affinity by the antibody. For example, other suitable metals include Zn(Zn 2+ ), Ca(Ca 2+ )or 209 Bi(Bi 2+ ) may be radioactive, and although the latter is radioactive, it is considered practically stable due to its very long half-life.

[0441] In a further embodiment, the present invention relates to a method for preparing a scavenger, comprising conjugating dextran or a dextran derivative to DOTAM or a functional variant or derivative thereof, wherein the method further comprises chelating DOTAM with Pb or another metal ion [e.g., Pb(II)] before and / or after conjugation of DOTAM or a functional variant thereof to dextran.

[0442] In a further embodiment, the present invention relates to a method for preparing a removal agent, Forming a conjugate by conjugating DOTAM or its functional variants or derivatives to dextran or a dextran derivative. Includes; Prior to conjugation, dextran or a dextran derivative is subjected to a filtration step to remove species below a molecular weight cutoff / threshold in the range of, for example, 50 kDa or more, 100 kDa or more, or 200 kDa or more, and if necessary, 50 kDa to 250 kDa or 50 kDa to 200 kDa, and if necessary, 100 kDa to 200 kDa, for example, species below 100 kDa, 150 kDa or 200 kDa, or The present invention relates to a method for preparing a scavenger, further comprising subjecting a conjugate to a filtration step to remove species below a molecular weight cutoff / threshold in the range of, for example, 50 kDa or more, 100 kDa or more, or 200 kDa or more, optionally 50 kDa to 250 kDa or 50 kDa to 200 kDa, or optionally 100 kDa to 200 kDa, for example, species below 100 kDa, 150 kDa, or 200 kDa.

[0443] As described above, the inventors have found it beneficial to apply a molecular weight cutoff to remove fragments below a certain size. The filtration method may be, for example, diafiltration. A knowledgeable reader will understand that the word "remove" in "remove species below molecular weight cutoff / threshold" is synonymous with "reduce the number," and that some residual low molecular weight species may remain depending on the specific filtration method used. The amount of species with molecular weights below the cutoff may be, for example, 5 wt% or less, 4 wt% or less, 3 wt% or less, 2 wt% or less, 1 wt% or less, 0.5 wt% or less, 0.4 wt% or less, 0.3 wt% or less, 0.2 wt% or less, 0.1% wt% or less, or 0.01 wt% or less, as a weight percentage of the remover. Preferably, the remover is essentially free of species with molecular weights below the cutoff / threshold after filtration.

[0444] Functional variants or derivatives of DOTAM may be as defined above, and may contain at least one R 1 The group acts as a linker. For example, a suitable (linker-(M-DOTAM)) group can be formed by reacting a compound of the following formula with the above aminodextran: TIFF2026062683000015.tif63170

[0445] The synthesis of this compound is described by Chappell et al., Nuclear Medicine and Biology, Vol. 27, pp. 93-100, 2000, and DOTAM derivatives are commercially available from Macrocyclics, Inc. (Plano, Texas).

[0446] DOTAM or its functional variants may be added in excess so that each dextran derivative has an average of more than one DOTAM. The average number of DOTAM or its functional variants on each dextran may be greater than 1, for example, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 50 or more, 100 or more, or preferably 40 or more. The inventors have found that improved removal can be achieved by using dextran conjugated with multiple M-DOTAM groups.

[0447] Preferably, the dextran has an average molecular weight of 200-800 kDa, optionally greater than 300, 350, 400 or 450 kDa, optionally less than 700, 650, 600 or 550 kDa, and optionally about 500 kDa.

[0448] A method for preparing a scavenging agent may also include a chelation step, which involves chelating DOTAM or a functional variant thereof with a metal ion. The metal ion may be a non-radioactive isotope, such as a non-radioactive isotope of Pb, Ca, or Zn, or 209 It may also be a substantially stable isotope such as Bi.

[0449] The chelation step is performed before the conjugation of DOTAM or its functional variant to dextran, and / or after the conjugation of DOTAM or its functional variant to dextran, and, if necessary, before the filtration step. Chelation of metal ions with DOTAM or its functional variant may be necessary to ensure proper binding of the bispecific antibody to the removal agent, for example, to ensure that DOTAM or its functional variant adopts the correct conformation for encounter with the antibody.

[0450] If the method includes a chelation step, the method preferably also includes a subsequent step of removing unbound metals. This can be achieved by adding a further chelating agent, which can then be separated from DOTAM or its functional variants bound to dextran during a filtration step. The further chelating agent is preferably different from DOTAM or its functional variants. Preferably, the further chelating agent has a lower molecular weight than the dextran-chelating agent conjugate to facilitate size-based separation. For example, the further chelating agent may be a polyaminocarboxylic acid or a salt thereof, such as ethylenediaminotetraacetic acid (EDTA).

[0451] Preferably, the method for preparing the chelating agent is: i) Forming a conjugate by conjugating DOTAM or its functional variants or derivatives to dextran or a dextran derivative; ii) Remove low molecular weight species from the product of step (i), if necessary; iii) Chelating the conjugate with metal ions, such as Pb, Bi, Zn, or Ca ions; iv) Adding further chelating agents to chelate unbound metal ions; and v) Perform a filtration step to remove species below the molecular weight cutoff / threshold. Includes.

[0452] DOTAM-chelated Pb radionuclides Pb radionuclides chelated with DOTAM or its functional variants can be used in any of the diagnostic, imaging, or therapeutic methods described herein. When used in such methods, it will be understood that the Pb radionuclides chelated with DOTAM or its functional variants are included in a composition. In one particular embodiment, the composition comprises a Pb radionuclides chelated with DOTAM or its functional variants and DOTAM or its functional variants that are not chelated with Pb radionuclides. Thus, in another embodiment, the present invention relates to such compositions and / or such compositions for use in any of the imaging or therapeutic methods described herein. The DOTAM-chelated Pb radionuclides referred to in such methods may also be in the form of the compositions described herein.

[0453] DOTAM or its functional variants that are not chelated with Pb radionuclides may also be unchelated DOTAM or its functional variants. When used in vivo, unchelated DOTAM or its functional variants can form complexes with environmentally derived metal ions, such as calcium ions. Such calcium ions chelated with DOTAM or its functional variants are pharmacologically inactive and can potentially block pharmacologically active Pb radionuclides chelated with DOTAM or its variants from targets in tumors, thus potentially reducing therapeutic efficacy, as well as / or standardized uptake values ​​for imaging and diagnosis.

[0454] The inventors have found that under specified conditions, quenching of unchelated DOTAM or its functional variants can increase the control of in vivo formulations of chelated Pb radionuclides, and / or avoid or reduce potential competition between pharmaceutically active and pharmaceutically inactive chelates. Therefore, in some embodiments, unchelated DOTAM or its functional variants with Pb radionuclides are DOTAM or its functional variants chelated with non-radioactive metal ions.

[0455] In some embodiments, the chelated Pb radionuclide is 212 It is Pb. In some embodiments, the chelated Pb radionuclide is 203 It is Pb.

[0456] When incorporated into DOTAM or its functional variants, it will be understood that the Pb radionuclide exists as a metal ion and its oxidation state changes depending on the specific element. Thus, a knowledgeable reader will understand that, for example, lead, Pb, or 206 It can be understood that the term Pb is intended to encompass the ionic form of the element, particularly Pb(II).

[0457] Non-radioactive metals present in the composition may be stable (non-radioactive) isotopes of lead, or stable or intrinsically stable isotopes of other metal ions. For example, other suitable metals include Gd, (Gd²⁺), Cu(Cu²⁺), Zn(Zn²⁺), Ca(Ca²⁺), or 209 It may also be Bi(Bi2+), the latter being radioactive, but considered practically stable due to its very long half-life. In some embodiments, the metal is Ca or Cu, and in some embodiments, the metal is Ca.

[0458] Functional variants or derivatives of DOTAM may be as defined above.

[0459] In a further embodiment, the present invention relates to a method for preparing a composition comprising a Pb radionuclide chelated with DOTAM or a functional variant thereof, i) To provide Pb radionuclides, ii) Chelating Pb radionuclides with DOTAM or its functional variants, iii) Chelating unchelated DOTAM or its functional variants with non-radioactive metal ions. Regarding methods including

[0460] The unchelated DOTAM or its functional variant in step iii) is the DOTAM or its functional variant that was not chelated with the Pb radionuclide in step ii).

[0461] The non-radioactive metal ion may be a Pb, Ca, Zn, Gd, or Cu ion. In some embodiments, the metal ion is a Ca or Cu ion. In some embodiments, the metal ion is a Ca ion, in particular Ca²⁺.

[0462] In some embodiments, the unchelated DOTAM or its functional variants remaining after step ii) are at least 90 mol%, at least 95 mol%, and at least 99 mol% of the DOTAM or its functional variants added to the Pb radionuclide. In one particular embodiment, the unchelated DOTAM or its functional variants remaining after step ii) are at least 99 mol%.

[0463] In some embodiments, the unchelated DOTAM or its functional variants remaining after step iii) is less than 5 mol%, less than 2 mol%, less than 1 mol%, less than 0.1 mol%, or less than 0.01 mol% of the DOTAM or its functional variants added to the Pb radionuclide. In one particular embodiment, the unchelated DOTAM or its functional variants remaining after step iii) is less than 1 mol%, less than 0.1 mol%, or less than 0.01 mol%.

[0464] The Pb radionuclide provided in step a) can be produced by introducing a radioactive material that decays the target Pb radionuclide and binds to a solid material into a generator. For example, 212 Such radioactive material in the production of Pb may be radium 224. The radionuclide of interest is then extracted from the generator into an aqueous solution which may contain radioactive and chemical impurities. The aqueous solution containing the Pb radionuclide of interest and impurities is purified by liquid chromatography on a column. Liquid chromatography on a column may be extraction chromatography or partition chromatography. Extraction or partition chromatography is based on the distribution of elements separated between an organic phase, or extract, and an aqueous phase, where the extract binds to an inert support and forms the stationary phase, and the aqueous phase is the mobile phase.

[0465] Extractive chromatography may use a stationary phase containing an ether crown, particularly dicyclohexano-1,8-crown-6 or dibenzo-1,8-crown-6, as the extract, in a solution of an organic diluent immiscible with water, typically a long hydrocarbon chain alcohol, in other words, a Cx chain or longer, in which the cyclohexyl or benzyl group is substituted with one or more C[C]2 alkyl groups having a linear or branched chain.

[0466] In particular, preferably, a stationary phase containing 4,4'(5')-di-er-butylcyclohexano-1,8-crown-6 as an extract, diluted in octan-1-ol, may be used. Such a stationary phase is typically obtained from a radium-224 generator. 212 In aqueous solutions containing a strong acid of 1.5 to 2.5 mol / L, corresponding to the type of aqueous solution used to extract Pb, more than 99% of the substance remains selectively. 212It has the advantage of having Pb present. This type of stationary phase is available, for example, in bottles, as well as packaged in ready-to-use columns or cartridges for chromatography by a company called TRISKEM International under the trade name "Pb Resin".

[0467] Alternatively, a solution containing the desired radionuclides and impurities can be purified using ion exchange chromatography, such as cation exchange chromatography.

[0468] 212 The method for producing and refining Pb is described in WO2013174949.

[0469] array The specific sequences described in this specification are provided in the following table. TIFF2026062683000016.tif253170TIFF2026062683000017.tif255170TIFF2026062683000018.tif255170TIFF2026062683000019.tif254170 TIFF2026062683000020.tif252170TIFF2026062683000021.tif255170TIFF2026062683000022.tif254170TIFF2026062683000023.tif177170

[0470] All patent and scientific document disclosures cited herein are explicitly incorporated in their entirety by reference.

[0471] The present invention will be further described here with reference to specific examples. It will be understood that various other embodiments can be carried out considering the general description provided above. [Examples]

[0472] Example 1: Explanation of Immunization Rabbit immunization A 1:1 mix of two enantiomers of Pb-DOTAM-alkyl-PEG4-KLH fractions (MS2-DOTAM KLH fraction 1 and MS2-DOTAM KLH fraction 2) was used to immunize New Zealand White rabbits or transgenic rabbits containing human immunoglobulin loci, as reported in WO2000 / 46251, WO2002 / 12437, WO2005 / 007696, WO2006 / 047367, US2007 / 0033661 and WO2008 / 027986. Each rabbit was immunized on day 0 by intradermal application with 500 ug of the immunogen mix emulsified with a complete Freund's adjuvant, and on days 7, 14, 28, and 56 by alternating intramuscular and subcutaneous application with 500 ug each. Subsequently, the rabbits received monthly subcutaneous immunization with 500 ug, and small blood samples were taken 7 days after immunization to determine serum titer. In the third and ninth months of immunization (5-7 days post-immunization), larger blood samples (10% of the estimated total blood volume) were taken, and peripheral mononuclear cells were isolated and used as a source of antigen-specific B cells in the B cell cloning process (Example 2).

[0473] Determination of serum titer (ELISA) Each of the two enantiomer Pb-DOTAM fractions (PJRD05.133F1 or PJRD05.133F2) was immobilized on a 96-well NUNC Maxisorp plate at 1 ug / ml, 100 ul / well in PBS, followed by: blocking of the plate with 2% Crotein C in PBS at 200 ul / well; application of serial dilutions of double-replicated antiserum in 0.5% Crotein C in PBS at 100 ul / well; and detection with HRP-conjugated donkey anti-rabbit IgG antibody (Jackson Immunoresearch / Dianova 711-036-152; 1 / 16000) and streptavidin-HRP, respectively, diluted in 0.5% Crotein C in PBS at 100 ul / well. For all steps, the plate was incubated at 37°C for 1 hour. Between all steps, the plate was washed three times with 0.05% Tween 20 in PBS. The signal was chromogenic by adding 100 μl / well of BM Blue POD substrate soluble form (Roche); and stopped by adding 100 μl / well of 1 M HCl. Absorbance was read at 450 nm compared to 690 nm as a reference. Titer was defined as the dilution of antiserum that yielded the maximum half signal.

[0474] Example 2: Cloning of B cells from rabbits Isolation of rabbit peripheral blood mononuclear cells (PBMCs) Blood samples were collected from immunized rabbits. Whole blood containing EDTA was diluted 2-fold with 1×PBS (PAA, Austria, Passing), and then centrifuged at high density using lympholyte mammal (Cedarlane Laboratories, Burlington, Ontario, Canada) according to the manufacturer's specifications. PBMCs were washed twice with 1×PBS.

[0475] EL-4 B5 medium RPMI 1640 (Pan Biotech, Germany, Eidenbach) supplemented with 10% FCS (Hyclone, Logan, Utah, USA), 2 mM glutamine, 1% penicillin / streptomycin solution (PAA, Passing, Austria), 2 mM sodium pyruvate, 10 mM HEPES (PAN Biotech, Eidenbach, Germany), and 0.05 mM β-mercaptoethanol (Gibco, Paisley, Scotland) was used.

[0476] Plate coating Sterile 6-well cell culture plates were coated overnight at 4°C with 2 μg / ml KLH in carbonate buffer (0.1 M sodium bicarbonate, 34 mM disodium hydrogencarbonate, pH 9.55). The plates were washed three times with sterile PBS before use. Sterile streptavidin-coated 6-well plates (Microcoat, Bernried, Germany) were coated at room temperature for 3 hours with a 1+1 enantiomer mixture of biotinylated TCMC-Pb-dPEC3-biotin isomers A (1 μg / ml) and B (1 μg / ml) in PBS. Prior to the panning step, these 6-well plates were washed three times with sterile PBS.

[0477] Macrophage / monocyte depletion PBMCs were seeded in sterile KLH-coated 6-well plates, and macrophages and monocytes were depleted by nonspecific adhesion, removing cells that bind to KLH. Each well was filled to capacity with 4 ml of medium and up to 6 × 10⁶ PBMCs derived from immunized rabbits, and bound for 1 hour at 37°C and 5% CO₂. Cells in the supernatant (peripheral blood lymphocytes (PBLs)) were used in the antigen panning step.

[0478] B cell enrichment in Pb-containing TCMC enantiomers Six-well plates coated with an enantiomer mixture of TCMC-Pb-dPEC3-biotin isomers A and B were seeded with up to 6 × 10 e6 PBLs per 4 ml of medium and bound at 37°C and 5% CO2 for 1 hour. Non-adherent cells were removed by carefully washing the wells 1 to 3 times with 1 × PBS. Remaining adherent cells were detached by trypsin at 37°C and 5% CO2 for 10 minutes. Trypsination was stopped with EL-4 B5 medium. Cells were kept on ice until immunofluorescence staining.

[0479] Immunofluorescence staining and flow cytometry Anti-IgG FITC (AbD Serotec, Düsseldorf, Germany) was used for single-cell sorting. For surface staining, cells from the depletion and enrichment steps were incubated with anti-IgG FITC antibody in PBS for 45 minutes in the dark at 4°C. After staining, PBMCs were washed twice with ice-cold PBS. Finally, PBMCs were resuspended in ice-cold PBS and immediately subjected to FACS analysis. Prior to FACS analysis, 5 μg / ml of propidium iodide (BD Pharmingen, San Diego, California, USA) was added to distinguish between dead and living cells.

[0480] Becton Dickinson FACSAria (BD Biosciences, USA), equipped with a computer and FACSDiva software, was used for single-cell sorting.

[0481] B cell culture Rabbit B cell cultures were prepared using the method described by Lightwood et al. (J Immunol Methods, 2006, 316:133~143). Briefly, a single selected rabbit B cell culture was incubated in a 96-well plate at 37°C for 7 days with 200 μl / well EL-4 B5 medium containing Pansorbin cells (1:100000) (Calbiochem (Merck), Darmstadt, Germany), 5% rabbit thymocyte supernatant (MicroCoat, Bernried, Germany), and gamma-irradiated mouse EL-4 B5 thymoma cells (5 × 10 e5 cells / well). The supernatant of the B cell culture was removed for screening, and the remaining cells were immediately collected and frozen at -80°C in 100 μl RLT buffer (Qiagen, Hilden, Germany).

[0482] Example 3: Expression of rabbit antibodies PCR amplification of the V-domain Total RNA was prepared from B cell lysates (resuspended in RLT buffer - Qiagen - Cat.N°79216) using the NucleoSpin 8 / 96 RNA Kit (Macherey & Nagel; 740709.4, 740698) according to the manufacturer's protocol. RNA was eluted in 60 μl of RNase-free water. Using 6 μl of RNA, cDNA was generated by reverse transcriptase reaction with Superscript III First-Strand Synthesis SuperMix (Invitrogen 18080-400) and oligo-dT primers according to the manufacturer's instructions. All steps were performed on a Hamilton ML Star System. Immunoglobulin heavy and light chain variable regions (VH and VL) were amplified using 4 μl of cDNA with AccuPrime Supermix (Invitrogen 12344-040) in a final volume of 50 μl, using primers rbHC.up and rbHC.do for the heavy chain and rbLC.up and rbLC.do for the light chain (Table 3). All forward primers were specific to the signal peptides (VH and VL, respectively), while the reverse primers were specific to the constant regions (VH and VL, respectively). PCR conditions for RbVH+RbVL were as follows: hot start at 94°C for 5 minutes; 35 cycles of 20 seconds at 94°C, 20 seconds at 70°C, 45 seconds at 68°C, and a final extension of 7 minutes at 68°C. TIFF2026062683000024.tif69170

[0483] 8 μl of the 50 μl PCR solution was loaded onto a 2% 48 E-gel (Invitrogen G8008-02). The positive PCR reaction was purified using the NucleoSpin Extract II kit (Macherey & Nagel; 740609250) according to the manufacturer's protocol and eluted into 50 μl of elution buffer. The entire purification step was performed on a Hamilton ML Starlet System.

[0484] Recombinant expression of rabbit monoclonal bivalent antibody For recombinant expression of rabbit monoclonal bivalent antibodies, PCR products encoding VH or VL were cloned as cDNA into expression vectors using the overhang cloning method (RS Haun et al., Biotechniques (1992) 13, 515-518; MZ Li et al., Nature Methods (2007) 4, 251-256). The expression vector contained an expression cassette consisting of a 5'CMV promoter containing intron A and a 3'BGH polyadenylated sequence. In addition to the expression cassette, the plasmid contained a pUC18-derived replication start site for plasmid amplification in Escherichia coli (E. coli) and a beta-lactamase gene conferring ampicillin resistance. Three variants of the base plasmid were used: one plasmid containing a rabbit IgG constant region designed to accept the VH region, and two additional plasmids containing rabbit or human kappa LC constant regions to accept the VL region. Linearized expression plasmids encoding the kappa or gamma constant region and VL / VH insert were amplified by PCR using duplicate primers. The purified PCR product was incubated with T4 DNA polymerase, thereby generating a single-stranded overhang. The reaction was stopped by adding dCTP. In the next step, the plasmid and insert were combined and incubated with recA to induce site-directed recombination. The recombinant plasmid was transformed into E. coli. The following day, grown colonies were harvested and tested for accurate recombinant plasmids by plasmid preparation, restriction analysis, and DNA sequencing. For antibody expression, isolated HC and LC plasmids were transiently co-transfected into 2 ml (96-well plate) FreeStyle HEK293-F cells (Invitrogen R790-07) using 239-Free transfection reagent (Novagen) according to the procedure suggested by the reagent supplier. The supernatant was collected after one week and delivered for purification.

[0485] Example 4: Selection of rabbit monoclonal antibodies The table below shows the characteristics of various monoclonal bivalent rabbit antibodies. PRIT-0128 was selected as a lead candidate due to its comparable binding to chelated Pb and Bi, reduced binding to other chelated metals, and high affinity (<100 pM).

[0486] The SET (Dissolution Equilibrium Titration) assay was performed as described below.

[0487] Assay plate preparation: A 384-well streptavidin plate (Nunc, Microcoat #11974998001) was incubated overnight at 4°C with 25 μl / well of DOTAM-biotin-isomer mix in PBS buffer at a concentration of 20 ng / ml.

[0488] Equilibrium of anti-DOTAM antibody samples with free DOTAM-metal chelates (Pb, Bi, Ca, Cu, Zn, Mg, Fe): 0.01 nM to 1 nM antibodies were titrated with the relevant DOTAM-metal chelates in 1:3, 1:2, or 1:1.7 dilution steps, starting with concentrations of 2500 nM, 500 nM, or 100 nM DOTAM-metal chelates. Samples were incubated overnight at 4°C in sealed REMP Storage polypropylene microplates (Brooks).

[0489] After overnight incubation, streptavidin plates were washed 3× with 90 μl of PBST per well. 15 μl of each sample was transferred from the equilibrium plate to the assay plate, incubated at RT for 15 minutes, followed by a 3×90 μl wash with PBST buffer. Detection was performed by adding 25 μl of goat anti-human IgG antibody-POD conjugate (Jackson, 109-036-088, 1:4000 in OSEP) followed by a 6×90 μl wash with PBST buffer. 25 μl of TMB substrate (Roche Diagnostics GmbH, catalog number: 11835033001) was added to each well. Measurements were performed at 370 / 492 nm using a Safire2 reader (Tecan).

[0490] material: 1. DOTAM-Biotin-Isomer Mix: The following mixture contains the following ingredients at a concentration of 20 ng / ml. - Pb-Dotam-Bn-biotin / TCMC-Pb-dPEG3-biotin, isomer A - Pb-Dotam-Bn-biotin / TCMC-Pb-dPEG3-biotin, isomer B - Pb-Dotam-alkyl-biotin isomer A - Pb-Dotam-alkyl-biotin isomer B 2. PBS: DPBS, PAN, P04-36500 3. BSA: Roche, 10735086001 4. Tween 20: Polysorbate 20 (USB, #20605, 500ml) 5.PBST:10×, Roche, #11666789001 / 0,1%Tween 20 6. OSEP:PBS (10x, Roche, #11666789001) / 0.5% BSA (Bovine serum albumin fraction V, fatty acid-free, Roche, #10735086001) / 0.05% Tween 20 TIFF2026062683000025.tif88170

[0491] Example 5: Molecular Biology Recombinant DNA techniques DNA was manipulated using standard methods as described in Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biological reagents were used according to the manufacturer's instructions.

[0492] Gene and oligonucleotide synthesis The desired gene segment was prepared by chemical synthesis at Geneart GmbH (Regensburg, Germany). The synthesized gene fragment was cloned into E. coli plasmids for propagation / amplification. The DNA sequences of the subcloned gene fragments were verified by DNA sequencing. Alternatively, short synthetic DNA fragments were assembled by annealing chemically synthesized oligonucleotides or by PCR. Each oligonucleotide was prepared by metabion GmbH (Planegg-Martinsried, Germany).

[0493] Protein determination The protein concentration of the purified polypeptide was determined by determining the optical density (OD) at 280 nm using the molar extinction coefficient calculated based on the polypeptide's amino acid sequence.

[0494] Plasmid generation for recombinant expression of antibody heavy or light chains Desired proteins were expressed by transient transfection of human embryonic kidney cells (HEK293). For the expression of the desired gene / protein (e.g., full-length antibody heavy chain, full-l...

Claims

1. An antibody comprising an antigen-binding site specific to DOTAM-lead (Pb) chelate, wherein the antigen-binding site comprises at least, a) Heavy chain CDR2 containing the amino acid sequence FIGSRRGDTYYASWAKG (SEQ ID NO: 2), or variants thereof having one, two, or up to three substitutions in SEQ ID NO: 2, wherein these substitutions do not include Phe50, Asp56, and Tyr58, and optionally also do not include Gly52 and / or Arg54; b) Heavy chain CDR3 containing the amino acid sequence ERDPYGGGAYPPHL (SEQ ID NO: 3), or a variant thereof having one, two, or up to three substitutions in SEQ ID NO: 3, wherein these substitutions do not include Glu95, Arg96, Asp97, Pro98, and optionally also Ala100C, Tyr100D, and / or Pro100E, and / or optionally also Tyr99; c) Light chain CDR1 containing the amino acid sequence QSSHSVYSDNDLA (SEQ ID NO: 4), or a variant thereof having one, two, or up to three substitutions in SEQ ID NO: 4, wherein these substitutions do not include Tyr28 and Asp32; d) A variant thereof, which has the amino acid sequence LGGYDDESDTYG (SEQ ID NO: 6) in its light chain CDR3, or SEQ ID NO: 6 having one, two, or up to three substitutions, wherein these substitutions do not include Gly91, Tyr92, Asp93, Thr95c, and Tyr96. Includes, Antibodies whose numbering system is based on Kabat technology.

2. The antibody according to claim 1, further comprising a heavy chain CDR1 and a light chain CDR2.

3. i) Heavy chain CDR1 containing the amino acid sequence GFSLSTYSMS (SEQ ID NO: 1) or variants thereof having up to 1, 2, or 3 substitutions, and optionally conserved substitutions, in SEQ ID NO: 1; and / or ii) A variant thereof having at least one, two, or three substitutions, and optionally a conservative substitution, in the light chain CDR2 containing the amino acid sequence QASKLAS (SEQ ID NO: 5) or SEQ ID NO:

5. The antibody according to claim 2, comprising:

4. The antigen-binding site is a) Heavy chain CDR1 containing the amino acid sequence GFSLSTYSMS (SEQ ID NO: 1) b) Heavy chain CDR2 containing the amino acid sequence FIGSRRGDTYYASWAKWG (SEQ ID NO: 2) c) Heavy chain CDR3 containing the amino acid sequence ERDPYGGGAYPPHL (SEQ ID NO: 3) d) Light chain CDR1 containing the amino acid sequence QSSHSVYSDNDLA (SEQ ID NO: 4) e) Light chain CDR2 containing amino acid sequence QASKLAS (SEQ ID NO: 5) f) Light chain CDR3 containing the amino acid sequence LGGYDDESDTYG (SEQ ID NO: 6) The antibody according to any one of claims 1 to 3, comprising at least one, two, three, four, five, or six CDRs selected from.

5. i) An antibody having a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 7 and a light chain variable domain containing the amino acid sequence of SEQ ID NO: 8; or i) An antibody having a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 9 and a light chain variable domain containing the amino acid sequence of SEQ ID NO: 10 The antibody according to any one of claims 1 to 4, which binds to the same epitope or overlapping epitopes of the Pb-DOTAM chelate.

6. The antibody according to any one of claims 1 to 5, which is human, chimeric, or humanized.

7. The antibody according to any one of claims 1 to 6, wherein the antigen-binding site comprises a heavy chain variable domain containing an amino acid sequence selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 9, or a variant thereof containing an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO: 7 or SEQ ID NO:

9.

8. The antibody according to any one of claims 1 to 7, wherein the antigen-binding site comprises a light chain variable domain containing an amino acid sequence selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 10, or a variant thereof containing an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO: 8 or SEQ ID NO:

10.

9. The antibody according to any one of claims 1 to 8, wherein the antigen-binding site comprises a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 7 and a light chain variable domain containing the amino acid sequence of SEQ ID NO:

8.

10. The antibody according to any one of claims 1 to 9, wherein the antigen-binding site comprises a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 9 and a light chain variable domain containing the amino acid sequence of SEQ ID NO:

10.

11. The antibody according to any one of claims 1 to 10, wherein the antigen-binding site binds to a Pb-DOTAM chelate with a Kd value of 100 pM, 50 pM, 20 pM, 10 pM, 5 pM, or 1 pM or less.

12. The antibody according to any one of claims 1 to 11, wherein the antigen-binding site is bound to a Pb-DOTAM chelate and a Bi-DOTAM chelate, and the ratio of the Kd values ​​of the Bi-DOTAM chelate to the Pb-DOTAM chelate is in the range of 0.1 to 10 or 1 to 10.

13. This refers to all antibodies, or Fv, Fab, Fab', Fab'-SH, F(ab'). ２ The antibody according to any one of claims 1 to 12, which is an antibody fragment selected from the group consisting of a diabody, a linear antibody, or a single-chain antibody molecule.

14. The antibody according to any one of claims 1 to 13, wherein the antibody is coupled to a portion that specifically binds to a target antigen.

15. The antibody according to claim 14, wherein the target antigen is a tumor-specific antigen.

16. A multispecific or bispecific antibody, optionally a multispecific or bispecific antibody according to any one of claims 17 to 51, as described in claim 14 or 15.

17. A multispecific or bispecific antibody comprising at least one antigen-binding site specific to Pb-DOTAM chelate and at least one antigen-binding site for a target antigen.

18. The multispecific or bispecific antibody according to claim 17, wherein the antigen-binding site specific to the Pb-DOTAM chelate is as defined in any one of claims 1 to 12.

19. The multispecific or bispecific antibody according to claim 17 or 18, wherein the target antigen is a tumor-specific antigen.

20. The multispecific or bispecific antibody according to claim 19, wherein the tumor-specific antigen is selected from the group consisting of CEA, HER2, and CD20.

21. The multispecific or bispecific antibody according to claim 20, wherein the tumor-specific antigen is carcinoembryonic antigen (CEA).

22. A multispecific or bispecific antibody comprises at least one antigen-binding site specific to Pb-DOTAM chelate and at least one antigen-binding site specific to CEA, wherein the CEA-specific antigen-binding site comprises a heavy chain containing at least one, two, or three heavy chain CDRs. d) The heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 11 e) The heavy chain CDR2 contains the amino acid sequence of SEQ ID NO: 12 f) The heavy chain CDR3 contains the amino acid sequence of SEQ ID NO: 13; and / or the antigen-binding site specific to the CEA includes at least one, two, or three light chain CDRs, a) Light chain CDR1 contains the amino acid sequence of SEQ ID NO: 14; b) Light chain CDR2 contains the amino acid sequence of SEQ ID NO: 15; c) The light chain CDR3 contains the amino acid sequence of SEQ ID NO: 16, A multispecific or bispecific antibody according to any one of claims 17 to 21.

23. The antigen-binding site for CEA is a) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 11; b) Heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 12; c) Heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 13; d) Light chain CDR1 containing the amino acid sequence of SEQ ID NO: 14; e) Light chain CDR2 containing the amino acid sequence of SEQ ID NO: 15; f) Light chain CDR3 containing the amino acid sequence of SEQ ID NO: 16 The multispecific or bispecific antibody according to claim 22, comprising at least one, two, three, four, five, or six (i.e., all) CDRs selected from the above.

24. A multispecific or bispecific antibody comprises at least one antigen-binding site specific to Pb-DOTAM chelate and at least one antigen-binding site specific to CEA, wherein the CEA-specific antigen-binding site is i) Heavy chain variable domains containing the amino acid sequence of SEQ ID NO: 17, or variants thereof containing amino acid sequences having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO: 17; and / or ii) A light chain variable domain containing the amino acid sequence of SEQ ID NO: 18, or a variant thereof containing an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:

18. A multispecific or bispecific antibody according to any one of claims 17 to 23, comprising:

25. The multispecific or bispecific antibody according to claim 24, wherein the antigen-binding site specific to CEA comprises a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 17 and / or a light chain variable domain containing the amino acid sequence of SEQ ID NO:

18.

26. The multispecific or bispecific antibody according to any one of claims 17 to 20, comprising at least one antigen-binding site specific to Pb-DOTAM chelate and at least one antigen-binding site specific to ERBB2, wherein the ERBB2-specific antigen-binding site comprises at least one, two, three, four, five, or six CDRs selected from (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 28; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 30; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 31; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 32; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:

33.

27. A multispecific or bispecific antibody comprises at least one antigen-binding site specific to Pb-DOTAM chelate and at least one antigen-binding site specific to ERBB2, wherein the antigen-binding site specific to ERBB2 is i) Heavy chain variable domains containing the amino acid sequence of SEQ ID NO: 34, or variants thereof containing amino acid sequences having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO: 34; and / or ii) A light chain variable domain containing the amino acid sequence of SEQ ID NO: 35, or a variant thereof containing an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:

35. A multispecific or bispecific antibody according to any one of claims 17 to 20 or 26, comprising:

28. The multispecific or bispecific antibody according to claim 27, wherein the antigen-binding site specific to ERBB2 comprises a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 34 and / or a light chain variable domain containing the amino acid sequence of SEQ ID NO:

35.

29. The multispecific or bispecific antibody according to any one of claims 17 to 20, wherein the multispecific or bispecific antibody comprises at least one antigen-binding site specific to Pb-DOTAM chelate and at least one antigen-binding site specific to CD20, and the CD20-specific antigen-binding site comprises at least one, two, three, four, five, or six CDRs selected from (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 39; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 40; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 41; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 42; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 43; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:

44.

30. A multispecific or bispecific antibody comprises at least one antigen-binding site specific to Pb-DOTAM chelate and at least one antigen-binding site specific to CD20, wherein the CD20-specific antigen-binding site is i) Heavy chain variable domains containing the amino acid sequence of SEQ ID NO: 45, or variants thereof containing amino acid sequences having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO: 45; and / or ii) A light chain variable domain containing the amino acid sequence of SEQ ID NO: 46, or a variant thereof containing an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:

46. A multispecific or bispecific antibody according to any one of claims 17 to 20 or 29, comprising:

31. The multispecific or bispecific antibody according to paragraph 30, wherein the antigen-binding site specific to CD20 comprises a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 45 and / or a light chain variable domain containing the amino acid sequence of SEQ ID NO:

46.

32. A multispecific or bispecific antibody according to any one of claims 17 to 31, comprising an Fc region.

33. The multispecific or bispecific antibody according to claim 32, wherein the Fc region is manipulated to reduce effector function.

34. An Fc region comprising substitution of one or more Fc region residues 234, 235, 238, 265, 269, 270, 297, 327 and / or 329, the multispecific or bispecific antibody according to claim 33.

35. The antibody comprises a full-length antibody containing first and second antibody heavy chains and first and second antibody light chains, wherein the first heavy chain and the first light chain assemble to form an antigen-binding site for a first antigen, and the second heavy chain and the second light chain assemble to form an antigen-binding site for a second antigen. A multispecific or bispecific antibody according to any one of claims 32 to 34, wherein either the first or second antigen is a Pb-DOTAM chelate and the other is a target antigen.

36. The multispecific or bispecific antibody according to claim 35, further comprising a further antigen-binding moiety to a first antigen.

37. A full-length antibody comprising first and second antibody heavy chains and first and second antibody light chains, wherein the first heavy chain and the first light chain assemble to form a Fab containing an antigen-binding site for a first antigen, and the second heavy chain and the second light chain assemble to form a cross-Fab containing an antigen-binding site for a second antigen; The multispecific or bispecific antibody according to claim 36, wherein either a first or second antibody heavy chain is fused via a linker to a polypeptide comprising CH1 and VH domains, and the first polypeptide assembles with the second polypeptide comprising CL and VL such that the first and second polypeptides assemble to form a Fab comprising an antigen-binding site for a first antigen.

38. The multispecific or bispecific antibody according to claim 37, wherein the N-terminus of a second antibody heavy chain is fused to the first polypeptide via a linker.

39. i) A full-length antibody containing an antigen-binding site for the first antigen; and ii) comprising at least a second heavy chain variable domain and a second light chain variable domain that together form an antigen-binding site for a second antigen, A multispecific or bispecific antibody according to any one of claims 32 to 34, wherein either the first or second antigen is a Pb-DOTAM chelate and the other is a target antigen.

40. The multispecific or bispecific antibody according to claim 39, comprising a full-length antibody having an antigen-binding site for a first antigen, wherein one N or C terminus of the heavy chain is linked to the first polypeptide via a polypeptide linker, and the first polypeptide associates with a second polypeptide to form a Fab or cross-Fab having a binding site for a second antigen.

41. i) A first polypeptide consisting of a VH domain and a CH1 domain, associated with a second polypeptide consisting of VL and CL domains; or ii) A first polypeptide consisting of a VL domain and a CH1 domain, associated with a second polypeptide consisting of VH and CL domains; or iii) comprising a first polypeptide consisting of a VH domain and a CL domain, which is associated with a second polypeptide consisting of a VL and a CH1 domain; The multispecific or bispecific antibody according to claim 40, wherein the first and second polypeptides together form an antigen-binding site for the second antigen.

42. The multispecific or bispecific antibody according to claim 41, comprising a full-length antibody containing an antigen-binding site for a first antigen, wherein one C-terminus of the heavy chain is linked to a first polypeptide consisting of a VL domain and a CH1 domain, and the second polypeptide consisting of a VH and CL domain is associated with the second polypeptide through a polypeptide linker.

43. a) A full-length antibody that specifically binds to the first antigen and consists of two antibody heavy chains and two antibody light chains; b) i) Antibody heavy chain variable domain (VH); or ii) Antibody heavy chain variable domain (VH) and antibody constant domain (CH1); or iii) Variable domain (VH) and constant domain (CL) of the antibody light chain A polypeptide consisting of, A polypeptide in which the N-terminus of the VH domain is fused to the C-terminus of one of the two heavy chains of the full-length antibody via a peptide linker; c) i) Antibody light chain variable domain (VL); or ii) Variable domain (VL) and constant domain (CL) of the antibody light chain; or iii) Variable domain (VL) and constant domain (CH1) of the antibody light chain A polypeptide consisting of, A polypeptide in which the N-terminus of the VL domain is fused to the other C-terminus of the two heavy chains of the full-length antibody via a peptide linker. Including; The antibody heavy chain variable domain of the peptide under (b) and the antibody light chain variable domain of the peptide under (c) together form an antigen-binding site for the second antigen. The multispecific or bispecific antibody according to claim 39, wherein either the first or second antigen is a Pb-DOTAM chelate and the other is a target antigen.

44. A multispecific or bispecific antibody according to any one of claims 35 to 43, wherein the first antigen is a target antigen and the second antigen is a Pb-DOTAM chelate.

45. The multispecific or bispecific antibody according to claim 44, wherein the first antigen is CEA.

46. a) A full-length antibody that specifically binds to CEA and consists of two antibody heavy chains and two antibody light chains, The heavy chain has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with the heavy chain of amino acids 1 to 450 of SEQ ID NO: 22 or 23; A full-length antibody whose light chain has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with the light chain of SEQ ID NO: 21; b) i) an antibody heavy chain variable domain (VH) having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the heavy chain variable domain of Sequence ID No. 7; or ii) The antibody heavy chain variable domain (VH) and antibody heavy chain constant domain (CH1) having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with respect to the heavy chain variable domain of Sequence ID No. 7; and iii) The antibody heavy chain variable domain (VH) and antibody light chain constant domain (CL) having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with respect to the heavy chain variable domain of Sequence ID No.

7. A polypeptide consisting of, A polypeptide in which the N-terminus of the VH domain is fused to the C-terminus of one of the two heavy chains of the full-length antibody via a peptide linker; and c) i) an antibody light chain variable domain (VL) having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the light chain variable domain of Sequence ID No. 8; or ii) The antibody light chain variable domain (VL) and antibody light chain constant domain (CL) having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the light chain variable domain of Sequence ID No. 8; or iii) The antibody light chain variable domain (VL) and antibody heavy chain constant domain (CH1) having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with respect to the light chain variable domain of Sequence ID No.

8. A polypeptide consisting of, The polypeptide comprises a VL domain whose N-terminus is fused to the other C-terminus of the two heavy chains of the full-length antibody via a peptide linker; The multispecific or bispecific antibody according to claim 45, wherein the antibody heavy chain variable domain of the peptide under (b) and the antibody light chain variable domain of the peptide under (c) together form an antigen-binding site for Pb-DOTAM chelate.

47. a) A full-length antibody that specifically binds to CEA and consists of two antibody heavy chains and two antibody light chains, The heavy chain has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with the heavy chain of amino acids 1 to 450 of SEQ ID NO: 19 or 20; A full-length antibody whose light chain has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with the light chain of SEQ ID NO: 21; b) i) an antibody heavy chain variable domain (VH) having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the heavy chain variable domain of Sequence ID No. 9; or ii) The antibody heavy chain variable domain (VH) and antibody heavy chain constant domain (CH1) having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the heavy chain variable domain of Sequence ID No. 9; or iii) The antibody heavy chain variable domain (VH) and antibody light chain constant domain (CL) having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with respect to the heavy chain variable domain of Sequence ID No.

9. A polypeptide consisting of, A polypeptide in which the N-terminus of the VH domain is fused to the C-terminus of one of the two heavy chains of the full-length antibody via a peptide linker; c) i) an antibody light chain variable domain (VL) having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the light chain variable domain of Sequence ID No. 10; or ii) The antibody light chain variable domain (VL) and antibody light chain constant domain (CL) having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with the light chain variable domain of Sequence ID No. 10; or iii) The antibody light chain variable domain (VL) and antibody heavy chain constant domain (CH1) having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with respect to the light chain variable domain of Sequence ID No.

10. A polypeptide consisting of, A polypeptide in which the N-terminus of the VL domain is fused to the other C-terminus of the two heavy chains of the full-length antibody via a peptide linker. Including; The multispecific or bispecific antibody according to claim 45, wherein the antibody heavy chain variable domain of the peptide under (b) and the antibody light chain variable domain of the peptide under (c) together form an antigen-binding site for Pb-DOTAM chelate.

48. i) A first heavy chain having an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the heavy chain of Sequence ID No. 22, ii) A second heavy chain having an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the heavy chain of Sequence ID No. 23, and iii) Two antibody light chains having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the light chain of Sequence ID No.

21. A multispecific or bispecific antibody according to claim 45, comprising:

49. i) A first heavy chain having the amino acid sequence of SEQ ID NO: 22; ii) A second heavy chain having the amino acid sequence of SEQ ID NO: 23; and iii) Two antibody light chains having the amino acid sequence of SEQ ID NO: 21 A multispecific or bispecific antibody according to claim 48, comprising:

50. i) A first heavy chain having an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the heavy chain of Sequence ID No. 19, ii) A second heavy chain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with the heavy chain of Sequence ID No. 20, iii) Two antibody light chains having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the light chain of Sequence ID No.

21. A multispecific or bispecific antibody according to claim 45, comprising:

51. i) A first heavy chain having the amino acid sequence of SEQ ID NO: 19; ii) A second heavy chain having the amino acid sequence of SEQ ID NO: 20; and iii) Two antibody light chains having the amino acid sequence of SEQ ID NO: 21 A multispecific or bispecific antibody according to claim 50, comprising:

52. An isolated polynucleotide or set of isolated polynucleotides encoding an antibody according to any one of claims 1 to 51.

53. A vector comprising a polynucleotide or a set of polynucleotides as described in claim 52.

54. A kit or composition comprising a set of vectors, comprising together the set of polynucleotides described in claim 52.

55. An expression vector, a set of the vectors described in claim 53 or claim 54.

56. A prokaryotic or eukaryotic host cell comprising an isolated polynucleotide or a set of isolated polynucleotides according to claim 52, which may optionally include the vector according to claim 53 or a set of vectors according to claim 54.

57. A method for producing an antibody according to any one of claims 1 to 51, comprising expressing an antibody from a host cell according to claim 56.

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