Antibodies that bind to cancer cells and target those cells with radioactive nucleotides.

Abstract

We provide peptide linkers and multi-domain proteins. [Solution] A peptide linker is provided, comprising y consecutive residues selected from the group consisting of Gly and Ser, wherein y = 5-100, 5-70, 5-60, 5-50 or 10-100, 10-70, 10-60 or 10-50, and the last serine is at position y-2 or y-3. The use of the peptide linker for joining the first and second domains of a multidomain protein is also provided. A multidomain protein linked by the peptide linker is also provided, preferably the multidomain protein is a bispecific antibody.

Description

[Technical Field]

[0001] Field of Invention The present invention relates to an antibody that binds to an antigen on a target cell and targets a radionuclide to that cell, and to a method of using the same.

[0002] The present invention also relates to a linker for connecting two domains of a multi-domain protein. [Background technology]

[0003] background Selective destruction of individual cells or specific cell types is often desirable in a variety of clinical situations. For example, a major goal of cancer treatment is to specifically destroy tumor cells while leaving healthy cells and tissues intact and undamaged.

[0004] In this regard, bispecific antibodies have been designed that bind to surface antigens on target cells with one "arm" and to the effector portion of drugs, etc., with a second "arm." A wide variety of bispecific formats have been developed, but the challenge of developing bispecific antibodies is by no means trivial.

[0005] Pre-targeted radioimmunotherapy (PRIT) uses antibody constructs that have affinity for tumor-associated antigens on the one hand and for radiolabeled compounds on the other. In the first step, the antibody is administered and localized to the tumor. Subsequently, the radiolabeled compound is administered. Because the radiolabeled compound is small, it can be rapidly delivered to the tumor, and the unbound compound is rapidly removed, reducing radiation exposure outside the tumor (Goldenberg et al Theranostics 2012, 2(5), 523-540). A similar procedure can be used for imaging. Pretargeting can use bispecific antibodies or systems using avidin-biotin, although the latter has the disadvantage that avidin / streptavidin is immunogenic.

[0006] Pre-targeted radioimmunotherapy or imaging methods generally utilize scavenging or blocking agents administered between the antibody administration step and the radiolabeled compound administration step. The purpose is to remove antibodies from the blood and / or block the binding sites of circulating antibodies to the radiolabeled compound (see, e.g., Karacay et al, Bioconj. Chem., 13(5), 1054-1070 (2002)). The use of scavenging or blocking agents allows for the administration of sufficient levels of radioactivity for efficient treatment while limiting harmful toxicity, but the timing and dosage must be carefully selected, as scavenging agents may introduce the risk of adverse effects such as immune responses. Therefore, the use of a scavenging phase is a complex aspect of pre-targeting methods. [Overview of the Initiative]

[0007] overview The present invention provides a set of antibodies useful in pretargeting methods, and a method for using them.

[0008] In one embodiment, the present invention is a set of antibodies, i) The antigen-binding site contains an antigen-binding portion that binds to an antigen expressed on the surface of a target cell, and the V portion of the antigen-binding site to the radiolabeled compound H It further includes domains, but the V of the antigen-binding site for the radiolabeled compound L The first antibody, which does not contain the domain, ii) The antigen-binding site contains an antigen-binding portion that binds to an antigen expressed on the surface of a target cell, and the V of the antigen-binding site to the radiolabeled compound L It further includes domains, but the V of the antigen-binding site for the radiolabeled compound H A second antibody that does not contain the domain and Includes, The V of the first antibody H Domain and the V of the second antibody L The present invention provides a set of antibodies in which the domains together can form a functional antigen-binding site for a radiolabeled compound.

[0009] In certain embodiments, the first antibody and the second antibody each contain an Fc domain. In some embodiments, each of the first antibody and the second antibody may contain a) an Fc domain, b) at least one antigen-binding moiety including a binding site for a target antigen, and c) a polypeptide containing either (but not both) a VL domain or a VH domain of the antigen-binding site for a radiolabeled compound, wherein the C-terminus of the antigen-binding moiety of (b) is fused to the N-terminus of one subunit of the Fc domain, and the C-terminus of the polypeptide of (c) is fused to the N-terminus of the other subunit of the Fc domain.

[0010] Therefore, the first antibody is, a) An Fc domain including a first subunit and a second subunit, b) An antigen-binding site including a binding site for the target antigen, c) A polypeptide comprising or consisting of an antibody heavy chain variable domain (VH) at the antigen-binding site for a radiolabeled compound. Includes or consists of The antigen-binding portion of (b) is fused to the N-terminus of the first subunit of the Fc domain of (a), and the polypeptide of (c) is fused to the N-terminus of the second subunit of the Fc domain of (a) by its C-terminus. The first antibody does not contain the VL domain of the antigen-binding site for the radiolabeled compound.

[0011] The second antibody is, a) An Fc domain including a first subunit and a second subunit, b) An antigen-binding site including a binding site for the target antigen, c) A polypeptide comprising or consisting of an antibody light chain variable domain (VL) at the antigen-binding site for a radiolabeled compound. Includes or consists of The antigen-binding portion of (b) is fused to the N-terminus of the first subunit of the Fc domain of (a), and the polypeptide of (c) is fused to the N-terminus of the second subunit of the Fc domain of (a) by its C-terminus. The second antibody does not contain the VH domain of the antigen-binding site for the radiolabeled compound.

[0012] Neither the first antibody nor the second antibody, by itself, contains a functional antigen-binding site for the radiolabeled compound. The first antibody has only the V H domain from the functional binding site for the radiolabeled compound and does not have the V L domain. The second antibody has only the V L domain and not the V H domain.

[0013] When the V H and V L domains of the first antibody and the second antibody associate, a functional antigen-binding site for the radiolabeled compound is formed. This can occur, for example, when the first antibody and the second antibody are bound to the same individual target cell or adjacent cells.

[0014] The first antibody and the second antibody described herein may be referred to herein as "single domain split antibodies", "SPLIT", "split antibodies", "half bodies" or "demi bodies". The V H and V L domains that together form an antigen-binding site capable of binding the radiolabeled compound are split between the two antibodies and do not exist as part of the same antibody.

[0015] The split domain format means that the radiolabeled compound cannot bind to the first antibody alone or to the second antibody alone. In blood, there is little or no stable association between the first antibody and the second antibody, and little or no stable binding of the radiolabeled compound.

[0016] Antigens expressed on the surface of target cells may be referred to herein as “target antigens,” “target cell antigens,” or “TA.” According to the present invention, the first and second antibodies may have binding sites for different target antigens or for the same target antigen. (To avoid misunderstanding, when it is stated that antibodies bind to the same target antigen, this means that they have binding sites that can bind to the same target antigen, and includes the possibility that antibodies may bind to two individual antigen molecules that are the same as each other). For example, in one embodiment, both the first and second antibodies bind to CEA.

[0017] In some embodiments, the first antibody and the second antibody may bind to the same epitope of the same target antigen (may have binding sites). In other embodiments, the first antibody may bind to a different epitope of the target antigen than the second antibody (may have binding sites).

[0018] In some embodiments, the first antibody and the second antibody may contain the same antigen-binding site for the target antigen. That is, they may have the same antigen-binding site. L Array and V H V may include an antigen-binding site that can bind to a target antigen, and which forms this antigen-binding site. L Array and V H The sequences are the same in the first antibody and the second antibody.

[0019] In some embodiments, each of the first and second antibodies is monovalent with respect to the target antigen. In other embodiments, each of the first and second antibodies is bivalent with respect to the target antigen. In some embodiments, they are each bivalent and monospecific with respect to the epitope. In other embodiments, each of the first and second antibodies is bispecific with respect to the target antigen, i.e., each of the first and second antibodies has binding sites to two different epitopes of the target antigen.

[0020] The presence of the Fc region offers advantages 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 may be observed with smaller fragments. The “split domain” format described herein may be particularly advantageous in this context because it mitigates the increased likelihood of association with radiolabeled compounds that would otherwise occur due to the prolonged presence of circulating antibodies.

[0021] In some embodiments, the Fc domain is modified to reduce or eliminate the effector function.

[0022] In another embodiment, the present invention provides a pharmaceutical composition comprising a set of antibodies described herein. In yet another embodiment, the present invention provides a kit comprising two distinct pharmaceutical compositions, each comprising one of the antibodies described herein (i.e., a first antibody and a second antibody, respectively).

[0023] In a further aspect, the present invention relates to a polynucleotide or set of polynucleotides encoding any of the antibodies or sets of antibodies described herein. In another aspect, the present invention relates to a vector or set of vectors comprising the polynucleotide(s) described herein, optionally an expression vector or set of expression vectors. For a further object, the present invention relates to a prokaryotic or eukaryotic host cell or set of host cells comprising the vector(s) of the present invention. Furthermore, a method for producing antibodies is provided, comprising culturing the host cell(s) to produce antibodies.

[0024] In some embodiments, the antibodies described herein are used in pre-targeted radioimmunotherapy (PRIT) or pre-targeted radioimaging methods.

[0025] In one embodiment, the present invention is a method for pre-targeted radioimmunotherapy, i) Administering the first antibody and the second antibody described above to a control, ii) Next, administer the radiolabeled compound to the subject. This provides a method that includes [something].

[0026] In another aspect, the present invention provides the above-described first antibody and second antibody for use in a method of treatment comprising administering the first antibody and second antibody to a subject, followed by administering a radiolabeled compound to the subject. In another aspect, the present invention provides the above-described first antibody for use in a method of treatment comprising administering the first antibody and second antibody to a subject, followed by administering a radiolabeled compound to the subject. In another aspect, the present invention provides the above-described second antibody for use in a method of treatment comprising administering the first antibody and second antibody to a subject, followed by administering a radiolabeled compound to the subject.

[0027] In another embodiment, the present invention is i) Administering the first antibody and the second antibody described herein, wherein the antibodies bind to the target antigen and are localized on the surface of cells expressing the target antigen. ii) Next, administer a radiolabeled compound, optionally, iii) Imaging tissues or organs where radionuclides are localized. The present invention provides a radiation imaging method that includes [a specific component].

[0028] In another aspect, the present invention provides a first antibody and a second antibody described herein for use in a diagnostic method performed on the body of a human or animal, the method being i) Administering the first antibody and the second antibody described herein, wherein the antibodies bind to the target antigen and are localized on the surface of cells expressing the target antigen. ii) Next, administer a radiolabeled compound, optionally, iii) Imaging tissues or organs where radionuclides are localized. Includes.

[0029] The imaging step may be followed by a step of forming a diagnosis and optionally a step of delivering that diagnosis to the target. In some embodiments, the method may further include determining an appropriate treatment and optionally administering that treatment to the target.

[0030] In each of the above methods / uses, the binding of the first and second antibodies to the same or adjacent target cells is due to the V of the antigen-binding site to the radiolabeled compound. H and V L This leads to the association of domains and the formation of a functional antigen-binding site for the radiolabeled compound. Therefore, after administration of the radiolabeled compound, the radiolabeled compound becomes V H and V L It binds to a functional antigen-binding site formed by the association of these molecules.

[0031] In any of the methods and uses described herein, the first antibody and the second antibody may be administered simultaneously or sequentially in any order.

[0032] In the art, PRIT or radiographic imaging methods often include a removal step. The removal step includes administering a drug between the administration of the antibody and the administration of the radiolabeled compound, the drug increasing the rate of antibody removal from the blood and / or blocking the binding of the radiolabeled compound to the antibody.

[0033] In one embodiment of the method and use described herein, the method does not include a removal step; that is, it does not include a step of administering a removal agent or blocking agent between the administration of the first and second antibodies and the administration of the radiolabeled compound (i.e., after the administration of the antibodies but before the administration of the radiolabeled compound). In another embodiment, optionally, no agents other than radiosensitizers, immunotherapeutic agents and / or chemotherapeutic agents are administered between the administration of the first and second antibodies and the administration of the radiolabeled compound. In yet another embodiment, no agents are administered between the administration of the first and second antibodies and the administration of the radiolabeled compound.

[0034] 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, immunotherapeutic agents and / or chemotherapeutic agents, and the radiosensitizers, immunotherapeutic agents or chemotherapeutic agents and antibodies may be administered simultaneously or sequentially in any order.

[0035] The methods of radioimaging and radioimmunotherapy described herein can be combined as desired, as will be discussed further herein.

[0036] In a further embodiment, the present invention is i) The first antibody and the second antibody described herein, ii) A radiolabeled compound that binds to the antigen-binding site formed by the association of the first antibody and the second antibody, We provide a kit that includes this.

[0037] The kit may optionally exclude (i.e., not contain) any removal or blocking agents as described herein.

[0038] Optionally, the kit may further contain a radiosensitizer, an immunotherapy agent, or a chemotherapeutic agent.

[0039] In some embodiments, the first antibody and the second antibody may be present in the same pharmaceutical composition. In other embodiments, the first antibody and the second antibody may be present in separate pharmaceutical compositions. In some embodiments, the radiolabeled compound may be present in a pharmaceutical composition separate from the antibody.

[0040] In another embodiment, the present invention relates to a novel peptide linker. The inventors have determined that in a peptide linker consisting of y amino acids, the Ser at the y position (i.e., the Ser as the last / C-terminal amino acid of the linker) can induce glycosylation of the y+2 amino acid (i.e., an amino acid located two residues from the last amino acid of the linker toward the C-terminus) depending on the properties of this y+2 amino acid. Therefore, it may be preferable that the last serine residue of the linker is located at the y-2 or y-3 position (i.e., the last serine residue of the linker is located at an amino acid two or three positions from the last amino acid of the linker toward the N-terminus). In some embodiments, the linker may consist of y consecutive amino acid residues selected from the group consisting of Gly and Ser, for example, y = 5 or more, for example y = 5 to 100, 5 to 70, 5 to 60 or 5 to 50; or y = 10 to 100; 10 to 70, 10 to 60 or 10 to 50; for example 15 to 31 or 15 to 30; for example y = 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25; the last serine is at position y-2 or y-3. (Therefore, serine may be present at position y-2 and glycine at positions y-1 and y; or serine may be present at position y-3 and glycine at positions y-2, y-1 and y). In some embodiments, y = 20 or 21 may be preferred. In some embodiments, the linker is (GxS)n(GGSGG) or (GxS)n(GGSGGG), where G=glycine, S=serine, x=4, and n=1-20, or 2-20, or 1-10, or 2-10, for example n=2, 3, 4, or 5, 6, 7, 8, or 9, for example n=2-5, or 2-4. For example, the linker may be GGGGSGGGGSGGGGSGGSGG (Sequence ID 150) or GGGGSGGGGSGGGGSGGSGGG (Sequence ID 151).

[0041] In another aspect of the present invention, the invention relates to the use of a peptide linker for joining two domains of a multidomain protein. In another aspect, the invention relates to a multidomain protein comprising at least a first domain and a second domain, wherein the first and second domains are linked by the linker. In another aspect, the invention relates to a method for linking two domains in a multidomain protein, comprising linking the domains via a linker. In some embodiments, the method may include expressing a multidomain protein from nucleic acids encoding a first domain, a linker, and a second domain, wherein the linker connects the first and second domains.

[0042] In some embodiments, the first and third domains may be independently selected from i) the Fc domain of the antibody; ii) the antigen-binding portion; and iii) the VL or VH domain (which can be seen, for example, in a split antibody or hemibody).

[0043] In some embodiments, the antigen-binding portion may be an antibody fragment such as Fv, Fab, cross-Fab, Fab', Fab'-SH, F(ab')2; a diabody; a linear antibody; a single-chain antibody molecule (e.g., scFv or scFab); or a single-domain antibody (dAb) such as VHH; or a non-antibody-binding skeleton such as DARPin (designed ankyrin repeat protein); an affibody; Sso7d; a monobody; or an antikalin.

[0044] In some embodiments, the multidomain protein may be a multispecific antibody, such as a bispecific antibody. The first and second domains may each be antibody fragments capable of binding to an antigen. In another embodiment, a linker may be used to ligate the antibody fragments to the Fc domain in the multispecific antibody.

[0045] In some embodiments, a multidomain protein may include a first domain which is an antigen-binding portion (such as an antibody fragment that can bind to an antigen) and a second domain which is either a VH domain or a VL domain. In some embodiments, a linker may be used to connect the antigen-binding portion (e.g., an antibody fragment) to the VH domain or VL domain. For example, in some embodiments, the C-terminus of the antibody fragment (or, for example, in the case of Fab, one of the chains of the antibody fragment containing two or more chains) is connected to the N-terminus of the VH domain or VL domain. In some embodiments, the multidomain protein may further include an Fc domain which includes a first subunit and a second subunit, and a linker may be used to connect the first or second subunit of the Fc domain to the VH domain or VL domain. In one embodiment, the C-terminus of the first or second subunit of the Fc domain is connected to the N-terminus of the VH domain or VL domain. In another embodiment, the C-terminus of the VH domain or VL domain is connected to the N-terminus of the first or second subunit of the Fc domain.

[0046] In some embodiments, the multidomain protein may be any of the antibodies described herein. [Brief explanation of the drawing]

[0047] [Figure 1] Figure 1 shows the schematic structures of the target antigen (TA)-DOTAM bispecific antibody (TA-DOTAM BsAb) and the exemplary TA-split-DOTAM-VH / VL antibody belonging to the comparative example. [Figure 2] Figure 2 is a schematic diagram showing the assembly of the split VH / VL DOTAM conjugate on tumor cells. The TA-split-DOTAM-VH / VL antibody does not significantly bind to 212Pb-DOTAM unless the two domains of the DOTAM conjugate bind to the tumor antigen (TA) on the targeted cell where they are assembled. [Figure 3] Figure 3 shows a schematic diagram of an example of a three-stage TA-PRIT concept, including the use of a removal agent. [Figure 4] Figure 4 shows a schematic diagram of an example of a two-stage TA-PRIT concept in which no removal agent is used. [Figure 5] Figure 5 shows the binding of the split antibody to MKN45 cells to demonstrate CEA binding ability. Antibody detection is performed using a human IgG-specific secondary antibody. [Figure 6] Figure 6 shows the binding of the split antibody to MKN45 cells to demonstrate DOTAM binding ability. Antibody detection is performed using Pb-DOTAM-FITC. [Figure 7A] Figure 7A shows an exemplary protocol for a two-step PRIT using CEA-split-DOTAM-VH / VL performed in SCID mice with SC BxPC3 tumors (h = hours, d = days, w = weeks). [Figure 7B] Figure 7B shows an exemplary three-step PRIT control protocol performed in SCID mice with SC BxPC3 tumors (h = hours, d = days, w = weeks). [Figure 8] Figure 8 shows the in vivo distribution of pre-targeted 212Pb-DOTAM 6 hours after injection, or using a standard 3-step PRIT, in SCID mice with SC BxPC3 tumors, using either CEA-split-DOTAM-VH alone, CEA-split-DOTAM-VL alone, or a combination of two complementary antibodies (%ID / g±SD, n=4). [Figure 9] Figure 9 shows the pharmacokinetics of CEA-split-DOTAM-VH / VL after IV injection in SCID mice. [Figure 10] Figure 10 shows the experimental design of Protocol 158, which includes CEA-PRIT in two steps (top) or three steps (bottom) in SCID mice with SC BxPC3 tumors. *Dose of CEA-split DOTAM BsAb adjusted to compensate for hole / hole impurities in the 2 / 4 construct. [Figure 11]Figure 11 shows the in vivo distribution of pre-targeted 212Pb-DOTAM in SCID mice (6-hour pi) with SC BxPC3 tumors. Distribution of 212Pb in tumor-bearing SCID mice 6 hours after injection of 212Pb-DOTAM pre-targeted by biparatopic combinations of CEA-DOTAM BsAb or CEA-split-DOTAM antibodies. Radioactivity content in organs and tissues is expressed as mean %ID / g ± SD (n=4). [Figure 12] Figure 12 shows the experimental schedule for Protocol 160 in SCID mice with SC BxPC3 tumors, including one cycle of 3-stage CEA-PRIT (top), 2-stage CEA-PRIT (middle), or 1-stage CEA-RIT. In vivo distribution (BD) scouts were euthanized 24 hours after radioactive injection, but the efficacy group mice were maintained and carefully monitored until they reached the termination criteria. [Figure 13] Figure 13 shows the in vivo distribution of pre-targeted 212Pb-DOTAM and 212Pb-DOTAM-CEA-DOTAM in SCID mice (24-hour pi) with SC BxPC3 tumors. The distribution of 212Pb in tumor-bearing SCID mice 24 hours after injection of CEA-DOTAM-pre-targeted 212Pb-DOTAM or pre-incubated 212Pb-DOTAM-CEA-DOTAM. Radioactivity content in organs and tissues is expressed as mean %ID / g ± SD (n=3). [Figure 14] Figure 14 shows the mean tumor growth with standard errors for the PRIT treatment group and control groups (A-E) in the BxPC3 model (n=10). Curves were truncated for n<5. The dotted vertical lines indicate 212Pb-DOTAM administration (20 μCi) to some or all groups, as per the study design. [Figure 15] Figure 15 shows the individual tumor growth curves for the PRIT-treated group and the control group (groups A-E) in the BxPC3 model (n=10). The dotted vertical line indicates administration of the 212Pb-labeled compound (20 μCi). [Figure 16]Figure 16 shows the mean weight loss of mice treated with CEA-PRIT and CEA-RIT (groups A-E, n=10) in the BxPC3 model. The curves were truncated for n<5. The dotted vertical lines indicate the administration of 212Pb-labeled compounds to some or all groups, as per the study design. [Figure 17] Figure 17 shows the experimental design of Protocol 175, which includes a two-step CEA-PRIT, in SCID mice with SC BxPC3 tumors that were sacrificed and necropsied 24 hours after 212Pb-DOTAM injection. The CEA-split-DOTAM-VH-AST dose was adjusted to compensate for whole / whole impurities. [Figure 18] Figure 18 shows the distribution of 212Pb in tumor-bearing SCID mice 24 hours after injection of 212Pb-DOTAM pre-targeted with CEA-split-DOTAM-VH / VL antibody (Protocol 175). Radioactivity content in organs and tissues is expressed as mean %ID / g ± SD (n=4). [Figure 19] Figure 19 shows the experimental design of Protocol 185, which includes a two-step CEA-PRIT, in SCID mice with SC BxPC3 tumors that were sacrificed and necropsied 6 hours after 212Pb-DOTAM injection. The CEA-split-DOTAM-VH-AST(CH1A1A) dose was adjusted to compensate for whole / whole impurities. [Figure 20] Figure 20 shows the distribution of 212Pb in tumor-bearing SCID mice 6 hours after injection of 212Pb-DOTAM pre-targeted with CEA-split-DOTAM-VH / VL antibody (Protocol 185). Radioactivity content in organs and tissues is expressed as mean %ID / g ± SD (n=5). [Figure 21]Figure 21 shows the distribution of CEA-split-DOTAM-VH / VL pairs (combinations of VH and VL antibodies) in two selected SC BxPC3 tumors 7 days after injection. A and B show tumor sections from mouse A3 injected with CEA-split-DOTAM-VH / VL targeting T84.66; A shows CEA expression and B shows the corresponding CEA-split-DOTAM-VH / VL distribution. C and D show tumor sections from mouse C5 injected with CEA-split-DOTAM-VH / VL targeting CH1A1A; C shows CEA expression and D shows the corresponding CEA-split-DOTAM-VH / VL distribution. [Figure 22] Figure 22 shows the experimental design of Protocol 189, which includes a two-step CEA-PRIT, in SCID mice with SC BxPC3 tumors that were sacrificed and necropsied 6 hours after 212Pb-DOTAM injection. The CEA-split-DOTAM-VH-AST(CH1A1A) dose was adjusted to compensate for whole / whole impurities. [Figure 23] Figure 23 shows the distribution of 212Pb in tumor-bearing SCID mice 6 hours after injection of 212Pb-DOTAM pre-targeted by a biparatopic pair of CEA-split-DOTAM-VH / VL antibodies (T84.66 and CH1A1A), compared to a positive control (CH1A1A only). Radioactivity content in organs and tissues is expressed as mean %ID / g ± SD. [Figure 24] Figure 24 shows the mean fluorescence intensity (MFI) determined by FACS for the SPLIT antibody. Pb-DOTA-FITC binding, as determined by FACS, can only be shown for co-incubation of both SPLIT antibodies with Pb-DOTA-FITC. Single SPLIT antibodies did not produce a significant signal. [Figure 25] Figures 25A to 25C show exemplary formats of split antibodies as described herein. [Figure 26]Figure 26 shows the results of Example 8, Experiment 1, which evaluated the binding of individual TA-split-DOTAM-VH antibodies and TA-split-DOTAM-VL antibodies captured on the chip to biotinylated DOTAM. [Figure 27] Figure 27 shows the results of Experiment 2 in Example 8, which evaluated the binding of DOTAM to individual TA-split-DOTAM-VH antibodies and TA-split-DOTAM-VL antibodies captured on the chip. [Figure 28] Figure 28 shows the results of Experiment 3 in Example 8, which evaluated the binding of DOTAM to TA-split-DOTAM-VH / VL antibodies (antibody pairs) captured on the chip. [Figure 29] Figure 29 shows the results of Experiment 1, Example 11, in which the binding of DOTAM to TA-split-DOTAM-VH / VL antibody (antibody pair) captured on a chip using immobilized anti-hFab was evaluated compared to a reference antibody. [Figure 30] Figure 30 shows the results of Example 11, Experiment 1, which evaluated the binding of DOTAM to individual TA-split-DOTAM-VH and TA-split-DOTAM-VL antibodies captured on a chip using immobilized anti-hFab, compared to binding to antibody pairs. [Figure 31] Figure 31 shows the results of Experiment 2 of Example 11, which evaluated the binding of DOTAM to TA-split-DOTAM-VH / VL antibody (antibody pair) captured on a chip using immobilized CEA. The upper image shows the interaction on the CEA target surface: 1. Strong dissociation of prodrugs A and B from the CEA target surface (monovalent CEA binding); 2. Injection of DOTAM and complexation of prodrugs A+B on the surface results in stronger binding of the active complex of prodrugs A+B+DOTAM and less dissociation from the CEA target surface; 3. Readout by monostreptavidin. The lower image shows data subject to "dual reference". In "dual reference", the inventors subtract dissociation from the CEA target surface to show only the net response of the interaction between DOTAM and prodrugs A / B. [Modes for carrying out the invention]

[0048] Detailed description of the invention I. Definition For the purposes of this specification, “acceptor human framework” means 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 a human consensus framework, as defined below. An acceptor human framework “derived” from a human immunoglobulin framework or a human consensus framework may contain the same amino acid sequence or may contain a modification of the amino acid sequence. In some embodiments, the number of amino acid modifications 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 the human consensus framework sequence.

[0049] "Affinity" refers to the sum of the strength of non-covalent interactions between a single binding site between a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between the members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y is generally expressed by the dissociation constant (K). D ) can be expressed by. Affinity can be measured by methods common in the art, including those described herein. Specific illustrative and exemplary methods for measuring binding affinity are described below.

[0050] An "affinity-matured" antibody is an antibody that, compared to an unmodified parent antibody, has one or more modifications in one or more complementarity-determining regions (CDRs), and such modifications improve the antibody's affinity for the antigen.

[0051] The term "antibody that binds to an antigen expressed on the surface of a target cell" refers to an antibody that can bind to the antigen with sufficient affinity to be useful as a diagnostic and / or therapeutic agent when the antibody targets the antigen. In one embodiment, the degree of antibody binding to unrelated non-antigen proteins is less than approximately 10% of the antibody binding to the antigen, as measured, for example, by surface plasmon resonance (SPR). In a particular embodiment, the antibody that binds to the antigen expressed on the surface of a target cell is ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 -8 M or less, for example, 10 -8 M~10 -13 M, for example 10 -9 M~10 -13 The dissociation constant (K) of M D ) has. The antibody is K D If the concentration is 1 μM or less, it is said to "specifically bind" to the antigen expressed on the surface of target cells. In certain embodiments, the antibody binds to an epitope of the antigen that is conserved among different species of that antigen.

[0052] The terms “antigen binding site for a radiolabeled compound” or “functional antigen binding site for a radiolabeled compound” refer to an antigen binding site, including VH and VL domains, that can bind to the radiolabeled compound with sufficient affinity to be useful as a diagnostic and / or therapeutic agent for associating the radiolabeled compound with the antibody. In one embodiment, the degree of binding of the antigen binding site to an unrelated non-antigen compound is less than approximately 10% of the binding of the antibody to the radiolabeled compound, as measured, for example, by surface plasmon resonance (SPR). In a particular embodiment, the antigen binding site that binds to the radiolabeled compound is less than 1 μM, less than 100 nM, less than 10 nM, less than 1 nM, less than 0.1 nM, less than 0.01 nM, or less than 0.001 nM (e.g., 10 -8 M or less, for example, 10 -8 M~10 -13 M, for example 10 -9 M~10- -13 The dissociation constant (K) of M D) has 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 K D It may be preferable to have a functional binding site with a K content of approximately 1 pM to 1 nM, for example, approximately 1 to 10 pM, 1 to 100 pM, 5 to 50 pM, 100 to 500 pM, or 500 pM to 1 nM. D It can bind to radiolabeled compounds. The antigen-binding site is a K2 cell with an antigen-binding site of 1 μM or less. D If it possesses this property, it is said to "specifically bind" to the radiolabeled compound.

[0053] The term "antibody" is used herein in its broadest sense and is not limited thereto, 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. Where used herein, the term "antibody" also encompasses individual hemibodies containing either a VH domain or a VL domain of the functional antigen-binding site.

[0054] An "antibody fragment" refers to a molecule other than an intact antibody, including a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments, though not limited to them, include Fv, Fab, cross-Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv and scFab); single-domain antibodies (dAb); and multispecific antibodies formed from antibody fragments. For a review of specific antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005). The term "Fab fragment" refers to a protein consisting of the VH and CH1 domains of the heavy chain and the VL and CL domains of the light chain of an immunoglobulin. A "Fab' fragment" differs from a Fab fragment by adding residues at the carboxyl terminus of the CH1 domain containing one or more cysteines from the antibody hinge region. For a description of the Fab and F(ab')2 fragments, which contain salvage receptor-binding epitope residues and have a longer in vivo half-life, please refer to U.S. Patent No. 5,869,046.

[0055] As used herein, references to “Fab fragments” are intended to include cross-Fab fragments or scFab fragments as well as conventional Fab fragments (i.e., those containing a light chain with VL and CL domains, and a heavy chain fragment with VH and CH1 domains).

[0056] The terms “cross-Fab fragment,” “xFab fragment,” or “crossover Fab fragment” refer to Fab fragments in which either the variable or constant regions of the heavy and light chains are exchanged. A cross-Fab fragment includes a polypeptide chain composed of a light chain variable region (VL) and a heavy chain constant region 1 (CH1), and a polypeptide chain composed of a heavy chain variable region (VH) and a light chain constant region (CL). For clarity, in a crossover Fab molecule in which the variable regions of the Fab light chain and Fab heavy chain are exchanged, the peptide chain containing the heavy chain constant region is referred to herein as the “heavy chain” of the crossover Fab molecule. Conversely, in a crossover Fab molecule in which the constant region of the Fab light chain and the constant region of the Fab heavy chain are exchanged, the peptide chain containing the heavy chain variable region is referred to herein as the “heavy chain” of the crossover Fab molecule.

[0057] As used herein, the term “single-chain” refers to a molecule containing amino acid monomers linearly linked by peptide bonds. A single-chain Fab molecule is a Fab molecule in which a Fab light chain and a Fab heavy chain are linked by a peptide linker to form a single peptide chain. In certain such embodiments, the C-terminus of the Fab light chain is connected to the N-terminus of the Fab heavy chain in the single-chain Fab molecule.

[0058] Asymmetric Fab arms can also be manipulated by introducing charged or uncharged amino acid mutations at the domain interface to direct correct Fab pairing. See, for example, International Publication 2016 / 172485.

[0059] A "single-stranded variable fragment" or "scFv" is a fusion peptide of the variable domains of the heavy chain (VH) and light chain (VL) of an antibody, linked by a linker. Specifically, the linker is a short polypeptide of 10–25 amino acids, usually rich in glycine for flexibility and serine or threonine for solubility, and can be linked at either the N-terminus of VH to the C-terminus of VL, or vice versa. This protein can retain the specificity of the original antibody despite the removal of the constant region and the introduction of a linker. For a review of scFv fragments, see, for example, Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269–315 (1994). See also International Publication No. 93 / 16185 and U.S. Publications No. 5,571,894 and No. 5,587,458.

[0060] The term "blocking agent" refers to a drug that blocks the binding of effector molecules, particularly radiolabeled compounds, to their functional binding sites. Generally, such blocking agents bind to the functional binding site of the effector molecule, for example, by specifically binding to that functional binding site.

[0061] The term "clearance agent" refers to a drug that increases the clearance rate of antibodies from the circulation of a target. Generally, clearance agents bind to antibodies, for example, specifically to antibodies.

[0062] As used herein, the terms “removal process” or “removal stage” encompass the use of either a blocking agent or a removal agent. Some agents can function as both removal agents and blocking agents.

[0063] The term "epitope" refers to a site on an antigen, whether proteinaceous or nonproteinaceous, to which an antibody binds. Epitopes can be formed from continuous amino acid stretch sites (linear epitopes) or from discontinuous amino acids (conformational epitopes), and are formed spatially close together, for example, due to the folding of the antigen (i.e., by tertiary folding of a proteinaceous antigen). Linear epitopes are typically still bound to antibodies even after exposure of proteinaceous antigens to denaturants, while conformational epitopes are typically destroyed by treatment with denaturants. Epitopes contain at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial structure.

[0064] Screening for antibodies that bind to a specific epitope (i.e., antibodies that bind to the same epitope) may be performed using methods commonly used in the art, such as, but not limited to, alanine scanning, peptide blotting (Meth.Mol.Biol.248(2004)443-463), peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of antigens (see Prot.Sci.9(2000)487-496), and cross-blocking (see "Antibodies", Harlow and Lane, Cold Spring Harbor Press, Cold Spring Harb., NY).

[0065] Antigen structure-based antibody profiling (ASAP), also known as modification-assisted profiling (MAP), allows for the classification of antibodies from a large number of monoclonal antibodies based on their binding profiles to chemically or enzymatically modified antigen surfaces, enabling them to specifically bind to an antigen (see, for example, U.S. Patent Application Publication 2004 / 0101920). Each classified antibody binds to the same epitope, which may be distinctly different from, or partially overlapping, a unique epitope from, another classified antibody.

[0066] Furthermore, competitive binding can be used to easily determine whether an antibody binds to the same epitope as a reference antibody, or whether it competes for binding to the reference antibody. For example, an antigen that "binds to the same epitope as the reference antibody" refers to an antibody that, in a competitive assay, blocks the binding of the reference antibody to that antigen by 50% or more, and conversely, the reference antibody, in a competitive assay, blocks the binding of the antibody to that antigen by 50% or more. Alternatively, for example, to determine whether an antibody binds to the same epitope as a reference antibody, the reference antibody can be bound to the antigen under saturated conditions. After removing excess reference antibody, the ability of the antibody in question to bind to the antigen is evaluated. If the antibody in question can bind to the antigen after saturated binding of the reference antibody, it can be concluded that the antibody in question binds to a different epitope than the reference antibody. However, if the antibody in question cannot bind to the antigen after saturated binding of the reference antibody, it is possible that the antibody in question binds to the same epitope as the reference antibody. Conventional experiments can be used to determine whether the antibody in question is binding to the same epitope or whether binding is simply being hindered for steric reasons (e.g., peptide mutation analysis, or binding analysis using ELISA, RIA, surface plasmon resonance, flow cytometry, or other quantitative or qualitative antibody binding assays available in the art). This assay should be performed in two setups, i.e., with both antibodies being saturated antibodies. If, in both setups, only the first (saturated) antibody can bind to the antigen, it can be concluded that the antibody in question and the reference antibody are competing for binding to the antigen.

[0067] In some embodiments, if competitive binding assays measure that a 1, 5, 10, 20, or 100-fold excess of one antibody inhibits the binding of the other by at least 50%, at least 75%, at least 90%, or even more than 99%, then the two antibodies are considered to bind to the same or overlapping epitopes. (See, for example, Junghans et al., Cancer Res. 50(1990) 1495-1502).

[0068] In some embodiments, two antibodies are considered to bind to the same epitope if substantially all of the amino acid mutations of an antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other antibody. Two antibodies are considered to have a “duplicate epitope” if only a subset of amino acid mutations that reduce or eliminate the binding of one antibody also reduces or eliminates the binding of the other antibody.

[0069] 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, while the remaining part of the heavy chain and / or light chain originates from a different source or species.

[0070] The "class" of an antibody refers to the type of constant domain or constant region held by its heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In certain embodiments, the antibody is the IgG1 isotype. In certain embodiments, the antibody is the IgG1 isotype with P329G, L234A, and L235A mutations to reduce the effector function of the Fc region. In other embodiments, the antibody is the IgG2 isotype. In certain embodiments, the antibody is the IgG4 isotype with the S228P mutation in the hinge region to improve the stability of the IgG4 antibody. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The light chain of an antibody can be assigned to one of two types, called kappa (κ) or lambda (λ), based on the amino acid sequence of its constant domain.

[0071] "Effector function" refers to the biological activities resulting from the Fc region of an antibody, which vary depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptor), and B cell activation.

[0072] An "effective amount" of a drug, e.g., a pharmaceutical composition, refers to an amount effective at dosages and for periods of time necessary to achieve the desired therapeutic or prophylactic result.

[0073] The term "tandem Fab" refers to an antibody that includes two Fab fragments connected via a peptide linker / tether. In some embodiments, the tandem Fab may include one Fab fragment and one cross-Fab fragment connected by a peptide linker / tether.

[0074] 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. In this specification, the term “Fc domain” is used to define the C-terminal region of an immunoglobulin that includes the constant regions of two heavy chains excluding the first constant region. Thus, the Fc domain refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, as well as the last three constant region immunoglobulin domains of IgE and IgM. This term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more amino acids, particularly one or two, from the C-terminus of the heavy chain. Thus, by expression of certain nucleic acid molecules encoding full-length heavy chains, antibodies produced by host cells may include full-length heavy chains or cleaved variants of full-length heavy chains. This may be the case where the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbered according to the EU index). Thus, a C-terminal lysine (Lys447) or C-terminal glycine (Gly446) and lysine (Lys447) may or may not be present in the Fc region. In one embodiment, the heavy chain containing the Fc region specified herein, as contained in the antibody of the present invention, includes a further C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to the EU index). In one embodiment, the heavy chain containing the Fc region specified herein, as contained in the antibody according to the present invention, includes a further C-terminal glycine residue (G446, numbered according to the EU index).Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. As used herein, a "subunit" of an Fc domain refers to one of the two polypeptides that form the dimeric Fc domain, i.e., a polypeptide that includes the C-terminal constant region of an immunoglobulin heavy chain that can stably associate with the other of the two polypeptides that form the dimeric Fc domain. For example, the subunits of an IgG Fc domain include the IgG CH2 and IgG CH3 constant domains.

[0075] "Framework" or "FR" refers to variable domain residues other than complementarity determining regions (CDRs). The FRs of a variable domain generally consist of four FR domains, FR1, FR2, FR3, and FR4. Thus, CDR and FR sequences generally occur in the following sequence in VH (or VL): FR1-CDR-H1 (CDR-L1)-FR2-CDR-H2 (CDR-L2)-FR3-CDR-H3 (CDR-L3)-FR4.

[0076] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody that has a structure substantially similar to a native antibody structure or that has a heavy chain containing an Fc region as defined herein.

[0077] "Fusing" means that the components are linked directly by a peptide bond or via one or more peptide linkers.

[0078] The terms “host cell,” “host cell line,” and “host cell culture” are used synonymously and refer to cells into which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include “transformers” and “transformed cells,” which, regardless of passage number, include primary transformed cells and their offspring. The offspring may not have exactly the same nucleic acid content as the parent cells and may contain mutations. The present invention includes the progeny of mutants having the same function or biological activity as those screened or selected for the original transformed cells.

[0079] A "human antibody" is defined as an antibody produced by a human or human cell, or an antibody that has an amino acid sequence corresponding to a non-human antibody that utilizes a sequence encoding a human antibody, such as the human antibody repertoire. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0080] The "Human Consensus Framework" is a framework representing the most commonly occurring amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from subgroups of variable domain sequences. Generally, the sequence subgroups are those described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup Kappa I, as described in Kabat et al. In another embodiment, for VH, the subgroup is subgroup Kappa III, as described in Kabat et al.

[0081] A “humanized” antibody refers to a chimeric antibody containing amino acid residues derived from non-human CDRs and amino acid residues derived from human FRs. In certain embodiments, a humanized antibody contains at least one, usually two, variable domains in all or nearly all CDRs corresponding to the variable domains of the non-human antibody, and in all or nearly all FRs corresponding to the variable domains of the human antibody. A humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody. The “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0082] As used herein, the terms “hypervariable region” or “HVR” refer to regions of an antibody variable domain that are hypervariable in sequence and determine antigen-binding specificity, such as “complementarity-determining regions” (CDRs).

[0083] Generally, an antibody contains six CDRs, three located in the VH (CDR-H1, CDR-H2, CDR-H3) and three located in the VL (CDR-L1, CDR-L2, CDR-L3). Illustrative CDRs as used herein include: (a) Hypervariable loops arising 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. 196:901-917 (1987)), (b) CDRs present in 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 Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and (c) Antigen contact occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) is an example (MacCallum et al. J.Mol.Biol.262:732-745 (1996)).

[0084] Unless otherwise specified, the CDR is determined in accordance with Kabat et al. above. Those skilled in the art will understand that the notation of the CDR may be determined in accordance with Chothia above, McCallum above, or any other scientifically recognized nomenclature system. Instead, the CDR-H1 sequences described herein may extend, for example, from Kabat 26 to Kabat 35 for the Pb-DOTAM binding variable domain.

[0085] In one embodiment, the CDR residues include those identified in the sequence listing or elsewhere in this specification.

[0086] Unless otherwise specified, HVR / CDR residues and other residues (e.g., FR residues) within the variable domain are numbered herein in accordance with Kabat et al. cited above.

[0087] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including but not limited to cytotoxic agents.

[0088] The "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (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 aspects, the individual or subject is a human.

[0089] The molecules described herein can be “isolated.” An “isolated” antibody is one that has been separated from its 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 a review of methods for evaluating antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0090] A "multidomain protein" is a protein that contains at least a first and a second protein domain. A protein domain is a substructure of a protein, is stable, and folds independently of the rest of the protein.

[0091] The terms “nucleic acid molecule” or “polynucleotide” include any compound and / or substance containing polymers of nucleotides. Each nucleotide is composed 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. Often, nucleic acid molecules are described by a base sequence, where the bases represent the primary structure (linear structure) of the nucleic acid molecule. The base sequence is typically represented 5' to 3'. In this specification, the term nucleic acid molecule includes deoxyribonucleic acid (DNA), e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers containing two or more of these molecules. Nucleic acid molecules may be linear or cyclic. In addition, the term nucleic acid molecule includes both sense strands and antisense strands, as well as both single-stranded and double-stranded forms. Furthermore, nucleic acid molecules described herein may include naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides, including derivatized sugar or phosphate backbone links or chemically modified residues, include modified nucleotide bases. Nucleic acid molecules also include DNA and RNA molecules 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, so that the mRNA can be injected into a target for antibody production in vivo. (For example, see Stadler et al, Nature Medicine 2017, published online 12 June 2017, doi:10.1038 / nm.4356 or EP 2 101 823 B1).

[0092] "Isolated" nucleic acids are nucleic acid molecules that have been separated from their natural environment. Isolated nucleic acids include nucleic acid molecules that were originally contained in cells but are located outside of chromosomes or at chromosomal locations different from their natural chromosomal locations.

[0093] "Antibody-encoding isolated nucleic acid" refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), and includes nucleic acid molecules(s) in a single vector or separate vectors, such nucleic acid molecules(s) located at one or more locations within a host cell.

[0094] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting that population are identical and / or bind to the same epitope, with the exception of possible mutant antibodies, such as those containing spontaneous mutations or arising during the production of a monoclonal antibody preparation, which are generally present in small amounts. In contrast to polyclonal antibody preparations, which typically contain various antibodies against various determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is against a single determinant on an antigen. Therefore, the modifier “monoclonal” indicates the characteristic of an antibody obtained from a substantially homogeneous collection of antibodies and should not be interpreted as requiring antibody production by any particular method. For example, monoclonal antibodies according to 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 described herein are described herein.

[0095] A "naked antibody" refers to an antibody that is not bound to a heterogeneous site (e.g., a cytotoxic site) or a radioactive label. Naked antibodies may be present in a pharmaceutical composition.

[0096] The term "native antibody" refers to immunoglobulin molecules that exist in native form with various structures. For example, native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, including those in which two identical light chains and two identical heavy chains are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable domain (VH), also called the variable heavy domain or heavy chain variable region, followed by three constant heavy domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable domain (VL), also called the variable light domain or light chain variable region, followed by a constant light (CL) domain.

[0097] The term "package insert" is used to refer to the instructions normally included in the commercial package of a therapeutic product and includes information regarding indications, use, dosage, administration, combination therapies, contraindications and / or warnings for such therapeutic products.

[0098] Percent (%) amino acid sequence identity with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences for alignment purposes and introducing gaps if necessary to achieve the highest possible sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for determining percent amino acid sequence identity can be achieved using various methods within the scope of the art, such as publicly available computer software like BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software, or FASTA program packages. Those skilled in the art can determine appropriate parameters for aligning the sequences, including any algorithm required to achieve the highest degree of alignment over the entire length of the sequences being compared. Alternatively, the percentage identity value can be generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and its source code is filed in the user documentation of the U.S. Copyright Office (Washington DC, 20559), registered under U.S. Copyright Registration No. TXU510087, and published internationally in publication No. 2001 / 007611.

[0099] However, for the purposes of this specification, the amino acid sequence identity percentage values ​​are generated using the ggsearch program in FASTA package version 36.3.8c, or subsequently using the BLOSUM50 comparison matrix. The FASTA program package is certified by WRPearson and DJLipman (1988), "Improved Tools for Biological Sequence Analysis," PNAS 85:2444-2448; WRPearson (1996), "Effective protein sequence comparison," Meth.Enzymol. 266:227-258; and Pearson et al. (1997), Genomics 46:24-36, and is publicly available at www.fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml or www.ebi.ac.uk / Tools / sss / fasta. Alternatively, the ggsearch(global protein:protein) program with default options (BLOSUM50;open:-10;ext:-2;Ktup=2) can be used to compare sequences using a public server accessible at fasta.bioch.virginia.edu / fasta_www2 / index.cgi, ensuring that a global rather than local alignment is performed. The amino acid identity percentage is given in the output alignment header.

[0100] The terms "pharmaceutical composition" or "pharmaceutical preparation" refer to a preparation in which the biological activity of the active ingredient contained in the preparation is effective, and which does not contain additional ingredients that are unacceptably toxic to the subject to which the pharmaceutical composition is to be administered.

[0101] A "pharmaceutically acceptable carrier" refers to a component in a pharmaceutical composition or preparation other than the active ingredient, which is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0102] As used herein, references to target antigens refer to any native target antigen from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses “full-length” unprocessed target antigens, as well as any form of target antigen resulting from intracellular processing. The term also encompasses naturally occurring variants of target antigens, such as splice variants or allele variants. For example, the target antigen CEA may have the amino acid sequence of human CEA, particularly UniProt (www.uniprot.org) accession number P06731 (version 119), or carcinoembryonic antigen-associated cell adhesion molecule 5 (CEACAM5) as shown in NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_004354.2. Another example of a target antigen is fibroblast-activating protein (FAP). The amino acid sequence of human FAP is shown in UniProt (www.uniprot.org) accession number Q12884 (version 149) or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_004451.2. Another example of a target antigen is GPRC5D (UniProt number Q9NZD1 (version 115); see NCBI RefSeq number NP_061124.1 for the human sequence).

[0103] The terms “split antibody,” “split antibody (plural),” “single-domain split antibody,” or “SPLIT PRIT” as used herein mean that the VH and VL domains, which together form an antigen-binding site capable of binding to a radiolabeled compound, are split between two antibodies and are not present as part of the same antibody (before in vivo assembly). “CEA-targeted SPLIT PRIT” refers to a split antibody that targets CEA. The term “SPLIT PRIT” may also be used interchangeably with “TA-split-DOTAM-VH / VL” (e.g., when “TA” or the target antigen is CEA, FAP, or GPRC5D). The term “CEA-targeted SPLIT PRIT” may also be used interchangeably with the term “CEA-split-DOTAM-VH / VL.”

[0104] As used herein, “treatment” (and its grammatical variations, e.g., “to treat” or “treating”) refers to a clinical intervention in an attempt to alter the natural course of a disease in the treated individual, and may be performed for preventive purposes or during the course of a clinicopathological disease. Desired effects of treatment include, but are not limited to, preventing the onset or recurrence of the disease, reducing symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, achieving remission or mitigation of symptoms, and achieving recovery or improving 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.

[0105] The term "variable region" or "variable domain" refers to a domain in the heavy or light chain of an antibody that is involved in the binding of the antibody to the antigen. The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) generally have similar structures, and each domain contains four conserved framework regions (FRs) and three complementarity-determining regions (CDRs). For example, Kindt et al., Kuby Immunology, 6. thSee WH.Freeman and Co., page 91 (2007). A single VH domain or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen may be isolated by screening a library of complementary VL domains or VH domains using the VH domain or VL domain of the antibody that binds to the antigen, respectively. See, for example, Portolano et al., J.Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0106] As used herein, the term “vector” refers to a nucleic acid molecule capable of replicating another nucleic acid to which it is linked. This term includes vectors as self-replicating nucleic acid structures, and vectors incorporated into the genome of a host cell into which they are introduced. Certain vectors can direct the expression of the nucleic acid to which they are functionally linked. Such vectors are referred to herein as “expression vectors.”

[0107] As used herein, the terms “Pb” or “lead” include its ion, for example, Pb(II). References to other metals also include their ions. Therefore, those skilled in the art will know, for example, lead, Pb, 212 Pb or 203 Understand that the term Pb is intended to encompass the ionic form of the element, particularly Pb(II).

[0108] II. Compositions and Methods In one embodiment, the present invention is based in part on a set of antibodies comprising a first antibody and a second antibody, each antibody capable of binding to an antigen on a target cell, but a functional antigen-binding site for an effector agent is formed only when the first antibody and the second antibody are associated with each other. The antibodies of the present invention are useful, for example, in pre-targeted immunotherapy methods and / or pre-targeted imaging. In a preferred embodiment, the method eliminates the step of administering a decontamination agent or blocking agent.

[0109] A. Target antigen Antigens expressed on the surface of target cells are also referred to herein as “target antigens” or “target cell antigens.” These terms are used interchangeably herein.

[0110] The present invention, with respect to treatment methods and products for use therein, is applicable to any condition treatable by cytotoxic activity targeting a patient's cells, e.g., diseased cells. Therefore, the target cells are any cells for which targeting cytotoxicity is desirable, e.g., any diseased cells. The treatment is preferably the treatment of tumors or cancer. However, the applicability of the present invention is not limited to tumors and cancer. For example, the treatment may also be the treatment of viral infections (by targeting infected cells) or T-cell-driven autoimmune diseases (by targeting T cells). 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, potentially treating T-cell-driven autoimmune diseases such as multiple sclerosis and graft-versus-host disease, as well as T-cell hematological malignancies currently undergoing clinical trials. Similarly, the methods of the present invention may be applicable to any cell type for which radiographic imaging is desirable, including but not limited to cancer cells or tumor cells.

[0111] Therefore, suitable target antigens may include cancer cell antigens, viral antigens, or microbial antigens.

[0112] Antigens are typically normal cell surface antigens that are expressed either in overexpression or abnormally. Ideally, target antigens are expressed only on diseased cells (such as tumor cells), but this is rarely observed in practice. As a result, target antigens are usually selected based on differential expression between diseased and healthy tissues.

[0113] The cell surface marker or target antigen may be, for example, a tumor-associated antigen.

[0114] 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 expressed alone or predominantly or overexpressed by tumor cells and / or cancer cells, or other cells in the tumor stroma such as cancer-associated fibroblasts, so that the antigen is associated with tumor(s) and / or cancer(s). Tumor-associated antigens may be further expressed by normal cells, non-tumor cells, or non-cancerous cells. However, in such cases, the expression of tumor-associated antigens by normal cells, non-tumor cells, 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 or express antigens at significantly higher levels compared to the expression of antigens by normal cells, non-tumor cells, or non-cancerous cells. Furthermore, tumor-associated antigens may be further expressed by cells in different stages of development or maturation. For example, tumor-associated antigens may be further expressed by embryonic or fetal cells, which are not typically found in adult hosts. Alternatively, tumor-associated antigens may be further expressed by stem cells or progenitor cells, which are not typically found in adult hosts.

[0115] Tumor-associated antigens may be antigens expressed by any cells of any cancer or tumor, including the cancers and tumors described herein. A tumor-associated antigen may be a tumor-associated antigen 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, a tumor-associated antigen may be a tumor-associated antigen of two or more types of cancer or tumors (for example, characteristic of two or more types). For example, a tumor-associated antigen may be expressed by both breast cancer cells and prostate cancer cells, and may not be expressed at all by normal cells, non-tumor cells, or non-cancerous cells.

[0116] Exemplary tumor-associated antigens to which the antibodies of the present invention can bind include, but are not limited to, melanoma-associated chondroitin sulfate proteoglycan (MCSP), mucin 1 (MUCl; tumor-associated epithelial mucin), preferentially expressed melanoma antigen (PRAME), carcinoembryonic antigen (CEA), prostate-specific membrane antigen (PSMA), PSCA, EpCAM, Trop2 (also known as trophoblast-2 or EGP-1), 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 another embodiment, the tumor antigens include differentiation antigens (CD)19, CD20, CD21, CD22, CD25, CD30, CD33 (sialic acid-binding 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), GPRC5D (G protein-coupled receptor class C group 5 member D), PSMA (prostate-specific membrane antigen), CA9=CAIX (carbonic anhydrase IX), Ll The group may be selected from CAM (neuronal cell adhesion molecule L1), endothialin, HER3 (activation conformation of epidermal growth factor receptor family member 3), Alkl / BMP9 complex (anaplastic lymphoma kinase 1 / bone morphogenetic protein 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 tube cancer, head and neck cancer, cervical cancer, and pancreatic cancer. CD22 is expressed in, for example, hairy 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, hairy 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.

[0117] 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 moiety (cell binding agent) may be an antibody selected from the group consisting of B3, RFB4, SS, SS1, MN, MB, HN1, HN2, HB21, and MORAb-009, and their antigen-binding moieties. Further exemplary targeted portions suitable for use in the chimeric molecule of the present invention include, for example, U.S. Patent No. 5,242,824 (anti-transferrin receptor); Patent Document No. 5,846,535 (anti-CD25); Patent Document No. 5,889,157 (anti-Lewis Y); Patent Document No. 5,981,726 (anti-Lewis Y); Patent Document No. 5,990,296 (anti-Lewis Y); Patent Document No. 7,081,518 (anti-mesoterin); Patent Document No. 7,355,012 (anti-CD22 and anti-CD25); Patent Document No. 7,368,110 (anti-mesoterin); Patent Document No. 7,470,775 (anti-CD30); Patent Document No. 7,521,054 (anti-CD25); and Patent Document No. 7,541,034 (anti-CD22); U.S. Patent Application Publication No. 2007 / 0189962 (anti-CD22); Frankel et al. This is disclosed in al., Clin. Cancer Res., 6:326-334 (2000), and Kreitman et al., AAPS Journal, 8(3):E532-E551 (2006) (these are incorporated herein by reference, respectively).

[0118] Further antibodies targeting specific tumor-associated antigens have been produced: 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 TMEFF2. Any of these or their antigen-binding fragments may be useful in the present invention, i.e., they may be incorporated into the antibodies described herein.

[0119] In some embodiments of the present invention, it may be preferable that the tumor-associated antigen is a carcinoembryonic antigen (CEA).

[0120] CEA is advantageous in relation to the present invention because it is internalized relatively slowly, and therefore a high percentage of antibodies remain available on the cell surface after initial treatment for binding to radionuclides. Other low-internalization targets / tumor-associated antigens may also be preferred. Other examples of tumor-associated antigens include CD20 or HER2. In further embodiments, the target may be EGP-1 (epithelial glycoprotein-1, also known as trophoblast-2), colon-specific antigen-p (CSAp), or pancreatic mucin MUC1. See, for example, 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;63:354-63), hPAM4, which binds to MUC1 (see also Gold et al Cancer Res. 2008:68:4819-26), valtuzumab, which binds to CD20 (see also Sharkey et al Cancer Res. 2008;68:5282-90), and hRS7, which binds to EGP-1 (see also Cubas et al Biochim Biophys Acta 2009;1796:309-14). Any of these, or their antigen-binding moieties, may be useful in the present invention, i.e., they may be incorporated into the antibodies described herein. An example of an antibody produced against CEA is T84.66 (shown in NCBI accession numbers CAA36980 (heavy chain) and CAA36979 (light chain), or in Sequence IDs 317 and 318 of International Publication No. 2016 / 075278), as well as its humanized and chimeric forms, e.g., T84.66-LCHA described in International Publication No. 2016 / 075278A1 and / or International Publication No. 2017 / 055389. Another example is the anti-CEA antibody CH1A1a, described in International Publication No. 2012 / 117002 and International Publication No. 2014 / 131712, and CEA hMN-14 (see also U.S. Patent Nos. 6,676,924 and 5,874,540).Another anti-CEA antibody is A5B7, described in MJBanfield et al, Proteins 1997, 29(2), 161-171. Humanized antibodies derived from the mouse antibody A5B7 are disclosed in International Publication No. 92 / 01059 and International Publication No. 2007 / 071422. See also concurrent pending application PCT / EP2020 / 067582. An example of a humanized version of A5B7 is A5H1EL1(G54A). Further exemplary antibodies against CEA are MFE23 and its humanized version, described in U.S. Patent No. 7626011 and / or concurrent pending application PCT / EP2020 / 067582. A further example of an antibody against CEA is 28A9. Any of these or their antigen-binding fragments may be useful in forming a CEA-binding moiety in the present invention.

[0121] FAP (fibroblast-activating protein alpha) or GPRC5D (G protein-coupled receptor class C group 5 member D) may also be preferred in some embodiments. FAP is an established target for imaging and therapy due to its widespread expression in the microenvironment of numerous tumor types (e.g., pancreatic cancer, breast cancer, and lung cancer) (Lindner, T., Loktev, A., Giesel, F. et al. Targeting of activated fibroblasts for imaging and therapy. EJNMMI radiopharm.chem. 4, 16 (2019)). Therefore, SPLIT PRIT using FAP as a target antigen on activated cancer-associated fibroblasts 212This is expected to result in the specific accumulation of Pb-DOTAM. Consequently, the emitted alpha radiation is expected to have adverse effects on immunosuppression of FAP-expressing malignancies, in addition to a limited direct tumor-killing effect on adjacent tumor cells. G protein-coupled receptor family group C5 member D (GPRC5D) is overexpressed on multiple myeloma plasma cells (Atamaniuk J, Gleiss A, Porpaczy E, Kainz B, Grunt TW, Raderer M, et al. Overexpression of G protein-coupled receptor 5D in the bone marrow is associated with poor prognosis in patients with multiple myeloma. Eur J Clin Invest. 2012;42:953-60.), and established subcutaneous (SC) in vivo models reflect the expression observed in multiple myeloma patients (e.g., OPM-2 and NCI-H 929) (Kodama T, Kochi Y, Nakai W, Mizuno H, Baba T, Habu K, et al. Anti-GPRC5D / CD3 bispecific T-cell-redirecting antibody for the treatment of multiple myeloma. Mol Cancer Ther. (2019) 18:1555-64). Therefore, the present inventors have found that SPLIT PRIT, which uses GPRC5D-split-DOTAM as the target antigen, 212 We predict that this will induce tumor-specific accumulation of Pb-DOTAM, followed by radiation-induced tumor cell death.

[0122] In some embodiments, the antibodies of the present invention can specifically bind to a target antigen (e.g., any target antigen discussed herein). In some embodiments, they may bind to ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 -7 M or less, for example, 10 -7 ~10 -13 , 10 -8M or less, for example, 10 -8 M~10 -13 M, for example 10 -9 M~10 -13 The dissociation constant (K) of M D They can be joined together.

[0123] In one embodiment, the first antibody and the second antibody may each bind to the same target antigen, which can be called "antigen A" (i.e., they have binding specificity to the same target antigen). They may each have binding specificity to the same epitope on antigen A. Alternatively, the first antibody may bind to a first epitope on antigen A, and the second antibody may bind to a different second epitope on antigen A. For example, in one embodiment, one antibody may bind to the T84.66 epitope of CEA, and the other may bind to the A5B7 epitope of CEA.

[0124] In some embodiments, one or both of the first and / or second antibodies may be bispecific to antigen A (i.e., each individual antibody may bind to two different epitopes of antigen A). The first antibody may include a first binding site and a second binding site that bind to a first and second epitope of antigen A, respectively, where the first and second epitopes are distinct from each other. Alternatively or additionally, the second antibody may include a first binding site and a second binding site that bind to a first and second epitope of antigen A, where the first and second epitopes are distinct from each other. In some embodiments, one or both of the epitopes bound by the first antibody may be different from one or both of the epitopes bound by the second antibody. In other embodiments, the two epitopes bound by the first antibody may be the same as the two epitopes bound by the second antibody.

[0125] In another embodiment, the first antibody and the second antibody may bind to different target antigens, which may be called antigen A and antigen B, respectively.

[0126] B. Radiolabeled compounds According to the present invention, the association of the first antibody and the second antibody forms a functional binding site for the effector molecule. The effector molecule according to the present invention is a radiolabeled compound containing a radioisotope, for example, a radiolabeled hapten.

[0127] In some embodiments, the effector molecule may contain a chelated radioisotope.

[0128] In some embodiments, the functional binding site to the effector molecule may be bound to a chelate containing a chelating agent and a radioisotope. In other embodiments, the antibody may be bound to a moiety conjugated with a chelated radioisotope, such as histamine-succinyl-glycine (HSG), digoxigenin, biotin, or caffeine.

[0129] The chelating agent may be, for example, a polydentate molecule such as an aminopolycarboxylic acid or aminopolythiocarboxylic acid, or a salt or functional variant thereof. The chelating agent may be, for example, a bidentate, tridentate, or tetradentate molecule. Examples of suitable metal chelating agents include EDTA (ethylenediaminetetraacetic acid, or a salt form such as CaNa2EDTA), DTPA (diethylenetriaminepentaacetic acid), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10tetraacetic acid), NOTA (2,2',2''-(1,4,7-triazanonanane-1,4,7-triyl)triacetic acid), IDA (iminodiacetic acid), MIDA ((methylimino)diacetic acid), TTHA (3,6,9,12-tetrakis(carboxymethyl)-3,6,9 12-Tetra-Azatetradecanediic acid), TETA(2,2',2'',2'''-(1,4,8,11-Tetraazacyclotetradecane-1,4,8,11tetrayl)tetraacetic acid), DOTAM(1,4,7,10-Tetrakis(carbamoylmethyl)-1,4,7,10-Tetraazacyclododecane), HEHA(1,4,7,10,13,16-Hexaazacyclohexadecane-1,4,7,10,13,16-Hexaacetic acid), available from Macrocyclics, Inc., Plano, Texas. ), NTA (nitrilotriacetic acid), EDDHA (ethylenediamine-N,N'-bis(2-hydroxyphenylacetic acid), BAL (2,3-dimercaptopropanol), DMSA (2,3-dimercaptosuccinic acid), DMPS (2,3-dimercapto-1-propanesulfonic acid), D-penicillamine (β-dimethylcysteine), MAG3 (mercaptoacetyltriglycine), Hynic (6-hydrazinopyridine-3-carboxylic acid), p-isothiocyanatobenzyl-desferrioxamine ( Examples include molecules that are labeled with zirconium for imaging purposes, and salts or functional variants / derivatives thereof that can chelate metals. In some embodiments, the chelating agent may preferably be DOTA or DOTAM or salts or functional variants / derivatives thereof that can chelate metals. Therefore, the chelating agent may be DOTA or DOTAM chelated with a radioactive isotope, or may contain DOTA or DOTAM.

[0130] The effector molecule may include, or consist of, functional variants or derivatives of the chelating agent described above, along with a radionuclide. Suitable variants / derivatives are somewhat different and have a structure that retains the ability to function as a chelating agent (i.e., retains sufficient activity for use in one or more of the purposes described herein). Functional variants / derivatives may also include the chelating agent described above conjugated to one or more additional parts or substituents, including small molecules, polypeptides, or carbohydrates. This conjugation may occur via one of the constituent carbons, for example, in the skeletal portion of the chelating agent. Suitable substituents may be heterocycles including, for example, hydrocarbon groups such as alkyl, alkenyl, aryl, or alkynyl; hydroxyl groups; alcohol groups; halogen atoms; nitro groups; cyano groups; sulfonyl groups; thiol groups; amine groups; oxo groups; carboxyl groups; thiocarboxyl groups; carbonyl groups; amide groups; ester groups; or heteroaryl groups. Substituents may include, for example, the following groups: 1 This may be one of the definitions of ". Small molecules may be, for example, dyes (Alexa 647 or Alexa 488, etc.), biotin or a biotin moiety, or a phenyl or benzyl moiety. Polypeptides may be, for example, oligopeptides, such as 2 or 3 amino acid oligopeptides. Exemplary carbohydrates include dextran, linear or branched polymers or copolymers (e.g., polyalkylenes, poly(ethylene-lysine), polymethacrylates, polyamino acids, poly or oligosaccharides, dendrimers). Derivatives may also include polymers of chelating compounds in which the above compounds are linked via a linker moiety. Derivatives may also include functional fragments of the above compounds that retain the ability to chelate metal ions.

[0131] Specific examples of derivatives include benzyl-EDTA and hydroxyethyl-thioulide-benzyl-EDTA, DOTA-benzene (e.g., (S-2-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecanetetraacetic acid)), DOTA-biotin, and DOTA-TyrLys-DOTA. In some embodiments of the present invention, the functional binding site formed by the association of the first antibody and the second antibody binds to a metal chelate containing DOTAM and a metal, such as lead (Pb). As stated above, "DOTAM" is a chemical name: It has the chemical name 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane and is a compound of the following formula. JPEG2026071214000002.jpg61170

[0132] In certain aspects and embodiments, the present invention may also utilize functional variants or derivatives of DOTAM incorporating metal ions. Suitable variants / derivatives of DOTAM have a structure that differs to some extent from the structure of DOTAM and retains the ability to function (i.e., retains sufficient activity for use in one or more purposes described herein). In such aspects and embodiments, DOTAM or a functional variant / derivative of DOTAM may be one of the active variants disclosed in International Publication No. 2010 / 099536. A suitable functional variant / derivative is given by the following formula: JPEG2026071214000003.jpg54170 (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 2~6Each alkynyl is optionally substituted with 1, 2, 3, or 4 independently selected R w groups, and 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, and C 1~7 heteroaryl-C 1~4 -alkyl are each optionally 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 of these is optionally substituted with 1, 2, or 3 independently selected R 1 groups, L 2 is C 2~4 linear alkylene, which is optionally substituted with an independently selected R 1 group and is optionally substituted with 1, 2, 3, or 4 groups independently selected from C 1~4 alkyl and / or C 1~4 haloalkyl, 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, diC1~6 Alkylcarbonylamino, C 1~6 Alkoxycarbonylamino, C 1~6 Alkoxycarbonyl-(C 1~6 Alkyl)amino, carbamyl, C 1~6 Alkylcarbamyl and di-C 1~6 Selected independently from alkylcarbamyls, Each D 1 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 Alkilen, C 1~8 Alkenylene and C 1~8 Selected independently from alkynylene, the C 1~8 Alkilen, C 1~8 Alkenylene and C 1~8 Alkynylene is selected from 1, 2, 3, or 4 independently selected R 4 It is arbitrarily substituted by the base, and the 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 Each alkyl group consists of 1, 2, 3, or 4 independently selected R 5 It is arbitrarily substituted by the base, Each D 2 It is either independent, does not exist, or C 1~20 A linear alkylene, and the C 1~20 -D 4 -It is arbitrarily replaced by the part, however C 1~20 At least one methylene unit in a linear alkylene is -D 4- Not arbitrarily replaced by the 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, 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 It is optionally substituted with one or more groups independently selected from alkylcarbamyl, Each D 3 H, halogen, cyano, nitro, hydroxyl, C 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 Each alkynyl is selected from 1, 2, 3, or 4 independently. 6It is arbitrarily substituted by the base, and the C 3~14 Cycloalkyl, C 3~14 Cycloalkyl-C 1~4 Alkyl, C 2~14 Heterocycloalkyl, 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 Each alkyl group consists of 1, 2, 3, or 4 independently selected R 7 It is arbitrarily substituted in the base, Each D 4 -O-, -S-, -NR 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, Each R 4 and R 6 These are halogens, cyano, nitro, hydroxyl, and C 1~4 Alkoxy, C 1~4 Haloalkoxy, C 1~4 Lukirchio, 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 1~4Alkylcarbonylamino, 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 Selected independently from alkylcarbamyls, each 5 These include halogens, cyano, cyanates, isothiocyanates, nitro, hydroxyl, and C. 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 Selected independently from alkylcarbamyls, Each R 7 These are halogens, cyano, nitro, hydroxyl, and C 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, C1~7 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 Each alkynyl is optionally substituted with 1, 2, 3, or 4 independently selected R' groups, and 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 Each alkyl group is optionally substituted with 1, 2, 3, or 4 independently selected R'' groups. Each R a , R b and R c H and C are independent of each other. 1~6Alkyl, 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 from alkyl groups, the C 1~6 Alkyl, C 1~6 Haloalkyl, C 2~6 Alkenil, C 2~6 Each alkynyl is selected from 1, 2, 3, or 4 independently. w The base is arbitrarily substituted, and 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 Each alkyl group consists of 1, 2, 3, or 4 independently selected R x It is arbitrarily substituted by the base, Each R o , R p , R q , R r , R s and R t H and C are independent of each other. 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 2~7Heterocycloalkyl-C 1~4 Alkyl, phenyl, phenyl-C 1~4 Alkyl, C 1~7 Heteroaryl, C 1~7 Heteroaryl-C 1~4 Selected from alkyl groups, the C 1~6 Alkyl, C 1~6 Haloalkyl, C 2~6 Alkenil, C 2~6 Each alkynyl is selected from 1, 2, 3, or 4 independently. y The base is arbitrarily substituted, and 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 Each alkyl group consists of 1, 2, 3, or 4 independently selected R z It is arbitrarily substituted in the base, Each R', R w and R y These are independently hydroxyl, cyano, nitro, and C. 1~4 Alkoxy, C 1~4 Haloalkoxy, amino, C 1~4 Alkylamino and di-C 1~4 Selected from alkylaminos, Each R'', R x and R z These are independently hydroxyl, halogen, cyano, nitro, and C 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 from alkylaminos, (However, this is subject to the condition that the valence of each atom in the arbitrarily substituted portion does not exceed that of the atom in the substituted portion.) It may be a compound of or a pharmaceutically acceptable salt thereof.

[0133] Appropriately, the functional variant / derivative of the above formula has an affinity for the antibody of the present invention equivalent to or greater than that of DOTAM, and a binding strength to Pb equivalent to or greater than that of DOTAM (where "affinity" is measured by the above dissociation constant). For example, the dissociation constant of the functional / variant derivative with the antibody or / Pb of the present invention 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.

[0134] Each R N H, C 1~6 Alkyl, or C 1~6 It may be a haloalkyl group; preferably H, C 1~4 Alkyl or C 1~4 It is a haloalkyl. Most preferably, each R N H is H.

[0135] In the case of DOTAM variants, 1, 2, 3, or most preferably each L 2 It is preferable that the C2 alkylene is present. Advantageously, C2 alkylene variants of DOTAM can have a particularly high affinity for Pb. 2 Any substituent of R 1 , C 1~4 Alkyl or C 1~4 It may be a haloalkyl. Appropriately, L 2 Any substituent of C 1~4 Alkyl or C 1~4 It may be a haloalkyl group.

[0136] Arbitrarily, each L 2 This may be an unsubstituted C2 alkylene-CH2CH2-.

[0137] Each L 1 Preferably C 1~4 Alkylenes, more preferably C1 alkylenes such as -CH2-.

[0138] Functional variants / derivatives of DOTAM are given by the following formula: JPEG2026071214000004.jpg62170 (In the formula, each Z is independently defined as R defined above) 1 The compound may be such that p, q, r, and s are 0, 1, or 2, and p+q+r+s is 1 or greater. Preferably, p, q, r, and s are 0 or 1, and / or p+q+r+s is 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).

[0139] Radioactive nuclides useful in this invention may include radioactive isotopes of metals such as lead (Pb), lutetium (Lu), or yttrium (Y).

[0140] Radionuclides that are particularly useful for imaging applications may be gamma-emitting radionuclides. For example, they are 203 Pb or 205 You can choose from Bi.

[0141] Radionuclides that are particularly useful for therapeutic purposes are those that are alpha or beta emitters. For example, they are, 212 Pb, 212 Bi, 213 Bi, 90 Y, 177 Lu, 225 Ac, 211 At, 227 Th, 223 You can select from Ra.

[0142] In some embodiments, DOTAM (or its salt or functional variant) may preferably be chelated with Pb or Bi, such as one of the above-mentioned Pb or Bi radioisotopes. In other embodiments, DOTA (or its salt or functional variant) may preferably be chelated with Lu or Y, such as one of the Lu or Y radioisotopes listed above.

[0143] In some embodiments, the method and use may include a combined therapeutic and imaging method utilizing a mixture of radioisotopes, for example, a therapeutic radioisotope and an imaging radioisotope. For example, these may be different radioisotopes of the same metal chelated with the same chelating agent. In one embodiment, the method is 203 Pb-DOTAM and 212 The method may include administering Pb-DOTAM as a mixture. In another embodiment, the method is 203 Pb or 205 The first cycle of dose measurement using gamma emitters such as Bi, and the subsequent 212 Pb, 212 Bi, 213 Bi, 90 Y, 177 Lu, 225 Ac, 211 At, 227 The or 223 This may include one or more treatments using an alpha or beta emitter such as Ra. Such methods are described further below.

[0144] In some embodiments, the functional binding site formed by the association of the first and second antibodies can bind to the Pb-DOTAM chelate.

[0145] In some embodiments, the functional binding site formed by the association of the first and second antibodies can specifically bind to the radiolabeled compound. In some embodiments, the functional binding site has a dissociation constant (Kd) to Pb-DOTAM and / or the target of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -7 M or less, for example, 10 -7 ~10 -13 , 10 -8 M or less, for example, 10 -8 M~10-13M, for example 10 -9 M~10 -13M) can bind to a radiolabeled compound, such as Pb-DOTAM chelate. In some embodiments, the functional binding site may preferably bind with a Kd value of binding affinity 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. For example, the functional binding site may bind to a metal chelate with a Kd of about 1 pM to 1 nM, for example, about 1 to 10 pM, 1 to 100 pM, 5 to 50 pM, 100 to 500 pM, or 500 pM to 1 nM.

[0146] Exemplary antigen-binding sites of C.DOTA In a particular embodiment of the present invention, the first antibody and the second antibody associate to form DOTA (or a functional derivative or variant thereof), for example, DOTA chelated with Lu or Y (for example, 177 Lu or 90 It forms a functional binding site for Y). For example, the functional binding site can bind to the radiolabeled compound with a Kd of approximately 1 pM to 1 nM, such as approximately 1 to 10 pM, 1 to 100 pM, 5 to 50 pM, 100 to 500 pM, or 500 pM to 1 nM.

[0147] C825 is 177 Lu and 90A known scFv has high affinity for DOTA-Bn(S-2-(4-aminobenzyl)-1,4,7,10tetraazacyclododecanetetraacetic acid) that forms a complex with a radioactive metal such as Y (see, for example, Cheal et al 2018, Theranostics 2018, and International Publication No. 2010099536, which are incorporated herein by reference). The CDR sequence of C825 and the VL and VH sequences are provided herein. In one embodiment, the heavy chain variable region that forms part of the antigen-binding site for the radiolabeled compound may include at least one, two, or all three CDRs selected from (a) CDR-H1 containing the amino acid sequence of 35, (b) CDR-H2 containing the amino acid sequence of 36, and (c) CDR-H3 containing the amino acid sequence of 37. In an alternative embodiment, CDR-H1 may have the sequence GFSLTDYGVH (SEQ ID NO: 148). The light chain variable region that forms part of the binding site for the radiolabeled compound may include at least one, two, or all three CDRs selected from (d) CDR-L1 containing the amino acid sequence of 38, (e) CDR-L2 containing the amino acid sequence of 39, and (f) CDR-L3 containing the amino acid sequence of 40.

[0148] In another embodiment, the heavy chain variable domain that forms part of the functional antigen-binding site for the radiolabeled compound (on the first antibody) includes the amino acid sequence of SEQ ID NO: 41, or a variant thereof that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO: 41. In a particular embodiment, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity includes substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the binding site including the sequence retains the ability to bind to DOTA complexed with Lu or Y at the affinity described herein, preferably. In a particular embodiment, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 41. In a particular embodiment, the substitutions, insertions, or deletions occur in the region outside the CDR (i.e., at the FR). Optionally, the antibody includes the VH sequence of SEQ ID NO: 41, which 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: 35 or sequence GFSLTDYGVH (SEQ ID NO: 148), (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 36, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 37.

[0149] Optionally, the light chain variable domains that form part of the functional antigen-binding site for the radiolabeled compound (on the second antibody) include the amino acid sequence of SEQ ID NO: 42, or variants thereof that have at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO: 42. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity includes substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the binding site including the sequence retains the ability to bind to DOTA complexed with Lu or Y at the affinity described herein. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 42. In certain embodiments, the substitutions, insertions, or deletions occur in the region outside the CDR (i.e., within the FR). Optionally, the antibody includes the VL sequence of SEQ ID NO: 42, which includes post-translational modifications of that sequence. In certain embodiments, the VL includes one, two, or three HVRs selected from (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 38, (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 39, and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 40.

[0150] Embodiments relating to the heavy chain variable region and the light chain variable region are explicitly intended to be used in combination. Thus, a functional antigen-binding site may be formed from the heavy chain variable region and the light chain variable region as defined above on the first antibody and the second antibody, respectively.

[0151] In any of the embodiments described above, the light chain variable region and the heavy chain variable region that form the binding site to the DOTA complex may be humanized. In one embodiment, the light chain variable region and the heavy chain variable region include the CDR of any of the embodiments described above, and further include an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0152] In some embodiments, the heavy chain variable domain may be extended by one or more C-terminal residues, such as one or more C-terminal alanine residues or one or more residues from the N-terminus of the CH1 domain, as will be discussed further below.

[0153] Exemplary antigen-binding sites of D.DOTAM In another specific embodiment of the present invention, the first antibody and the second antibody associate to form a functional antigen-binding site for Pb-DOTAM chelate (Pb-DOTAM).

[0154] An example of an antigen-binding site is described in International Publication No. 2019 / 201959, which is incorporated in its entirety herein by reference.

[0155] In certain embodiments, the functional antigen-binding site that binds to Pb-DOTAM may have one or more of the following characteristics: • Specifically binds to Pb-DOTAM and Bi-DOTAM; • It is selective for Pb-DOTAM (and optionally Bi-DOTAM) compared to other chelated metals such as Cu-DOTAM; • Binds to Pb-DOTAM with very high affinity; • It binds to the same Pb-DOTAM epitope as the antibody described in International Publication No. 2019 / 201959 (e.g., PRIT-0213 or PRIT-0214), and / or has the same contact residue as said antibody.

[0156] Radioactive isotopes of lead (Pb) are useful in diagnostic and therapeutic methods. Specific radioactive isotopes of lead that can be used in this invention include: 212 Pb and 203 Pb is one example.

[0157] Due to their combination of short path lengths and high linear energy transfer, alpha-particle emitters (radionuclides) cause less damage to surrounding tissues and have the potential for more specific tumor cell killing than beta-emitters. 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 can function as an in vivo generating agent for Bi, thereby 212 This effectively overcomes the short half-life of Bi (Yong and Brechbiel, Dalton Trans. 2001 June 21;40(23)6068-6076).

[0158] 203 Pb is useful as an imaging isotope. Therefore, 203 Antibodies bound to Pb-DOTAM may be useful for radioimmunoimaging (RII).

[0159] Generally, radioactive metals are used in chelated form. In certain embodiments 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. 2001 June 21;40(23)6068-6076; Chappell et al Nuclear Medicine and Biology, Vol.27, pp.93-100, 2000). Therefore, DOTAM is, 212 Pb and 203 It is particularly useful in combination with the aforementioned lead isotopes such as Pb.

[0160] In some embodiments, it may be preferable for the antibody to bind to Pb-DOTAM with a binding affinity Kd value of 100 pM, 50 pM, 20 pM, 10 pM, 5 pM, or 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. For example, the functional binding site may bind to the radiolabeled compound with a Kd of about 1 pM to 1 nM, for example, about 1 to 10 pM, 1 to 100 pM, 5 to 50 pM, 100 to 500 pM, or 500 pM to 1 nM.

[0161] In certain embodiments, the antibody further binds to Bi chelated with DOTAM. In some embodiments, it may be preferable for the antibody to bind to Bi-DOTAM (i.e., a chelate containing DOTAM complexed with bismuth, also referred herein as a "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. For example, the functional binding site may bind to the metal chelate with a Kd of about 1 pM to 1 nM, for example, about 1 to 10 pM, 1 to 100 pM, 5 to 50 pM, 100 to 500 pM, or 500 pM to 1 nM.

[0162] 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, such as the Kd ratio, may preferably be in the range of 0.1 to 10, for example, 1 to 10.

[0163] In one embodiment, the heavy chain variable region that forms part of the antigen-binding site for Pb-DOTAM may include at least one, two, or all three CDRs selected from (a) CDR-H1 containing the amino acid sequence of GFSLSTYSMS (SEQ ID NO: 1), (b) CDR-H2 containing the amino acid sequence of FIGSRGDTYYASWAKG (SEQ ID NO: 2), and (c) CDR-H3 containing the amino acid sequence of ERDPYGGGAYPPHL (SEQ ID NO: 3). The light chain variable region that forms part of the binding site for Pb-DOTAM may include at least one, two, or all three CDRs selected from (d) CDR-L1 containing the amino acid sequence of QSSHSVYSDNDLA (SEQ ID NO: 4), (e) CDR-L2 containing the amino acid sequence of QASKLAS (SEQ ID NO: 5), and (f) CDR-L3 containing the amino acid sequence of LGGYDDESDTYG (SEQ ID NO: 6).

[0164] In some embodiments, the antibody may comprise one or more CDR-H1, CDR-H2 and / or CDR-H3, or one or more 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. 1 to 6.

[0165] In some embodiments, antibodies may share the same contact residues as described herein, for example, these residues may be immutable. These residues may include: a) Heavy chain CDR2: Phe50, Asp56 and / or Tyr58, and optionally Gly52 and / or Arg54; b) Heavy chain CDR3: Glu95, Arg96, Asp97, Pro98, Tyr99, Ala100C and / or Tyr100D, and optionally Pro100E; c) Light chain CDR1:Tyr28 and / or Asp32; d) Light chain CDR3: Gly91, Tyr92, Asp93, Thr95c and / or Tyr96; e) Light chain CDR2: Gln50 (optional); Everything is numbered according to Kabat.

[0166] For example, in some embodiments, CDR-H2 may include the amino acid sequence FIGSRGDTYYASWAKG (SEQ ID NO: 2), or variants thereof having up to one, two, or three substitutions in SEQ ID NO: 2, where these substitutions do not include Phe 50, Asp 56 and / or Tyr 58, and optionally also do not include Gly 52 and / or Arg 54, all of which are numbered according to Kabat.

[0167] In some embodiments, CDR-H2 may be substituted at one or more positions as shown below. Here, and in the substitution table below, substitutions are based on germline residues (underlined) or by amino acids that are theoretically sterically compatible and also occur in the crystallization repertoire of that site. In some embodiments, the above residues may be fixed, and other residues may be substituted according to the table below: In other embodiments, substitution of any residue may be carried out according to the table below. JPEG2026071214000005.jpg111170

[0168] Optionally, CDR-H3 may include the amino acid sequence ERDPYGGGAYPPHL (SEQ ID NO: 3), or a variant thereof, having up to one, two, or three substitutions in SEQ ID NO: 3, where these substitutions do not include Glu95, Arg96, Asp97, or 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.

[0169] In certain embodiments, CDR-H3 may be substituted at one or more positions as 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 carried out according to the following table. JPEG2026071214000006.jpg97170

[0170] Optionally, CDR-L1 may contain the amino acid sequence QSSHSVYSDNDLA (SEQ ID NO: 4) or a variant thereof having up to one, two, or three substitutions in SEQ ID NO: 4, where these substitutions do not include Tyr28 and / or Asp32 (Kabat numbering).

[0171] In certain embodiments, CDR-L1 may be substituted at one or more positions as shown below. 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 carried out according to the table below. JPEG2026071214000007.jpg91170

[0172] Optionally, CDR-L3 may contain the amino acid sequence LGGYDDESDTYG (Sequence ID 6) or its variants having up to one, two, or three substitutions in SEQ ID NO: 6, excluding Gly91, Tyr92, Asp93, Thr95c, and / or Tyr96 (Kabat).

[0173] In certain embodiments, CDR-L3 may be substituted at the following positions, as shown below. (Many substitutions are possible as most residues are exposed to the solvent and not in contact with the antigen.) Also, 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 carried out according to the following table. JPEG2026071214000008.jpg131170

[0174] The antibody may optionally further include CDR-H1 or 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, optionally conservative substitutions.

[0175] Therefore, the heavy chain variable domain that forms part of the antigen-binding site for Pb-DOTAM is at least a) A heavy chain CDR2 containing the amino acid sequence FIGSRGDTYYASWAKG (SEQ ID NO: 2), or a variant thereof having up to 1, 2, or 3 substitutions in SEQ ID NO: 2, wherein these substitutions do not include Phe50, Asp56, and / or Tyr58, and optionally do not include Gly52, and / or Arg54, b) Heavy chain CDR3 comprising the amino acid sequence ERDPYGGGAYPPHL (SEQ ID NO: 3), or a variant thereof having up to 1, 2, or 3 substitutions in SEQ ID NO: 3, wherein these substitutions do not include Glu95, Arg96, Asp97, Pro98, optionally also omit Ala100C, Tyr100D and / or Pro100E, and / or optionally also omit Tyr99, and It may include.

[0176] In some embodiments, the heavy chain variable domain is optionally, c) A heavy chain CDR1 comprising the amino acid sequence GFSLSTYSMS (SEQ ID NO: 1), or a variant thereof having up to one, two, or three substitutions in SEQ ID NO: 1.

[0177] In another embodiment, the light chain variable domains that form part of the antigen-binding site for Pb-DOTAM are at least d) A light chain CDR1 comprising the amino acid sequence QSSHSVYSDNDLA (SEQ ID NO: 4), or a variant thereof having up to 1, 2, or 3 substitutions in SEQ ID NO: 4, wherein these substitutions do not include Tyr28 and Asp32, e) Light chain CDR3 comprising the amino acid sequence LGGYDDESDTYG (SEQ ID NO: 6), or a variant thereof having up to 1, 2, or 3 substitutions in SEQ ID NO: 6, wherein these substitutions do not include Gly91, Tyr92, Asp93, Thr95c, and Tyr96. Includes.

[0178] In some embodiments, the light chain variable domain is optionally, f) A light chain CDR2 comprising the amino acid sequence QASKLAS (SEQ ID NO: 5) or a variant thereof having at least one, two, or three substitutions in SEQ ID NO: 5, and optionally further comprising a light chain CDR2 that does not contain Gln50.

[0179] In any embodiment of the present invention, including a variant of the sequence containing the above-mentioned CDR (e.g., of a variable domain), the protein may be invariant in one or more of the above-mentioned CDR residues.

[0180] Optionally, the heavy chain variable domains that form part of the functional antigen-binding site for Pb-DOTAM (on the first antibody) include an amino acid sequence selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 9, or a variant thereof that includes 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. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity includes substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the binding site including the sequence retains the ability to bind to Pb-DOTAM with affinity preferably as described herein. The VH sequence may retain the above-mentioned invariant residues. 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, substitutions, insertions, or deletions occur in the region outside the CDR (i.e., at the FR). Optionally, the antibody includes the VH sequence of SEQ ID NO: 7 or SEQ ID NO: 9, which 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.

[0181] In further specific embodiments, the heavy chain variable domain that forms part of the functional antigen-binding site for Pb-DOTAM comprises the above amino acid sequence or variant sequence having an additional N-terminal amino acid, preferably an N-terminal amino acid from the original human germline sequence, i.e., an N-terminal amino acid corresponding to Q (glutamine) with respect to either SEQ ID NO: 7 or SEQ ID NO: 9. Other suitable residues at this position that enable the desired expression level of the construct can be identified by those skilled in the art. For example, in some embodiments, a suitable alternative amino acid may be another amino acid naturally present in the V germline gene at this position, e.g., E, K, R, S, T, A, L, or Y. In some embodiments, the alternative amino acid may be E. In other embodiments, a suitable alternative amino acid may be a conserved substitution such as D, N, or V.

[0182] Accordingly, in one embodiment, the heavy chain variable domain that forms part of the functional antigen-binding site for Pb-DOTAM (on the first antibody) includes the amino acid sequence of SEQ ID NO: 7, or a variant thereof having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO: 7, for example the above variant, and further includes an additional N-terminal residue. In another embodiment, the heavy chain variable domain that forms part of the functional antigen-binding site for Pb-DOTAM (on the first antibody) includes the amino acid sequence of SEQ ID NO: 9, or a variant thereof having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO: 9, for example the above variant, and further includes an additional N-terminal residue. In one embodiment, the additional N-terminal amino acid may be selected from the group consisting of Q, E, K, R, S, T, A, L, Y, D, N, or V, preferably Q. In another embodiment, the additional N-terminal amino acid may be selected from the group consisting of Q, E, K, R, S, T, A, L, or Y, or from the group consisting of Q or E. In yet another embodiment, the additional N-terminal amino acid may be selected from the group consisting of Q, D, N, or V.

[0183] In another embodiment, the heavy chain variable domain that forms part of the functional antigen-binding site for Pb-DOTAM (on the first antibody) includes or comprises the amino acid sequence of SEQ ID NO: 143, and includes post-translational modifications of that sequence. Alternatively, it may include variants thereof that have at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO: 143. In some embodiments, the N-terminal Q residue of SEQ ID NO: 143 may remain unchanged or may be replaced with another amino acid that results in a desired level of protein expression that can be identified by those skilled in the art. In one embodiment, the N-terminal Q residue may be replaced with a residue selected from the group consisting of E, K, R, S, T, A, L, Y, D, N, and V. In another embodiment, the N-terminal Q residue may be replaced with a residue selected from the group consisting of E, K, R, S, T, A, L, and Y. In another embodiment, the N-terminal Q residue may be replaced with E. In another embodiment, the N-terminal Q residue may be replaced with a residue selected from the group consisting of D, N, and V. In another embodiment, the heavy chain variable domain may include a variant of SEQ ID NO: 143 having one or more substitutions but no deletions; therefore, in one embodiment, it may differ from SEQ ID NO: 143 by only one or more substitutions and may have at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO: 143. The VH sequence may further retain invariant residues in the CDR as described above. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 143. In certain embodiments, mutations (e.g., substitutions, insertions, or deletions) occur in the outer region of the CDR (i.e., FR).

[0184] Optionally, the light chain variable domains that form part of the functional antigen-binding site for Pb-DOTAM (on the second antibody) include the amino acid sequence of SEQ ID NO: 8, or variants thereof that have at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO: 8. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity includes substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the anti-Pb-DOTAM binding site including the sequence retains the ability to bind to Pb-DOTAM with the affinity preferably described herein. The VL sequence may retain the above-mentioned invariant residues. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 8. In certain embodiments, substitutions, insertions, or deletions occur in the region outside the CDR (i.e., within the FR). Optionally, the anti-Pb-DOTAM antibody includes the VL sequence of SEQ ID NO: 8, which includes post-translational modifications of that sequence. In certain embodiments, the VL includes one, two, or three HVRs 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.

[0185] In further specific embodiments, the light chain variable domains that form part of the functional antigen-binding site for Pb-DOTAM include the above amino acid sequence or variant sequence having an additional N-terminal amino acid, preferably an N-terminal amino acid from the original human germline sequence, i.e., the N-terminal amino acid corresponding to A with respect to SEQ ID NO: 8. Other suitable residues at this position that enable the desired expression level of the construct can be identified by those skilled in the art. For example, in some embodiments, a suitable alternative amino acid may be another amino acid naturally present in the V germline gene at this position, e.g., D, N, E, Q, S, V, or L. In other embodiments, a suitable alternative amino acid may be a conserved substitution such as T, Y, K, or R.

[0186] Accordingly, in one embodiment, the light chain variable domain that forms part of the functional antigen-binding site for Pb-DOTAM (on the second antibody) includes the amino acid sequence of SEQ ID NO: 8, or a variant thereof having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO: 8, for example the above variant, and further includes an additional N-terminal residue. In one embodiment, the additional N-terminal amino acid may be selected from the group consisting of D, N, E, Q, S, A, V, L, T, Y, K, and R, preferably A or S. In another embodiment, the additional N-terminal amino acid may be selected from the group consisting of D, N, E, Q, S, A, V, and L or the group consisting of A and S. In yet another embodiment, the additional N-terminal amino acid may be selected from the group consisting of A, T, Y, K, and R.

[0187] In another embodiment, the light chain variable domain that forms part of the functional antigen-binding site for Pb-DOTAM (on the second antibody) includes or comprises the amino acid sequence of SEQ ID NO: 144, and includes post-translational modifications of that sequence. Alternatively, the light chain variable domain may include a variant thereof that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 144, and the N-terminal A residue of SEQ ID NO: 144 remains unchanged or is substituted with another suitable amino acid that can be identified by those skilled in the art. In one embodiment, the N-terminal A residue may be substituted with a residue selected from the group consisting of D, N, E, Q, S, A, V, L, T, Y, K, and R. In another embodiment, the N-terminal A residue may be substituted with a residue selected from the group consisting of D, N, E, Q, S, V, and L. In another embodiment, the N-terminal A residue may be substituted with S. In another embodiment, the N-terminal A residue may be substituted with a residue selected from the group consisting of D, N, and V. In another embodiment, the light chain variable domain may include a variant of SEQ ID NO: 144 having one or more substitutions but no deletions; therefore, in one embodiment, it may differ from SEQ ID NO: 144 by only one or more substitutions and may have at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO: 144. The VL sequence may further retain invariant residues in the CDR as described above. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 144. In certain embodiments, mutations (e.g., substitutions, insertions, or deletions) occur in the outer region of the CDR (i.e., FR).

[0188] Embodiments relating to the heavy chain variable region and the light chain variable region are explicitly intended to be used in combination. Thus, a functional antigen-binding site for Pb-DOTAM may be formed from the heavy chain variable region and the light chain variable region as defined above on the first antibody and the second antibody, respectively.

[0189] Optionally, the antigen-binding site specific to the Pb-DOTAM chelate may be formed from 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 their variants as defined above (including variants having the N-terminal extension considered above), and a light chain variable domain containing the amino acid sequence of SEQ ID NO: 8, or its variants as defined above (including variants having the N-terminal extension considered 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 its variant, and a light chain variable domain containing the amino acid sequence of SEQ ID NO: 8 or its variant (including post-translational modifications of these sequences). In another embodiment, it may include a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 9 or its variant, and a light chain variable domain containing the amino acid sequence of SEQ ID NO: 8 or its variant (including post-translational modifications of these sequences).

[0190] In another embodiment, the antigen-binding site specific to the Pb-DOTAM chelate may be formed from a heavy chain variable domain containing the amino acid sequence of 143, or a variant thereof as defined above, and a light chain variable domain containing the amino acid sequence of SEQ ID NO: 144, or a variant thereof as defined above.

[0191] In any of the embodiments described above, the light chain variable region and the heavy chain variable region forming the anti-Pb-DOTAM binding site may be humanized. In one embodiment, the light chain variable region and the heavy chain variable region comprise the CDR of any of the embodiments described above and further comprise an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework. In another embodiment, the light chain and / or heavy chain variable region comprises the CDR of any of the embodiments described above and further comprise framework regions derived from vk 1 39 and / or vh 2 26. In vk 1 39, in some embodiments, the reverse mutation may not be present. In vh 2 26, the germline Ala49 residue can be reversed to Gly49.

[0192] Exemplary antigen-binding sites of E.CEA In another specific embodiment of the present invention, which can be combined with the embodiments described above, the target antigen to which the first and / or second antibody binds may be CEA (carcinoembryonic antigen). Antibodies induced against CEA include T84.66 and its humanized and chimeric versions, e.g., T84.66-LCHA, CH1A1a as described in International Publication No. 2016 / 075278 and / or International Publication No. 2017 / 055389, anti-CEA antibodies as described in International Publication No. 2012 / 117002 and International Publication No. 2014 / 131712, and CEA hMN-14 or rabetsuzimab (e.g., as described in U.S. Patent No. 6,676,924 and U.S. Patent No. 5,874,540). Another exemplary antibody against CEA is A5B7 (e.g., described in MJBanfield et al, Proteins 1997, 29(2), 161-171), or the humanized antibody derived from mouse A5B7 described in International Publication No. 92 / 01059 and International Publication No. 2007 / 071422. See also concurrent pending application PCT / EP2020 / 067582. An example of a humanized version of A5B7 is A5H1EL1(G54A). Further exemplary antibodies against CEA are MFE23 and its humanized version described in U.S. Patent No. 7,626,011 and / or concurrent pending application PCT / EP2020 / 067582. A further example of an anti-CEA antibody is 28A9. Any of these or their antigen-binding fragments may be used in the present invention to form a CEA-binding moiety.

[0193] Optionally, the antigen-binding moiety that binds to CEA may have a K content of 1 nM or less, 500 pM or less, 200 pM or less, or 100 pM or less for monovalent binding. D They can be joined by value.

[0194] In some embodiments, the first and / or second antibody may bind to the CH1A1a epitope, A5B7 epitope, MFE23 epitope, T84.66 epitope, or 28A9 epitope of CEA.

[0195] In some embodiments, at least one of the first antibody and the second antibody binds to a CEA epitope that is not present on soluble CEA (sCEA). Soluble CEA is the portion of the CEA molecule that is cleaved by GPI phospholipase and released into the bloodstream. An example of an epitope not found on soluble CEA is the CH1A1A epitope. Optionally, one of the first and / or second antibodies binds to an epitope not present on soluble CEA, and the other binds to an epitope present on soluble CEA.

[0196] The CH1A1a epitopes and their parental mouse antibody PR1A3 are described in International Publication No. 2012 / 117002A1 and Durbin H. et al., Proc. Natl. Scad. Sci. USA, 91:4313-4317, 1994. Antibodies that bind to the CH1A1a epitope bind to the conformational epitopes in the B3 domain and GPI anchor of the CEA molecule. In one embodiment, the antibody binds to the same epitope as the CH1A1a antibody having VH in SEQ ID NO: 25 and VL in SEQ ID NO: 26 as described herein. The A5B7 epitope is described in concurrent pending application PCT / EP2020 / 067582. Antibodies that bind to the A5B7 epitope bind to the A2 domain of CEA, i.e., the domain containing the amino acid of SEQ ID NO: 141: PKPFITSNNSNPVEDEDAVALTCEPEIQNTTYLWWVNNQSLPVSPRLQLSNDNRTLTLLSVTRNDVGP YECGIQNKLSVDHSDPVILN(Sequence ID 141)

[0197] In one embodiment, the antibody binds to the same epitope as the A5B7 antibody having VH of SEQ ID NO: 49 and VL of SEQ ID NO: 50 as described herein.

[0198] In one embodiment, the antibody binds to the same epitope as T84.66 described in International Publication No. 2016 / 075278. The antibody may bind to the same epitope as the antibodies having VH in SEQ ID NO: 17 and VL in SEQ ID NO: 18 as described herein.

[0199] The MFE23 epitope is described in concurrent pending application PCT / EP2020 / 067582. The antibody that binds to the MFE23 epitope binds to the A1 domain of CEA, i.e., the domain containing the amino acids of SEQ ID NO: 142: PKPSISSNNSKPVEDKDAVAFTCEPETQDATYLWWVNNQSLPVSPRLQLSNGNRTLTLFNVTRNDTAS YKCETQNPVSARRSDSVILN (Sequence ID 142).

[0200] In one embodiment, the antibody may bind to the same epitopes as the antibodies having the VH domain of SEQ ID NO: 127 and the VL domain of SEQ ID NO: 128 as described herein.

[0201] In some embodiments, the first and / or second antibody may bind to the same CEA epitopes as the antibodies provided herein, for example, P1AD8749, P1AD8592, P1AE4956, P1AE4957, P1AF0709, P1AF0298, P1AF0710, or P1AF0711.

[0202] In some embodiments, the first antibody and the second antibody bind to the same CEA epitope. For example, both the first antibody and the second antibody may bind to the CH1A1a epitope, the A5B7 epitope, the MFE23 epitope, the T84.66 epitope, or the 28A9 epitope.

[0203] In some embodiments, both the first and second antibodies may have a CEA-binding sequence derived from CH1A1A (i.e., the CDR and / or VH / VL domain); or both the first and second antibodies may have a CEA-binding sequence derived from A5B7 or a humanized version thereof; or both the first and second antibodies may have a CEA-binding sequence derived from T84.66 or a humanized version thereof; or both the first and second antibodies may have a CEA-binding sequence derived from MFE23 or a humanized version thereof; or both the first and second antibodies may have a CEA-binding sequence derived from 28A9 or a humanized version thereof. Exemplary sequences are disclosed herein.

[0204] In other embodiments, the first and second antibodies bind to different epitopes of CEA. For example, i) one antibody may bind to the CH1A1A epitope, and the other antibody may bind to the A5B7 epitope, T84.66 epitope, MFE23 epitope, or 28A9 epitope; ii) one antibody may bind to the A5B7 epitope, and the other antibody may bind to the CH1A1A epitope, T84.66 epitope, MFE23 epitope, or 28A9 epitope; iii) one antibody may bind to the MFE23 epitope, and the other antibody may bind to the CH1A1A iv) One antibody may bind to the T84.66 epitope, the A5B7 epitope, the T84.66 epitope, or the 28A9 epitope; or v) One antibody may bind to the 28A9 epitope, and the other antibody may bind to the CH1A1A epitope, the A5B7 epitope, the MFE23 epitope, or the 28A9 epitope; or v) One antibody may bind to the 28A9 epitope, and the other antibody may bind to the CH1A1a epitope, the A5B7 epitope, the MFE23 epitope, or the T84.66 epitope.

[0205] In some embodiments, i) one antibody may have a CEA-binding sequence derived from CH1A1A (i.e., a CDR or VH / VL domain), and the other antibody may have a CEA-binding sequence derived from A5B7 or its humanized version, T84.66 or its humanized version, MFE23 or its humanized version, or 28A9 or its humanized version; ii) one antibody may have a CEA-binding sequence derived from A5B7 or its humanized version, and the other antibody may have a CEA-binding sequence derived from CH1A1A, T84.66 or its humanized version, MFE23 or its humanized version, or 28A9 or its humanized version; iii) one antibody may have a CEA-binding sequence derived from MFE23 or its humanized version iv) One antibody may have a CEA-binding sequence derived from CH1A1A, A5B7 or its humanized version, T84.66 or its humanized version, or 28A9 or its humanized version;v) One antibody may have a CEA-binding sequence derived from T84.66 or its humanized version, and the other antibody may have a CEA-binding sequence derived from CH1A1A, A5B7 or its humanized version, MFE23 or its humanized version, or 28A9 or its humanized version;v) One antibody may have a CEA-binding sequence derived from 28A9 or its humanized version, and the other antibody may have a CEA-binding sequence derived from CH1A1A, A5B7 or its humanized version, T84.66 or its humanized version, or MFE23 or its humanized version.

[0206] In a particular embodiment, one antibody may bind to the CH1A1A epitope and the other antibody may bind to the A5B7 epitope. The first antibody may have a CEA-binding sequence derived from antibody CH1A1A and the second antibody may have a CEA-binding sequence derived from A5B7 (including its humanized version), or the first antibody may have a CEA-binding sequence derived from antibody A5B7 (including its humanized version) and the second antibody may have a CEA-binding sequence derived from CH1A1A.

[0207] In another specific embodiment, one antibody may bind to the CH1A1A epitope and the other antibody may bind to the T84.66 epitope. The first antibody may have a CEA-binding sequence derived from antibody CH1A1A, and the second antibody may have a CEA-binding sequence derived from T84.66 (including its humanized version), or the first antibody may have a CEA-binding sequence derived from antibody T84.66 (including its humanized version) and the second antibody may have a CEA-binding sequence derived from CH1A1A. In some embodiments, the first antibody may bind to the T84.66 epitope and / or have an antigen-binding site as described in (i) below, and the second antibody may bind to the CH1A1A epitope and / or have an antigen-binding site as described in (ii) below.

[0208] Exemplary CEA-binding sequences i) to v) are disclosed below. These provide examples of CEA-binding sequences derived from i) T84.66, ii) CH1A1A, iii) A5B7, iv) 28A9, and v) MFE23 (or its humanized version).

[0209] i) In one embodiment, 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.

[0210] Optionally, 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.

[0211] Optionally, 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.

[0212] Optionally, the antigen-binding site that binds to CEA comprises 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 a VL domain containing at least one, at least two, or all three VL CDR sequences selected from (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 14, (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. Includes.

[0213] 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.

[0214] In any of the embodiments described above, the multispecific antibody can be humanized. In one embodiment, the anti-CEA antigen binding site comprises the same CDR as in any of the embodiments above, and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0215] 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 with 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 includes 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 above affinity. 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 occur in the region outside the HVR (i.e., at FR). Optionally, the antigen-binding site that binds to CEA includes the VH sequence of SEQ ID NO: 17, including post-translational modifications of the 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.

[0216] 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 with respect 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 includes 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 above affinity. 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 occur in the region outside the HVR (i.e., within the FR). Optionally, the antigen-binding site for CEA includes the VL sequence of SEQ ID NO: 18, including post-translational modifications of its 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.

[0217] In another embodiment, the antigen-binding site that binds to CEA includes VH in any of the embodiments provided above, and VL in any of the embodiments provided above. In one embodiment, the antibody includes the VH sequence and VL sequence of SEQ ID NO: 17 and SEQ ID NO: 18, respectively, including post-translational modifications of those sequences.

[0218] ii) In a further specific embodiment, 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: 19, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 20, (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 21, (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 22, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 23, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 24.

[0219] Optionally, 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: 19, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 20, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 21.

[0220] Optionally, 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: 22, (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 23, and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 24.

[0221] Optionally, the antigen-binding site that binds to CEA includes a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (a) (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 19, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 20, and (iii) CDR-H3 comprising the amino acid sequence selected from SEQ ID NO: 21, and a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from (b) (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 24.

[0222] In another embodiment, the antigen-binding sites that bind to CEA include (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 19, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 20, (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 21, (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 22, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 23, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 24.

[0223] In any of the embodiments described above, the multispecific antibody can be humanized. In one embodiment, the anti-CEA antigen binding site comprises the same CDR as in any of the embodiments above, and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0224] 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 with the amino acid sequence of SEQ ID NO: 25. In a particular embodiment, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity includes 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 above affinity. In a particular embodiment, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 25. In a particular embodiment, the substitutions, insertions, or deletions occur in the region outside the HVR (i.e., at FR). Optionally, the antigen-binding site that binds to CEA includes the VH sequence of SEQ ID NO: 25, including post-translational modifications of the 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: 19, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 20, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 21.

[0225] 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 with respect to the amino acid sequence of SEQ ID NO: 26. In a particular embodiment, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity includes 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 above affinity. In a particular embodiment, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 26. In a particular embodiment, the substitutions, insertions, or deletions occur in the region outside the HVR (i.e., within the FR). Optionally, the antigen-binding site for CEA includes the VL sequence of SEQ ID NO: 26, including post-translational modifications of its 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: 22, (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 23, and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 24.

[0226] In another embodiment, the antigen-binding site that binds to CEA includes VH in any of the embodiments provided above, and VL in any of the embodiments provided above. In one embodiment, the antibody includes the VH sequence and VL sequence of SEQ ID NO: 25 and SEQ ID NO: 26, respectively, including post-translational modifications of those sequences.

[0227] iii) In further specific embodiments, 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: 43, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 44, (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 45, (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 46, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 47, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 48. In some embodiments, CDR-H1 may have the sequence GFTFTDYYMN (SEQ ID NO: 149).

[0228] Optionally, 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: 43, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 44, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 45. In some embodiments, CDR-H1 may have the sequence GFTFTDYYMN (SEQ ID NO: 149).

[0229] Optionally, 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: 46, (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 47, and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 48.

[0230] Optionally, the antigen-binding site that binds to CEA includes a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (a) (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 43, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 44, and (iii) CDR-H3 comprising the amino acid sequence selected from SEQ ID NO: 45, and a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from (b) (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 46, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 47, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 48. In some embodiments, CDR-H1 may have the sequence GFTFTDYYMN (SEQ ID NO: 149).

[0231] In another embodiment, the antigen-binding site that binds to CEA includes (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 43, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 44, (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 45, (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 46, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 47, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 48. In some embodiments, CDR-H1 may have the sequence GFTFTDYYMN (SEQ ID NO: 149).

[0232] In any of the embodiments described above, the multispecific antibody can be humanized. In one embodiment, the anti-CEA antigen binding site comprises the same CDR as in any of the embodiments above, and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0233] 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 with the amino acid sequence of SEQ ID NO: 49. In a particular embodiment, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity includes 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 above affinity. In a particular embodiment, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 49. In a particular embodiment, the substitutions, insertions, or deletions occur in the region outside the HVR (i.e., at FR). Optionally, the antigen-binding site that binds to CEA includes the VH sequence of SEQ ID NO: 49, including post-translational modifications of the 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: 43 or the sequence GFTFTDYYMN (SEQ ID NO: 149), (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 44, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 45.

[0234] 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 with respect to the amino acid sequence of SEQ ID NO: 50. In a particular embodiment, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity includes 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 above affinity. In a particular embodiment, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 50. In a particular embodiment, the substitutions, insertions, or deletions occur in the region outside the HVR (i.e., within the FR). Optionally, the antigen-binding site for CEA includes the VL sequence of SEQ ID NO: 50, including post-translational modifications of its 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: 46, (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 47, and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 48.

[0235] In another embodiment, the antigen-binding site that binds to CEA includes VH in any of the embodiments provided above, and VL in any of the embodiments provided above. In one embodiment, the antibody includes the VH sequence and VL sequence of SEQ ID NO: 49 and SEQ ID NO: 50, respectively, including post-translational modifications of those sequences.

[0236] iv) In further specific embodiments, 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: 59, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 60, (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 61, (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 62, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 63, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 64.

[0237] Optionally, 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: 59, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 60, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 61.

[0238] Optionally, 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: 62, (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 63, and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 64.

[0239] Optionally, the antigen-binding site that binds to CEA includes a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (a) (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 59, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 60, and (iii) CDR-H3 comprising the amino acid sequence selected from SEQ ID NO: 61, and a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from (b) (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 62, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 63, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 64.

[0240] In another embodiment, the antigen-binding sites that bind to CEA include (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 59, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 60, (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 61, (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 62, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 63, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 64.

[0241] In any of the embodiments described above, the multispecific antibody can be humanized. In one embodiment, the anti-CEA antigen binding site comprises the same CDR as in any of the embodiments above, and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0242] 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 with the amino acid sequence of SEQ ID NO: 65. In a particular embodiment, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity includes 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 above affinity. In a particular embodiment, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 65. In a particular embodiment, the substitutions, insertions, or deletions occur in the region outside the HVR (i.e., at FR). Optionally, the antigen-binding site that binds to CEA includes the VH sequence of SEQ ID NO: 65, including post-translational modifications of the 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: 59, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 60, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 61.

[0243] 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 with respect to the amino acid sequence of SEQ ID NO: 66. In a particular embodiment, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity includes 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 above affinity. In a particular embodiment, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 66. In a particular embodiment, the substitutions, insertions, or deletions occur in the region outside the HVR (i.e., within the FR). Optionally, the antigen-binding site for CEA includes the VL sequence of SEQ ID NO: 66, including post-translational modifications of its 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: 62, (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 63, and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 64.

[0244] In another embodiment, the antigen-binding site that binds to CEA includes VH in any of the embodiments provided above, and VL in any of the embodiments provided above. In one embodiment, the antibody includes the VH sequence and VL sequence of SEQ ID NO: 65 and SEQ ID NO: 66, respectively, including post-translational modifications of those sequences.

[0245] v) In further specific embodiments, 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: 116, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 117 or 118, (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 119, (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 120, 121 or 122, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 123, 124 or 125, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 126.

[0246] The antigen-binding site that binds to CEA is, VH CDR sequences: (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 116; (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 117 or 118; and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 119; and / or The VL CDR sequence may include (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 120, 121, or 122; (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 123, 124, or 125; and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 126.

[0247] In one embodiment, the antigen-binding site for CEA includes a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 127, or (more preferably) an amino acid sequence selected from SEQ ID NOs: 129, 130, 131, 132, 133, or 134, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 128, or (more preferably) an amino acid sequence selected from SEQ ID NOs: 135, 136, 137, 138, 139, or 140.

[0248] In any of the embodiments described above, the multispecific antibody can be humanized. In one embodiment, the anti-CEA antigen binding site comprises the same CDR as in any of the embodiments above, and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0249] In certain embodiments, the antigen-binding domain that can bind to CEA is (a) A VH domain containing the amino acid sequence of SEQ ID NO: 129 and a VL domain containing the amino acid sequence of SEQ ID NO: 139, or (b) A VH domain containing the amino acid sequence of SEQ ID NO: 133 and a VL domain containing the amino acid sequence of SEQ ID NO: 139, or (c) A VH domain containing the amino acid sequence of SEQ ID NO: 130 and a VL domain containing the amino acid sequence of SEQ ID NO: 139, or (d) A VH domain containing the amino acid sequence of SEQ ID NO: 134 and a VL domain containing the amino acid sequence of SEQ ID NO: 138, or (e) A VH domain containing the amino acid sequence of SEQ ID NO: 133 and a VL domain containing the amino acid sequence of SEQ ID NO: 138, or (f) A VH domain containing the amino acid sequence of SEQ ID NO: 131 and a VL domain containing the amino acid sequence of SEQ ID NO: 138, or (g) VH domain containing the amino acid sequence of SEQ ID NO: 129 and VL domain containing the amino acid sequence of SEQ ID NO: 138 Includes.

[0250] 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 with respect to the amino acid sequences mentioned in a) to g) above. In a particular embodiment, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity includes 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 a particular embodiment, the substitutions, insertions, or deletions occur in the region outside the HVR (i.e., at FR).

[0251] 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 with respect to the amino acid sequences mentioned in a) to g) above. In a particular embodiment, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity includes 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 a particular embodiment, the substitutions, insertions, or deletions occur in the region outside the HVR (i.e., within the FR).

[0252] In another embodiment, the antigen-binding site that binds to CEA includes VH in any of the embodiments provided above, and VL in any of the embodiments provided above.

[0253] F. Exemplary antigen-binding sites for other targets In another specific embodiment of the present invention, which can be combined with the above embodiments (e.g., the binding site of DOTA or DOTAM), the target antigen to which the first and / or second antibody is bound may be GPRC5D or FAP.

[0254] Optionally, the antigen-binding moiety that binds to GPRC5D or FAP has a K content of 1 nM or less, 500 pM or less, 200 pM or less, or 100 pM or less for monovalent binding. D They can be joined by value.

[0255] An example of a GPRC5D binding sequence is shown below.

[0256] In one embodiment, the antigen-binding site that binds to GPRC5D 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: 67, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 68, (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 69, (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 70, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 71, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 72.

[0257] Optionally, the antigen-binding site that binds to GPRC5D 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: 67, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 68, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 69.

[0258] Optionally, the antigen-binding site that binds to GPRC5D 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: 70, (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 71, and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 72.

[0259] Optionally, the antigen-binding site for GPRC5D includes a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (a) (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 67, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 68, and (iii) CDR-H3 comprising the amino acid sequence selected from SEQ ID NO: 69, and a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from (b) (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 70, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 71, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 72.

[0260] In another embodiment, the antigen-binding sites that bind to GPRC5D include (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 67, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 68, (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 69, (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 70, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 71, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 72.

[0261] In any of the embodiments described above, the multispecific antibody can be humanized. In one embodiment, the anti-GPRC5D antigen binding site comprises the same CDR as in any of the embodiments above, and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0262] In another embodiment, the antigen-binding site that binds to GPRC5D 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 with the amino acid sequence of SEQ ID NO: 73. In a particular embodiment, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity includes 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 GPRC5D with the above affinity. In a particular embodiment, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 73. In a particular embodiment, the substitutions, insertions, or deletions occur in the region outside the HVR (i.e., at FR). Optionally, the antigen-binding site that binds to GPRC5D includes the VH sequence of SEQ ID NO: 73, including post-translational modifications of the 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: 67, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 68, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 69.

[0263] In another embodiment, the antigen-binding site that binds to GPRC5D includes a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 74. In a particular embodiment, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity includes 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 GPRC5D with the above affinity. In a particular embodiment, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 74. In a particular embodiment, the substitutions, insertions, or deletions occur in the region outside the HVR (i.e., within the FR). Optionally, the antigen-binding site for GPRC5D includes the VL sequence of SEQ ID NO: 74, including post-translational modifications of its sequence. In certain embodiments, the VL includes one, two, or three HVRs selected from (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 70, (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 71, and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 72.

[0264] In another embodiment, the antigen-binding site that binds to GPRC5D includes VH in any of the embodiments provided above, and VL in any of the embodiments provided above. In one embodiment, the antibody includes the VH sequence and VL sequence of SEQ ID NO: 73 and SEQ ID NO: 74, respectively, including post-translational modifications of those sequences.

[0265] An example of an FAP binding sequence is shown below.

[0266] In one embodiment, the antigen-binding site that binds to FAP 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: 75, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 76, (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 77, (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 78, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 79, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 80.

[0267] Optionally, the antigen-binding site that binds to FAP 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: 75, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 76, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 77.

[0268] Optionally, the antigen-binding site that binds to FAP 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: 78, (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 79, and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 80.

[0269] Optionally, the antigen-binding site that binds to FAP includes a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (a) (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 75, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 76, and (iii) CDR-H3 comprising the amino acid sequence selected from SEQ ID NO: 77, and a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from (b) (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 78, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 79, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 80.

[0270] In another embodiment, the antigen-binding sites that bind to FAP include (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 75, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 76, (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 77, (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 78, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 79, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 80.

[0271] In any of the embodiments described above, the multispecific antibody can be humanized. In one embodiment, the anti-FAP antigen binding site comprises the same CDR as in any of the embodiments above, and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0272] In another embodiment, the antigen-binding site that binds to FAP 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 with the amino acid sequence of SEQ ID NO: 81. In a particular embodiment, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity includes 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 FAP with the above affinity. In a particular embodiment, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 81. In a particular embodiment, the substitutions, insertions, or deletions occur in the region outside the HVR (i.e., at FR). Optionally, the antigen-binding site that binds to FAP includes the VH sequence of SEQ ID NO: 81, including post-translational modifications of the 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: 75, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 76, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 77.

[0273] In another embodiment, the antigen-binding site that binds to FAP includes a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 82. In a particular embodiment, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity includes 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 FAP with the above affinity. In a particular embodiment, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 82. In a particular embodiment, the substitutions, insertions, or deletions occur in the region outside the HVR (i.e., within the FR). Optionally, the antigen-binding site for FAP includes the VL sequence of SEQ ID NO: 82, including post-translational modifications of its sequence. In certain embodiments, the VL includes one, two, or three HVRs selected from (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 78, (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 79, and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 80.

[0274] In another embodiment, the antigen-binding site that binds to FAP includes VH in any of the embodiments provided above, and VL in any of the embodiments provided above. In one embodiment, the antibody includes the VH sequence and VL sequence of SEQ ID NO: 81 and SEQ ID NO: 82, respectively, including post-translational modifications of those sequences.

[0275] G. Antibody Format As described above, the present invention is i) A first antibody comprising an antigen-binding portion that binds to an antigen expressed on the surface of a target cell, and further comprising a VH domain of the antigen-binding site for a radiolabeled compound, but lacking a VL domain of the antigen-binding site for a radiolabeled compound, ii) A second antibody comprising an antigen-binding portion that binds to an antigen expressed on the surface of a target cell, further comprising a VL domain of the antigen-binding site for a radiolabeled compound, but lacking a VH domain of the antigen-binding site for a radiolabeled compound. Includes, This invention relates to a set of antibodies in which the VH domain of the first antibody and the VL domain of the second antibody are both capable of forming a functional antigen-binding site for a radiolabeled compound.

[0276] In some embodiments, the antigen-binding portion may be an antibody fragment such as Fv, Fab, cross-Fab, Fab', Fab'-SH, F(ab')2; a diabody; a linear antibody; a single-chain antibody molecule (e.g., scFv or scFab); or a single-domain antibody (dAb) such as VHH; or a non-antibody-binding skeleton such as DARPin (designed ankyrin repeat protein); an affibody; Sso7d; a monobody; or an antikalin.

[0277] In embodiments of the present invention, the first antibody and the second antibody may each contain an Fc domain. The presence of the Fc region offers advantages 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 smaller fragments.

[0278] In certain embodiments, the Fc domain is an IgG Fc domain. In specific embodiments, the Fc domain is an IgG1 Fc domain. In another specific embodiment, the Fc domain is an IgG4 Fc domain. In yet another specific embodiment, the Fc domain is an IgG4 Fc domain containing the amino acid substitution S228P (Kabat numbering). In certain embodiments, the Fc domain is a human Fc domain.

[0279] In some embodiments, it may be preferable to manipulate the Fc region to reduce or eliminate effector function. This may involve substitution of one or more 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 substitution of Pro 329 with Gly, substitution of Leu 234 with Ala, and / or substitution of Leu 235 with Ala (numbering follows the EU index).

[0280] In some embodiments, when the first antibody and the second antibody associate, they preferably form an antibody complex that is monovalent to the radiolabeled compound, i.e., the two associated antibodies provide a monovalent binding site to the radiolabeled compound. Thus, the first antibody may contain only one VH domain of the antigen-binding site to the radiolabeled compound, and the second antibody may contain only one VL domain of the antigen-binding site to the radiolabeled compound, and as a result, they together form only one complete functional binding site to the radiolabeled compound.

[0281] Fusion may occur directly or indirectly, for example, via a peptide linker. In some embodiments, fusion may be via a linker. For example, the Fc region may be fused to the antigen-binding region via a hinge region or another suitable linker. Similarly, the connection between the VL or VH domain of the antigen-binding site for a radiolabeled compound and the rest of the antibody structure may be via a linker. In some embodiments, the linker may be a peptide of 2 to 20 amino acids. In other embodiments, the linker may be a peptide of at least 5 or at least 10 amino acids, for example 5-100, 5-70, 5-60 or 5-50; or 10-100, 10-70, 10-60 or 10-50, for example 25-50 amino acids. In some embodiments, the linker may preferably be 15-30 amino acid long, for example 15-25, for example 16, 17, 18, 19, 20, 21, 22, 23 or 24 amino acid long. The linker may be a rigid linker or a flexible linker. In some embodiments, the linker is a flexible linker containing or consisting of Thr, Ser, Gly and / or Ala residues. For example, the linker may contain or consist of Gly and Ser residues. In some embodiments, the linker may have a repeating motif such as (Gly-Gly-Gly-Gly-Ser)n, where n is, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the peptide linker is (GxS)n or (GxS)nGm, where G = glycine, S = serine, and (x=3, n=3, 4, 5, or 6, and m=0, 1, 2, or 3) or (x=4, n=2, 3, 4, or 5, and m=0, 1, 2, or 3), for example, x=4 and n=2 or 3, for example, x=4, n=2. Other suitable peptide linkers include, for example, (G4S)n, (SG4)n, (G4S)n, or G4(SG4)n peptide linkers, where "n" is generally a number from 1 to 10, typically from 2 to 4.In another embodiment, the peptide linker is (GxS)n or (GxS)nGm, where G = glycine, S = serine, and (x = 3, n = 3, 4, 5 or 6, and m = 0, 1, 2 or 3) or (x = 4, n = 2, 3, 4 or 5, and m = 0, 1, 2 or 3), for example, x = 4 and n = 2 or 3, for example, x = 4, n = 2. In some embodiments, the linker may be or include the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 31). In another embodiment, the linker may be or include GGGGSGGGGSGGGGSGGSGG (SEQ ID NO: 150), GGGGSGGGGSGGGGSGGSGGS (SEQ ID NO: 152), or GGGGSGGGGSGGGGSGGSGGG (SEQ ID NO: 151). Another exemplary peptide linker is EPKSC(D)-(G4S)2. Furthermore, when the antigen-binding portion fuses to the N-terminus of the Fc domain subunit, it may be fused with or without an additional peptide linker via the immunoglobulin hinge region or a portion thereof. Other linkers may be used and can be identified by those skilled in the art.

[0282] As discussed above, the inventors have determined that in a peptide linker consisting of y amino acids, the Ser at the y position (i.e., the Ser as the last / C-terminal amino acid of the linker) can induce glycosylation of the y+2 amino acids (i.e., amino acids located two residues from the last amino acid of the linker toward the C-terminus) depending on the properties of these y+2 amino acids. Therefore, it may be preferable that the last serine residue of the linker is located at the y-2 or y-3 position (i.e., the last serine residue of the linker is located at an amino acid two or three positions from the last amino acid of the linker toward the N-terminus). Suitable linkers are any of the novel linkers described herein. In some specific embodiments, the linker may consist of y consecutive amino acid residues selected from the group consisting of Gly and Ser, for example, y = 5-100, 5-70, 5-60, 5-50, 10-100, 10-70, 10-60, or 10-50, for example 15-31 or 15-30, for example 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, where the last serine is at position y-2 or y-3. (Therefore, serine may be present at position y-2 and glycine at positions y-1 and y; or serine may be present at position y-3 and glycine at positions y-2, y-1 and y). In some embodiments, y = 20 or 21 may be preferred. In some embodiments, the linker is (GxS)n(GGSGG) or (GxS)n(GGSGGG), where G = glycine, S = serine, x = 4, and n = 1-20, 2-20, 1-10, 2-10, for example 2, 3, 4, 5, 6, 7, 8, or 9, for example n = 2-5 or n = 2-4. For example, the linker may be GGGGSGGGGSGGGGSGGSGG (Sequence ID 150) or GGGGSGGGGSGGGGSGGSGGG (Sequence ID 151).

[0283] As described above, in some embodiments, the first and / or second antibodies may each be polyvalent, for example, bivalent, with respect to the target antigen (e.g., tumor-associated antigen). This may have the advantage of increasing binding activity. Antibodies may be polyvalent, for example, bivalent, and each may be monospecific to a specific epitope (which may be the same epitope for the first and second antibodies, or different for the first and second antibodies). Therefore, in some embodiments, the first antibody may comprise i) two or more antigen-binding sites, e.g., antibody fragments, containing antigen-binding sites specific to the same epitope of the target antigen; ii) either (but not both) the VL domain or the VH domain of the antigen-binding site for the radiolabeled compound; and iii) an Fc region. The second antibody may comprise i) two or more antigen-binding sites, e.g., antibody fragments, containing antigen-binding sites specific to the same epitope of the target antigen; ii) either (but not both) the VL domain or the VH domain of the antigen-binding site for the radiolabeled compound; and iii) an Fc region. As described above, the epitope may be the same for the first antibody and the second antibody, or it may be different for the first antibody and the second antibody.

[0284] For example, each of the first and second antibodies may contain a tandem Fab, i.e., two Fab fragments connected via a peptide tether (Fab-tether-Fab), where the first Fab is connected to the N-terminus of the second Fab via its C-terminus.

[0285] In such embodiments, the correct assembly of the light chain and its respective heavy chain can be assisted by using cross-mab technology. For example, in one embodiment, each antibody may contain a tandem Fab comprising one Fab and one cross-Fab, where one fragment selected from the Fab and cross-Fab is specific to a first epitope and the other is specific to a second epitope.

[0286] In any of the tandem Fab embodiments (including those involving cross-Fab), the peptide tether / linker connecting the Fab fragments in the first antibody and the second antibody may be any linker as described herein. In some embodiments, the peptide may be a peptide having an amino acid sequence having a length of at least 5 amino acids, preferably 5 to 100 amino acids, and more preferably 10 to 50 amino acids. In one embodiment, the peptide linker is (GxS)n or (GxS)nGm, where G = glycine, S = serine, and (x = 3, n = 3, 4, 5 or 6, and m = 0, 1, 2 or 3) or (x = 4, n = 2, 3, 4 or 5, and m = 0, 1, 2 or 3), preferably x = 4 and n = 2 or 3, more preferably x = 4, n = 2. In one embodiment, the peptide tether is (G4S)2. In other embodiments, the linker may be any novel linker as described herein.

[0287] In certain embodiments, each of the first and second antibodies may comprise a) an Fc domain, b) at least one antigen-binding moiety including an antigen-binding site for a target antigen, and c) a polypeptide comprising either (but not both) a VL domain or a VH domain of the antigen-binding site for a radiolabeled compound, wherein the antigen-binding moiety of (b) is fused to the N-terminus of one chain of the Fc domain, and the C-terminus of the polypeptide of (c) is fused to the N-terminus of the other chain of the Fc domain. In some embodiments, the C-terminus of the antigen-binding moiety of (b) is fused to the N-terminus of the other chain of the Fc domain.

[0288] Therefore, the first antibody is, a) An Fc domain including a first subunit and a second subunit, b) An antigen-binding site including a binding site for the target antigen, c) A polypeptide comprising or consisting of an antibody heavy chain variable domain (VH) at the antigen-binding site for a radiolabeled compound. Includes or consists of The antigen-binding portion of (b) is fused to the N-terminus of the first subunit of the Fc domain of (a), and the polypeptide of (c) is fused to the N-terminus of the second subunit of the Fc domain of (a) by its C-terminus. The first antibody does not contain the VL domain of the antigen-binding site for the radiolabeled compound.

[0289] The second antibody is, a) An Fc domain including a first subunit and a second subunit, b) An antigen-binding site including a binding site for the target antigen, c) A polypeptide comprising or consisting of an antibody light chain variable domain (VL) at the antigen-binding site for a radiolabeled compound. Includes or consists of The antigen-binding portion of (b) is fused to the N-terminus of the first subunit of the Fc domain of (a), and the polypeptide of (c) is fused to the N-terminus of the second subunit of the Fc domain of (a) by its C-terminus. The second antibody does not contain the VH domain of the antigen-binding site for the radiolabeled compound.

[0290] This format avoids the free C-terminus of the VH domain at the split antigen-binding site, thus reducing the likelihood of anti-drug antibody responses, including those involving existing human anti-VH (HAVH) autoantibodies.

[0291] In some embodiments, the antigen-binding portion may preferably be an antibody fragment. In some embodiments, the antigen-binding portion may preferably be Fab.

[0292] In the first and / or second antibody, the antigen-binding portion is preferably a Fab, which may be fused to the N-terminus of a first subunit of the Fc domain by the C-terminus of its heavy chain. In some embodiments, the Fab fragment comprises a light chain containing a VL domain and a CL domain, and a heavy chain fragment containing a VH domain and a CH1 domain, the C-terminus of the CH1 domain being fused to the N-terminus of a first subunit of the Fc domain.

[0293] The fusion of the antigen-binding portion in (b), for example, the antibody fragment, is preferably via a hinge region. The fusion of the polypeptide in (c) may be via a linker positioned between the C-terminus of the polypeptide and the N-terminus of the Fc region, and / or via part or all of the upper hinge region (e.g., Asp221 and the C-terminus of the residue thereon, according to the EU numbering index). In one embodiment, the antibody fragment in (b) may be a Fab fragment. In one embodiment, in the first antibody, the polypeptide in (c) consists of the VH domain of the antigen-binding site for the radiolabeled compound, and in the second antibody, the polypeptide in (c) consists of the VL domain of the antigen-binding site for the radiolabeled compound.

[0294] Therefore, in one embodiment, the first antibody is i) A complete light chain, ii) A complete heavy chain, iii) Additional Fc chains, iv) A polypeptide containing or comprising a VH domain at the antigen-binding site for a radiolabeled compound Includes or consists of The light chain of (i) and the heavy chain of (ii) together provide an antigen-binding site for the target antigen, and a polypeptide containing or consisting of a VH domain of the antigen-binding site for the radiolabeled compound is fused to the N-terminus of (iii) preferably via a linker at its C-terminus.

[0295] The second antibody is, v) A complete light chain, vi) A complete heavy chain, vii) Additional Fc chains, viii) A polypeptide comprising or consisting of a VL domain, which is an antigen-binding site for a radiolabeled compound. Includes or consists of The light chain of (v) and the heavy chain of (vi) both provide an antigen-binding site for the target antigen, and a polypeptide containing or consisting of a VL domain of the antigen-binding site for the radiolabeled compound is fused to the N-terminus of (vii) preferably via a linker at its C-terminus.

[0296] The linker may include any flexible linker known to those skilled in the art or described herein, for example, a novel linker as described herein, e.g., linker GGGGSGGGGSGGGGSGGGGG (SEQ ID NO: 150). The linker may further include a portion of the entire upper hinge region, for example, extending from Asp221 to the initiation of the Fc chain (for example, in Cys226).

[0297] Polypeptides containing a VH domain or VL domain at the antigen-binding site for a radiolabeled compound may preferably not contain a constant region (e.g., CH1 or CL). In some embodiments, this may consist of a VH domain or VL domain at the antigen-binding site for a radiolabeled compound.

[0298] In some embodiments, the first and second antibodies are monospecific and monovalent with respect to the target antigen; that is, the antibody contains only one antigen-binding portion (such as an antibody fragment) that includes an antibody-binding site for the target antigen.

[0299] In other embodiments, they may comprise two or more antigen-binding moieties (e.g., antibody fragments). These may be linked in tandem. For example, component (b) may comprise a tandem Fab comprising a first Fab fragment and a second Fab fragment, wherein the first Fab fragment is linked to the N-terminus of the second Fab fragment via a peptide linker by its C-terminus (the C-terminus of the second Fab fragment is linked to the N-terminus of the first subunit of the Fc domain of (a)). In some embodiments, the first Fab fragment binds to a first epitope of target antigen A, and the second Fab fragment binds to a second epitope of target antigen A. Optionally, one of the Fab fragments selected from the first and second fragments is a conventional Fab, and the other is a cross-Fab or scFab.

[0300] The correct assembly of heterodimeric heavy chains can be assisted by the knob-into-hole technique.

[0301] As used herein, the term “full-length antibody” means an antibody comprising two “full-length antibody heavy chains” and two “full-length antibody light chains.” A “full-length antibody heavy chain” can be a polypeptide comprising, in the direction from the N-terminus to the C-terminus, an 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), and is abbreviated as VH-CH1-HR-CH2-CH3, and optionally, in the case of a lower class IgE antibody, an antibody heavy chain constant domain 4 (CH4). 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,” and therefore, the heavy chain may exchange the VH domain with a VL domain or the CH1 domain with a CL domain. A "full-length antibody light chain" can be a polypeptide consisting of a variable antibody light chain domain (VL) and a constant antibody light chain domain (CL) in the direction from the N-terminus to the C-terminus, and is abbreviated as VL-CL. 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 constant antibody light chain domain (CL) can be κ (kappa) or γ (lambda). Two full-length antibody chains are linked together via interpolypeptide 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 antibody according to the present invention may be derived from a single species, e.g., human, or it may be a chimeric antibody or a humanized antibody. The full-length antibodies described herein each comprise two antigen-binding sites formed by a VH and VL pair, which, in some embodiments, may both specifically bind to the same antigen or to different antigens. The C-terminus of the heavy or light chain of the full-length antibody indicates the last amino acid of the C-terminus of the heavy or light chain.

[0302] The N-terminus of the antibody heavy chain variable domain (VH) of the polypeptide under b) and the antibody light chain variable domain (VL) of the polypeptide under d) indicates the last amino acid at the N-terminus of the VH domain or the VL domain.

[0303] Any of the heterodimers described herein can also be produced using known techniques for producing multispecific antibodies. These include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)) and "knob-in-hole" operations (see, e.g., U.S. Patent No. 5,731,168 and Atwell et al., J.Mol.Biol.270:26 (1997)). Other methods include manipulating the electrostatic steering effect to produce antibody Fc-heterodimer molecules (see, e.g., International Publication No. 2009 / 089004); crosslinking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980 and Brennan et al., Science, 229:81 (1985)); using leucine zippers (see, e.g., Kostelny et al., J.Immunol., 148(5):1547-1553 (1992) and International Publication No. 2011 / 034605); and using common light chain techniques to avoid the problem of light chain mispairing (see, e.g., International Publication No. 98 / 50431).

[0304] The CH3 domains of full-length antibodies like those described above can be modified by the "knob-into-holes" technique, which is described in detail with several examples in, for example, International Publication No. 96 / 027011, Ridgway, JB, et al., Protein Eng 9(1996)617-621; and Merchant, AM, et al., Nat Biotechnol 16(1998)677-681. In this method, the interaction surface of the two CH3 domains is modified to increase heterodimerization of both heavy chains containing these two CH3 domains. Each of the two CH3 domains (of the two heavy chains) can be a "knob" and the other a "hole". For example, one includes what is called a "knob mutation" (T366W and optionally either S354C or Y349C) according to EU index numbering, while the other includes so-called "hole mutations" (T366S, L368A, and Y407V and optionally Y349C or S354C) (e.g., Carter, P. et al., Immunotechnol. 2 (1996) 73).

[0305] The introduction of disulfide crosslinks can be used, either additionally or alternatively, to stabilize heterodimers (Merchant, AM, et al., Nature Biotech 16(1998) 677-681; Atwell, S., et al., J. Mol. Biol. 270(1997) 26-35) and increase yields.

[0306] Therefore, in some embodiments, the first and / or second antibodies are 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 meet at an interface including the original interface between the antibody CH3 domains, the interface being modified to promote antibody formation, the modification being characterized by: a) The CH3 domain of one heavy chain is modified so that, within its original interface, the CH3 domain of the one heavy chain that fits into the original interface of the CH3 domain of the other heavy chain in the antibody is modified such that amino acid residues are replaced with amino acid residues having a larger side chain volume, thereby creating a raised portion within the interface of the CH3 domain of the one heavy chain that can be positioned within the cavity within the interface of the CH3 domain of the other heavy chain. and b) The CH3 domain of the other heavy chain is modified so that, within the original interface of the second CH3 domain intersecting the original interface of the first CH3 domain in the antibody, amino acid residues are replaced with amino acid residues having a smaller side chain volume, thereby creating a cavity within the interface of the second CH3 domain where a projection within the interface of the first CH3 domain can be positioned.

[0307] The amino acid residue having a larger side chain volume may optionally 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 optionally be selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V).

[0308] Optionally, 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 a disulfide bridge can be formed between both CH3 domains.

[0309] The multispecific (e.g., biparatopic) antibodies of the present invention may contain amino acid substitutions in the Fab molecules (including cross-Fab molecules) that are particularly effective in reducing the incorrect pairing of light chains with 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) binding arms (see, in whole, PCT International Publication 2015 / 150447, incorporated herein by reference, particularly the examples therein). The ratio of the desired multispecific antibody to undesirable byproducts, particularly the Bence-Jones type byproducts that occur in one of their 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 molecules (sometimes referred to herein as "charge modification").

[0310] Accordingly, 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 having charge modification as described herein and a light chain constant domain CL domain having charge modification as described herein.

[0311] Charge modification can be performed using either a conventional Fab molecule(s) included in the antibody of the present invention, or a crossover Fab molecule(s) included in the antibody of the present invention (but not both). In certain embodiments, charge modification is performed using a conventional Fab molecule(s) included in the antibody of the present invention.

[0312] In some embodiments, in a Fab or cross-Fab comprising a charge-modified light chain constant domain CL and a charge-modified heavy chain constant domain CH1, the charge modification of the light chain constant domain CL is located at position 124 and optionally at position 123 (numbering according to Kabat), and the charge modification of the heavy chain constant domain CH1 is located at position 147 and / or 213 (numbering according to Kabat EU index). 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) (numbering according to Kabat) (in one preferred embodiment, independently by lysine (K)), and in the heavy chain constant domain CH1, the amino acid at position 147 and / or the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

[0313] H. Exemplary antibody The embodiments and models relating to target binding (e.g., CEA binding, FAP binding, or GPRC5D binding), and the embodiments and models relating to DOTA binding, can be combined in several embodiments. That is, it may be preferable that the first antibody and the second antibody each contain a binding site for CEA, FAP, or GPRC5D, for example, containing one of the sequences described above, and that the first antibody and the second antibody associate to form a binding site for a DOTA chelate having one of the sequences described above. The embodiments and models relating to CEA binding, FAP, or GPRC5D and / or DOTA binding can be combined with the preferred formats of the antibodies described above, that is, in any of the preferred formats, the portion that binds to the target antigen may contain the CDR or variable region sequence described above, and / or the portion that binds to the radionuclide-labeled compound may be a DOTA binder having the CDR and / or variable region sequence described above.

[0314] Similarly, the embodiments and forms relating to target binding (e.g., CEA binding, FAP binding, or GPRC5D binding), and the embodiments and forms relating to Pb-DOTAM binding, can be combined in several embodiments. That is, it may be preferable that the first antibody and the second antibody each contain a binding site for CEA, FAP, or GPRC5D, for example, containing one of the sequences described above, and that the first antibody and the second antibody associate to form a binding site for a Pb-DOTAM chelate having one of the sequences described above. The embodiments and forms relating to CEA binding, FAP, or GPRC5D and / or Pb-DOTAM binding can be combined with preferred forms of the antibodies described above, that is, in any of the preferred forms, the portion that binds to the target antigen may contain a CDR or variable region sequence as described above, and / or the portion that binds to a radionuclide-labeled compound may be a Pb-DOTAM binder having a CDR and / or variable region sequence as described above.

[0315] In some embodiments, the first antibody is a) An Fc domain including a first subunit and a second subunit, b) An antibody fragment containing an antibody binding site for CEA, GPRC5D, FAP, and a target antigen optionally selected from CEA, c) A polypeptide comprising or consisting of an antibody heavy chain variable domain (VH) at the antigen-binding site for Pb-DOTAM, wherein the heavy chain variable domain comprises the heavy chain CDR of SEQ ID NOs. 1-3, and / or the heavy chain variable domain has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with SEQ ID NO. 143. It may include or consist of, The antibody fragment in (b) is fused to the N-terminus of the first subunit of the Fc domain in (a), and the polypeptide in (c) is fused to the N-terminus of the second subunit of the Fc domain in (a) by its C-terminus. The first antibody does not contain the VL domain of the antigen-binding site for the radiolabeled compound.

[0316] In the case of the antibody heavy chain variable domain (VH) of the antigen-binding site for Pb-DOTAM, the inventors have determined that an N-terminal residue such as N-terminal Q can be added to SEQ ID NO: 7, which may result in good expression. The antigen-binding site of DOTAM was originally developed from rabbit antibodies, and during the humanization process, the N-terminal residue of the human germline sequence was removed to resemble the rabbit sequence. The N-terminal Q residue represents the re-insertion of the original N-terminal residue from the human germline sequence. Therefore, if the heavy chain variable domain is a variant of SEQ ID NO: 143, the N-terminal Q residue may remain unchanged or may be replaced with another suitable amino acid that gives a good level of protein expression, as can be identified by those skilled in the art. In one embodiment, the N-terminal Q residue may be replaced with a residue selected from the group consisting of E, K, R, S, T, A, L, Y, D, N, and V. In another embodiment, the N-terminal Q residue may be replaced with a residue selected from the group consisting of E, K, R, S, T, A, L, and Y. In yet another embodiment, the N-terminal Q residue may be replaced with E. In another embodiment, the N-terminal Q residue may be replaced with a residue selected from the group consisting of D, N, and V.

[0317] The second antibody is, a) An Fc domain including a first subunit and a second subunit, b) An antibody fragment containing an antibody binding site for CEA, GPRC5D, FAP, and a target antigen optionally selected from CEA, c) A polypeptide comprising or consisting of an antibody light chain variable domain (VL) of an antigen-binding site for Pb-DOTAM, wherein the light chain variable domain comprises the CDR of SEQ ID NOs. 4-6, and / or the light chain variable domain has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO. 8, and / or the light chain variable domain has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO. 144. It may include or consist of, The antibody fragment in (b) is fused to the N-terminus of the first subunit of the Fc domain in (a), and the polypeptide in (c) is fused to the N-terminus of the second subunit of the Fc domain in (a) by its C-terminus. The second antibody does not contain the VH domain of the antigen-binding site for the radiolabeled compound.

[0318] Regarding the antibody light chain variable domain (VL) of the antigen-binding site for Pb-DOTAM, the inventors have determined that an additional N-terminal residue, such as N-terminal A or S, can be added to SEQ ID NO: 8, resulting in good expression. The N-terminal A residue represents the re-insertion of the original N-terminal residue from the human germline sequence. When the antibody light chain variable domain (VL) of the antigen-binding site for Pb-DOTAM is a variant of SEQ ID NO: 144, the N-terminal A residue may remain unchanged or be substituted with another suitable amino acid that can be identified by those skilled in the art. In one embodiment, the N-terminal A residue may be substituted with a residue selected from the group consisting of D, N, E, Q, S, A, V, L, T, Y, K, and R. In another embodiment, the N-terminal A residue may be substituted with a residue selected from the group consisting of D, N, E, Q, S, V, and L. In another embodiment, the N-terminal A residue may be substituted with S. In another embodiment, the N-terminal A residue may be substituted with a residue selected from the group consisting of D, N, and V.

[0319] In one particular embodiment, the first antibody is i) A complete light chain, ii) A complete heavy chain, iii) Additional Fc chains, iv) A polypeptide comprising or consisting of a VH domain of an antigen-binding site for Pb-DOTAM, wherein the heavy chain variable domain comprises the heavy chain CDR of SEQ ID NOs. 1-3, and / or the heavy chain variable domain has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with SEQ ID NO. 143. May include, The light chain of (i) and the heavy chain of (ii) together provide an antigen-binding site for a target antigen (e.g., CEA, GPRC5D, or FAP, optionally CEA), and a polypeptide containing or consisting of the VH domain of the antigen-binding site for the radiolabeled compound is fused to the N-terminus of (iii) preferably via a linker at its C-terminus.

[0320] The second antibody is, v) A complete light chain, vi) A complete heavy chain, vii) Additional Fc chains, viii) A polypeptide comprising or consisting of a VL domain of an antigen-binding site for Pb-DOTAM, wherein the light chain variable domain comprises the CDR of SEQ ID NOs. 4-6, and / or the light chain variable domain has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO. 8, and / or the light chain variable domain has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO. 144, and It may include or consist of, The light chain of (v) and the heavy chain of (vi) together provide an antigen-binding site for a target antigen (e.g., CEA, GPRC5D, or FAP, optionally CEA), and a polypeptide containing or consisting of the VL domain of the antigen-binding site for the radiolabeled compound is fused to the N-terminus of (vii) by its C-terminus, preferably via a linker.

[0321] In any of the above embodiments, the first antibody may have a CEA-binding sequence (i.e., a CDR or VH / VL domain) derived from the antibody CH1A1A.

[0322] For example, the complete light chain may contain the CDRs of SEQ ID NOs. 22-24 and / or a light chain variable domain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO. 26. In some embodiments, it may have at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO. 34.

[0323] The complete heavy chain may contain the CDRs of SEQ ID NOs. 19-21, and / or the complete heavy chain may contain variable domains having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with SEQ ID NO. 25.

[0324] In another specific embodiment, the first antibody may have a CEA-binding sequence (i.e., a CDR or VH / VL domain) derived from antibody A5B7 (including its humanized version).

[0325] For example, the complete light chain may contain the CDRs of SEQ ID NOs. 46-48 and / or light chain variable domains having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO. 50. In some embodiments, it may have at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO. 54.

[0326] In some embodiments, the complete heavy chain may include the CDRs of SEQ ID NOs. 43-45 and / or include variable domains having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO. 49.

[0327] In another specific embodiment, the first antibody may have a CEA-binding sequence (i.e., a CDR or VH / VL domain) derived from antibody T84.66 (including its humanized version).

[0328] For example, the complete light chain may contain the CDRs of SEQ ID NOs. 14-16 and / or a light chain variable domain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO. 18. In some embodiments, it may have at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO. 89.

[0329] In some embodiments, the complete heavy chain may include the CDRs of SEQ ID NOs. 11-13 and / or include variable domains having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO. 17.

[0330] In another specific embodiment, the first antibody may have a CEA-binding sequence (i.e., a CDR or VH / VL domain) derived from antibody 28A9 (including its humanized version).

[0331] For example, the complete light chain may contain the CDRs of SEQ ID NOs. 62-64 and / or contain a light chain variable domain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO. 66. In some embodiments, it may have at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO. 96.

[0332] In some embodiments, the complete heavy chain may include the CDRs of SEQ ID NOs. 59-61 and / or include variable domains having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO. 65.

[0333] In some embodiments, the second antibody may have a CEA-binding sequence (i.e., a CDR or VH / VL domain) derived from the antibody CH1A1A.

[0334] For example, the complete light chain may contain the CDRs of SEQ ID NOs. 22-24 and / or a light chain variable domain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO. 26. In some embodiments, it may have at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO. 34.

[0335] In some embodiments, the complete heavy chain comprises the CDRs of SEQ ID NOs. 19-21 and / or a variable domain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO. 25.

[0336] In another specific embodiment, the second antibody may have a CEA-binding sequence (i.e., a CDR or VH / VL domain) derived from A5B7 (including its humanized version).

[0337] For example, the complete light chain may contain the CDRs of SEQ ID NOs. 46-48 and / or light chain variable domains having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO. 50. In some embodiments, it may have at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO. 58.

[0338] In some embodiments, the complete heavy chain comprises the CDRs of SEQ ID NOs. 43-45 and / or a variable domain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO. 49.

[0339] In another specific embodiment, the second antibody may have a CEA-binding sequence (i.e., a CDR or VH / VL domain) derived from antibody T84.66 (including its humanized version).

[0340] For example, the complete light chain may contain the CDRs of SEQ ID NOs. 14-16 and / or a light chain variable domain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO. 18. In some embodiments, it may have at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO. 89.

[0341] In some embodiments, the complete heavy chain may include the CDRs of SEQ ID NOs. 11-13 and / or include variable domains having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO. 17.

[0342] In another specific embodiment, the second antibody may have a CEA-binding sequence (i.e., a CDR or VH / VL domain) derived from antibody 28A9 (including its humanized version).

[0343] For example, the complete light chain may contain the CDRs of SEQ ID NOs. 62-64 and / or contain a light chain variable domain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO. 66. In some embodiments, it may have at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO. 96.

[0344] In some embodiments, the complete heavy chain may include the CDRs of SEQ ID NOs. 59-61 and / or include variable domains having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO. 65.

[0345] In some embodiments, the first antibody and the second antibody bind to the same CEA epitope. For example, both the first antibody and the second antibody may have a CEA-binding sequence derived from antibody CH1A1A; or both the first antibody and the second antibody may have a CEA-binding sequence derived from A5B7 (including its humanized version); or both the first antibody and the second antibody may have a CEA-binding sequence derived from T84.66 (including its humanized version); or both the first antibody and the second antibody may have a CEA-binding sequence derived from 28A9 (including its humanized version); or both the first antibody and the second antibody may have a CEA-binding sequence derived from MFE23 (including its humanized version). In some embodiments, it may be preferable that the light chain polypeptide of (ii) has the same sequence as the light chain of (v).

[0346] In other embodiments, as discussed above, the first and second antibodies bind to different epitopes of CEA. For example, the first antibody may have a CEA-binding sequence derived from antibody CH1A1A and the second antibody may have a CEA-binding sequence derived from A5B7, or the first antibody may have a CEA-binding sequence derived from antibody A5B7 and the second antibody may have a CEA-binding sequence derived from CH1A1A.

[0347] In further specific embodiments, the target may be, for example, a CEA having a CEA-binding sequence derived from the antibody CH1A1A, and the format may be as shown in Figure 25C. Optionally, the first antibody and the second antibody associate to form a functional antigen-binding site for Pb-DOTAM chelate (Pb-DOTAM).

[0348] Therefore, in one example, i) The first antibody comprises the first heavy chain of SEQ ID NO: 112, the second heavy chain of SEQ ID NO: 114, and the light chain of SEQ ID NO: 115. ii) The second antibody comprises the first heavy chain of SEQ ID NO: 112, the second heavy chain of SEQ ID NO: 113, and the light chain of SEQ ID NO: 115.

[0349] In a preferred embodiment, i) The first antibody comprises the first heavy chain of SEQ ID NO: 112, the second heavy chain of SEQ ID NO: 146, and the light chain of SEQ ID NO: 115. ii) The second antibody comprises the first heavy chain of SEQ ID NO: 112, the second heavy chain of SEQ ID NO: 145, and the light chain of SEQ ID NO: 115.

[0350] I. 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 may 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 of such residues. Any combination of deletions, insertions, and substitutions can be carried out so as to reach the final construct, insofar as the final construct has the desired characteristics (e.g., antigen binding).

[0351] Substitution, insertion, and deletion variants In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for mutagenesis by substitution include HVR(CDR) and FR. Conservative substitutions are shown in Table 1 under the heading "Preferred Substitutions." More substantial variations 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 retained / improved antigen binding, decreased immunogenicity, or reduction or elimination of ADCC or CDC. [Table 1]

[0352] Amino acids can be classified according to their general side-chain properties. (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.

[0353] Non-conservative substitutions would involve swapping one member of these classes with one of another.

[0354] Certain substitution mutants involve substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized antibody or a human antibody). Generally, the resulting mutant(s) selected for further study will have modifications (e.g., improvements) (e.g., increased affinity, decreased immunogenicity) of specific biological properties compared to the parent antibody, and / or substantially retain specific biological properties of the parent antibody. Exemplary substitution mutants are affinity-matured antibodies and can be readily generated, for example, using phage display-based affinity maturation techniques as described herein. Briefly, one or more CDR residues are mutated, the mutant antibody is displayed to a phage, and it is screened for specific biological activities (e.g., binding affinity).

[0355] To improve antibody affinity, modifications (e.g., substitutions) may be made in the CDR, for example. Such modifications may be made in CDR "hot spots," i.e., residues encoded by codons that frequently undergo mutations during the somatic cell maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and / or residues that come into contact with the antigen, and the resulting mutant VH or VL is tested for binding affinity. Affinity maturation by secondary library construction and reselection from there is described, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some aspects of affinity maturation, diversity is introduced into the variable genes selected for maturation by one of various methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then constructed. Next, this library is screened to identify antibody variants with the desired affinity. Another method for introducing diversity involves CDR-directed methods in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR 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.

[0356] In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, provided that such alterations do not substantially reduce the antibody's ability to bind to the antigen. For example, conservative alterations that do not substantially reduce binding affinity (e.g., conservative substitutions as provided herein) may be made in a CDR. Such alterations may, for example, be outside the antigen-contact residue in the CDR. In the specific variant VH and VL sequences described above, each CDR is either unaltered or has one, two, or three or fewer amino acid substitutions.

[0357] 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, 244:1081-1085. This method identifies target residues or groups (e.g., charged residues such as arg, asp, his, lys, and glu) and substituted 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 are functionally sensitive to the initial substitution. Alternatively, or additionally, the crystal structure of the antigen-antibody complex may be used to identify contact points between the antibody and antigen. Such contact residues and adjacent residues may be targeted or removed as candidate substitutions. Mutants may be screened to determine whether they possess the desired properties.

[0358] Amino acid insertions include amino-terminus and / or carboxyl-terminus fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of one or more amino acid residues. An example of terminal insertion is an antibody with an N-terminal methionyl residue. Other insertion variants of antibody molecules include the fusion of the N-terminus or C-terminus of an antibody to an enzyme (e.g., ADEPT (for antibody-directed enzyme prodrug therapy)) or polypeptide, which increases the serum half-life of the antibody.

[0359] Glycosylated mutants In certain embodiments, the antibody provided herein is modified to increase or decrease the degree of glycosylation of the antibody. The addition or deletion of glycosylation sites to an antibody can be conveniently achieved by modifying the amino acid sequence so that one or more glycosylation sites are created or removed.

[0360] If the antibody contains an Fc region, the oligosaccharide attached to the antibody may be modified. Native antibodies produced by mammalian cells typically contain branched, bibranched oligosaccharides that are commonly bound to Asn297 of the CH2 domain of the Fc region by an N-bond. See, for example, Wright et al. TIBTECH 15:26-32 (1997). Oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose bound to the GlcNAc of the "stem" of the bibranched oligosaccharide structure. In some embodiments, the modification of the oligosaccharide in the antibody of the present invention may be carried out to generate antibody variants having specific improved properties.

[0361] In one embodiment, an antibody variant is provided having an oligosaccharide structure lacking a non-fucosylated oligosaccharide, i.e., fucose binding (direct or indirect) to the Fc region. Such a non-fucosylated oligosaccharide (also called "afucosylated" oligosaccharide) is in particular an N-linked oligosaccharide lacking a fucose residue to which a first GlcNAc is bound in the stem of a bibranched oligosaccharide structure. In one embodiment, an antibody variant is provided having an increased proportion of non-fucosylated oligosaccharides in the Fc region compared to a native or parent antibody. For example, the proportion of non-fucosylated oligosaccharides may be at least about 20%, at least about 40%, at least about 60%, at least about 80%, or possibly about 100% (i.e., no fucosylated oligosaccharides are present). The proportion of non-fucosylated oligosaccharides is the (average) amount of fucose-less oligosaccharides relative to the total of all oligosaccharides bound to Asn297 (e.g., complex, hybrid, and high-mannose structures), as measured by MALDI-TOF mass spectrometry, for example, as described in International Publication No. 2006 / 082515. Asn297 refers to the asparagine residue located at approximately position 297 of the Fc region (EU numbering of Fc region residues); however, Asn297 may also be located upstream or downstream of position 297, i.e., approximately ±3 amino acids between positions 294 and 300, due to minor sequence changes in the antibody. Such antibodies with an increased proportion of non-fucosylated oligosaccharides in the Fc region may have improved FcγRIIIa receptor binding and / or improved effector function, particularly improved ADCC function. For example, see U.S. Patent Application Publication No. 2003 / 0157108; U.S. Patent Application Publication No. 2004 / 0093621.

[0362] Examples of cell lines capable of producing antibodies with reduced fucosylation include Lec13CHO cells with insufficient protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Patent Application Publication No. 2003 / 0157108; and International Publication No. 2004 / 056312, particularly Example 11), and knockout cell lines, such as FUT8 of the alpha-1,6-fucosyltransferase gene, and knockout CHO cells (e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614-622 (2004); Kanda, Y. et al.). Examples include cells in which GDP-fucose synthesis or transporter protein activity is reduced or eliminated (see, for example, U.S. Patent Publication No. 2004259150, U.S. Patent Publication No. 2005031613, U.S. Patent Publication No. 2004132140, and U.S. Patent Publication No. 2004110282).

[0363] In a further embodiment, the antibody variant is provided with a bifurcated oligosaccharide, for example, 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, as described above. Examples of such antibody variants are described, for example, in Umana et al., Nat Biotechnol 17, 176-180 (1999); Ferrara et al., Biotechn Bioeng 93, 851-861 (2006); International Publication No. 99 / 54342; International Publication No. 2004 / 065540; and International Publication No. 2003 / 011878.

[0364] Antibody variants having at least one galactose residue of an oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Examples of such antibody variants are described, for example, in International Publications 1997 / 30087; 1998 / 58964; and 1999 / 22764.

[0365] Antibodies may preferably be modified to reduce the degree of glycosylation. In some embodiments, antibodies may be deglycosylated or deglycosylated. Antibodies may include substitutions at N297, for example, N297D / A.

[0366] Fc region mutant In certain embodiments, one or more amino acid modifications are introduced into the Fc region of the antibody provided herein, thereby creating an Fc region variant. 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.

[0367] 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 aspects, the present invention conceives antibody variants that, having some, but not all, effector functions, are desirable candidates for applications where the in vivo half-life of the antibody is important, but certain effector functions (e.g., complement-dependent cell-mediated cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC)) are unnecessary or detrimental.

[0368] In vitro and / or in vivo cytotoxicity assays may be performed to confirm reduced / deficient CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to confirm that an antibody lacks FcγR binding (and therefore may lack ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for ADCC mediation, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression in hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (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, for example, 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); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays may be employed (see, for example, ACTI® non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA) and 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 or additionally, the ADCC activity of the molecule of interest can be evaluated in vivo in animal models, such as those disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay may also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity.For example, see the C1q and C3c-conjugated ELISAs in International Publication Nos. 2006 / 029879 and 2005 / 100402. To evaluate complement activation, a CDC assay can be performed (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)). The determination of FcRn binding and in vivo clearance / half-life can also be performed using methods known in the art (see, for example, Petkova, S B et al., Int'l.Immunol.18(12):1759-1769(2006); see International Publication No. 2013 / 120929Al).

[0369] Antibodies with reduced effector function include those containing one or more substitutions (e.g., P329G) at residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region (U.S. Patent No. 6,737,056). Such Fc mutants include those having substitutions at two or more amino acid positions 265, 269, 270, 297, and 327, and include the so-called "DANA" Fc mutant in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581).

[0370] In certain embodiments, the antibody variant includes an Fc region having one or more amino acid substitutions that reduce FcγR binding, for example, Fc regions at positions 234 and 235 (EU-numbered residues). 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, International Publication No. 2012 / 130831.) In another embodiment, the substitutions are L234A, L235A, and D265A (LALA-DA) in the Fc region derived from the human IgG1 Fc region. Alternative replacements include L234F and / or L235E, which may be optionally combined with D265A and / or P329G and / or P331S.

[0371] In other embodiments, it may be possible to use IgG subtypes with reduced effector function, such as IgG4 or IgG2. Specific antibody variants exhibiting improved or reduced binding to FcR have been described. (See, for example, U.S. Patent No. 6,737,056; International Publication No. 2004 / 056312; and Shields et al., J. Biol. Chem. 9(2):6591-6604(2001).)

[0372] In some embodiments, the modification is made in the Fc region resulting in a modified (i.e., improved or reduced, preferably reduced) C1q binding and / or complement-dependent cytotoxicity (CDC), as described, for example, in U.S. Patent No. 6,194,551, International Publication No. 99 / 51642, Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0373] In certain embodiments, the antibody variant includes an Fc region having one or more amino acid substitutions that reduce FcRn binding, e.g., mutations at Fc region positions 253 and / or 310 and / or 435 (EU-numbered residues). 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) 5497-5508.

[0374] In certain embodiments, the antibody variant includes an Fc region having one or more amino acid substitutions that reduce FcRn binding, e.g., mutations at positions 310 and / or 433 and / or 436 (EU-numbered 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, International Publication No. 2014 / 177460A1). For example, in some embodiments, normal FcRn binding may be used.

[0375] For other examples of Fc region variants, see Duncan & Winter, Nature 322:738-40 (1988), U.S. Patent Nos. 5,648,260, 5,624,821, and International Publication No. 94 / 29351.

[0376] The C-terminus of the heavy chain of the full-length antibody described herein may be a complete C-terminus ending with 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. The C-terminus of the heavy chain may be a shortened C-terminal PG. In one of all embodiments reported herein, an antibody comprising a heavy chain containing the C-terminal CH3 domain as specified herein contains a C-terminal glycine residue (G446, amino acid position in EU index numbering). This is still clearly encompassed by the terms “full-length antibody” or “full-length heavy chain” as used herein.

[0377] antibody derivative In certain embodiments, the antibodies provided herein may be further modified to include additional non-proteinoid moieties known and readily available in the art. Suitable sites 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 (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous 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 attached to an antibody varies, and if multiple polymers are attached, they may be the same molecule or different molecules. In general, the number and / or types of polymers used for derivatization are not limited but can be determined based on considerations including the specific properties or functions of the antibody being improved, and whether the antibody derivative will be used for therapeutic purposes under defined conditions.

[0378] J. Recombination methods and compositions Antibodies may be produced, for example, using recombinant methods and compositions described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid or a set of isolated nucleic acids encoding a set of antibodies described herein is provided.

[0379] For example, a set of nucleic acids may include the following nucleic acids that encode the first antibody: i) A nucleic acid encoding the first heavy chain of a first antibody, wherein the first heavy chain comprises the heavy chain of a full-length antibody that specifically binds to a target antigen; ii) A nucleic acid encoding a second heavy chain of a first antibody, wherein the second heavy chain comprises, from the N-terminus to the C-terminus, a VH domain for antigen binding to a radiolabeled compound, an optional linker, and an Fc subunit (e.g., CH2-CH3); iii) The nucleic acid encoding the light chain of the first antibody.

[0380] The set of nucleic acids according to the present invention may additionally or alternatively include the following nucleic acids that encode a second antibody: iv) A nucleic acid encoding the first heavy chain of a second antibody, wherein the first heavy chain comprises the heavy chain of a full-length antibody that specifically binds to a target antigen; v) A nucleic acid encoding a second heavy chain of a second antibody, wherein the second heavy chain comprises, from the N-terminus to the C-terminus, a VL domain for antigen binding to a radiolabeled compound, an optional linker, and an Fc subunit (e.g., CH2-CH3); vi) The nucleic acid encoding the light chain of the second antibody.

[0381] In some embodiments, specific ones of these nucleic acids may be the same as one another. For example, the nucleic acid of (iii) may be the same as that of (vi), such that the entire set contains only five different nucleic acid sequences.

[0382] Nucleic acids may be contained in one or more nucleic acid molecules or expression vectors.

[0383] Therefore, in further embodiments, one or more vectors (e.g., expression vectors) containing such nucleic acids are provided. In one embodiment, each heavy chain and light chain is expressed from an individual plasmid.

[0384] In further embodiments, a host cell or set of host cells containing such nucleic acid(s) or vector(s) is provided. In one embodiment, a first host cell expressing a first antibody is provided, and a second host cell expressing a second antibody is provided.

[0385] In one such embodiment, a first host cell contains (e.g., is transformed with) one or more vectors that collectively encode the nucleic acids of a first antibody. A second host cell contains (e.g., is transformed with) one or more vectors that collectively encode the nucleic acids of a second antibody.

[0386] In one embodiment, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell, or a lymphoid cell (such as a Y0, NS0, or Sp20 cell). In one embodiment, a method for producing an antibody according to the present invention is provided, which includes culturing a host cell containing the nucleic acid encoding the antibody under conditions suitable for antibody expression, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0387] Regarding the recombinant production of antibodies, for example, the nucleic acids encoding the aforementioned antibodies 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).

[0388] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as 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, KA, In: Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, antibodies may be isolated from bacterial cell paste in the soluble fraction and further purified.

[0389] In addition to prokaryotes, eukaryotes such as filamentous fungi and yeasts are suitable as cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains with "humanized" glycosylation pathways, resulting in the production of antibodies with partially or completely human glycosylation patterns. See Gerngross, TU, Nat. Biotech. 22(2004) 1409-1414 and Li, H. et al., Nat. Biotech. 24(2006) 210-215.

[0390] Furthermore, host cells suitable for the expression of (glycosylated) antibodies are derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains have been identified, and these may be used in combination with insect cells, particularly for the transfection of Spodoptera frugiperda cells.

[0391] 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 antibody production in transgenic plants).

[0392] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension may be useful. Other examples of useful mammalian host cell lines include: monkey kidney CV1 cell line (COS-7) transformed with SV40; human embryonic kidney cell lines (e.g., 293 or 293T cells, as described by Graham, FL et al., J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells, as described by Mather, JP, Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical tumor cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor cells (MMT060562); TRI cells (e.g., Mather, JP et al., Annals) These include MRC5 cells and FS4 cells, as described in NYAcad.Sci.383(1982)44-68. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells (Urlaub, G. et al., Proc. Natl. Acad.Sci. USA 77(1980)4216-4220), including DHFR-CHO cells, as well as myeloma cell lines, such as Y0, NS0, and Sp2 / 0. For a review of specific mammalian host cells suitable for antibody production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0393] In one embodiment, the host cell is a eukaryote, for example, a Chinese hamster ovary (CHO) cell or a lymphocyte (e.g., Y0, NS0, Sp20 cell).

[0394] K. assay The antibodies provided herein may be identified, screened, or characterized for their physical / chemical properties and / or biological activity by various assays known in the art.

[0395] In one embodiment, the antibody of the present invention is tested for its antigen-binding activity by known methods such as ELISA or Western blotting.

[0396] antibody affinity In certain embodiments, the antibodies provided herein have a range 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) Dissociation constant (K) for the target antigen as otherwise described herein. D ) has.

[0397] In certain embodiments, the antigen-binding site for the radiolabeled compound is 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (for example, 10 -8 M or less, for example, 10 -8 M~10 -13 M, for example 10 -9 M~10 -13 The dissociation constant (K) of the radiolabeled compound M D ) has. In some embodiments, K DThe K content is 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 as otherwise described herein. For example, the functional binding site is K content of about 1 pM to 1 nM, for example, about 1 to 10 pM, 1 to 100 pM, 5 to 50 pM, 100 to 500 pM, or 500 pM to 1 nM. D It can bind to radiolabeled compounds / metal chelates.

[0398] In one embodiment, K D This is measured by a radiolabeled antigen-binding assay (RIA). In one embodiment, the RIA is performed using a Fab version of the antibody of interest and its antigen. For example, the solution binding affinity of Fab to the antigen is measured in the presence of a titration system of the unlabeled antigen at a minimum concentration. 125 I) Fab is equilibrated with a labeled antigen, and then the bound antigen is captured on a plate coated with anti-Fab antibody for measurement (see, for example, Chen et al., J.Mol.Biol.293:865-881 (1999)). To establish the assay conditions, a MICROTITER® multiwell plate (Thermo Scientific) is 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 the anti-VEGF antibody Fab-12 in Presta et al., Cancer Res. 57:4593-4599 (1997)). Then incubate the Fab of interest overnight; however, incubation may be continued for a longer period (e.g., about 65 hours) to reach equilibrium. Subsequently, transfer the mixture to a capture plate for incubation at room temperature (e.g., 1 hour). Next, 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 scintillant (MICROSCINT-20®; Packard) and count the plate with a TOPCOUNT® gamma counter (Packard) for 10 minutes. Select the concentration of each Fab that yields less than 20% of maximum binding for use in competitive binding assays.

[0399] According to another embodiment, K DThis is measured using the BIACORE® surface plasmon resonance assay. For example, assays using BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) are performed at 25°C to ~10 response units (RUs) using an immobilized antigen CM5 chip. In one embodiment, a carboxymethylated dextran biosensor chip (CM5, BIACORE) is activated with N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the instructions of those skilled in the art. The antigen is diluted in 10 mM sodium acetate, pH 4.8, and then diluted to 5 μg / mL (approximately 0.2 μM) before injection at a flow rate of 5 μl / min to achieve approximately 10 response units (RUs) of the binding protein. After antigen injection, 1 M ethanolamine is injected to block unreacted groups. For dynamic measurement, Fab's 2-fold serial dilutions (0.78 nM to 500 nM) are injected at a flow rate of approximately 25 μL / min into PBS containing 0.05% polysorbate 20 (TWEEN-20®) surfactant (PBST) at 25°C. The association rate (kon) and dissociation rate (koff) are calculated by simultaneously fitting the association sensorgram and dissociation sensorgram using a simple 1:1 Langmuir coupling model (BIACORE® Evaluation Software version 3.2). The equilibrium dissociation constant (K) is calculated. D) is calculated as the ratio koff / kon. For example, see Chen et al. J.Mol.Biol.293:865-881(1999). If the on rate exceeds 10⁶ M⁻¹ s⁻¹ by the surface plasmon resonance assay described above, the on rate can be determined by using a fluorescence quenching technique, which measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm, emission = 340 nm, 16 nm band passthrough) of 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) at 25°C in the presence of an increased concentration of antigen, measured with a spectrophotometer such as an Aviv Instruments spectrophotometer equipped with a stop flow or an 8000 series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with a stirring cuvette.

[0400] In another embodiment, K D This is measured using a SET (Solution Equilibrium Titration) assay. In this assay, the test antibody is typically applied 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 the antigen-coated surface and detected with labeled / tagged anti-species antibodies, generally using electrochemiluminescence (e.g., Haenel et al Analytical Biochemistry 339(2005)182-184).

[0401] 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 antibody samples containing free antigen, 0.01 nM to 1 nM antibodies are 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 samples are incubated overnight at 4°C in sealed REMP-stored polypropylene microplates (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 room temperature for 15 minutes, followed by a 3 × 90 μl wash with 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), followed by a 6 × 90 μl wash with PBST buffer. 25 μl of TMB substrate (Roche Diagnostics GmbH, catalog number: 11835033001) is added to each well. Measurement is performed using a Safire 2 reader (Tecan) at 370 / 492 nm.

[0402] In another embodiment, K DThis is measured using the KinExA (kinetic exclusion) assay. In this assay, the antigen is typically titrated to a constant concentration of antibody binding sites, the sample is equilibrated, and then rapidly drawn through a flow cell into which the free antibody binding sites are captured on antigen-coated beads, and the antigen-saturated antibody complex is washed away. The bead-captured antibody is then detected with a labeled anti-species antibody, such as a fluorescently labeled anti-species antibody (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 the running buffer supplemented with 1 mg / ml BSA ("sample buffer"). The flow rate is 0.25 ml / min. A constant volume of antibody with a binding site concentration of 5 pM is titrated with the antigen by two-fold serial dilutions starting at 100 pM (concentration range 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 the KinExA system at a volume of 5 ml, allowing the unbound antibody to be captured by the beads without disturbing the equilibrium of the solution. The captured antibody is detected using a 250 ng / ml Dylight 650 (copyright) conjugate anti-human Fc fragment-specific secondary antibody in sample buffer. Each sample is measured in two sequences for all equilibrium experiments. KD is obtained from a 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.

[0403] L. Treatment methods and compositions A set of antibodies as described herein may be used in therapeutic methods. In one embodiment, a set of antibodies described herein for use as a pharmaceutical is provided. In a particular embodiment, a set of antibodies for use in a treatment method is provided.

[0404] As described above, in some embodiments, the antibody set according to the present invention is suitable for any treatment in which it is desirable to deliver a radionuclide to target cells. For example, a set of antibodies described herein is provided for use in a pre-targeted radioimmunotherapy method, such as in cancer treatment.

[0405] In certain embodiments, the present invention provides a set of antibodies for use in a method of pre-targeted radioimmunotherapy in an individual, comprising administering an effective amount of the set of antibodies to the individual. The “individual” in any of the above embodiments is preferably a human.

[0406] As described above, the treatment may be for any condition that can be treated by cytotoxic activity targeting 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. Optionally, the targeting may also be for T cells for the treatment of T cell-driven autoimmune diseases or T cell hematological cancers. Thus, 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 drugs for multiple sclerosis and graft-versus-host disease.

[0407] As used herein, the term "cancer" includes lymphoma, lymphocytic leukemia, lung cancer, non-small cell lung (NSCL) cancer, bronchiolalveolar cell lung cancer, bone cancer, pancreatic cancer including pancreatic ductal adenocarcinoma (PDAC), skin cancer, head and neck cancer, melanoma of the skin or eye, uterine cancer, ovarian cancer, anal cancer, stomach cancer, gastric cancer, colorectal cancer which may be colon cancer and / or rectal cancer, breast cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney or ureteral cancer, renal cell carcinoma, renal pelvis carcinoma, mesothelioma, hepatocyte cancer This includes both solid tumors and hematological malignancies, such as cholangiocarcinoma, neoplasms of the central nervous system (CNS), axial tumors of the spinal cord, gliomas of the brainstem, glioblastoma multiforme, astrocytoma, Schwann cell tumor, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma and Ewing sarcoma (including refractory versions of any of the above cancers), checkpoint inhibitor-treated versions of any of the above cancers, or combinations of one or more of the above cancers.

[0408] Methods for targeting cells, tissues, or organs with radioactive isotopes for therapeutic purposes are: i) Administering the first antibody and the second antibody described herein to a target (in either order simultaneously or sequentially), wherein the antibodies bind to the target antigen, localize to the surface of cells expressing the target antigen, and the association of the first antibody and the second antibody forms a functional binding site for the radiolabeled compound. and, ii) The next step is to administer a radiolabeled compound, which includes administering a radiolabeled compound that binds to a functional binding site for the radiolabeled compound.

[0409] Radiolabeled compounds are labeled with radioactive isotopes that are cytotoxic to cells. Suitable radioactive isotopes include alpha and beta emitters, as mentioned above.

[0410] In pre-targeted radioimmunotherapy methods using bispecific antibodies (i.e., not “split” antibodies according to the present invention), it is common practice to administer a scavenger or blocking agent between the administration of the antibody and the administration of the radiolabeled compound. The scavenger binds to the antibody and increases the rate of clearance from the body. Scavengers include anti-idiotype antibodies. Blocking agents are typically drugs that bind to the antigen-binding site of the radiolabeled compound but are not radiolabeled themselves. For example, if the radiolabeled compound contains a chelating agent supported by a radioactive isotope of a particular chemical element (e.g., a metal), the blocking agent may contain the same chelating agent supported by a non-radioactive isotope of the same element (e.g., a metal), or it may contain an unsupported chelating agent or a chelating agent supported by a different non-radioactive moiety (e.g., a non-radioactive isotope of a different element), as long as it can still be bound by the antigen-binding site. In some cases, the blocking agent may further contain a moiety that increases the size and / or hydrodynamic radius of the molecule. These interfere with the ability of molecules to access tumors without interfering with the ability of molecules to bind to antibodies in circulation. Exemplary parts include hydrophilic polymers. The part may be a polymer or copolymer 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 part may be an unstructured peptide or protein, such as XTEN polypeptide (unstructured hydrophilic protein polymer), homoamino acid polymer (HAP), proline-alanine-serine polymer (PAS), elastin-like peptide (ELP), or gelatin-like protein (GLK). Further exemplary parts include proteins such as albumin, e.g., bovine serum albumin, or IgG. Suitable molecular weights for the part / polymer may be, for example, at least 50 kDa, for example, in the range of 50 kDa to 2000 kDa. For example, the molecular weight can be 200-800 kDa, arbitrarily greater than 300, 350, 400 or 450 kDa, arbitrarily less than 700, 650, 600 or 550 kDa, and arbitrarily about 500 kDa.

[0411] In certain embodiments of the present invention, there is no step of administering a scavenging agent or blocking agent to the subject. In certain embodiments, there is no step of administering any agent that binds to the first or second antibody between the administration of the antibody and the administration of the radiolabeled compound. In certain embodiments, there is no step of administering any agent between the administration of the antibody and the radiolabeled compound, except for a compound optionally selected from chemotherapeutic agents, immunotherapeutic agents and radiosensitizers. In some embodiments, no agent is administered between the administration of the antibody and the administration of the radiolabeled compound. In some embodiments, there may be no injection or infusion of any other agent into the subject between the administration of the antibody and the administration of the radiolabeled compound.

[0412] In some embodiments, the method may be a two-step method of pre-targeted radioimmunotherapy comprising, i) the step of administering a set of antibodies (the first antibody and the second antibody may be administered simultaneously or sequentially in either order), and ii) the step of subsequently administering a radiolabeled compound. The treatment may comprise multiple cycles of such treatment, i.e., multiple cycles of these two steps. An exemplary treatment cycle duration is 28 days, with the set of antibodies administered on day 1 of the cycle, and the radiolabeled compound optionally administered on day 1, 2, 3, 4, 5, 6, 7, or 8 of the cycle, for example, on day 7. The number of treatment cycles may vary. In one embodiment, there may be 4, 5, or 6 treatment cycles.

[0413] The inventors have surprisingly determined that it is possible to obtain therapeutically effective uptake of radiolabeled compounds into tumors while avoiding excessive accumulation of radioactivity in normal tissues using the antibodies according to the present invention. In fact, in examples, the level of radioactivity accumulation in non-target tissues was found to be lower than that of the three-step PRIT method using bispecific antibodies and a removal step, but with a simpler procedure.

[0414] In some embodiments, the radiolabeled compound can be administered to the subject after a suitable period of time has been given for the first and second antibodies to localize to the target cells. For example, in some embodiments, the radiolabeled compound may be administered to the subject immediately after the first and second antibodies, or at least 4 hours, 8 hours, 1 day, or 2 days after the first and second antibodies. Optionally, the radiolabeled compound may be administered up to 3, 5, or 7 days after the first and second antibodies. In a particular embodiment, the radiolabeled compound may be administered to the subject 2 to 7 days after the first and second antibodies.

[0415] 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 chemotherapeutic agents, and the chemotherapeutic agents and antibodies may be administered simultaneously or sequentially in any order. Additionally or alternatively, they may be administered in combination with one or more immunotherapeutic agents, and the immunotherapeutic agents and antibodies may be administered simultaneously or sequentially in any order.

[0416] In some embodiments, the antibodies described herein may be administered additionally or alternatively in combination with a radiosensitizer. The radiosensitizer and the antibody may be administered simultaneously or sequentially in any order.

[0417] The antibodies of the present invention (and any further therapeutic agents, e.g., radiolabeled compounds) may be administered by any suitable means, including parenteral, intrapulmonary and intranasal, and, if desired for local treatment, intrafocal administration. Parenteral administration includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Administration may be by any suitable route, e.g., injection, e.g., intravenous or subcutaneous injection.

[0418] In some embodiments, as described above, one or more dosimetry cycles can be used before one or more treatment cycles. A dosimetry cycle may include i) administering a set of antibodies (the first and second antibodies may be administered simultaneously or sequentially in either order), and ii) subsequently administering an imaging-suitable compound radiolabeled with a gamma emitter (the radiolabeled compound binds to a functional binding site for the radiolabeled compound). The compound may be the same as the compound used in subsequent treatment cycles, except that it is labeled with a gamma emitter rather than an alpha or beta emitter. For example, in one embodiment, the radiolabeled compound used in the dosimetry cycle is 203 Pb-DOTAM is a radiolabeled compound used in the treatment cycle. 212 This may be Pb-DOTAM. The patient may undergo imaging to determine the uptake of the compound into the tumor and / or to estimate the absorbed dose of the compound. This information may be used to estimate the expected radiation exposure in subsequent treatment steps and to adjust the dose of the radiolabeled compound used in the treatment steps to a safe level.

[0419] M. Pharmaceutical Products The first antibody and the second antibody described herein may be formulated into a single pharmaceutical composition or separate pharmaceutical compositions. Therefore, in further embodiments, the present invention provides a pharmaceutical composition comprising a pharmaceutical composition comprising the first antibody and the second antibody of the present invention, or a pharmaceutical formulation comprising the first antibody of the present invention, and a second pharmaceutical composition comprising the second antibody of the present invention, for use in, for example, any therapeutic or diagnostic method described herein. In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition further comprises at least one further therapeutic agent, as described below, for example.

[0420] The pharmaceutically acceptable preparations of antibodies described herein may be prepared in the form of lyophilized preparations or aqueous solutions by mixing such antibodies having a desired degree of purity with one or more pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)).

[0421] Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosage and concentration used, and are not limited to, but include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl, or benzyl alcohol, alkylparabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); and low molecular weight (approximately 10 residues). The materials include polypeptides (less than 1 / 2), proteins such as serum albumin, gelatin, or immunoglobulin, 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 as specified herein further include interstitial drug dispersants such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein such as rHuPH20 (HYLENEX®, Halozyme, Inc.). Specific exemplary sHASEGPs 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 (e.g., chondroitinases).

[0422] An example of a lyophilized antibody composition is described in U.S. Patent No. 6,267,958. Examples of aqueous antibody compositions are described in U.S. Patent No. 6,171,586 and International Publication No. 2006 / 044908, the latter of which comprises a histidine-acetate buffer.

[0423] The formulations described herein may also contain two or more active ingredients necessary for the specific indication being treated, preferably having complementary activities that do not adversely affect each other. For example, as discussed above, it may be desirable to further provide chemotherapeutic agents, immunotherapeutic agents and / or radiosensitizers. Such active ingredients are preferably present in combination in amounts effective for the intended purpose.

[0424] The active ingredient may be encapsulated in microcapsules prepared, for example, by coacervation technology or interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively), or encapsulated in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions. Such technologies are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980).

[0425] Sustained-release formulations may be prepared. Preferred examples of sustained-release preparations include a semipermeable matrix of a solid hydrophobic polymer containing an antibody, where these matrices are in the form of molded articles, such as films or microcapsules.

[0426] Preparations used for in vivo administration are generally sterilized. Sterilization can be easily achieved, for example, by filtration using a sterile filtration membrane.

[0427] N. Methods and compositions for diagnosis and detection The antibody sets described herein may also be used in diagnostic or imaging methods, preferably pre-targeted radioimmunoimaging methods or methods comprising pre-targeted radioimmunoimaging. Thus, the present invention provides diagnostic and imaging methods. The present invention further provides the antibody sets in the imaging methods described herein for use in diagnostic methods performed on a subject, e.g., the body of a human or animal, and the use of the antibody sets described herein (i.e., the first antibody and the second antibody described herein).

[0428] The imaging method is suitable for imaging the presence and / or distribution of target antigens in the body. For example, this method may be a method for imaging cells expressing antigens associated with a disease (e.g., any of the disease symptoms described above). Optionally, this method is for imaging tumors or cancers. This method may be for the purpose of diagnosing subjects suspected of having proliferative disorders such as cancer or infectious diseases.

[0429] In some embodiments, it may be preferable that the subject be human.

[0430] Methods for targeting radioisotopes to tissues or organs for imaging or diagnostic purposes are: i) Administering the first antibody and the second antibody described herein to a target (in either order simultaneously or sequentially), wherein the antibodies bind to the target antigen, localize to the surface of cells expressing the target antigen, and the association of the first antibody and the second antibody forms a functional binding site for the radiolabeled compound. and, ii) The next step is to administer a radiolabeled compound, which may include administering a radiolabeled compound that binds to a functional binding site for the radiolabeled compound.

[0431] Optionally, this method is iii) Further includes imaging the tissue or organ where the radiolabeled compound is localized or is expected to be localized.

[0432] Optionally, the method may further include one or more steps of forming a diagnosis, delivering the diagnosis to the target, and / or determining and / or administering appropriate treatment based on the diagnosis.

[0433] In another embodiment, the method of the present invention may include imaging the tissue or organ of a subject, and the subject is i) The first antibody and the second antibody described herein (simultaneously or sequentially, in any order) which bind to a target antigen, localize to the surface of a cell expressing the target antigen, and whose association with the first antibody and the second antibody forms a functional binding site for a radiolabeled compound, and ii) The patient has previously been administered a radiolabeled compound, wherein the radiolabeled compound binds to an antigen-binding site for the radiolabeled compound formed by the association of the first antibody and the second antibody.

[0434] In the imaging and / or diagnostic methods described herein, the radiolabeled compound is labeled with a radioisotope suitable for imaging. Suitable radioisotopes include gamma emitters as described above.

[0435] In conventional pre-targeted radiation imaging methods, it is common practice to administer a removal agent or blocking agent, such as the one described above, between the administration of the antibody and the administration of the radiolabeled compound.

[0436] In certain embodiments of the present invention, there is no step of administering a removal agent or blocking agent. In certain embodiments, there is no step of administering any agent that binds to the first or second antibody between the administration of the antibody and the administration of the radiolabeled compound. In certain embodiments, there is no step of administering any agent between the administration of the antibody and the radiolabeled compound, except for a compound optionally selected from chemotherapeutic agents, immunotherapeutic agents and radiosensitizers. In some embodiments, no agent is administered between the administration of the antibody and the administration of the radiolabeled compound. In some embodiments, there may be no injection or infusion of any other agent into the subject between the administration of the antibody and the administration of the radiolabeled compound.

[0437] In some embodiments, the radiolabeled compound can be administered to the subject after a suitable period of time has been given for the first and second antibodies to localize to the target cells. For example, in some embodiments, the radiolabeled compound may be administered to the subject immediately after the first and second antibodies, or at least 4 hours, 8 hours, 1 day, or 2 days after the first and second antibodies. Optionally, the radiolabeled compound may be administered up to 3, 5, or 7 days after the first and second antibodies. In a particular embodiment, the radiolabeled compound may be administered to the subject 2 to 7 days after the first and second antibodies.

[0438] In some embodiments, the imaging method may be a pre-targeted radiation imaging method comprising, or essentially comprising, the steps of i) administering a set of antibodies (the first antibody and the second antibody may be administered simultaneously or sequentially in either order), ii) subsequently administering a radiolabeled compound, and iii) imaging the tissue or organ of interest. The diagnostic method may or may essentially comprise the steps of said and a subsequent step of forming a diagnosis, which may then be delivered to the patient, used as a basis for administering selected and / or treatment regimens.

[0439] The target antigen may be any target antigen discussed herein. In some embodiments, the target antigen may be a tumor-specific antigen discussed above, and imaging may be a tumor or a method of imaging a tumor. The individual may be known to have a tumor or may be suspected to have a tumor.

[0440] For example, this method can be used for lung cancer, non-small cell lung (NSCL) cancer, bronchioloalveolar cell lung cancer, bone cancer, pancreatic cancer including PDAC, skin cancer, head and neck cancer, melanoma of the skin or eye, uterine cancer, ovarian cancer, colorectal cancer which may be colon cancer and / or rectal cancer, cancer of the anal region, stomach cancer, gastric cancer. This may be a method for imaging tumors in individuals who have or are suspected of having a combination of one or more of the above cancers, including cancers such as breast cancer, uterine cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney or ureteral cancer, renal cell carcinoma, renal pelvis carcinoma, mesothelioma, hepatocellular carcinoma, cholangiocarcinoma, neoplasms of the central nervous system (CNS), spinal axial tumor, brainstem glioma, glioblastoma multiforme, astrocytoma, Schwann cell tumor, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma and Ewing's sarcoma (including refractory versions of any of the above cancers), or a checkpoint inhibitor-treated version of any of the above cancers, or a combination of one or more of the above cancers.

[0441] Further embodiments of the present invention Further embodiments of the present invention are described below in the following numbered paragraphs.

[0442] 1. A set of antibodies, i) A first antibody comprising an antigen-binding portion that binds to an antigen expressed on the surface of a target cell, and further comprising a VH domain of the antigen-binding site for a radiolabeled compound, but lacking a VL domain of the antigen-binding site for a radiolabeled compound, ii) A second antibody comprising an antigen-binding portion that binds to an antigen expressed on the surface of a target cell, further comprising a VL domain of the antigen-binding site for a radiolabeled compound, but lacking a VH domain of the antigen-binding site for a radiolabeled compound. Includes, A set of antibodies in which the VH domain of the first antibody and the VL domain of the second antibody are both capable of forming a functional antigen-binding site for a radiolabeled compound.

[0443] 2. A set of antibodies as described in paragraph 1, wherein the first antibody and the second antibody each contain i) an antibody fragment containing an antigen-binding site specific to an antigen expressed on the surface of a target cell, and ii) either the VL domain or the VH domain of the antigen-binding site for a radiolabeled compound.

[0444] 3. A set of antibodies as described in paragraph 2, wherein the antibody fragment is selected from at least one Fv, scFv, Fab, or cross-Fab fragment.

[0445] 4. A set of antibodies described in any one of paragraphs 1 to 3, wherein the first antibody and the second antibody each further contain an Fc domain.

[0446] 5. A set of antibodies as described in paragraph 4, wherein the first antibody and the second antibody each comprise i) an antibody fragment containing an antigen-binding site specific to an antigen expressed on the surface of a target cell, ii) an Fc region, and iii) either a VL domain or a VH domain of an antigen-binding site for a radiolabeled compound fused to the Fc region.

[0447] 6. A set of antibodies described in paragraph 4 or 5, wherein the Fc domain is modified to reduce or eliminate effector function.

[0448] 7. A set of antibodies according to any one of paragraphs 4 to 6, wherein each of the first and second antibodies comprises a) an Fc domain, b) at least one antigen-binding moiety including an antigen-binding site for a target antigen, and c) a polypeptide including either a VL domain or a VH domain of the antigen-binding site for a radiolabeled compound, wherein the antigen-binding moiety of (b) is fused to the N-terminus of one chain of the Fc domain, and the C-terminus of the polypeptide of (c) is fused to the N-terminus of the other chain of the Fc domain.

[0449] A set of antibodies as described in paragraph 7, wherein the antigen-binding portion of 8.(b) is an antibody fragment such as scFv, Fv, Fab, or cross Fab.

[0450] A set of antibodies described in paragraph 8, wherein the antigen-binding portion of 9.(b) is Fab.

[0451] 10. The first antibody, i) A complete light chain, ii) A complete heavy chain, iii) Additional Fc chains, iv) A polypeptide containing or comprising a VH domain at the antigen-binding site for a radiolabeled compound Includes, Both the light chain of (i) and the heavy chain of (ii) provide an antigen-binding site for the target antigen, and a polypeptide containing or consisting of a VH domain of the antigen-binding site for the radiolabeled compound is fused to the N-terminus of (iii) via a linker through its C-terminus. The second antibody, v) A complete light chain, vi) A complete heavy chain, vii) Additional Fc chains, viii) A polypeptide comprising or consisting of a VL domain, which is an antigen-binding site for a radiolabeled compound. Includes, A set of antibodies according to any one of paragraphs 7-9, wherein the light chain of (v) and the heavy chain of (vi) together provide an antigen-binding site for a target antigen, and a polypeptide comprising or consisting thereof a VL domain of the antigen-binding site for a radiolabeled compound is fused to the N-terminus of (vii) via a linker by its C-terminus.

[0452] 11. A set of antibodies described in any one of paragraphs 1 to 10, wherein the radiolabeled compound comprises a radiolabeled DOTA, or a salt or functional variant thereof.

[0453] 12. A set of antibodies described in any one of paragraphs 1 to 11, wherein the radiolabeled compound is a DOTA radiolabeled with a Lu or Y radioisotope, or a salt or functional variant thereof.

[0454] 13. A set of antibodies according to paragraph 11 or 12, wherein the VH domain of the antigen-binding site for the radiolabeled compound comprises (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 35, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 36, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 37.

[0455] 14. A set of antibodies according to any one of paragraphs 11 to 13, wherein the VH domain of the antigen-binding site for the radiolabeled compound comprises the amino acid sequence of SEQ ID NO: 41, or a variant thereof having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO: 41.

[0456] 15. A set of antibodies described in any one of paragraphs 11 to 14, wherein the VL domain of the antigen-binding site for the radiolabeled compound comprises (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 38, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 39, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 40.

[0457] 16. A set of antibodies according to any one of paragraphs 11 to 15, wherein the VL domain of the antigen-binding site for a radiolabeled compound comprises the amino acid sequence of SEQ ID NO: 42, or a variant thereof having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO: 42.

[0458] 17. A set of antibodies described in any one of paragraphs 1-10, wherein the radiolabeled compound contains Pb-DOTAM.

[0459] 18. A set of antibodies described in paragraph 17, wherein the functional binding site to Pb-DOTAM binds at binding affinity KD values ​​of 100 pM, 50 pM, 20 pM, 10 pM, 5 pM, or 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.

[0460] 19. A set of antibodies described in paragraph 17 or 18, wherein the functional binding site for Pb-DOTAM binds to Pb-DOTAM and Bi-DOTAM.

[0461] 20. The VH domain of the antigen-binding site for the radiolabeled compound, a) A heavy chain CDR2 containing the amino acid sequence FIGSRGDTYYASWAKG (SEQ ID NO: 2), or a variant thereof having up to 1, 2, or 3 substitutions in SEQ ID NO: 2, wherein these substitutions do not include Phe50, Asp56, and / or Tyr58, and optionally do not include Gly52, and / or Arg54, b) A heavy chain CDR3 comprising the amino acid sequence ERDPYGGGAYPPHL (SEQ ID NO: 3), or a variant thereof having up to 1, 2, or 3 substitutions in SEQ ID NO: 3, wherein these substitutions do not include Glu95, Arg96, Asp97, Pro98, optionally also omit Ala100C, Tyr100D and / or Pro100E, and / or optionally also omit Tyr99, Optionally, c) A set of antibodies according to any one of paragraphs 17 to 19, comprising a heavy chain CDR1 which is a heavy chain CDR1 having the amino acid sequence GFSLSTYSMS (SEQ ID NO: 1), or a variant thereof having up to one, two, or three substitutions in SEQ ID NO: 1.

[0462] 21. The VH domain of the antigen-binding site for the radiolabeled compound is (a) CDR-H1 containing the amino acid sequence GFSLSTYSMS (SEQ ID NO: 1), (b) CDR-H2 containing the amino acid sequence FIGSRGDTYYASWAKG (SEQ ID NO: 2), and (c) CDR-H3 containing the amino acid sequence ERDPYGGGAYPPHL (SEQ ID NO: 3). The set of antibodies described in paragraph 30, including

[0463] 22. The VH domain of the antigen-binding site for the radiolabeled compound, i) The amino acid sequence of SEQ ID NO: 143, or ii) A variant of SEQ ID NO: 143 having an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 143, wherein the N-terminal Q residue is either unchanged or substituted with a residue selected from the group consisting of E, K, R, S, T, A, L, Y, D, N and V, or iii) A variant of SEQ ID NO: 143 that differs from SEQ ID NO: 143 by only one or more substitutions and has an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 143, and optionally the N-terminal Q residue remains unchanged. A set of antibodies described in any one of paragraphs 17-21, including the set of antibodies described in any one of paragraphs 17-21.

[0464] 23. A set of antibodies according to any one of paragraphs 17-21, wherein the VH domain of the antigen-binding site for the radiolabeled compound comprises the amino acid sequence of SEQ ID NO: 7, or a variant thereof having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO: 7, and optionally further comprises an additional N-terminal residue, such as Q, or a residue selected from the group consisting of E, K, R, S, T, A, L, Y, D, N, and V.

[0465] 24. A set of antibodies according to any one of paragraphs 17 to 21, wherein the VH domain of the antigen-binding site for a radiolabeled compound comprises the amino acid sequence of SEQ ID NO: 9, or a variant thereof having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO: 9, and optionally further comprises an N-terminal residue, such as Q, or a residue selected from the group consisting of E, K, R, S, T, A, L, Y, D, N, and V.

[0466] 25. The VL domain of the antigen-binding site for the radiolabeled compound, d) A light chain CDR1 comprising the amino acid sequence QSSHSVYSDNDLA (SEQ ID NO: 4), or a variant thereof having up to 1, 2, or 3 substitutions in SEQ ID NO: 4, wherein these substitutions do not include Tyr28 and Asp32, e) A light chain CDR3 comprising the amino acid sequence LGGYDDESDTYG (SEQ ID NO: 6), or a variant thereof having up to 1, 2, or 3 substitutions in SEQ ID NO: 6, wherein these substitutions do not include Gly91, Tyr92, Asp93, Thr95c, and Tyr96, Optionally, f) A light chain CDR2 comprising the amino acid sequence QASKLAS (SEQ ID NO: 5) or a variant thereof having at least one, two, or three substitutions in SEQ ID NO: 5, and optionally a light chain CDR2 that does not contain Gln50. A set of antibodies described in any one of paragraphs 17-24, including the set of antibodies described in any one of paragraphs 17-24.

[0467] 26. A set of antibodies as described in paragraph 25, wherein the VL domain of the antigen-binding site for the radiolabeled compound comprises (d) CDR-L1 containing the amino acid sequence of QSSHSVYSDNDLA (SEQ ID NO: 4), (e) CDR-L2 containing the amino acid sequence of QASKLAS (SEQ ID NO: 5), and (f) CDR-L3 containing the amino acid sequence of LGGYDDESDTYG (SEQ ID NO: 6).

[0468] 27. The VL domain of the antigen-binding site for the radiolabeled compound, i) The amino acid sequence of SEQ ID NO: 144, or ii) A variant of SEQ ID NO: 144 having an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 144, wherein the N-terminal A residue is either unchanged or substituted with another amino acid selected from the group consisting of D, N, E, Q, S, A, V, L, T, Y, K and R, or iii) A variant of SEQ ID NO: 144 that differs from SEQ ID NO: 144 by only one or more substitutions and has an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 144, and optionally the N-terminal A residue remains unchanged. A set of antibodies described in any one of paragraphs 17-26, including [the specified antibody].

[0469] 28. A set of antibodies according to any one of paragraphs 17-26, wherein the VL domain of the antigen-binding site for the radiolabeled compound comprises the amino acid sequence of SEQ ID NO: 8, or a variant thereof having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO: 8, and optionally further comprising additional N-terminal residues (such as A), or residues selected from the group consisting of D, N, E, Q, S, A, V, L, T, Y, K, and R.

[0470] 29. A set of antibodies described in any one of paragraphs 1 to 28, wherein the first antibody and the second antibody bind to the same target antigen.

[0471] 30. The set of antibodies described in paragraph 29, wherein the first antibody and the second antibody bind to the same ep...

Claims

1. It is a set of antibodies, i) A first antibody comprising an antibody fragment containing an antigen-binding site specific to an antigen expressed on the surface of a target cell, and a polypeptide containing an Fc domain and a VH domain of an antigen-binding site for a radiolabeled compound, wherein the C-terminus of the antibody fragment is fused to the N-terminus of one chain of the Fc domain, and the C-terminus of the polypeptide is fused to the N-terminus of the other chain of the Fc domain, and the first antibody does not contain a VL domain of an antigen-binding site for the radiolabeled compound, and ii) A second antibody comprising an antibody fragment containing an antigen-binding site specific to an antigen expressed on the surface of a target cell, and a polypeptide containing an Fc domain and a VL domain of the antigen-binding site for the radiolabeled compound, wherein the C-terminus of the antibody fragment is fused to the N-terminus of one chain of the Fc domain, and the C-terminus of the polypeptide is fused to the N-terminus of the other chain of the Fc domain, and the second antibody does not contain the VH domain of the antigen-binding site for the radiolabeled compound. Includes, A set of antibodies in which the VH domain of the first antibody and the VL domain of the second antibody are both capable of forming a functional antigen-binding site for the radiolabeled compound.

2. The antibody set according to claim 1, wherein the antibody fragment is selected from at least one Fv, scFv, Fab, or cross-Fab fragment.

3. The antibody set according to claim 2, wherein the antibody fragment is Fab.

4. A set of antibodies according to any one of claims 1 to 3, wherein the Fc domain is modified to reduce or eliminate effector function.

5. The first antibody described above, i) A complete light chain, ii) A complete heavy chain, iii) Additional Fc chains, iv) A polypeptide comprising or consisting of the VH domain of the antigen-binding site to the radiolabeled compound Includes, The light chain of (i) and the heavy chain of (ii) both provide an antigen-binding site for the target antigen, and the polypeptide, which includes or consists of the VH domain of the antigen-binding site for the radiolabeled compound, is fused to the N-terminus of (iii) via a linker at its C-terminus. The second antibody described above, v) A complete light chain, vi) A complete heavy chain, Vii) Additional Fc chains, viiii) A polypeptide comprising or consisting of the VL domain of the antigen-binding site to the radiolabeled compound Includes, A set of antibodies according to any one of claims 1 to 4, wherein the light chain of (v) and the heavy chain of (vi) together provide an antigen-binding site for a target antigen, and the polypeptide comprising or consisting of the VL domain of the antigen-binding site for the radiolabeled compound is fused to the N-terminus of (vii) via a linker at its C-terminus.

6. A set of antibodies according to any one of claims 1 to 5, wherein the radiolabeled compound comprises radiolabeled DOTA, or a salt or functional variant thereof.

7. The antibody set according to any one of claims 1 to 6, wherein the radiolabeled compound is DOTA radiolabeled with a Lu or Y radioisotope, or a salt or functional variant thereof.

8. A set of antibodies according to claim 6 or 7, wherein the VH domain of the antigen-binding site for the radiolabeled compound comprises (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 35, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 36, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO:

37.

9. A set of antibodies according to any one of claims 6 to 8, wherein the VH domain of the antigen-binding site for the radiolabeled compound includes the amino acid sequence of SEQ ID NO: 41, or a variant thereof having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO:

41.

10. A set of antibodies according to any one of claims 6 to 9, wherein the VL domain of the antigen-binding site for the radiolabeled compound comprises (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 38, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 39, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO:

40.

11. A set of antibodies according to any one of claims 6 to 10, wherein the VL domain of the antigen-binding site for the radiolabeled compound includes the amino acid sequence of SEQ ID NO: 42, or a variant thereof having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO:

42.

12. A set of antibodies according to any one of claims 1 to 5, wherein the radiolabeled compound comprises Pb-DOTAM.

13. The functional binding site for Pb-DOTAM has a binding affinity of K Ｄ A set of antibodies according to claim 12, which binds at values ​​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.

14. A set of antibodies according to claim 12 or claim 13, wherein the functional binding site for Pb-DOTAM binds to Pb-DOTAM and Bi-DOTAM.

15. The VH domain of the antigen-binding site for the radiolabeled compound, a) A heavy chain CDR2 comprising the amino acid sequence FIGSRRGDTYYASWAKG (SEQ ID NO: 2), or a variant thereof 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, b) A heavy chain CDR3 comprising the amino acid sequence ERDPYGGGAYPPHL (SEQ ID NO: 3), or a variant thereof having up to one, two, or three substitutions in SEQ ID NO: 3, wherein these substitutions do not include Glu95, Arg96, Asp97, Pro98, optionally also omit Ala100C, Tyr100D and / or Pro100E, and / or optionally also omit Tyr99, Optionally, c) Heavy chain CDR1 containing the amino acid sequence GFSLSTYSMS (SEQ ID NO: 1), or a variant thereof having up to 1, 2, or 3 substitutions in SEQ ID NO: 1, and A set of antibodies according to any one of claims 12 to 14, including the set of antibodies described in any one of claims 12 to 14.

16. The antibody set according to claim 15, wherein the VH domain of the antigen-binding site for the radiolabeled compound comprises (a) CDR-H1 containing the amino acid sequence of GFSLSTYSMS (SEQ ID NO: 1), (b) CDR-H2 containing the amino acid sequence of FIGSRRGDTYYASWAKG (SEQ ID NO: 2), and (c) CDR-H3 containing the amino acid sequence of ERDPYGGGAYPPHL (SEQ ID NO: 3).

17. The VH domain of the antigen-binding site for the radiolabeled compound, i) The amino acid sequence of SEQ ID NO: 143, or ii) A variant of SEQ ID NO: 143 containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 143, wherein the N-terminal Q residue is either unchanged or replaced with a residue selected from the group consisting of E, K, R, S, T, A, L, Y, D, N and V, or iii) A variant of SEQ ID NO: 143 that differs from SEQ ID NO: 143 by only one or more substitutions and has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 143, wherein the N-terminal Q residue remains unchanged. A set of antibodies according to any one of claims 12 to 16, including the set of antibodies described in any one of claims 12 to 16.

18. A set of antibodies according to any one of claims 12 to 16, wherein the VH domain of the antigen-binding site for the radiolabeled compound comprises the amino acid sequence of SEQ ID NO: 7, or a variant thereof comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO: 7, and optionally further comprises an additional N-terminal residue, for example Q, or a residue selected from the group consisting of E, K, R, S, T, A, L, Y, D, N, and V.

19. A set of antibodies according to any one of claims 12 to 16, wherein the VH domain of the antigen-binding site for the radiolabeled compound includes the amino acid sequence of SEQ ID NO: 9, or a variant thereof having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO: 9, and optionally further includes an N-terminal residue, for example Q, or a residue selected from the group consisting of E, K, R, S, T, A, L, Y, D, N, and V.

20. The VL domain of the antigen-binding site for the radiolabeled compound, d) A light chain CDR1 comprising the amino acid sequence QSSHSVYSDNDLA (SEQ ID NO: 4), or a variant thereof having up to one, two, or three substitutions in SEQ ID NO: 4, wherein these substitutions do not include Tyr28 and Asp32, e) A light chain CDR3 comprising the amino acid sequence LGGYDDESDTYG (SEQ ID NO: 6), or a variant thereof having up to one, two, or three substitutions in SEQ ID NO: 6, wherein these substitutions do not include Gly91, Tyr92, Asp93, Thr95c, and Tyr96, Optionally, f) A light chain CDR2 comprising the amino acid sequence QASKLAS (SEQ ID NO: 5) or a variant thereof having at least one, two, or three substitutions in SEQ ID NO: 5, and optionally a light chain CDR2 that does not contain Glun50. A set of antibodies according to any one of claims 12 to 19, including the set of antibodies described in any one of claims 12 to 19.

21. A set of antibodies according to claim 20, wherein the VL domain of the antigen-binding site for the radiolabeled compound comprises (d) CDR-L1 containing the amino acid sequence of QSSHSVYSDNDLA (SEQ ID NO: 4), (e) CDR-L2 containing the amino acid sequence of QASKLAS (SEQ ID NO: 5), and (f) CDR-L3 containing the amino acid sequence of LGGYDDESDTYG (SEQ ID NO: 6).

22. The VL domain of the antigen-binding site for the radiolabeled compound, i) The amino acid sequence of SEQ ID NO: 144, or ii) A variant of SEQ ID NO: 144 having an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 144, wherein the N-terminal A residue is either unchanged or substituted with another amino acid selected from the group consisting of D, N, E, Q, S, A, V, L, T, Y, K and R, or iii) A variant of SEQ ID NO: 144 that differs from SEQ ID NO: 144 by only one or more substitutions and has an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 144, wherein the N-terminal A residue remains unchanged. A set of antibodies according to any one of claims 12 to 21, including the set of antibodies described in any one of claims 12 to 21.

23. A set of antibodies according to any one of claims 12 to 21, wherein the VL domain of the antigen-binding site for the radiolabeled compound includes the amino acid sequence of SEQ ID NO: 8, or a variant thereof having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO: 8, and optionally further includes an additional N-terminal residue (such as A), or a residue selected from the group consisting of D, N, E, Q, S, A, V, L, T, Y, K, and R.

24. A set of antibodies according to any one of claims 1 to 23, wherein the first antibody and the second antibody bind to the same target antigen.

25. The antibody set according to claim 24, wherein the first antibody and the second antibody bind to the same epitope of the target antigen.

26. The antibody set according to claim 24, wherein the first antibody binds to an epitope of the target antigen that is different from the second antibody.

27. A set of antibodies according to any one of claims 1 to 26, wherein the antigen expressed on the surface of target cells is a tumor-associated antigen.

28. A set of antibodies according to any one of claims 1 to 27, wherein the antigen expressed on the surface of target cells is selected from the group consisting of carcinoembryonic antigen (CEA), CD20, HER2, EGP-1 (also known as trophoblast-2, epithelial glycoprotein-1), colon-specific antigen-p (CSAp), pancreatic mucin MUC1, GPRC5D, and FAP.

29. A set of antibodies according to any one of claims 1 to 28, wherein the antigen expressed on the surface of target cells is selected from the group consisting of CEA, GPRC5D, and FAP.

30. A set of antibodies according to any one of claims 1 to 29, wherein the antigen expressed on the surface of target cells is CEA.

31. The first antibody includes an antigen-binding site that binds to CEA, and the antigen-binding site is A heavy chain variable region comprising (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 19, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 20, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 21, and (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 22, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 23, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 24, comprising a light chain variable region. A set of antibodies according to claim 30, including the set of antibodies described in claim 30.

32. The set of antibodies according to claim 30 or 31, wherein the first antibody includes an antigen-binding site for CEA, comprising a VH sequence which comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 25, or a variant thereof which has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO:

25.

33. A set of antibodies according to any one of claims 30 to 32, wherein the first antibody includes an antigen-binding site for CEA, comprising a VL sequence which comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 26, or a variant thereof which has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO:

26.

34. A set of antibodies according to claim 30, wherein the first antibody includes an antigen-binding site that binds to CEA, comprising a heavy chain variable region including (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 43, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 44, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 45, and a light chain variable region including (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 46, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 47, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO:

48.

35. The set of antibodies according to claim 30 or 34, wherein the first antibody comprises an antigen-binding site for CEA, the first antibody comprising a VH sequence comprising an amino acid sequence selected from the group comprising SEQ ID NO: 49, or a variant thereof having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO:

49.

36. A set of antibodies according to any one of claims 30, 34, or 35, wherein the first antibody includes an antigen-binding site for CEA, comprising a VL sequence which comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 50, or a variant thereof which has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO:

50.

37. The first antibody includes an antigen-binding site that binds to CEA, and the antigen-binding site is A heavy chain variable region comprising (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, and (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, comprising a light chain variable region. A set of antibodies according to claim 30, including the set of antibodies described in claim 30.

38. The antibody set according to claim 30 or 37, wherein the first antibody includes an antigen-binding site for CEA, comprising a VH sequence which comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 17, or a variant thereof which has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO:

17.

39. The set of antibodies according to claim 30, 37, or 38, wherein the first antibody includes an antigen-binding site for CEA, comprising a VL sequence which comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 18, or a variant thereof which has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO:

18.

40. The first antibody includes an antigen-binding site that binds to CEA, and the antigen-binding site is A heavy chain variable region comprising (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 59, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 60, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 61, and (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 62, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 63, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 64, comprising a light chain variable region. A set of antibodies according to claim 30, including the set of antibodies described in claim 30.

41. A set of antibodies according to claim 30 or claim 40, wherein the first antibody comprises an antigen-binding site for CEA, the first antibody comprising a VH sequence comprising an amino acid sequence selected from the group comprising SEQ ID NO: 65, or a variant thereof comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO:

65.

42. A set of antibodies according to claim 30, 40, or 41, wherein the first antibody comprises an antigen-binding site for CEA, the VL sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 66, or a variant thereof having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO:

66.

43. A set of antibodies according to claim 40, wherein the first antibody includes an antigen-binding site that binds to CEA, comprising a heavy chain variable region including (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 116, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 117 or 118, (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 119, and (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 120, 121 or 122, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 123, 124 or 125, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO:

126.

44. A set of antibodies according to claim 30 or 43, wherein the first antibody includes an antigen-binding site that binds to CEA, comprising a heavy chain variable region (VH) containing an amino acid sequence selected from SEQ ID NOs. 129, 130, 131, 132, 133, or 134, or a sequence having 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity therewith, and a light chain variable region (VL) containing an amino acid sequence selected from SEQ ID NOs. 135, 136, 137, 138, 139, or 140, or a sequence having 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity therewith.

45. The first antibody includes an antigen-binding site that binds to CEA, and the antigen-binding site is (a) A VH domain containing the amino acid sequence of SEQ ID NO: 129 and a VL domain containing the amino acid sequence of SEQ ID NO: 139, or (b) A VH domain containing the amino acid sequence of SEQ ID NO: 133 and a VL domain containing the amino acid sequence of SEQ ID NO: 139, or (c) A VH domain containing the amino acid sequence of SEQ ID NO: 130 and a VL domain containing the amino acid sequence of SEQ ID NO: 139, or (d) A VH domain containing the amino acid sequence of SEQ ID NO: 134 and a VL domain containing the amino acid sequence of SEQ ID NO: 138, or (e) A VH domain containing the amino acid sequence of SEQ ID NO: 133 and a VL domain containing the amino acid sequence of SEQ ID NO: 138, or (f) A VH domain containing the amino acid sequence of SEQ ID NO: 131 and a VL domain containing the amino acid sequence of SEQ ID NO: 138, or (g) VH domain containing the amino acid sequence of SEQ ID NO: 129 and VL domain containing the amino acid sequence of SEQ ID NO: 138 A set of antibodies according to claim 30, 43, or 44, comprising:

46. The second antibody includes an antigen-binding site that binds to CEA, and the antigen-binding site is A heavy chain variable region comprising (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 19, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 20, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 21, and (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 22, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 23, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 24, comprising a light chain variable region. A set of antibodies according to any one of claims 30 to 45, including the set of antibodies described in any one of claims 30 to 45.

47. A set of antibodies according to any one of claims 30 to 46, wherein the second antibody includes an antigen-binding site for CEA, comprising a VH sequence which comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 25, or a variant thereof which has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO:

25.

48. A set of antibodies according to any one of claims 30 to 47, wherein the second antibody includes an antigen-binding site for CEA, comprising a VL sequence which comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 26, or a variant thereof which has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO:

26.

49. A set of antibodies according to any one of claims 30 to 45, wherein the second antibody includes an antigen-binding site that binds to CEA, comprising a heavy chain variable region including (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 43, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 44, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 45, and a light chain variable region including (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 46, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 47, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO:

48.

50. A set of antibodies according to any one of claims 30 to 45 or 49, wherein the second antibody comprises an antigen-binding site for CEA, the VH sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 49, or a variant thereof having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO:

49.

51. A set of antibodies according to any one of claims 30 to 45, 49, or 50, wherein the second antibody includes an antigen-binding site for CEA, comprising a VL sequence which comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 50, or a variant thereof which has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO:

50.

52. The second antibody includes an antigen-binding site that binds to CEA, and the antigen-binding site is A heavy chain variable region comprising (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, and (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, comprising a light chain variable region. A set of antibodies according to any one of claims 30 to 45, including the set of antibodies described in any one of claims 30 to 45.

53. A set of antibodies according to any one of claims 30 to 45 or 52, wherein the second antibody includes an antigen-binding site for CEA, comprising a VH sequence which comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 17, or a variant thereof which has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO:

17.

54. A set of antibodies according to any one of claims 30 to 45, 52, or 53, wherein the second antibody includes an antigen-binding site for CEA, comprising a VL sequence which comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 18, or a variant thereof which has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO:

18.

55. The second antibody includes an antigen-binding site that binds to CEA, and the antigen-binding site is A heavy chain variable region comprising (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 59, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 60, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 61, and (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 62, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 63, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 64, comprising a light chain variable region. A set of antibodies according to any one of claims 30 to 45, including the set of antibodies described in any one of claims 30 to 45.

56. A set of antibodies according to any one of claims 30 to 45 or 55, wherein the second antibody includes an antigen-binding site for CEA, comprising a VH sequence which comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 65, or a variant thereof which has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO:

65.

57. The antibody set according to claim 30 to 45, 55 or 56, wherein the second antibody comprises an antigen-binding site for a CEA that includes a VL sequence containing an amino acid sequence selected from the group consisting of SEQ ID NO: 66, 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:

66.

58. A set of antibodies according to claims 30 to 45, wherein the second antibody includes an antigen-binding site that binds to CEA, comprising a heavy chain variable region including (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 116, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 117 or 118, (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 119, and (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 120, 121 or 122, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 123, 124 or 125, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO:

126.

59. A set of antibodies according to claims 30 to 45 or 58, wherein the second antibody includes an antigen-binding site that binds to CEA, comprising a heavy chain variable region (VH) containing an amino acid sequence selected from SEQ ID NOs. 129, 130, 131, 132, 133, or 134, or a sequence having 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity therewith, and a light chain variable region (VL) containing an amino acid sequence selected from SEQ ID NOs. 135, 136, 137, 138, 139, or 140, or a sequence having 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity therewith.

60. The second antibody includes an antigen-binding site that binds to CEA, and the antigen-binding site is (a) A VH domain containing the amino acid sequence of SEQ ID NO: 129 and a VL domain containing the amino acid sequence of SEQ ID NO: 139, or (b) A VH domain containing the amino acid sequence of SEQ ID NO: 133 and a VL domain containing the amino acid sequence of SEQ ID NO: 139, or (c) A VH domain containing the amino acid sequence of SEQ ID NO: 130 and a VL domain containing the amino acid sequence of SEQ ID NO: 139, or (d) A VH domain containing the amino acid sequence of SEQ ID NO: 134 and a VL domain containing the amino acid sequence of SEQ ID NO: 138, or (e) A VH domain containing the amino acid sequence of SEQ ID NO: 133 and a VL domain containing the amino acid sequence of SEQ ID NO: 138, or (f) A VH domain containing the amino acid sequence of SEQ ID NO: 131 and a VL domain containing the amino acid sequence of SEQ ID NO: 138, or (g) VH domain containing the amino acid sequence of SEQ ID NO: 129 and VL domain containing the amino acid sequence of SEQ ID NO: 138 A set of antibodies according to claims 30 to 45, 58, or 59, comprising:

61. The antibody set according to claim 30, wherein the first antibody is the antibody described in any one of claims 31 to 33, and the second antibody is the antibody described in any one of claims 46 to 48.

62. i) The first antibody comprises the first heavy chain of SEQ ID NO: 112, the second heavy chain of SEQ ID NO: 146, and the light chain of SEQ ID NO:

115. ii) The antibody set according to claim 1, wherein the second antibody comprises the first heavy chain of SEQ ID NO: 112, the second heavy chain of SEQ ID NO: 145, and the light chain of SEQ ID NO:

115.

63. A set of nucleic acids that expresses a set of antibodies according to any one of claims 1 to 62.

64. An expression vector or a set of expression vectors comprising the set of nucleic acids described in claim 63.

65. A host cell or a set of host cells comprising the expression vector or set of expression vectors described in claim 64.

66. A method for pre-targeting radioimmunotherapy, i) Administering the antibody set described in any one of claims 1 to 62, wherein the first antibody and the second antibody are administered simultaneously or sequentially in any order, the antibodies bind to the target antigen and localize to the surface of cells expressing the target antigen, and the association of the first antibody and the second antibody forms a functional binding site for the radiolabeled compound. and, ii) Next, administering a radiolabeled compound, wherein the radiolabeled compound binds to a functional binding site for the radiolabeled compound. Methods that include...

67. The method according to claim 66, wherein the method does not include the step of administering a removal agent or a blocking agent.

68. The method according to claim 66 or 67, wherein the subject is a human.

69. The method according to any one of claims 66 to 67, wherein the target antigen is a cancer-related antigen or a tumor-related antigen, and the method is a method of radioimmunotherapy for a tumor or cancer.

70. A set of antibodies according to any one of claims 1 to 62 for use in a method for pretargeting radioimmunotherapy according to any one of claims 66 to 69.

71. A method for targeting a tissue or organ with a radioactive isotope for radiation imaging, i) Administering a set of antibodies according to any one of claims 1 to 62, wherein the first antibody and the second antibody are administered simultaneously or sequentially in any order, the antibodies bind to a target antigen and localize to the surface of cells expressing the target antigen, and the association of the first antibody and the second antibody forms a functional binding site for the radiolabeled compound. and, ii) Next, administering a radiolabeled compound, wherein the radiolabeled compound binds to the functional binding site for the radiolabeled compound. Methods that include...

72. The method according to claim 71, wherein the method does not include the step of administering a removal agent or a blocking agent.

73. The method according to claim 71 or 72, further comprising the step of imaging.

74. The method according to claim 73, wherein the target antigen is a cancer-related antigen or tumor-related antigen, and the method is a method for imaging a tumor or cancer.

75. A peptide linker comprising y consecutive residues selected from the group consisting of Gly and Ser, wherein y = 5-100, 5-70, 5-60, 5-50 or 10-100, 10-70, 10-60 or 10-50, and the last serine is at position y-2 or y-3.

76. The peptide linker according to claim 75, wherein y = 10 to 50.

77. The peptide linker according to claim 75, wherein y = 15 to 31 or 15 to 30.

78. The peptide linker according to claim 77, wherein y = 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25.

79. The peptide linker according to claim 78, wherein y = 20 or 21.

80. The peptide linker according to claim 75, comprising the sequence (GxS)n(GGSGG) or (GxS)n(GGSGGGG), wherein G = glycine, S = serine, x = 4, and n = 1 to 20, 2 to 20, 1 to 10, or 2 to 10.

81. The peptide linker according to claim 80, wherein n = 2, 3, 4, or 5.

82. The peptide linker according to claim 80, wherein n = 2, 3, or 4.

83. A peptide linker according to claim 75, comprising the sequence GGGGGSGGGGGSGGGGGGGG (SEQ ID NO: 150) or GGGGGSGGGGGGGGGG (SEQ ID NO: 151).

84. Use of a peptide linker according to any one of claims 75 to 83 for joining a first domain and a second domain of a multi-domain protein.

85. A multidomain protein comprising at least a first domain and a second domain, wherein the first domain and the second domain are linked by a peptide linker according to any one of claims 75 to 83.

86. The use according to claim 84, or the multidomain protein according to claim 85, wherein the multidomain protein is an antibody.

87. The use according to claim 84, or the multidomain protein according to claim 85, wherein the multidomain protein is a bispecific antibody.

88. The use according to claim 84, or the multi-domain protein according to claim 85, wherein the first domain is an antigen-binding portion and the second domain is a VH domain or a VL domain.

89. Use of the multidomain-binding protein according to claim 88, wherein the antigen-binding portion is an antibody fragment.

90. The use according to claim 84, or the multi-domain protein according to claim 85, wherein the first domain is an Fc domain and the second domain is a VH domain or a VL domain.

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