Antibodies that bind to cancer cells and target radionuclides to said cells

Abstract

To provide a set of antibodies that bind to an antigen on a target cell and target a radionuclide to the cell, and an agent comprising the set of antibodies.SOLUTION: I) a first antibody that binds to an antigen expressed on the surface of a target cell, wherein said first antibody further comprises a VH domain of a functional antigen binding site for a radiolabeled compound, but does not comprise a VL domain of a functional antigen binding site for a radiolabeled compound; and ii) a second antibody that binds to said antigen expressed on the surface of a target cell, wherein said second antibody comprises: A second antibody which further comprises a VL domain of a functional antigen binding site for a radiolabelled compound, but does not comprise a VH domain of a functional antigen binding site for a radiolabelled compound; wherein said VH domain of the first antibody and said VL domain of the second antibody are capable of together forming a functional antigen binding site for a radiolabelled compound.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to antibodies that bind to antigens on target cells and target radionuclides to said cells, and methods of using the antibodies. [Background technology]

[0002] Monoclonal antibodies have been developed to target drugs to cancer cells, and the potential exists for conjugating toxic drugs to antibodies that bind to tumor-associated antigens to result in more specific tumor killing with reduced damage to surrounding tissue.

[0003] Pretargeting radioimmunotherapy (PRIT) uses an antibody construct that has 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. Then, the radiolabeled compound is administered. Because the radiolabeled compound is a small molecule, it can be rapidly delivered to the tumor and rapidly eliminated, thereby reducing the amount of radiation exposure outside the tumor (Goldenberg et al., Theranostics, 2012, 2(5), 523-540). Similar procedures can also be used for imaging. Pretargeting can use bispecific antibodies or avidin-biotin systems, but the latter has the disadvantage that avidin / streptavidin is immunogenic.

[0004] Pretargeting radioimmunotherapy or imaging generally uses a removal agent or blocking agent, which is administered between the step of administering antibody and the step of administering radiolabeled compound.The purpose is to remove antibody from blood and / or block the binding site of circulating antibody to radiolabeled compound (see, for example, Karacay et al., Bioconj.Chem., 13(5), 1054-1070(2002)).The use of a removal agent or blocking agent allows for the administration of a sufficient level of radioactivity for effective treatment while limiting harmful toxicity, but the timing and dosage must be carefully selected.Therefore, the use of a removal phase is a complex aspect of pretargeting method. Summary of the Invention

[0005] The present invention provides a set of antibodies useful in pretargeting methods and methods of using the antibodies.

[0006] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: i) a first antibody that binds to an antigen expressed on the surface of a target cell, the first antibody further comprising a VH domain of an antigen-binding site for a radiolabeled compound, but not comprising a VL domain of an antigen-binding site for a radiolabeled compound; and ii) a second antibody that binds to an antigen expressed on the surface of a target cell, the second antibody further comprising a VL domain of an antigen-binding site for a radiolabeled compound, but not comprising a VH domain of an antigen-binding site for a radiolabeled compound. Includes; the VH domain of the first antibody and the VL domain of the second antibody are capable of together forming a functional antigen-binding site for a radiolabeled compound; A set of antibodies is provided.

[0007] Neither the first antibody nor the second antibody contains a functional antigen-binding site for the radiolabeled compound. The first antibody has only a VH domain derived from the functional binding site for the radiolabeled compound, but no VL domain. The second antibody has only a VL domain, but no VH domain.

[0008] A functional antigen-binding site for a radiolabeled compound is formed when the VH domain of a first antibody associates with the VL domain of a second antibody, which can occur, for example, when the first and second antibodies bind to the same individual target cell or to adjacent cells.

[0009] As used herein, the first and second antibodies described herein may be referred to as "single domain split antibodies," "split antibodies," or "demibodies." The VH and VL domains, which together form the antigen binding site capable of binding a radiolabeled compound, are distributed between the two antibodies and are not part of the same antibody.

[0010] The split-domain format means that the radiolabeled compound cannot bind to the first antibody on its own or to the second antibody on its own: there is little or no stable association between the first and second antibodies in blood, and therefore little or no stable binding to the radiolabeled compound.

[0011] Herein, an antigen expressed on the surface of a target cell may be referred to as a "target antigen" or "TA." In accordance with the present invention, the first antibody and the second antibody described above have binding sites for the same target antigen (for the avoidance of doubt, when it is stated that an antibody binds to the same target antigen, this means that the antibody has a binding site capable of binding to the same target antigen, including the possibility that the antibody may bind to two separate antigen molecules that are the same as each other). For example, in one embodiment, both the first antibody and the second antibody bind to CEA.

[0012] In some embodiments, the first antibody and the second antibody may bind to (have a binding site for) the same epitope of the target antigen, while in other embodiments, the first antibody may bind to (have a binding site for) a different epitope of the target antigen than the second antibody.

[0013] In some embodiments, the first antibody and the second antibody may comprise the same antigen-binding site for the target antigen, i.e., the first antibody and the second antibody may comprise an antigen-binding site capable of binding to the target antigen comprising a VL sequence and a VH sequence, and the VL sequence and VH sequence forming the antigen-binding site are the same in the first antibody and the second antibody.

[0014] In some embodiments, each of the first and second antibodies is bivalent with respect to the target antigen. In some embodiments, each of the first and second antibodies is bivalent and monospecific with respect to an epitope. In other embodiments, each of the first and second antibodies is biparatopic with respect to the target antigen, i.e., each of the first and second antibodies has binding sites for two different epitopes of the target antigen.

[0015] In some embodiments, it may be preferable for the first antibody and / or the second antibody to comprise an Fc region. The presence of an Fc region has benefits in the context of radioimmunotherapy and radioimaging, for example, extending the circulating half-life of the protein and / or resulting in higher tumor uptake than observed with small fragments. The "split-domain" formats described herein may be particularly advantageous in this context, as they mitigate the increased likelihood of association with radiolabeled compounds that would otherwise result from the extended presence of circulating antibodies.

[0016] In some embodiments, the Fc domain is modified to reduce or eliminate effector function.

[0017] In another aspect, the present invention provides a pharmaceutical composition comprising the set of antibodies described herein. In another aspect, the present invention provides a kit comprising two separate pharmaceutical compositions, each comprising one of the antibodies described herein (i.e., each of the first antibody and the second antibody).

[0018] In a further aspect, the present invention relates to a polynucleotide or set of polynucleotides encoding any of the antibodies or set of antibodies described herein. In another aspect, the present invention relates to a vector or set of vectors, optionally an expression vector or set of expression vectors, comprising said one or more polynucleotides. In a further object, the present invention relates to a prokaryotic or eukaryotic host cell or set of host cells comprising the vector or set of vectors of the present invention. Additionally, a method of producing an antibody is provided, comprising culturing a host cell such that the antibody is produced.

[0019] In some embodiments, the antibodies described herein are used in pretargeted radioimmunotherapy (PRIT) or pretargeted radioimaging methods.

[0020] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: i) administering to a subject a first antibody and a second antibody described above; and ii) subsequently administering to said subject a radiolabeled compound. The present invention provides a pretargeting radioimmunotherapy method comprising:

[0021] In another aspect, the present invention provides a first antibody and a second antibody described above for use in a method of treatment comprising administering the first antibody and the second antibody to a subject and subsequently administering a radiolabeled compound to the subject. In another aspect, the present invention provides a first antibody described above for use in a method of treatment comprising administering the first antibody and the second antibody to a subject and subsequently administering a radiolabeled compound to the subject. In another aspect, the present invention provides a second antibody described above for use in a method of treatment comprising administering the first antibody and the second antibody to a subject and subsequently administering a radiolabeled compound to the subject.

[0022] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: i) administering to a subject a first antibody and a second antibody described herein, wherein the antibodies bind to a target antigen and localize to the surface of a cell expressing the target antigen, and wherein the first antibody and the second antibody described herein; ii) subsequently administering a radiolabeled compound; and optionally, iii) Imaging the tissue or organ in which the radionuclide is localized The present invention provides a method of radiological imaging, comprising:

[0023] In another aspect, the present invention provides a diagnostic method performed on the human or animal body, comprising: i) administering to a subject a first antibody and a second antibody described herein, wherein the antibodies bind to a target antigen and localize to the surface of a cell expressing the target antigen, and wherein the first antibody and the second antibody described herein; ii) subsequently administering a radiolabeled compound; and optionally, iii) Imaging the tissue or organ in which the radionuclide is localized The present invention provides a first antibody and a second antibody described herein for use in a diagnostic method comprising:

[0024] The imaging step is followed by forming a diagnosis and, optionally, delivering the diagnosis to the subject. In some embodiments, the method can further include determining an appropriate treatment and, optionally, administering the treatment to the subject.

[0025] In each of the above methods / uses, binding of the first antibody and the second antibody to the same or adjacent target cells results in association of the VH and VL domains of the antigen-binding site for the radiolabeled compound and the formation of a functional antigen-binding site for the radiolabeled compound. Thus, after administration of the radiolabeled compound, the radiolabeled compound binds to the functional antigen-binding site formed by the association of VH and VL.

[0026] 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.

[0027] In the art, PRIT or radioimaging methods often involve a clearing step, which involves administering an agent between the administration of the antibody and the administration of the radiolabeled compound, which agent increases the rate of clearance of the antibody from the blood and / or blocks the binding of the radiolabeled compound to the antibody.

[0028] In certain embodiments of the methods and uses described herein, the methods do not include a removal step. That is, the methods do not include a step of administering a removal or blocking agent between the administration of the first and second antibodies and the administration of the radiolabeled compound (i.e., after administration of the antibodies but before administration of the radiolabeled compound). In another embodiment, no agent other than an optional radiosensitizer, immunotherapeutic agent, and / or chemotherapeutic agent is administered between the administration of the first and second antibodies and the administration of the radiolabeled compound. In another embodiment, no agent is administered between the administration of the first and second antibodies and the administration of the radiolabeled compound.

[0029] In some embodiments, the antibodies described herein may be administered as part of a combination therapy. For example, the antibodies described herein may be administered in combination with one or more radiosensitizers, immunotherapeutics, and / or chemotherapeutics; the radiosensitizers, immunotherapeutics, or chemotherapeutics and the antibodies may be administered simultaneously or sequentially, in any order.

[0030] The radioimaging methods and radioimmunotherapy described herein may optionally be combined as discussed further herein.

[0031] In a further aspect, the present invention provides a method for producing a pharmaceutical composition comprising: i) a first antibody and a second antibody described herein; ii) a radiolabeled compound that binds to the antigen-binding site formed by the association of the first antibody with the second antibody; A kit comprising:

[0032] Optionally, the kit may exclude (ie, may not include) a clearing agent or a blocking agent, as described herein.

[0033] Optionally, the kit can further comprise a radiosensitizing agent, an immunotherapeutic agent, or a chemotherapeutic agent.

[0034] 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 is present in a pharmaceutical composition separate from the antibody. [Brief explanation of the drawings]

[0035] [Figure 1] FIG. 1 shows the schematic structures of a target antigen (TA)-DOTAM bispecific antibody (TA-DOTAM BsAb) belonging to the comparative example, and an exemplary TA-split-DOTAM-VH / VL antibody according to the present invention. [Figure 2]Schematic diagram showing assembly of split-VH / VL DOTAM binders on tumor cells. TA-split-DOTAM-VH / VL antibodies will not significantly bind 212Pb-DOTAM unless they are bound to a tumor antigen (TA) on the target cell where the two domains of the DOTAM binder assemble. [Figure 3] FIG. 1 shows an overview of an example of a three-step TA-PRIT concept with the use of a remover. [Figure 4] FIG. 1 shows an overview of an example of a two-step TA-PRIT concept where no remover is used. [Figure 5] Binding of split antibodies to MKN45 cells confirms their CEA binding ability. Antibody detection is performed using a secondary antibody specific for human IgG. [Figure 6] Binding of split antibodies to MKN45 cells confirms DOTAM binding ability. Antibody detection is performed using Pb-DOTAM-FITC. [Figure 7A] FIG. 1 shows an exemplary protocol (h=hours, d=days, w=weeks) for two-step PRIT with CEA-split-DOTAM-VH / VL performed in SCID mice bearing SC BxPC3 tumors. [Figure 7B] FIG. 1 shows an exemplary protocol (h=hours, d=days, w=weeks) for a three-step PRIT control performed in SCID mice bearing SC BxPC3 tumors. [Figure 8] Figure 1 shows the biodistribution of pretargeted 212Pb-DOTAM in SCID mice bearing SC BxPC3 tumors 6 hours after injection of 212Pb-DOTAM pretargeted with CEA-split-DOTAM-VH alone, CEA-split-DOTAM-VL alone, or a combination of two complementary antibodies, or using standard three-step PRIT (ID% per g ± SD, n=4). [Figure 9] FIG. 1 shows the pharmacokinetics of CEA-split-DOTAM-VH / VL after IV injection in SCID mice. [Figure 10] Figure 1 shows the experimental design of protocol 158, involving two (top) or three (bottom) steps of CEA-PRIT in SCID mice bearing SC BxPC3 tumors. *The dose of CEA split DOTAM BsAb was adjusted to compensate for hole / hole impurities in the 2 / 4 construct. [Figure 11] Figure 1 shows the biodistribution of pretargeted Pb-DOTAM in SCID mice bearing SC BxPC3 tumors (6 hours after injection). Distribution of Pb in tumor-bearing SCID mice 6 hours after injection of Pb-DOTAM pretargeted with CEA-DOTAM BsAb or a dual paratope combination of CEA-split-DOTAM antibodies. The content of radioactive material in organs and tissues is expressed as the mean ID% ± SD per g (n = 4). [Figure 12] Figure 1 shows the experimental schedule for protocol 160, which includes one cycle of three-step CEA-PRIT (top), two-step CEA-PRIT (middle), or one-step CEA-RIT in SCID mice bearing SC BxPC3 tumors. Biodistribution (BD) probes were euthanized 24 hours after radioactive material injection, whereas mice in the efficacy group were maintained and closely monitored until they reached the termination criteria. [Figure 13] Figure 1 shows the biodistribution of pretargeted Pb-DOTAM and Pb-DOTAM-CEA-DOTAM in SCID mice bearing SC BxPC3 tumors (24 hours after injection). Distribution of Pb in tumor-bearing SCID mice 24 hours after injection of CEA-DOTAM-pretargeted Pb-DOTAM or preincubated Pb-DOTAM-CEA-DOTAM. The content of radioactive material in organs and tissues is expressed as the mean ID% ± SD per g (n = 3). [Figure 14]Figure 1 shows mean tumor growth (n=10) with standard error for PRIT-treated groups and controls (Groups A-E) in the BxPC3 model. Curves were truncated at n<5. Vertical dotted lines indicate administration of 212Pb-DOTAM (20 μCi) for some or all groups according to study design. [Figure 15] Figure 1 shows individual tumor growth curves (n=10) for PRIT-treated groups and controls (Groups A-E) in the BxPC3 model. The vertical dotted line indicates administration of 212Pb-labeled compound (20 μCi). [Figure 16] Figure 1 shows the mean weight loss (Groups A-E, n=10) for mice treated with CEA-PRIT and CEA-RIT in the BxPC3 model. The curves were truncated at n<5. The vertical dotted lines indicate administration of 212Pb-labeled compound for some or all groups according to the study design. [Figure 17] Figure 1 shows the experimental design of protocol 175, which involves two-step CEA-PRIT in SCID mice bearing SC BxPC3 tumors, with sacrifice and necropsy 24 hours after injection of 212Pb-DOTAM. The dose of CEA-split-DOTAM-VH-AST was adjusted to compensate for hole / hole impurities. [Figure 18] Figure 1 shows the distribution of Pb in tumor-bearing SCID mice 24 hours after injection of Pb-DOTAM pretargeted with CEA-split-DOTAM-VH / VL antibody (Protocol 175). Organ and tissue radioactive content is expressed as mean ID% ± SD per g (n = 4). [Figure 19] Figure 1 shows the experimental design of protocol 185, which involves two-step CEA-PRIT in SCID mice bearing SC BxPC3 tumors, with sacrifice and necropsy 6 hours after injection of 212Pb-DOTAM. The dose of CEA-split-DOTAM-VH-AST(CH1A1A) was adjusted to compensate for hole / hole impurities. [Figure 20]Figure 1 shows the distribution of Pb in tumor-bearing SCID mice 6 hours after injection of Pb-DOTAM pretargeted with CEA-split-DOTAM-VH / VL antibody (Protocol 185). Organ and tissue radioactive content is expressed as mean ID% ± SD per g (n = 5). [Figure 21] Figure 1 shows the distribution of CEA-split-DOTAM-VH / VL pairs (combinations of VH and VL antibodies) within two selected SC BxPC3 tumors 7 days after injection. A and B show sections of a tumor from mouse A3 injected with CEA-split-DOTAM-VH / VL targeting T84.66, where A shows CEA expression and B shows the distribution of the corresponding CEA-split-DOTAM-VH / VL. C and D show sections of a tumor from mouse C5 injected with CEA-split-DOTAM-VH / VL targeting CH1A1A, where C shows CEA expression and D shows the distribution of the corresponding CEA-split-DOTAM-VH / VL. [Figure 22] Figure 1 shows the experimental design of protocol 189, which involves two-step CEA-PRIT in SCID mice bearing SC BxPC3 tumors, with sacrifice and necropsy 6 hours after injection of 212Pb-DOTAM. The dose of CEA-split-DOTAM-VH-AST(CH1A1A) was adjusted to compensate for hole / hole impurities. [Figure 23] Figure 1 shows the distribution of Pb in tumor-bearing SCID mice 6 hours after injection of Pb-DOTAM pretargeted by the dual paratope pair of CEA-split-DOTAM-VH / VL antibodies (T84.66 and CH1A1A) compared to the positive control (CH1A1A alone). Organ and tissue radioactive content is expressed as mean ID% ± SD per g. [Figure 24]Figure 1 shows the mean fluorescence intensity (MFI) determined by FACS for split antibodies. Binding of Pb-DOTA-FITC, determined by FACS, can be demonstrated only for co-incubation of both split antibodies with Pb-DOTA-FITC. A single split antibody did not produce a significant signal. [Figure 25A-C] FIG. 1 shows exemplary formats of the antibodies described herein. [Figure 26] FIG. 1 shows the results from experiment 1 of Example 11, assessing the binding of individual TA-split-DOTAM-VH and TA-split-DOTAM-VL antibodies to biotinylated DOTAM captured on a chip. [Figure 27] FIG. 1 shows the results from experiment 2 of Example 11, assessing the binding of DOTAM to individual TA-split-DOTAM-VH and TA-split-DOTAM-VL antibodies captured on a chip. [Figure 28] FIG. 1 shows the results from experiment 3 of Example 11, assessing the binding of DOTAM to TA-split-DOTAM-VH / VL antibodies (antibody pairs) captured on a chip. DETAILED DESCRIPTION OF THE INVENTION

[0036] I. Definition For purposes herein, a "human acceptor framework" is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. A human acceptor framework "derived from" a human immunoglobulin framework or a human consensus framework can comprise the same amino acid sequence of a human immunoglobulin framework or a human consensus framework and may contain amino acid sequence changes. In some embodiments, the number of amino acid changes 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 human VL acceptor framework is identical in sequence to the human immunoglobulin VL framework sequence or the human VL consensus framework sequence.

[0037] "Affinity" refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity," as used herein, refers to the intrinsic binding affinity, reflecting a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed by a dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary methods for measuring binding affinity are described below.

[0038] An "affinity matured" antibody refers to an antibody with one or more alterations in one or more complementarity determining regions (CDRs) compared to a parent antibody that does not possess such alterations, which alterations result in an improvement in the affinity of the antibody for the antigen.

[0039] The term "antibody that binds to an antigen expressed on the surface of a target cell" refers to an antibody that is capable of binding to the antigen with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting the antigen. In one embodiment, the extent of binding of the antibody to an unrelated, non-antigenic protein is less than about 10% of the binding of the antibody to the antigen, as measured, for example, by surface plasmon resonance (SPR). In certain embodiments, an antibody that binds to an antigen expressed on the surface of a target cell has an affinity of ≦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, e.g., 10 -8 M~10 -13 M, e.g., 10 -9 M~10 -13 An antibody has a dissociation constant (KD) of 1 μM or less. An antibody is said to "specifically bind to" an antigen expressed on the surface of a target cell if the antibody has a KD of 1 μM or less. In certain embodiments, the antibody binds to an epitope of the antigen that is conserved among the antigens from different species.

[0040] The term "antigen-binding site for a radiolabeled compound" or "functional antigen-binding site for a radiolabeled compound" refers to an antigen-binding site comprising VH and VL domains capable of binding a radiolabeled compound with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent for associating the radiolabeled compound with the antibody. In one embodiment, the extent of binding of the antigen-binding site to an unrelated, non-antigenic compound is less than about 10% of the binding of the antibody to the radiolabeled compound, as measured, for example, by surface plasmon resonance (SPR). In certain embodiments, the antigen-binding site that binds to a radiolabeled compound has an affinity of ≦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, e.g., 10 -8 M~10 -13 M, e.g., 10 -9 M~10 -13It may be preferable for an antigen-binding site that binds to a radiolabeled compound to have a K of 100 pM, 50 pM, 20 pM, 10 pM, 5 pM, 1 pM or less, e.g., 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, a functional binding site may bind to a radiolabeled compound with a K of about 1 pM to 1 nM, e.g., about 1 to 10 pM, 1 to 100 pM, 5 to 50 pM, 100 to 500 pM, or 500 pM to 1 nM. An antigen-binding site is said to "specifically bind" a radiolabeled compound if the antigen-binding site has a K of 1 μM or less.

[0041] As used herein, the term "antibody" is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0042] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, 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 (dAbs); and multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology, 23:1126-1136 (2005). Thus, the term "Fab fragment" refers to an antibody fragment containing a light chain comprising a VL domain and a CL domain, and a heavy chain fragment comprising a VH domain and a CH1 domain. "Fab' fragments" differ from Fab fragments by the addition of one or more cysteine-containing residues from the antibody hinge region at the carboxy terminus of the CH1 domain. For a discussion of Fab and F(ab')2 fragments that contain salvage receptor-binding epitope residues and extend in vivo half-life, see U.S. Patent No. 5,869,046. The term "crossover Fab fragment" or "xFab fragment" or "crossover Fab fragment" refers to a Fab fragment in which the variable or constant region of the heavy chain has been swapped with the variable or constant region of the light chain. A crossover Fab fragment contains a polypeptide chain composed of a light chain variable region (VL) and heavy chain constant region 1 (CH1), and a polypeptide chain composed of a heavy chain variable region (VH) and light chain constant region (CL). Asymmetric Fab arms can also be engineered by introducing charged or uncharged amino acid mutations at the domain interface to direct proper Fab pairing. See, e.g., WO2016 / 172485.

[0043] A "single-chain variable fragment" or "scFv" is a fusion protein of the variable domains of an antibody's heavy chain (VH) and light chain (VL) connected by a peptide linker. In particular, the linker is a short polypeptide of 10 to 25 amino acids, typically rich in glycine for flexibility and serine or threonine for solubility, and can connect the N-terminus of the VH to the C-terminus of the VL, or vice versa. This protein retains the specificity of the original antibody despite the removal of the constant region and the introduction of the linker. For a review of scFv fragments, see, e.g., Plückthun, "Pharmacology of Monoclonal Antibodies," Vol. 113, edited by Rosenburg and Moore (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458.

[0044] The term "blocking agent" refers to an agent that blocks the binding of an effector molecule, particularly a radiolabeled compound, to a functional binding site for the effector molecule. Generally, the blocking agent binds to, e.g., specifically binds to, a functional binding site for the effector molecule.

[0045] The term "clearing agent" refers to an agent that increases the rate of clearance of an antibody from the circulation of a subject. Generally, a clearing agent binds to an antibody, e.g., specifically binds to an antibody.

[0046] As used herein, the term "removal step" or "removal phase" encompasses the use of a blocking agent or a removing agent. Some agents can function as both a removing agent and a blocking agent.

[0047] The term "epitope" refers to a site on a proteinaceous or non-proteinaceous antigen to which an antibody binds. Epitopes can be formed from both a series of contiguous amino acids (linear epitopes) and noncontiguous amino acids that are spatially adjacent, for example, due to antigen folding, i.e., tertiary folding of a protein antigen (conformational epitopes). Linear epitopes typically remain bound by antibodies even after exposure of the protein antigen to denaturing agents, whereas conformational epitopes are typically destroyed upon treatment with denaturing agents. An epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation.

[0048] Screening for antibodies that bind to a specific epitope (i.e., antibodies that bind to the same epitope) can be done using methods routine in the art, such as, but not limited to, alanine scanning, peptide blotting (see 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)).

[0049] ASAP (Antigen Structure-based Antibody Profiling), also known as MAP (Modification-Assisted Profiling), allows for the binning of multiple monoclonal antibodies that specifically bind to an antigen based on the binding profile of each of the multiple antibodies to a chemically or enzymatically modified antigen surface (see, e.g., US2004 / 0101920). Antibodies within each bin bind to the same epitope, which may be a unique epitope that is significantly different from or partially overlaps with epitopes represented by other bins.

[0050] Competitive binding can also be used to easily determine whether an antibody binds to the same epitope as a reference antibody or competes with the reference antibody for binding. For example, an "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competitive assay; conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competitive assay. Also, for example, to determine whether an antibody binds to the same epitope as a reference antibody, the reference antibody is bound to the antigen under saturating conditions. After removing excess reference antibody, the antibody's ability to bind to the antigen is evaluated. If the antibody in question is able to bind to the antigen after saturating 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 is not able to bind to the antigen after saturating binding of the reference antibody, the antibody in question may bind to the same epitope as the reference antibody. To determine whether the antibodies in question bind to the same epitope or are simply prevented from binding due to steric reasons, routine experiments (such as peptide mutagenesis and binding analysis using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art) can be used. This assay should be performed in two settings, namely, in a setting where both antibodies are saturating antibodies. In both settings, if only the first (saturating) antibody can bind to the antigen, it can be concluded that the antibody in question and the reference antibody compete for binding to the antigen.

[0051] In some embodiments, two antibodies are considered to bind the same or overlapping epitope if a 1-, 5-, 10-, 20-, or 100-fold excess of one antibody inhibits binding of the other antibody by at least 50%, at least 75%, at least 90%, or even 99% or more, as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res., 50 (1990), 1495-1502).

[0052] In some embodiments, two antibodies are considered to bind to the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other antibody. Two antibodies are considered to have "overlapping epitopes" if only a subset of amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other antibody.

[0053] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0054] The "class" of an antibody refers to the type of constant domain or constant region carried by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In certain embodiments, the antibody is of the IgG1 isotype. In certain embodiments, the antibody is of the IgG1 isotype with mutations P329G, L234A, and L235A to reduce the effector function of the Fc region. In other embodiments, the antibody is of the IgG2 isotype. In certain embodiments, the antibody is of the IgG4 isotype with a mutation S228P in the hinge region to improve the stability of the IgG4 antibody. The heavy chain constant domains corresponding to the 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) and lambda (λ), based on the amino acid sequence of its constant domain.

[0055] "Effector function" refers to a biological activity attributable to the Fc region of an antibody, which varies with antibody isotype. Examples of antibody effector functions include binding to C1q and complement-dependent cytotoxicity (CDC); binding to Fc receptors; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0056] An "effective amount" of an agent, eg, a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic or prophylactic result.

[0057] The term "tandem Fab" refers to an antibody comprising two Fab fragments connected via a peptide linker / tether. In some embodiments, a tandem Fab can comprise one Fab fragment and one crossover Fab fragment connected by a peptide linker / tether.

[0058] As used herein, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. The term "Fc region" includes native-sequence Fc regions and variant Fc regions. In one embodiment, the Fc region of a human IgG heavy chain extends from Cys226 or Pro230 of the heavy chain to the carboxyl terminus. However, antibodies produced by host cells may undergo post-translational cleavage of one or more amino acids, particularly one or two amino acids, from the C-terminus of the heavy chain. Thus, antibodies produced by host cells through expression of a specific nucleic acid molecule encoding a full-length heavy chain can include full-length heavy chains and truncated variants of the full-length heavy chain. This may be the case when the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447; numbering according to the EU index). Thus, the C-terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (Lys447) of the Fc region may or may not be present. In one embodiment, a heavy chain comprising an Fc region as designated herein, comprised within an antibody in accordance with the invention, comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447; numbering according to the EU index). In one embodiment, a heavy chain comprising an Fc region as designated herein, comprised within an antibody in accordance with the invention, comprises an additional C-terminal glycine residue (G446; numbering according to the EU index). Unless otherwise specified herein, the numbering of amino acid residues within an Fc region or constant region is according to the EU numbering system, also referred to as the EU index, as set forth in Kabat et al., "Sequences of Proteins of Immunological Interest," 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0059] "Framework" or "FR" refers to variable domain residues other than the complementarity-determining regions (CDRs). The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the CDR and FR sequences generally appear in the following order in VH (or VL): FR1-CDR-H1 (CDR-L1)-FR2-CDR-H2 (CDR-L2)-FR3-CDR-H3 (CDR-L3)-FR4.

[0060] As used herein, the terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably to refer to an antibody having a heavy chain that has a structure substantially similar to a native antibody structure or that contains an Fc region as defined herein.

[0061] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," including the primary transformed cell and progeny derived therefrom regardless of the number of passages. Progeny cells may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Also included herein are mutant progeny cells that have the same function or biological activity as screened or selected for in the originally transformed cell.

[0062] A "human antibody" is an antibody that possesses an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or derived from a non-human source that utilizes a human antibody repertoire or other human antibody coding sequence. This definition of a human antibody specifically excludes humanized antibodies, which contain non-human antigen-binding residues.

[0063] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is derived from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup such as the subgroups in Kabat et al., "Sequences of Proteins of Immunological Interest," 5th ed., NIH Publication No. 91-3242, Bethesda, MD (1991), vols. 1-3. In one embodiment, for the VL, the subgroup is subgroup kappa I, such as the subgroups in Kabat et al., supra. In one embodiment, for the VH, the subgroup is subgroup III, such as the subgroups in Kabat et al., supra.

[0064] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues derived from non-human CDRs and amino acid residues derived from human FRs. In certain embodiments, a humanized antibody will comprise at least one variable domain, typically substantially all of two variable domains, in which all or substantially all of the CDRs correspond to the CDRs of a non-human antibody and all or substantially all of the FRs correspond to the FRs of a human antibody. A humanized antibody can optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0065] The term "hypervariable region" or "HVR" as used herein refers to each of the regions of an antibody variable domain, e.g., the "complementarity-determining regions" ("CDRs"), that are hypervariable in sequence and determine antigen-binding specificity.

[0066] Generally, antibodies comprise six CDRs: three in the VH (CDR-H1, CDR-H2, CDR-H3) and three in the VL (CDR-L1, CDR-L2, CDR-L3). Exemplary CDRs herein are: (a) Hypervariable loops, occurring 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 occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., "Sequences of Proteins of Immunological Interest," 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and (c) Antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al., J. Mol. Biol. 262:732-745 (1996)). Includes.

[0067] Unless otherwise indicated, CDRs are determined according to Kabat et al., supra. One of skill in the art will understand that CDR designations may also be determined according to Chothia, supra, McCallum, supra, or any other scientifically accepted terminology system. Alternatively, the CDR-H1 sequence described herein may extend from Kabat 26 to Kabat 35, for example, for the variable domain that binds Pb-DOTAM.

[0068] In one embodiment, the CDR residues include those identified in the sequence listing or elsewhere herein.

[0069] Unless otherwise indicated, HVR / CDR residues and other residues within the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

[0070] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecule(s), including, but not limited to, a cytotoxic agent.

[0071] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is human.

[0072] The molecules described herein may be "isolated" molecules. An "isolated" antibody is one that has been separated from components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, as determined, for example, by electrophoretic methods (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic methods (e.g., ion exchange or reverse-phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr., B848:79-87 (2007).

[0073] The terms "nucleic acid molecule" or "polynucleotide" include any compound and / or substance comprising a polymer of nucleotides. Each nucleotide consists of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Nucleic acid molecules are described by the sequence of bases, which often represent the primary (linear) structure of the nucleic acid molecule. The sequence of bases is typically represented 5' to 3'. As used herein, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers comprising two or more of these molecules. Nucleic acid molecules can be linear or circular. In addition, the term nucleic acid molecule includes both sense and antisense strands, as well as both single-stranded and double-stranded forms. Furthermore, the nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases in which the sugar or phosphate backbone linkage is derivatized, or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for direct expression of the antibodies of the present invention in vitro and / or in vivo, e.g., in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors may be unmodified or modified. For example, mRNA can 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 subject to generate antibodies in vivo (see, e.g., Stadler et al., Nature Medicine, 2017, published online June 12, 2017, doi:10.1038 / nm.4356 or EP2101823B1).

[0074] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acid typically contains a nucleic acid molecule, but includes nucleic acid molecules contained within a cell where the nucleic acid molecule is present extrachromosomally or in a chromosomal location that is different from its natural chromosomal location.

[0075] An "isolated nucleic acid encoding an antibody" refers to a nucleic acid molecule comprising one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of an antibody, such nucleic acid molecule(s) in a single vector or in separate vectors, and such nucleic acid molecule(s) present in one or more locations within a host cell.

[0076] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies; i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible variant antibodies containing, for example, naturally occurring mutations or mutations that arise during the production of a monoclonal antibody preparation, which generally occur in minor amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody obtained from a substantially homogeneous antibody population and is not intended to require production of the antibody by any particular method. For example, monoclonal antibodies in accordance with the present invention can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of human immunoglobulin genes; such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0077] A "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The naked antibody may be present in a pharmaceutical composition.

[0078] "Native antibodies" refer to naturally occurring immunoglobulin molecules with varying structures. For example, native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable domain (VH), also called a heavy chain variable domain or heavy chain variable region, followed by three heavy chain constant domains (CH1, CH2, and CH3). Similarly, from N- to C-terminus, each light chain has a variable domain (VL), also called a light chain variable domain or light chain variable region, followed by a light chain constant (CL) domain.

[0079] The term "package insert" is used to refer to instructions typically included in commercial packaging of a pharmaceutical product that contain information about the indications, usage, dosage, administration, concomitant therapy, contraindications, and / or warnings regarding the use of such pharmaceutical product.

[0080] "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 to achieve the maximum percent sequence identity, introducing gaps, if necessary, and excluding conservative substitutions for alignment purposes from the sequence identity. Alignment for determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, using publicly available computer software, such as BLAST software, BLAST-2 software, Clustal W software, Megalign (DNASTAR) software, or the FASTA program package. Those skilled in the art can determine the appropriate parameter sequence, including any algorithm for aligning sequences, required to achieve maximum alignment across the entire length of the sequences being compared. Alternatively, percent sequence identity values ​​can be generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and the source code together with user documentation has been submitted to the U.S. Copyright Office, Washington, DC 20559, where it is registered under U.S. Copyright Registration No. TXU510087 and described in WO2001 / 007611.

[0081] Unless otherwise indicated, for purposes of this specification, percent amino acid sequence identity values ​​are generated using the ggsearch program of the FASTA package version 36.3.8c, followed by the BLOSUM50 comparison matrix. The FASTA program package was developed by W.R. Pearson and D.J. Lipman (1988), "Improved Tools for Biological Sequence Analysis," PNAS, 85:2444-2448; W.R. Pearson (1996), "Effective protein sequence comparison," Meth. Enzymol., 266:227-258; and Pearson et al. (1997), Genomics, 46:24-36, and is available at www.fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml or www.ebi.ac.uk / Tools / sss / fasta. Alternatively, the public server accessible at fasta.bioch.virginia.edu / fasta_www2 / index.cgi can be used to compare sequences using the ggsearch(global protein:protein) program and default options (BLOSUM50; open:-10; ext:-2; Ktup=2) to ensure global, rather than local, alignments. Percent amino acid identity is shown in the alignment header of the output.

[0082] The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a preparation that is in a form that allows the biological activity of the active ingredient contained therein to be effective, and that does not contain additional components that are unacceptably toxic to the subject to which the pharmaceutical composition is administered.

[0083] A "pharmaceutically acceptable carrier" refers to an ingredient, other than an active ingredient, in a pharmaceutical composition or formulation that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, additives, stabilizers, or preservatives.

[0084] As used herein, unless otherwise indicated, a reference to a target antigen refers to any natural target antigen from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term target antigen encompasses unprocessed, "full-length" target antigens as well as any form of target antigen resulting from intracellular processing. The term target antigen also encompasses naturally occurring variants of target antigens, such as splice variants or allelic variants. For example, the target antigen CEA may have the amino acid sequence of human CEA, particularly carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), as set forth in UniProt (www.uniprot.org) Accession Number: P06731 (version 119) or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq: NP_004354.2. Another example of a target antigen is fibroblast activation 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 (for the human sequence, see UniProt accession number: Q9NZD1 (version 115); NCBI RefSeq accession number: NP_061124.1).

[0085] As used herein, the terms "split antibody," "split antibody," "single-domain split antibody," or "SPLIT PRIT" refer to an antibody in which the VH and VL domains, which together form an antigen-binding site capable of binding a radiolabeled compound, are distributed between two antibodies and are not present as part of the same antibody (prior to in vivo assembly). A "CEA-targeting SPLIT PRIT" refers to a split antibody that targets CEA. The term "SPLIT PRIT" may also be used interchangeably with the term "TA-split-DOTAM-VH / VL" (e.g., when the "TA" or target antigen is CEA, FAP, or GPRC5D). The term "CEA-targeting SPLIT PRIT" may also be used interchangeably with the term "CEA-split-DOTAM-VH / VL."

[0086] As used herein, the term "treatment" (and grammatical variations thereof, such as "treating" or "treating") refers to clinical intervention in an attempt to alter the natural course of disease in the individual being treated, and may be performed for the prevention of, or during the course of, a clinical condition. Desired effects of treatment include preventing the onset or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliating the condition, and remission or improved prognosis. In some embodiments, the antibodies of the invention are used to delay the onset of disease or slow the progression of disease.

[0087] The term "variable region" or "variable domain" refers to the domain of an antibody's heavy or light chain that is involved in binding to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have a similar structure, with each domain containing four conserved framework regions (FR) and three complementarity-determining regions (CDR) (see, for example, Kindt et al., "Kuby Immunology," 6th ed., W.H. Freeman and Co., p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen can be isolated using a VH or VL domain derived from an antibody that binds to the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol., 150:880-887 (1993); Clarkson et al., Nature, 352:624-628 (1991).

[0088] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it has been linked. The term "vector" includes vectors as self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0089] As used herein, the term "Pb" or "lead" includes its ions, e.g., Pb(II). References to other metals also include their ions. Thus, those skilled in the art will recognize that, e.g., lead, Pb, 212 Pb, or 203 It is understood that the term Pb is intended to encompass the ionic form of the element, particularly Pb(II).

[0090] 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, wherein each antibody can bind to the antigen on target cell, but the functional antigen binding site for effector agent is only formed when the first antibody and the second antibody are associated with each other.The antibody of the present invention is useful for, for example, pre-targeting immunotherapy and / or pre-targeting imaging.In a preferred embodiment, the method does not require the step of administering removal agent or blocking agent.

[0091] A. Target antigen Herein, an antigen expressed on the surface of a target cell is also referred to as a "target antigen."

[0092] Insofar as the present invention relates to treatment methods and pharmaceuticals for use in treatment methods, the present invention is applicable to any condition treatable by cytotoxic activity targeted to patient cells, e.g., diseased cells. Thus, target cells are any cells, e.g., any diseased cells, for which targeting of cytotoxicity is desired. The treatment is preferably tumor or cancer treatment. However, the applicability of the present invention is not limited to tumors and cancer. For example, the treatment can also be the treatment of viral infections (by targeting infected cells) or T cell-driven autoimmune diseases (by targeting T cells). Immunotoxins directed against viral antigens expressed on the surface of infected cells have been explored for various viral infections, such as HIV, rabies, and EBV. Cai and Berger, 2011, Antiviral Research, 90(3):143-50, used a PE38-containing immunotoxin for targeted killing of cells infected with Kaposi's sarcoma-associated herpesvirus. Additionally, Resimmune® (A-dmDT390-bisFv(UCHT1)) selectively kills human malignant T cells and transiently depletes normal T cells, and is believed to have potential for the treatment of T cell-driven autoimmune diseases such as multiple sclerosis and graft-versus-host disease, as well as T cell hematological cancers undergoing clinical trials. Similarly, the methods of the present invention may be applicable to any cell type for which radioimaging is desired, including, but not limited to, cancer or tumor cells.

[0093] Thus, suitable target antigens can include cancer cell antigens, viral antigens, or microbial antigens.

[0094] The antigens are typically normal cell surface antigens that are overexpressed or expressed abnormally. Ideally, the target antigen would be expressed only in diseased cells (such as tumor cells), but this is rarely observed in practice. As a result, target antigens are typically selected based on differential expression between diseased and healthy tissues.

[0095] The cell surface marker or target antigen can be, for example, a tumor-associated antigen.

[0096] As used herein, the term "tumor-associated antigen" or "tumor-specific antigen" refers to any molecule (e.g., protein, peptide, lipid, carbohydrate, etc.) that is exclusively or primarily expressed or overexpressed by tumor cells and / or cancer cells, or other tumor stromal cells, such as cancer-associated fibroblasts, such that the antigen is associated with tumor(s) and / or cancer(s). In addition, tumor-associated antigens can also be 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 that by tumor cells or cancer cells. In this regard, tumor or cancer cells may overexpress the antigen, or may express the antigen at a significantly higher level than that expressed by normal cells, non-tumor cells, or non-cancerous cells. In addition, tumor-associated antigens can also be expressed by cells with different developmental or maturational states. For example, tumor-associated antigens may also be expressed by embryonic or fetal cells that are not found in the normal adult host. Alternatively, in addition, tumor-associated antigens may be expressed by stem or progenitor cells that are not found in the normal adult host.

[0097] A tumor-associated antigen can be an antigen expressed by any cell of any cancer or tumor, including the cancers and tumors described herein. A tumor-associated antigen can 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 can be a tumor-associated antigen of (e.g., characteristic of) more than one type of cancer or tumor. For example, a tumor-associated antigen can be expressed by both breast cancer cells and prostate cancer cells, but not at all by normal cells, non-tumor cells, or non-cancerous cells.

[0098] Exemplary tumor-associated antigens to which the antibodies of the invention may bind include, but are not limited to, mucin 1 (MUC1; tumor-associated epithelial mucin), PRAME (preferentially expressed antigen of melanoma), carcinoembryonic antigen (CEA), prostate-specific membrane antigen (PSMA), PSCA, EpCAM, Trop2 (trophoblast 2, also known as 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, tyrosine kinase-related protein (TRP) I, and TRP2. In another embodiment, the tumor antigen is cluster of differentiation (CD) 19, CD20, CD21, CD22, CD25, CD30, CD33 (sialic acid-binding Ig-like lectin 3, a myeloid cell surface antigen), CD79b, CD123 (interleukin-3 receptor alpha), transferrin receptor, EGF receptor, mesothelin, cadherin, Lewis The mesothelin may be selected from the group consisting of Y, glypican 3, FAP (fibroblast activation protein alpha), GPRC5D (G protein-coupled receptor class C group 5 member D), PSMA (prostate-specific membrane antigen), CA9=CAIX (carbonic anhydrase IX), LlCAM (neural cell adhesion molecule L1), endosialin, HER3 (epidermal growth factor receptor family member 3 activated conformation), Alk1 / BMP9 complex (anaplastic lymphoma kinase 1 / bone morphogenetic protein 9), TPBG=5T4 (trophoblast glycoprotein), ROR1 (receptor tyrosine kinase-like surface antigen), HER1 (epidermal growth factor receptor activated conformation), and CLL1 (C-type lectin domain family 12 member A). Mesothelin is expressed, for example, in 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, for example, in hairy cell leukemia, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), non-Hodgkin's lymphoma, small lymphocytic lymphoma (SLL), and acute lymphocytic leukemia (ALL).CD25 is expressed in leukemia and lymphoma, including hairy cell leukemia and Hodgkin's lymphoma. Lewis Y antigen is expressed in bladder cancer, breast cancer, ovarian cancer, colorectal cancer, esophageal cancer, gastric cancer, lung cancer, and pancreatic cancer. CD33 is expressed in acute myeloid leukemia (AML), chronic myelomonocytic leukemia (CML), and myeloproliferative disorders.

[0099] Exemplary 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 variants thereof), and antibodies against the Lewis Y antigen (e.g., B3 and its variants). In this regard, the targeting moiety (cell binding agent) can be an antibody selected from the group consisting of B3, RFB4, SS, SS1, MN, MB, HN1, HN2, HB21, and MORAb-009, and antigen-binding portions thereof. Further exemplary targeting moieties suitable for use in the chimeric molecules of the present invention are described, for example, in U.S. Pat. Nos. 5,242,824 (anti-transferrin receptor); 5,846,535 (anti-CD25); 5,889,157 (anti-Lewis Y); 5,981,726 (anti-Lewis Y); 5,990,296 (anti-Lewis Y); Y); 7,081,518 (anti-mesothelin); 7,355,012 (anti-CD22 and anti-CD25); 7,368,110 (anti-mesothelin); 7,470,775 (anti-CD30); 7,521,054 (anti-CD25); and 7,541,034 (anti-CD22); U.S. Patent Application Publication No. 2007 / 0189962 (anti-CD22); Frankel et al., Clin. Cancer Res., 6:326-334 (2000); and Kreitman et al., AAPS Journal, 8(3):E532-E551 (2006).

[0100] Additional antibodies have been raised against target-specific tumor-associated antigens, including 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, Mucl6, and TMEFF2. Any of these antibodies, or antigen-binding fragments thereof, may be useful in the present invention, i.e., may be incorporated into the antibodies described herein.

[0101] In some embodiments of the present invention, it may be preferred that the tumor-associated antigen is carcinoembryonic antigen (CEA).

[0102] CEA is advantageous in the context of the present invention because it is internalized relatively slowly, and therefore a high percentage of the antibody remains available on the surface of the cell for binding to the radionuclide after initial treatment. Other low-internalization targets / tumor-associated antigens may also be preferred. Other examples of tumor-associated antigens include CD20 or HER2. In still 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)), which is incorporated herein by reference. This reference also describes antibodies such as mu9, 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 antibodies, or antigen-binding portions thereof, may be useful in the present invention, i.e., may be incorporated into the antibodies described herein. One example of an antibody raised against CEA is T84.66 (heavy chain, NCBI accession number: CAA36980, and light chain, CAA36979, or SEQ ID NOs: 317 and 318 in WO2016 / 075278), and its humanized and chimeric forms, such as T84.66-LCHA, described in WO2016 / 075278A1 and / or WO2017 / 055389. Another example is CH1A1a, an anti-CEA antibody described in WO2012 / 117002 and WO2014 / 131712, and CEA hMN-14 (see also US6676924 and US5874540).Another anti-CEA antibody is A5B7, described in MJ Banfield et al., Proteins, 1997, 29(2), 161-171. Humanized antibodies derived from the murine antibody A5B7 are disclosed in WO92 / 01059 and WO2007 / 071422. See also co-pending application PCT / EP2020 / 067582. An example of a humanized version of A5B7 is A5H1EL1(G54A). A further exemplary antibody against CEA is MFE23, and its humanized form, described in US7626011 and / or co-pending application PCT / EP2020 / 067582. Yet another example of an antibody against CEA is 28A9. Any of these antibodies, or their antigen-binding fragments, may be useful in forming a CEA-binding portion in the present invention.

[0103] In some embodiments, FAP (fibroblast activation protein alpha) or GPRC5D (G protein-coupled receptor class C group 5 member D) may also be preferred. FAP is an established target for imaging and therapy due to its widespread expression in the microenvironment of many tumor types, such as 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-split-DOTAM-VH / VL antibody can be used to detect activated cancer-associated fibroblasts. 212It would be expected that this would result in the specific accumulation of Pb-DOTAM. As a result, alpha-emitting radiation would be expected to have a negative impact on immune suppression by FAP-expressing malignancies, in addition to limiting the direct tumor-killing effect on adjacent tumor cells. G protein-coupled receptor family C5 group 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, such as OPM-2 and NCI-H929, reflect the expression found in multiple myeloma patients (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, we expect that SPLIT PRIT using GPRC5D-split-DOTAM-VH / VL antibodies will lead to tumor-specific accumulation of 212Pb-DOTAM followed by irradiation-induced tumor cell death.

[0104] In some embodiments, antibodies of the invention may specifically bind to a target antigen (e.g., any of the target antigens discussed herein). In some embodiments, antibodies of the invention may specifically bind to a target antigen (e.g., any of the target antigens discussed herein) with a specificity of ≦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, e.g., 10 -7 ~10 -13 , 10-8 M or less, e.g., 10 -8 M~10 -13 M, e.g., 10 -9 M~10 -13 It can bind with a dissociation constant (KD) of 1 M.

[0105] The first antibody and the second antibody each bind to the same target antigen, which may be referred to as "antigen A" (i.e., the first antibody and the second antibody have binding specificity for the same target antigen). The first antibody and the second antibody may have binding specificity for the same epitope on antigen A. Alternatively, the first antibody may be capable of binding 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 of the antibodies may be capable of binding to the T84.66 epitope of CEA, and the other may bind to the A5B7 epitope of CEA.

[0106] In some embodiments, one or both of the first antibody and / or the second antibody may be biparatopic with respect to antigen A (i.e., each individual antibody may bind to two different epitopes of antigen A). The first antibody may comprise a first binding site and a second binding site that bind to a first epitope and a second epitope of antigen A, respectively, where the first epitope and the second epitope are different from each other. Alternatively, or in addition, the second antibody may comprise a first binding site and a second binding site that bind to a first epitope and a second epitope of antigen A, where the first epitope and the second epitope are different 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 a first antibody may be the same as the two epitopes bound by a second antibody.

[0107] B. Radiolabeled compounds According to the present invention, the association of the first and second antibodies forms a functional binding site for an effector molecule. The effector molecule according to the present invention is a radiolabeled compound containing a radioisotope, e.g., a radiolabeled hapten.

[0108] In some embodiments, the effector molecule may comprise a chelated radioisotope.

[0109] In some embodiments, the functional binding site for the effector molecule may bind to a chelate comprising a chelator and a radioisotope, hi other embodiments, the antibody may bind to a moiety conjugated to a chelated radioisotope, e.g., histamine-succinyl-glycine (HSG), digoxigenin, biotin, or caffeine.

[0110] The chelating agent can be, for example, a multidentate molecule such as an aminopolycarboxylic acid or aminopolythiocarboxylic acid, or a salt or functional variant thereof. The chelating agent can be, for example, a bidentate chelating agent, a tridentate chelating agent, or a tetradentate chelating agent. Examples of suitable metal chelating agents include EDTA (ethylenediaminetetraacetic acid or salt forms such as CaNa2EDTA), DTPA (diethylenetriaminepentaacetic acid), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), NOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), and the like. (2,2',2''-(1,4,7-triazanonane-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-azatetradecanedioic acid), TETA (2,2',2'',2'''-(1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetramethyl-2,2',2''' ... (1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane), 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; commercially available from Macrocyclics, Inc., Plano, Texas), NTA (nitrilotriacetic acid), EDDHA (ethylenediaminetetraacetic acid), These include diamine-N,N'-bis(2-hydroxyphenylacetic acid), BAL (2,3-dimercaptopropanol), DMSA (2,3-dimercaptosuccinic acid), DMPS (2,3-dimercapto-1-propanesulfonic acid), D-penicillamine (B-dimethylcysteine), MAG3 (mercaptoacetyltriglycine), Hynic (6-hydrazinopyridine-3-carboxylic acid), p-isothiocyanatobenzyl-desferrioxamine (e.g., zirconium-labeled for imaging), and molecules containing salts or functional variants / derivatives thereof capable of chelating metals. In some embodiments, the chelating agent may preferably be DOTA or DOTAM, or a salt or functional variant / derivative thereof capable of chelating metals.Thus, the chelating agent may be or include DOTA or DOTAM, with the radioisotope chelated thereto.

[0111] The effector molecule can comprise or consist of a functional variant or derivative of the above-described chelators in combination with a radionuclide. Suitable variants / derivatives have a limited structural difference while retaining the ability to function as a chelator (i.e., retain sufficient activity to be used for one or more of the purposes described herein). Functional variants / derivatives can also include the above-described chelators conjugated to one or more additional moieties or substituents, including small molecules, polypeptides, or carbohydrates. This conjugation can occur, for example, through one of the constituent carbons in the backbone portion of the chelator. Suitable substituents can be, for example, alkyl, alkenyl, aryl, or alkynyl; hydroxy groups; alcohol groups; halogen atoms; nitro groups; cyano groups; sulfonyl groups; thiol groups; amine groups; oxo groups; carboxy groups; thiocarboxy groups; carbonyl groups; amide groups; ester groups; or hydrocarbon groups such as heterocycles, including heteroaryl groups. The substituent can be, for example, one of the substituents defined for the "R" group below. The small molecule can be, for example, a dye (such as Alexa 647 or Alexa 488), biotin or a biotin moiety, or a phenyl or benzyl moiety. The polypeptide can be, for example, an oligopeptide, e.g., an oligopeptide of two or three amino acids. Exemplary carbohydrates include dextrans, linear or branched polymers or copolymers (e.g., polyalkylenes, poly(ethylene-lysine), polymethacrylates, polyamino acids, polysaccharides or oligosaccharides, dendrimers). Derivatives can also include multimers of chelator compounds in which the above-specified compounds are linked via linker moieties. Derivatives can also include functional fragments of the above compounds that retain the ability to chelate metal ions.

[0112] Specific examples of derivatives include benzyl-EDTA and hydroxyethyl-thiouride-benzyl EDTA, DOTA-benzene (e.g., (S-2-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecanetetraacetic acid), DOTA-biotin, and DOTA-TyrLys-DOTA.

[0113] In some embodiments of the present invention, the functional binding site formed by association of the first and second antibodies binds to a metal chelate comprising DOTAM and a metal, such as lead (Pb). As mentioned above, "DOTAM" has the following formula: The compound in TIFF2026000913000002.tif63170 has the chemical name: It has 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane.

[0114] In certain aspects and embodiments, the present invention may also use functional variants or derivatives of DOTAM that incorporate metal ions. Suitable variants / derivatives of DOTAM have a structure that differs to a limited extent from the structure of DOTAM and retain the ability to function (i.e., retain sufficient activity to be used for one or more of the purposes described herein). In such aspects and embodiments, DOTAM or functional variants / derivatives of DOTAM may be one of the active variants disclosed in WO2010 / 099536. Suitable functional variants / derivatives have the following formula: TIFF2026000913000003.tif65170, or a pharmaceutically acceptable salt thereof [wherein: provided that the valence of each atom in an optionally substituted moiety is not exceeded. R Nis H, C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 cycloalkyl, C3-C7 cycloalkyl-C1-C4 alkyl, C2-C7 heterocycloalkyl, C2-C7 heterocycloalkyl-C1-C4 alkyl, phenyl, phenyl-C1-C4 alkyl, C1-C7 heteroaryl, and C1-C7 heteroaryl-C1-C4 alkyl, wherein C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, and C2-C6 alkynyl are each selected from one, two, three, or four independently selected R w groups; wherein said C3-C7 cycloalkyl, C3-C7 cycloalkyl-C1-C4 alkyl, C2-C7 heterocycloalkyl, C2-C7 heterocycloalkyl-C1-C4 alkyl, phenyl, phenyl-C1-C4 alkyl, C1-C7 heteroaryl, and C1-C7 heteroaryl-C1-C4 alkyl are each optionally substituted with one, two, three, or four independently selected R x and optionally substituted by a group; L 1 independently, each of which may be one, two, or three independently selected R 1 C1-C6 alkylene, C1-C6 alkenylene, or C1-C6 alkynylene, optionally substituted by a group; L 2 is an independently selected R 1 C2-C4 straight chain alkylene optionally substituted with 1, 2, 3, or 4 groups independently selected from C1-C4 alkyl, and / or C1-C4 haloalkyl; R 1 independently, 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 Alkyl sulfonyl, amino, C1~6 Alkylamino, Di-C 1~6 Alkylamino, C 1~4 Alkylcarbonyl, carboxy, C 1~6 Alkoxycarbonyl, C 1~6 Alkylcarbonylamino, di-C 1~6 Alkylcarbonylamino, C 1~6 Alkoxycarbonylamino, C 1~6 Alkoxycarbonyl-(C 1~6 Alkyl)amino, carbamyl, C 1~6 Alkylcarbamyl, and di-C 1~6 alkylcarbamyl; Each D 1 independently, C6-C 10 Aryl-C1-C4 alkyl, C1-C9 heteroaryl-C1-C4 alkyl, C3-C 10 cycloalkyl-C1-C4 alkyl, C2-C9 heterocycloalkyl-C1-C4 alkyl, C1-C8 alkylene, C1-C8 alkenylene, and C1-C8 alkynylene, wherein said C1-C8 alkylene, C1-C8 alkenylene, and C1-C8 alkynylene are selected from one, two, three, or four independently selected R 4 may be substituted by a group; 10 Aryl-C1-C4 alkyl, C1-C9 heteroaryl-C1-C4 alkyl, C3-C 10 Cycloalkyl-C1-C4 alkyl, C2-C9 heterocycloalkyl-C1-C4 alkyl each have one, two, three, or four independently selected R 5 and optionally substituted by a group; Each D 2 are independently absent or C1-C 20 linear alkylene [wherein the C1-C 20 The 1 to 6 non-adjacent methylene groups of the linear alkylene are 20 At least one methylene unit in the linear alkylene is optionally -D 4 - a moiety [wherein said C1-C 20Straight chain alkylenes can be substituted with halogen, cyano, nitro, hydroxyl, 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, carboxy, 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 each optionally replaced by an independently selected -D4- moiety, provided that none of the -D4- moieties is replaced by an -D4- moiety, which may be substituted with one or more groups independently selected from alkylcarbamyl; Each D 3 are independently H, halogen, cyano, nitro, hydroxyl, C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C 14 Cycloalkyl, C3-C 14 Cycloalkyl-C1-C4 alkyl, C2-C 14 Heterocycloalkyl, C2-C 14 Heterocycloalkyl-C1-C4 alkyl, C6-C 14 Aryl, C6-C 14 Aryl-C1-C4 alkyl, C1-C 13 Heteroaryl, C1-C 13 heteroaryl-C1-C4 alkyl, wherein said C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, and C2-C6 alkynyl are each selected from one, two, three, or four independently selected R 6 may be substituted by a group; 14 Cycloalkyl, C3-C 14Cycloalkyl-C1-C4 alkyl, C2-C 14 Heterocycloalkyl, C2-C 14 Heterocycloalkyl-C1-C4 alkyl, C6-C 14 Aryl, C6-C 14 Aryl-C1-C4 alkyl, C1-C 13 Heteroaryl, C1-C 13 Heteroaryl-C1-C4 alkyl is each selected from one, two, three, or four independently selected R 7 and optionally substituted by a group; Each D 4 are independently -O-, -S-, and -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 from; Each R 4 and R 6 are independently halogen, cyano, nitro, hydroxyl, C 1~4 Alkoxy, C 1~4 Haloalkoxy, C 1~4 Alkylthio, C 1~4 Alkylsulfinyl, C 1~4 Alkyl sulfonyl, amino, C 1~4 Alkylamino, Di-C 1~4 Alkylamino, C 1~4 Alkylcarbonyl, carboxy, C 1~4 Alkoxycarbonyl, C 1~4 Alkylcarbonylamino, di-C 1~4 Alkylcarbonylamino, C 1~4Alkoxycarbonylamino, C 1~4 Alkoxycarbonyl-(C 1~4 Alkyl)amino, carbamyl, C 1~4 Alkylcarbamyl, and di-C 1~4 alkylcarbamyl; Each R 5 are independently halogen, cyano, cyanate, isothiocyanate, nitro, hydroxyl, C 1~4 Alkyl, C 2~4 Alkenyl, C 2~4 Alkynyl, C 1~4 Alkoxy, C 1~4 Haloalkoxy, C 1~4 Alkylthio, C 1~4 Alkylsulfinyl, C 1~4 Alkyl sulfonyl, amino, C 1~4 Alkylamino, Di-C 1~4 Alkylamino, C 1~4 Alkylcarbonyl, carboxy, 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 alkylcarbamyl; Each R 7 are independently selected from halogen, cyano, nitro, hydroxyl, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 cycloalkyl, C3-C7 cycloalkyl-C1-C4 alkyl, C2-C7 heterocycloalkyl, C2-C7 heterocycloalkyl-C1-C4 alkyl, phenyl, phenyl-C1-C4 alkyl, C1-C7 heteroaryl, C1-C7 heteroaryl-C1-C4 alkyl, -OR O , -SR O , -S(=O)R P , -S(=O)2R P , -S(=O)NR s Rt , -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 wherein said C1-C6 alkyl, C2-C6 alkenyl, and C2-C6 alkynyl are each optionally substituted with one, two, three, or four independently selected R' groups; and said C3-C7 cycloalkyl, C3-C7 cycloalkyl-C1-C4 alkyl, C2-C7 heterocycloalkyl, C2-C7 heterocycloalkyl-C1-C4 alkyl, phenyl, phenyl-C1-C4 alkyl, C1-C7 heteroaryl, and C1-C7 heteroaryl-C1-C4 alkyl are each optionally substituted with one, two, three, or four independently selected R'' groups; Each R a , R b , and R c are independently selected from H, C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 cycloalkyl, C3-C7 cycloalkyl-C1-C4 alkyl, C2-C7 heterocycloalkyl, C2-C7 heterocycloalkyl-C1-C4 alkyl, phenyl, phenyl-C1-C4 alkyl, C1-C7 heteroaryl, and C1-C7 heteroaryl-C1-C4 alkyl, wherein said C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, and C2-C6 alkynyl each represent one, two, three, or four independently selected R wgroups; wherein said C3-C7 cycloalkyl, C3-C7 cycloalkyl-C1-C4 alkyl, C2-C7 heterocycloalkyl, C2-C7 heterocycloalkyl-C1-C4 alkyl, phenyl, phenyl-C1-C4 alkyl, C1-C7 heteroaryl, and C1-C7 heteroaryl-C1-C4 alkyl are each optionally substituted with one, two, three, or four independently selected R x and optionally substituted by a group; Each R O , R P , R q , R r , R s , and R t are independently selected from H, C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 cycloalkyl, C3-C7 cycloalkyl-C1-C4 alkyl, C2-C7 heterocycloalkyl, C2-C7 heterocycloalkyl-C1-C4 alkyl, phenyl, phenyl-C1-C4 alkyl, C1-C7 heteroaryl, and C1-C7 heteroaryl-C1-C4 alkyl, wherein said C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, and C2-C6 alkynyl each represent one, two, three, or four independently selected R y groups; wherein said C3-C7 cycloalkyl, C3-C7 cycloalkyl-C1-C4 alkyl, C2-C7 heterocycloalkyl, C2-C7 heterocycloalkyl-C1-C4 alkyl, phenyl, phenyl-C1-C4 alkyl, C1-C7 heteroaryl, and C1-C7 heteroaryl-C1-C4 alkyl are each optionally substituted with one, two, three, or four independently selected R z and optionally substituted by a group; Each R', R w , and R y are independently hydroxyl, cyano, nitro, C1-C4 alkoxy, C1-C4 haloalkoxy, amino, C1-C4 alkylamino, and diC1-C4 alkylamino Selected from; Each R'', R x, and R z are independently hydroxyl, halogen, cyano, nitro, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 alkoxy, C1-C4 haloalkoxy, amino, C1-C4 alkylamino, and di-C1-C4 alkylamino Select from It is possible.

[0115] Suitably, functional variants / derivatives of the above formula have an affinity for the antibody of the invention that is equal to or greater than that of DOTAM, and have a binding strength ("affinity" as measured by the dissociation constant described above) for Pb that is equal to or greater than that of DOTAM. For example, the dissociation constant of a functional variant / derivative for an antibody / Pb of the invention may be 1.1-fold or less, 1.2-fold or less, 1.3-fold or less, 1.4-fold or less, 1.5-fold or less, or 2-fold or less than the dissociation constant of DOTAM for the same antibody / Pb.

[0116] Each R N may be H, C1-C6 alkyl, or C1-C6 haloalkyl; preferably, H, C1-C4 alkyl, or C1-C4 haloalkyl. Most preferably, each R N is H.

[0117] Referring to DOTAM variants, one, two, three, or, most preferably, each L 2 is preferably a C2 alkylene. C2 alkylene variants of DOTAM are particularly advantageous as they may have a high affinity for Pb. 2 Optional substituents for 1 , C1-C4 alkyl, or C1-C4 haloalkyl. Suitably, L 2 The optional substituents on may be C1-C4 alkyl or C1-C4 haloalkyl.

[0118] Optionally, each L2 can be an unsubstituted C2 alkylene -CH2CH2-.

[0119] Each L 1 is preferably C1-C4 alkylene, more preferably C1 alkylene such as -CH2-.

[0120] Functional variants / derivatives of DOTAM have the following formula: TIFF2026000913000004.tif62170 [wherein each Z is independently R as defined above] 1 and p, q, r, and s are 0, 1, or 2, and p+q+r+s is 1 or greater. Preferably, p, q, r, and s are 0 or 1, and / or p+q+r+s is 1. For example, a compound can have p+q+r+s=1, where Z is a p-SCN-benzyl moiety (such compounds are commercially available from Macrocyclics, Inc., Plano, Texas). The compound may be:

[0121] Radionuclides useful in the present invention can include radioactive isotopes of metals such as lead (Pb), lutetium (Lu), or yttrium (Y).

[0122] Radionuclides that are particularly useful in imaging applications can be those that are gamma emitters. For example, the radionuclide can be: 203 Pb or 205 Bi.

[0123] Radionuclides that are particularly useful in therapeutic applications can be those that are alpha-emitters or beta-emitters. For example, the radionuclide can be: 212 Pb, 212 Bi, 213 Bi, 90Y , 177 Lu, 225 Ac, 211 At, 227 Th, 223 Ra.

[0124] In some embodiments, it may be preferable for DOTAM (or a salt or functional variant thereof) to be chelated with Pb or Bi, such as one of the radioisotopes of Pb or Bi listed above. In other embodiments, it may be preferable for DOTA (or a salt or functional variant thereof) to be chelated with Lu or Y, such as one of the radioisotopes of Lu or Y listed above.

[0125] In some embodiments, the methods and uses can include combined therapy and imaging methods using a mixture of radioisotopes, for example a mixture of a radioisotope suitable for therapy and a radioisotope suitable for imaging. For example, these can be different radioisotopes of the same metal chelated with the same chelator. In one embodiment, the method comprises: 203 Pb-DOTAM, and 212 In another embodiment, the method comprises administering Pb-DOTAM as a mixture. 203 Pb or 205 Following the first dosimetry cycle using a gamma emitter such as Bi, 212 Pb, 212 Bi, 213 Bi, 90Y , 177 Lu, 225 Ac, 211 At, 227 Th, or 223 It may involve one or more rounds of treatment with an alpha or beta emitter, such as Ra. Such methods are further described below.

[0126] In some embodiments, the functional binding site formed by association of the first and second antibodies is capable of binding to a Pb-DOTAM chelate.

[0127] In some embodiments, the functional binding site formed by association of the first and second antibodies is capable of specifically binding a radiolabeled compound, such as a Pb-DOTAM chelate, with a specific binding affinity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., ≦10 ...) to the Pb-DOTAM and / or target. -7 M or less, e.g., 10 -7 ~10 -13 , 10 -8 M or less, e.g., 10 -8 M~10 -13 M, e.g., 10 -9 M~10 -13 The functional binding site may bind with a dissociation constant (KD) of about 100 pM, 50 pM, 20 pM, 10 pM, 5 pM, 1 pM or less, e.g., 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 binding affinity. For example, the functional binding site may bind to a metal chelate with a Kd of about 1 pM to 1 nM, e.g., about 1 to 10 pM, 1 to 100 pM, 5 to 50 pM, 100 to 500 pM, or 500 pM to 1 nM.

[0128] C. Exemplary antigen binding sites for DOTA In one particular embodiment of the invention, the first and second antibodies are associated with DOTA (or a functional derivative or variant thereof), e.g., Lu or Y (e.g., 177 Lu or 90 Y) to form a functional binding site for the chelated DOTA. For example, the functional binding site may bind to the radiolabeled compound with a Kd of about 1 pM to 1 nM, e.g., about 1 to 10 pM, 1 to 100 pM, 5 to 50 pM, 100 to 500 pM, or 500 pM to 1 nM.

[0129] What is C825? 177 Lu and 90C825 is a known scFv with high affinity for DOTA-Bn (S-2-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecanetetraacetic acid) complexed with a radiometal such as Y (see, e.g., Cheal et al., 2018; Theranostics, 2018; and WO2010099536, incorporated herein by reference). The C825 CDR and VL and VH sequences are presented herein. In one embodiment, the heavy chain variable region that forms part of the antigen-binding site for the radiolabeled compound can comprise at least one, two, or all three CDRs selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 35; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 36; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 37. In an alternative embodiment, CDR-H1 can have the sequence of GFSLTDYGVH (SEQ ID NO: 148). The light chain variable region that forms part of the binding site for the radiolabeled compound can comprise at least one, two, or all three CDRs selected from: (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 38; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 39; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 40.

[0130] In another embodiment, the heavy chain variable domain (on the first antibody) that forms part of the functional antigen-binding site for the radiolabeled compound comprises the amino acid sequence of SEQ ID NO: 41, or a variant thereof, comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO: 41. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but a binding site comprising this sequence retains the ability to bind DOTA complexed with Lu or Y, preferably with the affinity described herein. In certain embodiments, a total of 1 to 10 amino acids within SEQ ID NO: 41 have been substituted, inserted, and / or deleted. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., within the FRs). Optionally, the antibody comprises a VH sequence within SEQ ID NO: 41, including post-translational modifications of this sequence. In certain embodiments, the VH comprises one, two, or three CDRs selected from (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 35 or the sequence of GFSLTDYGVH (SEQ ID NO: 148), (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 36, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 37.

[0131] Optionally, the light chain variable domain (on the second antibody) that forms part of the functional antigen-binding site for the radiolabeled compound comprises the amino acid sequence of SEQ ID NO: 42, or a variant thereof, including an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO: 42. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but a binding site comprising this sequence retains the ability to bind DOTA complexed with Lu or Y, preferably with the affinity described herein. In certain embodiments, a total of 1 to 10 amino acids in SEQ ID NO: 42 have been substituted, inserted, and / or deleted. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., within the FRs). Optionally, the antibody comprises a VL sequence within SEQ ID NO: 42, including post-translational modifications of this sequence. In certain embodiments, the VL comprises one, two, or three CDRs selected from (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 38; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 39; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 40.

[0132] The embodiments relating to heavy and light chain variable regions are expressly contemplated in combination, and thus a functional antigen-binding site can be formed from the heavy chain variable region defined above and the light chain variable region defined above in each of the first and second antibodies.

[0133] In any of the above embodiments, the light and heavy chain variable regions that form the binding site for the DOTA complex may be humanized. In one embodiment, the light and heavy chain variable regions comprise the CDRs of any of the above embodiments and further comprise a human acceptor framework, e.g., a human immunoglobulin framework or a human consensus framework.

[0134] 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 discussed further below.

[0135] D. Exemplary antigen binding sites for DOTAM In another specific embodiment of the invention, the first antibody and the second antibody associate to form a functional antigen binding site chelate to Pb-DOTAM (Pb-DOTAM).

[0136] In certain embodiments, a functional antigen binding site that binds Pb-DOTAM has the following characteristics: ·Specific binding properties of Pb-DOTAM and Bi-DOTAM; · Selectivity for Pb-DOTAM (and, optionally, Bi-DOTAM) compared to other chelated metals, such as Cu-DOTAM; · Extremely high affinity binding to Pb-DOTAM; the property of binding to the same epitope on Pb-DOTAM as an antibody described herein, e.g., PRIT-0213 or PRIT-0214, and / or having the same contact residues as said antibody; The ion channel may have one or more of:

[0137] Radioisotopes of Pb are useful in diagnostics and therapy. Specific radioisotopes of lead that may be useful in the present invention are: 212 Pb and 203 Contains Pb.

[0138] Radionuclides that are alpha particle emitters, due to a combination of short path lengths and high linear energy transfer, cause less damage to surrounding tissue than beta emitters and have the potential for more specific tumor cell killing. 212 Bi is an alpha particle emitter, but its short half-life precludes its direct use. 212 Pb is 212The parent radionuclide of Bi, 212 It is used as a source of Bi, which allows 212 This can effectively overcome the short half-life of Bi (Yong and Brechbiel, Dalton Trans., 2001, June 21, 40(23) 6068-6076).

[0139] 203 Pb is a useful imaging isotope. 203 Antibodies conjugated to Pb-DOTAM may have utility in radioimmunoimaging (RII).

[0140] Generally, radiometals are used in chelated form. In certain embodiments of the present invention, DOTAM is used as the chelating agent. DOTAM is a stable chelating agent for Pb(II) (Yong and Brechbiel, Dalton Trans., June 21, 2001, 40(23) 6068-6076; Chappell et al., Nuclear Medicine and Biology, Vol. 27, pp. 93-100, 2000). Thus, DOTAM is 212 Pb and 203 This is particularly useful when accompanied by isotopes of lead, such as Pb, discussed above.

[0141] In some embodiments, it may be preferred that the antibody bind to Pb-DOTAM with a binding affinity Kd value of 100 pM, 50 pM, 20 pM, 10 pM, 5 pM, 1 pM or less, e.g., 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, a functional binding site may bind to a radiolabeled compound with a Kd of about 1 pM to 1 nM, e.g., about 1 to 10 pM, 1 to 100 pM, 5 to 50 pM, 100 to 500 pM, or 500 pM to 1 nM.

[0142] In certain embodiments, the antibody additionally binds Bi chelated with DOTAM. In some embodiments, it may be preferable for the antibody to bind Bi-DOTAM (i.e., a chelate comprising DOTAM complexed with bismuth, also referred to herein as a "Bi-DOTAM chelate") with a binding affinity Kd value of 1 nM, 500 pM, 200 pM, 100 pM, 50 pM, 10 pM, or less, e.g., 9 pM, 8 pM, 7 pM, 6 pM, 5 pM, or less. For example, a functional binding site may bind to a metal chelate with a Kd of about 1 pM to 1 nM, e.g., about 1 to 10 pM, 1 to 100 pM, 5 to 50 pM, 100 to 500 pM, or 500 pM to 1 nM.

[0143] In some embodiments, the antibody may bind to Bi-DOTAM and Pb-DOTAM with similar affinity. For example, it may be preferable that the affinity ratio for Bi-DOTAM / Pb-DOTAM, e.g., the ratio of Kd values, is in the range of 0.1 to 10, e.g., 1 to 10.

[0144] In one embodiment, the heavy chain variable region forming part of the antigen-binding site for Pb-DOTAM can comprise at least one, two, or all three CDRs selected from (a) CDR-H1 comprising the amino acid sequence of GFSLSTYSMS (SEQ ID NO: 1); (b) CDR-H2 comprising the amino acid sequence of FIGSRGDTYYASWAKG (SEQ ID NO: 2); and (c) CDR-H3 comprising the amino acid sequence of ERDPYGGGAYPPHL (SEQ ID NO: 3). The light chain variable region forming part of the antigen-binding site for Pb-DOTAM can comprise at least one, two, or all three CDRs selected from (d) CDR-L1 comprising the amino acid sequence of QSSHSVYSDNDLA (SEQ ID NO: 4); (e) CDR-L2 comprising the amino acid sequence of QASKLAS (SEQ ID NO: 5); and (f) CDR-L3 comprising the amino acid sequence of LGGYDDESDTYG (SEQ ID NO: 6).

[0145] In some embodiments, the antibody may include one or more of CDR-H1, CDR-H2, and / or CDR-H3, or one or more of CDR-L1, CDR-L2, and / or CDR-L3, each having, for example, one, two, or three substitutions compared to the amino acid sequences of SEQ ID NOs: 1-6.

[0146] In some embodiments, the antibodies may share the same contact residues as those described herein; for example, these residues may be invariant. These residues may include the following: all numbered according to Kabat: a) within the heavy chain CDR2, Phe50, Asp56, and / or Tyr58 may be included, and optionally Gly52 and / or Arg54 may also be included; b) within the heavy chain CDR3, may include Glu95, Arg96, Asp97, Pro98, Tyr99, Ala100C, and / or Tyr100D, and may optionally also include Pro100E; c) Tyr28 and / or Asp32 can be included within the light chain CDR1; d) the light chain CDR3 can include Gly91, Tyr92, Asp93, Thr95c, and / or Tyr96; e) Within the light chain CDR2, Gln50 can optionally be included.

[0147] For example, in some embodiments, CDR-H2 can comprise the amino acid sequence of FIGSRGDTYYASWAKG (SEQ ID NO: 2), or a variant thereof having up to one, two, or three substitutions within SEQ ID NO: 2, where these substitutions do not include Phe50, Asp56, and / or Tyr58, and optionally also do not include Gly52 and / or Arg54, all numbered according to Kabat.

[0148] In some embodiments, CDR-H2 may be substituted at one or more positions as shown below. Herein and in the substitution table below, substitutions are based on germline residues (underlined) or by amino acids that theoretically sterically fit and also occur at this position in the crystal repertoire. In some embodiments, the residues mentioned above are fixed and other residues may be substituted according to the table below; in other embodiments, substitution of any residue may be made according to the table below. TIFF2026000913000005.tif149170

[0149] Optionally, CDR-H3 can comprise the amino acid sequence of ERDPYGGGAYPPHL (SEQ ID NO: 3), or a variant thereof having up to one, two, or three substitutions within SEQ ID NO: 3, where the substitutions do not include Glu95, Arg96, Asp97, Pro98, and optionally also do not include Ala100C, Tyr100D, and / or Pro100E, and / or optionally also do not include Tyr99. For example, in some embodiments, the substitutions do not include Glu95, Arg96, Asp97, Pro98, Tyr99, Ala100C, and Tyr100D.

[0150] In certain embodiments, CDR-H3 may be substituted at one or more positions as shown below: In some embodiments, the above-mentioned residues are fixed and other residues may be substituted according to the table below, and in other embodiments, substitution of any residue may be made according to the table below. TIFF2026000913000006.tif132170

[0151] Optionally, CDR-L1 can comprise the amino acid sequence of QSSHSVYSDNDLA (SEQ ID NO: 4) or a variant thereof having up to one, two, or three substitutions within SEQ ID NO: 4, where these substitutions do not include Tyr28 and / or Asp32 (Kabat numbering).

[0152] In certain embodiments, CDR-L1 may be substituted at one or more positions as shown below: Again, in some embodiments, the above-mentioned residues may be fixed and other residues may be substituted according to the table below, and in other embodiments, substitution of any residue may be made according to the table below. TIFF2026000913000007.tif124170

[0153] Optionally, CDR-L3 can comprise the amino acid sequence of LGGYDDESDTYG (SEQ ID NO: 6), or a variant thereof having up to one, two, or three substitutions within SEQ ID NO: 6, where these substitutions do not include Gly91, Tyr92, Asp93, Thr95c, and / or Tyr96 (Kabat).

[0154] In certain embodiments, CDR-L3 may be substituted at the following positions, as shown below (most residues are solvent exposed and do not make contacts with the antigen, so many substitutions are possible): Again, in some embodiments, the above-mentioned residues may be fixed and other residues may be substituted according to the table below, and in other embodiments, substitutions of any residue may be made according to the table below. TIFF2026000913000008.tif216170

[0155] The antibody may optionally further comprise a CDR-H1 or CDR-L2 having the sequence of SEQ ID NO: 1 or SEQ ID NO: 5, respectively, or a variant thereof having at least one, two, or three substitutions, optionally conservative substitutions.

[0156] Thus, the heavy chain variable domain that forms part of the antigen binding site for Pb-DOTAM comprises at least: a) a heavy chain CDR2 comprising the amino acid sequence of FIGSRGDTYYASWAKG (SEQ ID NO: 2), or a variant thereof having up to one, two, or three substitutions within 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 of ERDPYGGGAYPPHL (SEQ ID NO: 3), or a variant thereof having up to one, two, or three substitutions within SEQ ID NO: 3, wherein these substitutions do not include Glu95, Arg96, Asp97, Pro98, and optionally also do not include Ala100C, Tyr100D, and / or Pro100E, and / or optionally also do not include Tyr99. may include:

[0157] In some embodiments, the heavy chain variable domain additionally optionally comprises: c) a heavy chain CDR1 comprising the amino acid sequence of GFSLSTYSMS (SEQ ID NO: 1), or a variant thereof having up to one, two, or three substitutions within SEQ ID NO: 1; and a heavy chain CDR1 which is

[0158] In some embodiments, the heavy chain variable domain additionally comprises a C-terminal alanine (e.g., Ala 114 according to the Kabat numbering system) to avoid binding of existing antibodies that recognize the free VH region. As reported in Holland MC et al., J. Clin Immunol (2013), HAVH autoantibodies do not bind to intact IgG, or IgG fragments (fAbs or modified VH molecules) containing the same VH framework sequence, or VK domain antibodies, so the free C-terminus is thought to be important for binding of HAVH (human anti-VH domain) autoantibodies to VH domain antibodies. Cordy JC et al., Clinical and Experimental Immunology (2015) mention the presence of cryptic epitopes in the C-terminal epitopes of VH dAbs that are not naturally accessible to HAVH antibodies within intact IgG molecules.

[0159] Thus, where an antibody comprises a free VH region (a VH region that is not fused at its C-terminus to any other domain), the sequence may be extended by one or more C-terminal residues. The extension may prevent binding of an antibody that recognizes the free VH region. For example, the extension may be by 1 to 10 residues, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues. In one embodiment, the VH sequence may be extended by one or more C-terminal alanine residues. The VH sequence may also be extended by 1 to 10 residues from the N-terminal portion of a CH1 domain, e.g., from the N-terminus of a CH1 domain, e.g., from the CH1 domain of human IgG1 (the first 10 residues of the CH1 domain of human IgG1 are ASTKGPSVFP (SEQ ID NO: 149) and, therefore, in one embodiment, 1 to 10 residues may be taken from the N-terminus of this sequence). For example, in one embodiment, the peptide sequence AST (corresponding to the first three residues of the CH1 domain of IgG1) is added to the C-terminus of the VH region.

[0160] In another embodiment, the light chain variable domain that forms part of the antigen binding site for Pb-DOTAM comprises at least: d) a light chain CDR1 comprising the amino acid sequence of 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 of LGGYDDESDTYG (SEQ ID NO: 6), or a variant thereof having up to one, two, or three substitutions within SEQ ID NO: 6, wherein these substitutions exclude Gly91, Tyr92, Asp93, Thr95c, and Tyr96.

[0161] In some embodiments, the light chain variable domain additionally optionally comprises: f) a light chain CDR2 comprising the amino acid sequence of QASKLAS (SEQ ID NO: 5) or a variant thereof having at least one, two, or three substitutions within SEQ ID NO: 5, optionally excluding Gln50; light chain CDR2 Includes.

[0162] In any embodiment of the invention comprising a variant of a sequence comprising the above-identified CDRs (e.g., of a variable domain), the protein may be unaltered at one or more of the above-identified CDR residues.

[0163] Optionally, the heavy chain variable domain that forms part of the functional antigen-binding site for Pb-DOTAM (on the first antibody) comprises an amino acid sequence selected from the group consisting of SEQ ID NO:7 and SEQ ID NO:9, or variants thereof, including amino acid sequences having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:7 or SEQ ID NO:9. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the binding site comprising this sequence retains the ability to bind to Pb-DOTAM, preferably with the affinity described herein. The VH sequence may retain the invariant residues identified above. In certain embodiments, a total of 1 to 10 amino acids in SEQ ID NO: 7 or SEQ ID NO: 9 have been substituted, inserted, and / or deleted. In certain embodiments, the substitutions, insertions, or deletions occur within regions outside the CDRs (i.e., within the FRs). Optionally, the antibody comprises a VH sequence in SEQ ID NO: 7 or SEQ ID NO: 9, optionally including a post-translational modification of this sequence with a C-terminal Ala. In certain embodiments, the VH comprises one, two, or three CDRs selected from (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3.

[0164] In some variants of some embodiments mentioned above, SEQ ID NO: 7 or 9 may be extended by one or more additional C-terminal residues, for example by one or more alanine residues, optionally a single alanine residue. Thus, for example, in one particular variant, the sequence of SEQ ID NO: 7 is: VTLKESGPVLVKPTETLTLTCTVSGFSLSTYSMSWIRQPPGKALEWLGFIGSRGDTYYASWAKGRLTISKDTSKSQVVLTMTNMDPVDTATYYCARERDPYGGGAYPPHLWGRGTLVTVSSA (SEQ ID NO: 150) can be expanded so that

[0165] In other embodiments, the extension may be an N-terminal portion of the CH1 domain described above, e.g., an extension of 1 to 10 residues from the N-terminus of the CH1 domain, e.g., from the CH1 domain of human IgG1. For example, the extension may be an extension of the sequence of the peptide AST.

[0166] Optionally, the light chain variable domain forming part of the functional antigen-binding site for Pb-DOTAM (on the second antibody) comprises the amino acid sequence of SEQ ID NO:8 or a variant thereof, including an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:8. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an anti-Pb-DOTAM binding site comprising this sequence retains the ability to bind to Pb-DOTAM, preferably with the affinity described herein. The VL sequence may retain the invariant residues identified above. In certain embodiments, a total of 1 to 10 amino acids within SEQ ID NO:8 are substituted, inserted, and / or deleted. In certain embodiments, the substitutions, insertions, or deletions occur within regions outside the CDRs (i.e., within the FRs). Optionally, the anti-Pb-DOTAM antibody comprises a VL sequence within SEQ ID NO: 8, including post-translational modifications of this sequence. In certain embodiments, the VL comprises one, two, or three CDRs selected from (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

[0167] The embodiments relating to heavy and light chain variable regions are expressly contemplated in combination, and thus a functional antigen-binding site for Pb-DOTAM can be formed from the heavy chain variable region defined above and the light chain variable region defined above in each of the first and second antibodies.

[0168] Optionally, an antigen-binding site specific for a Pb-DOTAM chelate may be formed from a heavy chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 7 or SEQ ID NO: 9, or a variant thereof as defined above (including variants with a C-terminal extension as discussed above), and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof, as defined above. For example, an antigen-binding site specific for a Pb-DOTAM chelate may comprise a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof, and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof, including post-translational modifications of these sequences. In another embodiment, an antigen-binding site specific for a Pb-DOTAM chelate may comprise a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 9, or a variant thereof (including variants with a C-terminal extension as discussed above), including post-translational modifications of these sequences, and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof.

[0169] In any of the above embodiments, the light and heavy chain variable regions that form the anti-Pb-DOTAM binding site may be humanized. In one embodiment, the light and heavy chain variable regions comprise the CDRs of any of the above embodiments and further comprise a human acceptor framework, e.g., a human immunoglobulin framework or a human consensus framework. In another embodiment, the light and / or heavy chain variable regions comprise the CDRs of any of the above embodiments and further comprise framework regions derived from vk 1-39 and / or vh 2-26. In some embodiments, for vk 1-39, there may be no backmutation. For vh 2-26, the germline Ala49 residue may be backmutated to Gly49.

[0170] E. Exemplary antigen-binding sites for CEA In another specific embodiment of the present invention, which can be combined with the embodiments discussed above, the target antigen to which the first and second antibodies bind can be CEA (carcinoembryonic antigen). Antibodies raised against CEA include T84.66 and its humanized and chimeric forms, such as T84.66-LCHA, described in WO2016 / 075278A1 and / or WO2017 / 055389, CH1A1a, an anti-CEA antibody described in WO2012 / 117002 and WO2014 / 131712, and CEA hMN-14 or labetuzumab (e.g., as described in US6676924 and US5874540). Another exemplary antibody against CEA is A5B7 (described, for example, in MJ Banfield et al., Proteins, 1997, 29(2), 161-171), or a humanized antibody derived from murine A5B7 described in WO92 / 01059 and WO2007 / 071422. See also co-pending application PCT / EP2020 / 067582. An example of a humanized version of A5B7 is A5H1EL1(G54A). A further exemplary antibody against CEA is MFE23, and humanized forms thereof, described in US7626011 and / or co-pending application PCT / EP2020 / 067582. Yet a further example of an anti-CEA antibody is 28A9. Any of these antibodies, or antigen-binding fragments thereof, can be used to form a CEA-binding portion in the present invention.

[0171] Optionally, an antigen-binding site that binds CEA may bind with a Kd value for monovalent binding of 1 nM or less, 500 pM or less, 200 pM or less, or 100 pM or less.

[0172] In some embodiments, the first antibody and / or the second antibody may bind to the CH1A1a epitope, the A5B7 epitope, the MFE23 epitope, the T84.66 epitope, or the 28A9 epitope of CEA.

[0173] 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 a portion of the CEA molecule that is cleaved by GPI phospholipase and released into the blood. An example of an epitope that is not found on soluble CEA is the CH1A1A epitope. Optionally, one of the first antibody and / or the second antibody binds to an epitope that is not present on soluble CEA, and the other binds to an epitope that is present on soluble CEA.

[0174] The epitopes of CH1A1a and its parent murine antibody, PR1A3, are described in WO2012 / 117002A1 and Durbin H. et al., Proc. Natl. Scad. Sci. USA, 91:4313-4317, 1994. Antibodies that bind to the CH1A1a epitope bind to a conformational epitope within 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 of SEQ ID NO: 25 and VL of SEQ ID NO: 26 herein. The A5B7 epitope is described in the co-pending application PCT / EP2020 / 067582. Antibodies that bind to the A5B7 epitope bind to the A2 domain of CEA, i.e., SEQ ID NO: 154: PKPFITSNNSNPVEDEDAVALTCEPEIQNTTYLWWVNNQSLPVSPRLQLSNDNRTLTLLSVTRNDVGP YECGIQNKLSVDHSDPVILN (SEQ ID NO: 154) It binds to a domain containing the amino acid In one aspect, the antibody binds to the same epitope as the A5B7 antibody herein having a VH of SEQ ID NO:49 and a VL of SEQ ID NO:50.

[0175] In one embodiment, the antibody binds to the same epitope as T84.66 described in WO2016 / 075278. The antibody may bind to the same epitope as the antibody having VH of SEQ ID NO: 17 and VL of SEQ ID NO: 18 herein.

[0176] The MFE23 epitope is described in co-pending application PCT / EP2020 / 067582. Antibodies that bind to the MFE23 epitope are those that bind to the A1 domain of CEA, i.e., SEQ ID NO: 155: PKPSISSNNSKPVEDKDAVAFTCEPETQDATYLWWVNNQSLPVSPRLQLSNGNRTLTLFNVTRNDTASYKCETQNPVSARRSDSVILN (SEQ ID NO: 155) It binds to a domain containing the amino acid In one aspect, the antibody may bind to the same epitope as an antibody herein having a VH domain of SEQ ID NO:167 and a VL domain of SEQ ID NO:168.

[0177] In some embodiments, the first antibody and / or the second antibody may bind to the same CEA epitope as an antibody presented herein, e.g., P1AD8749, P1AD8592, P1AE4956, P1AE4957, P1AF0709, P1AF0298, P1AF0710, or P1AF0711.

[0178] In some embodiments, the first antibody and the second antibody bind to the same epitope of CEA as each other. Thus, for example, the first antibody and the second antibody can both bind to the CH1A1a epitope, the A5B7 epitope, the MFE23 epitope, the T84.66 epitope, or the 28A9 epitope.

[0179] In some embodiments, both the first antibody and the second antibody may have a CEA-binding sequence (i.e., CDR and / or VH / VL domain) derived from CH1A1A; both the first antibody and the second antibody may have a CEA-binding sequence derived from A5B7 or a humanized version thereof; both the first antibody and the second antibody may have a CEA-binding sequence derived from T84.66 or a humanized version thereof; both the first antibody and the second antibody may have a CEA-binding sequence derived from MFE23 or a humanized version thereof; or both the first antibody and the second antibody may have a CEA-binding sequence derived from 28A9 or a humanized version thereof. Exemplary sequences are disclosed herein.

[0180] In other embodiments, the first antibody and the second antibody bind to different epitopes of CEA. Thus, for example, i) one antibody can bind to the CH1A1A epitope, and the other antibody can bind to the A5B7 epitope, the T84.66 epitope, the MFE23 epitope, or the 28A9 epitope; ii) one antibody can bind to the A5B7 epitope, and the other antibody can bind to the CH1A1A epitope, the T84.66 epitope, the MFE23 epitope, or the 28A9 epitope; iii) one antibody can bind to the MFE23 epitope, and the other antibody can bind to the CH1A1A epitope. iv) one antibody may be capable of binding to the T84.66 epitope and the other antibody may bind to the CH1A1A epitope, the A5B7 epitope, the MFE23 epitope, or the 28A9 epitope; v) one antibody may be capable of binding 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.

[0181] In some embodiments, i) one antibody may have a CEA-binding sequence (i.e., CDR or VH / VL domain) derived from CH1A1A, and the other may have a CEA-binding sequence derived from A5B7 or a humanized version thereof, T84.66 or a humanized version thereof, MFE23 or a humanized version thereof, or 28A9 or a humanized version thereof; ii) one antibody may have a CEA-binding sequence derived from A5B7 or a humanized version thereof, and the other may have a CEA-binding sequence derived from CH1A1A, T84.66 or a humanized version thereof, MFE23 or a humanized version thereof, or 28A9 or a humanized version thereof; iii) one antibody may have a CEA-binding sequence derived from MFE23 or a humanized version thereof. and the other may have a CEA-binding sequence derived from CH1A1A, A5B7 or a humanized version thereof, T84.66 or a humanized version thereof, or 28A9 or a humanized version thereof; iv) one antibody may have a CEA-binding sequence derived from T84.66 or a humanized version thereof, and the other may have a CEA-binding sequence derived from CH1A1A, A5B7 or a humanized version thereof, MFE23 or a humanized version thereof, or 28A9 or a humanized version thereof; v) one antibody may have a CEA-binding sequence derived from 28A9 or a humanized version thereof, and the other may have a CEA-binding sequence derived from CH1A1A, A5B7 or a humanized version thereof, T84.66 or a humanized version thereof, or MFE23 or a humanized version thereof.

[0182] In one particular embodiment, one antibody can bind to the CH1A1A epitope and the other can bind to the A5B7 epitope. The first antibody can have a CEA-binding sequence derived from antibody CH1A1A, and the second antibody can have a CEA-binding sequence derived from A5B7 (including humanized versions thereof); the first antibody can have a CEA-binding sequence derived from antibody A5B7 (including humanized versions thereof), and the second antibody can have a CEA-binding sequence derived from CH1A1A.

[0183] In another specific embodiment, one antibody can bind to the CH1A1A epitope and the other can bind to the T84.66 epitope. The first antibody can have a CEA-binding sequence derived from antibody CH1A1A, and the second antibody can have a CEA-binding sequence derived from T84.66 (including humanized versions thereof); the first antibody can have a CEA-binding sequence derived from antibody T84.66 (including humanized versions thereof), and the second antibody can have a CEA-binding sequence derived from CH1A1A. In some embodiments, the first antibody can bind to the T84.66 epitope and / or have an antigen-binding site described in (i) below, and the second antibody can bind to the CH1A1A epitope and / or have an antigen-binding site described in (ii) below.

[0184] Exemplary CEA-binding sequences i) through v) are disclosed below, which provide examples of CEA-binding sequences derived from i) T84.66, ii) CH1A1A, iii) A5B7, iv) 28A9, and v) MFE23 (or from humanized versions thereof).

[0185] i) In one embodiment, the antigen-binding site that binds to CEA may comprise at least one, two, three, four, five, or six CDRs selected from (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 11; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 12; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 13; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 14; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 15; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 16.

[0186] Optionally, the antigen-binding site that binds to CEA may comprise at least one, at least two, or all three VH CDR sequences selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 11; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 12; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 13.

[0187] Optionally, the antigen-binding site that binds to CEA comprises at least one, at least two, or all three VL CDR sequences selected from: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 14; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 15; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 16.

[0188] Optionally, the antigen-binding site that binds to CEA comprises: (a) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 11, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 12, and (iii) CDR-H3 comprising an amino acid sequence selected from SEQ ID NO: 13; and (b) a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 14, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 15, and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 16.

[0189] In another embodiment, the antigen binding site that binds to CEA comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 11; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 12; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 13; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 14; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 15; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 16.

[0190] In any of the above embodiments, the multispecific antibody may be humanized. In one embodiment, the anti-CEA antigen-binding site comprises the CDRs of any of the above embodiments and further comprises a human acceptor framework, e.g., a human immunoglobulin framework or a human consensus framework.

[0191] In another embodiment, the antigen-binding site that binds to CEA comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 17. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the antigen-binding site comprising this sequence retains the ability to bind to CEA, preferably with the affinity specified above. In certain embodiments, a total of 1 to 10 amino acids within SEQ ID NO: 17 are substituted, inserted, and / or deleted. In certain embodiments, the substitutions, insertions, or deletions occur within a region outside the HVR (i.e., within the FR). Optionally, the antigen-binding site that binds to CEA comprises a VH sequence within SEQ ID NO: 17, including post-translational modifications of this sequence. In certain embodiments, the VH comprises one, two, or three CDRs selected from (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 11, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 12, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 13.

[0192] In another embodiment, the antigen-binding site that binds to CEA comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 18. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the antigen-binding site comprising this sequence retains the ability to bind to CEA, preferably with the affinity specified above. In certain embodiments, a total of 1 to 10 amino acids within SEQ ID NO: 18 are substituted, inserted, and / or deleted. In certain embodiments, the substitutions, insertions, or deletions occur within a region outside the HVR (i.e., within the FR). Optionally, the antigen-binding site against CEA comprises a VL sequence within SEQ ID NO: 18, including post-translational modifications of this sequence. In certain embodiments, the VL comprises one, two, or three CDRs selected from (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 14; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 15; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 16.

[0193] In another embodiment, the antigen-binding site that binds to CEA comprises a VH of any of the above-presented embodiments and a VL of any of the above-presented embodiments. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 17 and SEQ ID NO: 18, respectively, including post-translational modifications of these sequences.

[0194] ii) In a further specific embodiment, the antigen-binding site that binds to CEA may comprise at least one, two, three, four, five, or six CDRs selected from (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 19; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 20; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 21; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 24.

[0195] Optionally, the antigen-binding site that binds to CEA may comprise at least one, at least two, or all three VH CDR sequences selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 19; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 20; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 21.

[0196] Optionally, the antigen-binding site that binds to CEA comprises at least one, at least two, or all three VL CDR sequences selected from: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22; (b) 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.

[0197] Optionally, the antigen-binding site that binds to CEA comprises: (a) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 19, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 20, and (iii) CDR-H3 comprising an amino acid sequence selected from SEQ ID NO: 21; and (b) a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from (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 (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 24.

[0198] In another embodiment, the antigen binding site that binds to CEA comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 19; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 20; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 21; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 24.

[0199] In any of the above embodiments, the multispecific antibody may be humanized. In one embodiment, the anti-CEA antigen-binding site comprises the CDRs of any of the above embodiments and further comprises a human acceptor framework, e.g., a human immunoglobulin framework or a human consensus framework.

[0200] In another embodiment, the antigen-binding site that binds to CEA comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 25. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the antigen-binding site comprising this sequence retains the ability to bind to CEA, preferably with the affinity specified above. In certain embodiments, a total of 1 to 10 amino acids within SEQ ID NO: 25 are substituted, inserted, and / or deleted. In certain embodiments, the substitutions, insertions, or deletions occur within a region outside the HVR (i.e., within the FR). Optionally, the antigen-binding site that binds to CEA comprises a VH sequence within SEQ ID NO: 25, including post-translational modifications of this sequence. In certain embodiments, the VH comprises one, two, or three CDRs selected from (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 19, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 20, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 21.

[0201] In another embodiment, the antigen-binding site that binds to CEA comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 26. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the antigen-binding site comprising this sequence retains the ability to bind to CEA, preferably with the affinity specified above. In certain embodiments, a total of 1 to 10 amino acids within SEQ ID NO: 26 are substituted, inserted, and / or deleted. In certain embodiments, the substitutions, insertions, or deletions occur within a region outside the HVR (i.e., within the FR). Optionally, the antigen-binding site against CEA comprises a VL sequence within SEQ ID NO: 26, including post-translational modifications of this sequence. In certain embodiments, the VL comprises one, two, or three CDRs selected from (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23; and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 24.

[0202] In another embodiment, the antigen-binding site that binds to CEA comprises a VH of any of the above-presented embodiments and a VL of any of the above-presented embodiments. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 25 and SEQ ID NO: 26, respectively, including post-translational modifications of these sequences.

[0203] iii) In a further specific embodiment, the antigen-binding site that binds to CEA can comprise at least one, two, three, four, five, or six CDRs selected from (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 43; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 44; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 45; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 46; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 47; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 48. In some embodiments, CDR-H1 can have the sequence GFTFTDYYMN (SEQ ID NO: 151).

[0204] Optionally, the antigen-binding site that binds to CEA can comprise at least one, at least two, or all three VH CDR sequences selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 43; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 44; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 45. In some embodiments, CDR-H1 can have the sequence GFTFTDYYMN (SEQ ID NO: 151).

[0205] Optionally, the antigen-binding site that binds to CEA comprises at least one, at least two, or all three VL CDR sequences selected from: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 46; (b) 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.

[0206] Optionally, the antigen-binding site that binds to CEA comprises: (a) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 43, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 44, and (iii) CDR-H3 comprising an amino acid sequence selected from SEQ ID NO: 45; and (b) a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from (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 (iii) 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: 151).

[0207] In another aspect, the antigen-binding site that binds to CEA comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 43; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 44; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 45; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 46; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 47; and (f) 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: 151).

[0208] In any of the above embodiments, the multispecific antibody may be humanized. In one embodiment, the anti-CEA antigen-binding site comprises the CDRs of any of the above embodiments and further comprises a human acceptor framework, e.g., a human immunoglobulin framework or a human consensus framework.

[0209] In another embodiment, an antigen-binding site that binds to CEA comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 49. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an antigen-binding site comprising this sequence retains the ability to bind to CEA, preferably with the affinity specified above. In certain embodiments, a total of 1 to 10 amino acids within SEQ ID NO: 49 are substituted, inserted, and / or deleted. In certain embodiments, the substitutions, insertions, or deletions occur within a region outside the HVR (i.e., within the FR). Optionally, the antigen-binding site that binds to CEA comprises a VH sequence within SEQ ID NO: 49, including post-translational modifications of this sequence. In certain embodiments, the VH comprises one, two, or three CDRs selected from (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 43 or the sequence of GFTFTDYYMN (SEQ ID NO: 151), (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 44, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 45.

[0210] In another embodiment, the antigen-binding site that binds to CEA comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 50. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the antigen-binding site comprising this sequence retains the ability to bind to CEA, preferably with the affinity specified above. In certain embodiments, a total of 1 to 10 amino acids within SEQ ID NO: 50 are substituted, inserted, and / or deleted. In certain embodiments, the substitutions, insertions, or deletions occur within a region outside the HVR (i.e., within the FR). Optionally, the antigen-binding site against CEA comprises a VL sequence within SEQ ID NO: 50, including post-translational modifications of this sequence. In certain embodiments, the VL comprises one, two, or three CDRs selected from (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 46; (b) 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.

[0211] In another embodiment, the antigen-binding site that binds to CEA comprises a VH of any of the above-presented embodiments and a VL of any of the above-presented embodiments. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 49 and SEQ ID NO: 50, respectively, including post-translational modifications of these sequences.

[0212] iv) In yet a further specific embodiment, the antigen-binding site that binds to CEA may comprise at least one, two, three, four, five, or six CDRs selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 59; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 60; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 61; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 62; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 63; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 64.

[0213] Optionally, the antigen-binding site that binds to CEA may comprise at least one, at least two, or all three VH CDR sequences selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 59; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 60; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 61.

[0214] Optionally, the antigen-binding site that binds to CEA comprises at least one, at least two, or all three VL CDR sequences selected from: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 62; (b) 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.

[0215] Optionally, the antigen-binding site that binds to CEA comprises: (a) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 59, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 60, and (iii) CDR-H3 comprising an amino acid sequence selected from SEQ ID NO: 61; and (b) a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from (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 (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 64.

[0216] In another embodiment, the antigen binding site that binds to CEA comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 59; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 60; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 61; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 62; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 63; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 64.

[0217] In any of the above embodiments, the multispecific antibody may be humanized. In one embodiment, the anti-CEA antigen-binding site comprises the CDRs of any of the above embodiments and further comprises a human acceptor framework, e.g., a human immunoglobulin framework or a human consensus framework.

[0218] In another embodiment, the antigen-binding site that binds to CEA comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 65. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the antigen-binding site comprising this sequence retains the ability to bind to CEA, preferably with the affinity specified above. In certain embodiments, a total of 1 to 10 amino acids within SEQ ID NO: 65 are substituted, inserted, and / or deleted. In certain embodiments, the substitutions, insertions, or deletions occur within a region outside the HVR (i.e., within the FR). Optionally, the antigen-binding site that binds to CEA comprises a VH sequence within SEQ ID NO: 65, including post-translational modifications of this sequence. In certain embodiments, the VH comprises one, two, or three CDRs selected from (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 59, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 60, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 61.

[0219] In another embodiment, the antigen-binding site that binds to CEA comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 66. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the antigen-binding site comprising this sequence retains the ability to bind to CEA, preferably with the affinity specified above. In certain embodiments, a total of 1 to 10 amino acids within SEQ ID NO: 66 are substituted, inserted, and / or deleted. In certain embodiments, the substitutions, insertions, or deletions occur within a region outside the HVR (i.e., within the FR). Optionally, the antigen-binding site against CEA comprises a VL sequence within SEQ ID NO: 66, including post-translational modifications of this sequence. In certain embodiments, the VL comprises one, two, or three CDRs selected from (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 62; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 63; and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 64.

[0220] In another embodiment, the antigen-binding site that binds to CEA comprises a VH of any of the above-presented embodiments and a VL of any of the above-presented embodiments. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 65 and SEQ ID NO: 66, respectively, including post-translational modifications of these sequences.

[0221] v) In yet a further specific embodiment, the antigen-binding site that binds to CEA may comprise at least one, two, three, four, five, or six CDRs selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 156; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 157 or 158; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 159; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 160, 161, or 162; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 163, 164, or 165; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 166.

[0222] Optionally, the antigen binding site that binds to CEA is a VH CDR sequence, wherein the VH CDR sequence is (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 156; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 157 or 158; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 159; and / or VL CDR sequences, which are: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 160, 161, or 162; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 163, 164, or 165; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 166. may include:

[0223] In one embodiment, the antigen-binding site against CEA comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 167, or (more preferably) selected from SEQ ID NOs: 169, 170, 171, 172, 173, or 174, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 168, or (more preferably) selected from SEQ ID NOs: 175, 176, 177, 178, 179, or 180.

[0224] In any of the above embodiments, the multispecific antibody may be humanized. In one embodiment, the anti-CEA antigen-binding site comprises the CDRs of any of the above embodiments and further comprises a human acceptor framework, e.g., a human immunoglobulin framework or a human consensus framework.

[0225] In a particular embodiment, the antigen binding domain capable of binding to CEA comprises: (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 169 and a VL domain comprising the amino acid sequence of SEQ ID NO: 179, or (b) a VH domain comprising the amino acid sequence of SEQ ID NO: 173 and a VL domain comprising the amino acid sequence of SEQ ID NO: 179; or (c) a VH domain comprising the amino acid sequence of SEQ ID NO: 170 and a VL domain comprising the amino acid sequence of SEQ ID NO: 179; or (d) a VH domain comprising the amino acid sequence of SEQ ID NO: 174 and a VL domain comprising the amino acid sequence of SEQ ID NO: 178; or (e) a VH domain comprising the amino acid sequence of SEQ ID NO: 173 and a VL domain comprising the amino acid sequence of SEQ ID NO: 178; or (f) a VH domain comprising the amino acid sequence of SEQ ID NO: 171 and a VL domain comprising the amino acid sequence of SEQ ID NO: 178; or (g) a VH domain comprising the amino acid sequence of SEQ ID NO: 169, and a VL domain comprising the amino acid sequence of SEQ ID NO: 178 Includes.

[0226] In another embodiment, the antigen-binding site that binds to CEA comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence mentioned in a) to g) above. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the antigen-binding site comprising this sequence retains the ability to bind to CEA, preferably with the affinity specified above. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted. In certain embodiments, the substitutions, insertions, or deletions occur within a region outside the HVR (i.e., within the FR).

[0227] In another embodiment, the antigen-binding site that binds to CEA comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence mentioned in a) to g) above. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the antigen-binding site comprising this sequence retains the ability to bind to CEA, preferably with the affinity specified above. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted. In certain embodiments, the substitutions, insertions, or deletions occur within a region outside the HVR (i.e., within the FR).

[0228] In another embodiment, the antigen binding site that binds to CEA comprises a VH of any of the above presented embodiments and a VL of any of the above presented embodiments.

[0229] F. Exemplary Antigen Binding Sites for Other Targets In another specific embodiment of the invention, which may be combined with the embodiments discussed above (e.g., binding sites for DOTA or DOTAM), the target antigen to which the first and second antibodies bind may be GPRC5D or FAP.

[0230] Optionally, an antigen-binding site that binds to GPRC5D or FAP may bind with a Kd value for monovalent binding of 1 nM or less, 500 pM or less, 200 pM or less, or 100 pM or less.

[0231] Below, exemplary GPRC5D binding sequences are described.

[0232] In one embodiment, the antigen binding site that binds to GPRC5D may comprise at least one, two, three, four, five, or six CDRs selected from (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 67; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 68; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 69; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 70; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 71; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 72.

[0233] Optionally, the antigen-binding site that binds to GPRC5D may comprise at least one, at least two, or all three VH CDR sequences selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 67; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 68; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 69.

[0234] Optionally, the antigen binding site that binds to GPRC5D comprises at least one, at least two, or all three VL CDR sequences selected from: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 70; (b) 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.

[0235] Optionally, the antigen-binding site that binds to GPRC5D comprises: (a) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 67, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 68, and (iii) a CDR-H3 comprising an amino acid sequence selected from SEQ ID NO: 69; and (b) a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 70, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 71, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 72.

[0236] In another aspect, the antigen binding site that binds to GPRC5D comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 67; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 68; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 69; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 70; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 71; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 72.

[0237] In any of the above embodiments, the multispecific antibody may be humanized. In one embodiment, the anti-GPRC5D antigen-binding site comprises the CDRs of any of the above embodiments and further comprises a human acceptor framework, e.g., a human immunoglobulin framework or a human consensus framework.

[0238] In another embodiment, an antigen-binding site that binds to GPRC5D comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 73. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an antigen-binding site comprising this sequence retains the ability to bind to GPRC5D, preferably with the affinity specified above. In certain embodiments, a total of 1 to 10 amino acids within SEQ ID NO: 73 are substituted, inserted, and / or deleted. In certain embodiments, the substitutions, insertions, or deletions occur within a region outside the HVR (i.e., within the FR). Optionally, the antigen-binding site that binds to GPRC5D comprises a VH sequence within SEQ ID NO: 73, including post-translational modifications of this sequence. In certain embodiments, the VH comprises one, two, or three CDRs selected from (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 67, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 68, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 69.

[0239] In another embodiment, an antigen-binding site that binds to GPRC5D comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 74. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an antigen-binding site comprising this sequence retains the ability to bind to GPRC5D, preferably with the affinity specified above. In certain embodiments, a total of 1 to 10 amino acids within SEQ ID NO: 74 are substituted, inserted, and / or deleted. In certain embodiments, the substitutions, insertions, or deletions occur within a region outside the HVR (i.e., within the FR). Optionally, the antigen-binding site for GPRC5D comprises a VL sequence within SEQ ID NO: 74, including post-translational modifications of this sequence. In certain embodiments, the VL comprises one, two, or three CDRs selected from (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 70; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 71; and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 72.

[0240] In another embodiment, the antigen-binding site that binds to GPRC5D comprises a VH in any of the above-presented embodiments and a VL in any of the above-presented embodiments. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 73 and SEQ ID NO: 74, respectively, including post-translational modifications of these sequences.

[0241] Below, exemplary FAP-binding sequences are described.

[0242] In one embodiment, the antigen-binding site that binds to the FAP may comprise at least one, two, three, four, five, or six CDRs selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 75; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 76; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 77; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 78; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 79; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 80.

[0243] Optionally, the antigen-binding site that binds to the FAP can comprise at least one, at least two, or all three VH CDR sequences selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 75; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 76; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 77.

[0244] Optionally, the antigen-binding site that binds to the FAP comprises at least one, at least two, or all three VL CDR sequences selected from: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 78; (b) 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.

[0245] Optionally, the antigen-binding site that binds to the FAP comprises: (a) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 75, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 76, and (iii) CDR-H3 comprising an amino acid sequence selected from SEQ ID NO: 77; and (b) a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from (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 (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 80.

[0246] In another embodiment, the antigen binding site that binds to the FAP comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 75; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 76; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 77; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 78; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 79; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 80.

[0247] In any of the above embodiments, the multispecific antibody may be humanized. In one embodiment, the anti-FAP antigen-binding site comprises the CDRs of any of the above embodiments and further comprises a human acceptor framework, e.g., a human immunoglobulin framework or a human consensus framework.

[0248] In another embodiment, an antigen-binding site that binds to a FAP comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 81. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an antigen-binding site comprising this sequence retains the ability to bind to a FAP, preferably with the affinity specified above. In certain embodiments, a total of 1 to 10 amino acids within SEQ ID NO: 81 are substituted, inserted, and / or deleted. In certain embodiments, the substitutions, insertions, or deletions occur within a region outside the HVR (i.e., within the FR). Optionally, the antigen binding site that binds to the FAP comprises a VH sequence within SEQ ID NO: 81, including post-translational modifications of this sequence. In certain embodiments, the VH comprises one, two, or three CDRs selected from (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 75, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 76, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 77.

[0249] In another embodiment, an antigen-binding site that binds to a FAP comprises a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 82. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an antigen-binding site comprising this sequence retains the ability to bind to a FAP, preferably with the affinity specified above. In certain embodiments, a total of 1 to 10 amino acids within SEQ ID NO: 82 are substituted, inserted, and / or deleted. In certain embodiments, the substitutions, insertions, or deletions occur within a region outside the HVR (i.e., within the FR). Optionally, the antigen-binding site against the FAP comprises a VL sequence within SEQ ID NO: 82, including post-translational modifications of this sequence. In certain embodiments, the VL comprises one, two, or three CDRs selected from: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 78; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 79; and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 80.

[0250] In another embodiment, the antigen binding site that binds to the FAP comprises a VH in any of the above-presented embodiments and a VL in any of the above-presented embodiments. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 81 and SEQ ID NO: 82, respectively, including post-translational modifications of these sequences.

[0251] G. Antibody Formats As described above, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis. i) a first antibody that binds to an antigen expressed on the surface of a target cell, the first antibody further comprising a VH domain of an antigen-binding site for a radiolabeled compound, but not comprising a VL domain of an antigen-binding site for a radiolabeled compound; and ii) a second antibody that binds to an antigen expressed on the surface of a target cell, the second antibody further comprising a VL domain of an antigen-binding site for a radiolabeled compound, but not comprising a VH domain of an antigen-binding site for a radiolabeled compound. Includes; the VH domain of the first antibody and the VL domain of the second antibody are capable of together forming a functional antigen-binding site for a radiolabeled compound; Concerning a set of antibodies.

[0252] In some embodiments, the first antibody and the second antibody can each comprise an Fc domain. The presence of an Fc region has benefits in the context of radioimmunotherapy and radioimaging, for example, extending the circulating half-life of the protein and / or resulting in higher tumor uptake than observed with small fragments.

[0253] In some embodiments, if an Fc region is present, it may be preferable that the Fc region be engineered to reduce or eliminate effector function. This may include substitution of one or more of Fc region residues 234, 235, 238, 265, 269, 270, 297, 327, and / or 329, e.g., one or more of 234, 235, and / or 329. In some embodiments, the Fc region may be engineered to include a Pro329 to Gly substitution, a Leu234 to Ala substitution, and / or a Leu235 to Ala substitution (numbering according to the EU index).

[0254] In some embodiments, as discussed above, when the VH domain of the antigen-binding site for a radiolabeled compound is free at its C-terminus (e.g., not fused to another domain via its C-terminus), the VH domain of the antigen-binding site for a radiolabeled compound may be extended by one or more residues to avoid binding of HAVH autoantibodies. For example, the extension may be by 1 to 10 residues, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues. In one embodiment, the VH domain of the antigen-binding site for a radiolabeled compound may be extended by one or more alanine residues, optionally by one alanine residue. The VH sequence may also be extended by 1 to 10 residues from the N-terminal portion of the CH1 domain, e.g., from the N-terminus of the CH1 domain, e.g., from the CH1 domain of human IgG1 (the first 10 residues of the CH1 domain of human IgG1 are ASTKGPSVFP (SEQ ID NO: 149), so in one embodiment, 1 to 10 residues may be taken from the N-terminus of this sequence). For example, in one embodiment, the peptide sequence AST (corresponding to the first three residues of the CH1 domain of IgG1) is added to the C-terminus of the VH region. In some embodiments, the first antibody and / or the second antibody may each be multivalent, e.g., bivalent, with respect to the target antigen (e.g., a tumor-associated antigen). This has the advantage of increasing avidity.

[0255] In some embodiments, when the first antibody and the second antibody are associated, it may be preferable that the first antibody and the second antibody form a monovalent antibody complex for the radiolabeled compound. Thus, the first antibody can comprise only one VH domain of the antigen-binding site for the radiolabeled compound, and the second antibody can comprise only one VL domain of the antigen-binding site for the radiolabeled compound, so that the first antibody and the second antibody together form only one complete functional binding site for the radiolabeled compound.

[0256] Each antibody may comprise: i) at least one antibody fragment comprising an antigen-binding site specific for the target antigen; ii) a VL or VH domain comprising an antigen-binding site for the radiolabeled compound; and iii) optionally, an Fc region. The antibody fragment may be, for example, at least one Fv fragment, scFv fragment, Fab fragment, or cross-Fab fragment comprising an antigen-binding site specific for the target antigen. The antibody fragment may be fused to a) the VL or VH domain comprising the antigen-binding site for the radiolabeled compound, or b) if the antibody comprises an Fc region, the Fc region may be fused to the VL or VH domain comprising the antigen-binding site for the radiolabeled compound. In some embodiments, the C-terminus of the Fc region is fused to the N-terminus of the VL or VH domain.

[0257] Fusion can be direct or indirect. In some embodiments, fusion can be via a linker. For example, an Fc region can be fused to an antibody fragment via a hinge region or another suitable linker. Similarly, the VL or VH domain of the antigen-binding site for a radiolabeled compound can be connected to the remainder of the antibody structure via a linker. The linker can be a peptide of at least 5 amino acids, preferably 5 to 100, more preferably 10 to 50 or 25 to 50 amino acids. The linker can be a rigid or flexible linker. In some embodiments, the linker is a flexible linker comprising or consisting of Thr, Ser, Gly, and / or Ala residues. For example, the linker can comprise or consist of Gly and Ser residues. In some embodiments, the linker can have a repeat 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), e.g., x=4, and n=2 or 3, e.g., x=4, n=2. In some embodiments, the linker can be or include the sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 31). Other linkers may also be used and can be identified by one of skill in the art.

[0258] In one particular embodiment, the first antibody is a) an scFv fragment that binds to a target antigen; and b) i) an antibody heavy chain variable domain (VH); or ii) an antibody heavy chain variable domain (VH) and an antibody heavy chain constant domain, wherein the C-terminus of the VH domain is fused to the N-terminus of the constant domain; A polypeptide comprising or consisting of: a polypeptide fused by the N-terminus of the VH domain, preferably via a peptide linker, to the C-terminus of the scFv fragment It may comprise or consist of:

[0259] The second antibody is c) a second scFv fragment that binds to the target antigen; and d) i) an antibody light chain variable domain (VL); or ii) an antibody light chain variable domain (VL) and an antibody light chain constant domain, wherein the C-terminus of the VL domain is fused to the N-terminus of the constant domain; A polypeptide comprising or consisting of: a polypeptide fused by the N-terminus of the VL domain, preferably via a peptide linker, to the C-terminus of the scFv fragment It may comprise or consist of:

[0260] The antibody heavy chain variable domain (VH) of the first antibody and the antibody light chain variable domain (VL) of the second antibody, when the two antibodies associate, combine to form a functional antigen-binding site for a radiolabeled compound.

[0261] Optionally, the polypeptide of portion b(i) can additionally comprise one or more residues, optionally one or more alanine residues, optionally a single alanine residue, at the C-terminus of the VH domain. Optionally, the additional residues can be 1 to 10 residues from the N-terminal portion of the CH1 domain described above, e.g., the CH1 domain of human IgG1, e.g., from the N-terminus of the CH1 domain. For example, the additional residue can be AST.

[0262] The heavy and light chain variable domains of the scFv that recognize the target antigen can be connected by a peptide tether. Such a peptide tether can comprise 1 to 25 amino acids, preferably 12 to 20 amino acids, and preferably 12 to 16 or 15 to 20 amino acids. The tethers described above can comprise one or more (G3S) and / or (G4S) motifs, particularly one, two, three, four, five, or six (G3S) and / or (G4S) motifs, preferably three or four (G3S) and / or (G4S) motifs, and more preferably three or four (G4S) motifs.

[0263] Optionally, the first antibody can consist essentially of or consist of the above-listed components (a) and (b), and the second antibody can consist essentially of the above-listed components (c) and (d). In either case, the first antibody does not contain an antibody light chain variable domain (VL) capable of associating with component (b) of the first antibody to form a functional antigen-binding site for a radiolabeled compound; and the second antibody does not contain an antibody heavy chain variable domain (VH) capable of associating with component (d) of the second antibody to form a functional antigen-binding site for a radiolabeled compound.

[0264] In another specific embodiment, the first antibody is a) a Fab fragment that binds to a target antigen, and b) i) an antibody heavy chain variable domain (VH) of the antigen-binding site for a radiolabeled compound, or ii) an antibody heavy chain variable domain (VH) and an antibody heavy chain constant domain of an antigen binding site for a radiolabeled compound, wherein the C-terminus of the VH domain is fused to the N-terminus of the constant domain; A polypeptide comprising or consisting of: a polypeptide fused by the N-terminus of the VH domain, preferably via a peptide linker, to the C-terminus of the CL domain or CH1 domain of the Fab fragment It may comprise or consist of:

[0265] The second antibody is c) a Fab fragment that binds to a target antigen, and d) iii) an antibody light chain variable domain (VL) of the antigen-binding site for a radiolabeled compound; or iv) an antibody light chain variable domain (VL) and an antibody light chain constant domain of an antigen binding site for a radiolabeled compound, with the C-terminus of the VL domain fused to the N-terminus of the constant domain; A polypeptide comprising or consisting of: a polypeptide fused by the N-terminus of the VL domain, preferably via a peptide linker, to the C-terminus of the CL domain or CH1 domain of the Fab fragment It may comprise or consist of:

[0266] The antibody heavy chain variable domain (VH) of the polypeptide of (b) and the antibody light chain variable domain (VL) of the polypeptide of (d) together form a functional antigen-binding site for a radiolabeled compound (i.e., when the two antibodies associate).

[0267] Optionally, the polypeptide of portion b(i) can additionally comprise one or more residues, optionally one or more alanine residues, optionally a single alanine residue, at the C-terminus of the VH domain. Optionally, the additional residues can be 1 to 10 residues from the N-terminal portion of the CH1 domain described above, e.g., the CH1 domain of human IgG1, e.g., from the N-terminus of the CH1 domain. For example, the additional residue can be AST.

[0268] Optionally, the first antibody can consist essentially of or consist of the above-listed components (a) and (b), and the second antibody can consist essentially of the above-listed components (c) and (d). In either case, the first antibody does not contain an antibody light chain variable domain (VL) capable of associating with component (b) of the first antibody to form a functional antigen-binding site for a radiolabeled compound; and the second antibody does not contain an antibody heavy chain variable domain (VH) capable of associating with component (d) of the second antibody to form a functional antigen-binding site for a radiolabeled compound.

[0269] The chain of the Fab fragment fused to the polypeptide can be selected independently for the first antibody and the second antibody. Thus, in one embodiment, the polypeptide (b) is fused to the C-terminus of the CH1 domain of the Fab fragment of the first antibody, and the polypeptide (d) is fused to the C-terminus of the CH1 domain of the Fab fragment of the second antibody. In another embodiment, the polypeptide (b) is fused to the C-terminus of the CL domain of the Fab fragment of the first antibody, and the polypeptide (d) is fused to the C-terminus of the CL domain of the Fab fragment of the second antibody. In another embodiment, the polypeptide (b) is fused to the C-terminus of the CH1 domain of the Fab fragment of the first antibody, and the polypeptide (d) is fused to the C-terminus of the CL domain of the Fab fragment of the second antibody. In a further embodiment, the polypeptide (b) is fused to the C-terminus of the CL domain of the Fab fragment of the first antibody, and the polypeptide (d) is fused to the C-terminus of the CH1 domain of the Fab fragment of the second antibody.

[0270] As mentioned above, in some embodiments, the first antibody and / or the second antibody may each be multivalent, e.g., bivalent, with respect to the target antigen (e.g., tumor-associated antigen). This has the advantage of increasing avidity. The antibodies can be multivalent, e.g., bivalent, and each may be monospecific with respect to a particular epitope (which may be the same epitope for the first antibody and the second antibody, or different epitopes for the first antibody and the second antibody). Thus, in some embodiments, the first antibody may comprise: i) two or more antibody fragments comprising antigen-binding sites specific for the same epitope of the target antigen; ii) a VL domain or a VH domain (but not both) of an antigen-binding site for a radiolabeled compound; and iii) optionally an Fc region. The second antibody can comprise: i) two or more antibody fragments comprising antigen-binding sites specific for the same epitope of the target antigen; ii) a VL domain or a VH domain (but not both) of an antigen-binding site for a radiolabeled compound; and iii) optionally, an Fc region. As stated above, the epitopes can be the same for the first and second antibodies, or different for the first and second antibodies.

[0271] For example, the first and second antibodies can each comprise a tandem Fab, i.e., two Fab fragments connected via a peptide tether (Fab-tether-Fab), in which the first Fab is connected via its C-terminus to the N-terminus of the second Fab.

[0272] In one embodiment, the first antibody is a) a tandem Fab comprising two Fab fragments, wherein a first Fab fragment and a second Fab fragment bind to the same target antigen ("target antigen A"), the epitope bound by the first Fab fragment is the same as the epitope bound by the second Fab fragment, and the first Fab fragment and the second Fab fragment are connected via a peptide tether, wherein the first Fab is connected via its C-terminus to the N-terminus of the second Fab; and b) i) an antibody heavy chain variable domain (VH); or ii) an antibody heavy chain variable domain (VH) and an antibody constant domain (CH1) in which the C-terminus of the VH domain is fused to the N-terminus of the CH1 domain; A polypeptide comprising or consisting of: a polypeptide fused by the N-terminus of the VH domain, preferably via a peptide linker, to the C-terminus of the CL domain or CH1 domain of the second Fab fragment; and the second antibody comprises c) a tandem Fab comprising two Fab fragments, wherein a first Fab fragment and a second Fab fragment bind to target antigen A, the epitope to which the first Fab fragment binds is the same as the epitope to which the second Fab fragment binds, and the first Fab fragment and the second Fab fragment are connected via a peptide tether, and the first Fab is connected via its C-terminus to the N-terminus of the second Fab; and d) i) an antibody light chain variable domain (VL); or ii) an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL) in which the C-terminus of the VH domain is fused to the N-terminus of the constant domain; A polypeptide comprising or consisting of: a polypeptide fused by the N-terminus of the VL domain, preferably via a peptide linker, to the C-terminus of the CL domain or CH1 domain of the second Fab fragment; Includes.

[0273] The antibody heavy chain variable domain (VH) of portion b (in the first antibody) and the antibody light chain variable domain (VL) of portion (d) (in the second antibody) together, i.e., when the two antibodies associate, form a functional antigen-binding site for a radiolabeled compound.

[0274] Optionally, the polypeptide of portion b(i) can additionally comprise one or more residues, optionally one or more alanine residues, optionally a single alanine residue, at the C-terminus of the VH domain. Optionally, the additional residues can be 1 to 10 residues from the N-terminal portion of the CH1 domain described above, e.g., the CH1 domain of human IgG1, e.g., from the N-terminus of the CH1 domain. For example, the additional residue can be AST.

[0275] The chain of the Fab tandem fused to the polypeptide (i.e., whether the polypeptide is fused to the CL domain or the CH1 domain of the second Fab fragment) can be selected independently for the first antibody and the second antibody.

[0276] As described above, the first Fab fragment of the Fab tandem is connected to the N-terminus of the second Fab fragment. In one embodiment, the C-terminus of the heavy chain fragment of the first Fab fragment is connected to the N-terminus of the heavy chain or light chain fragment of the second Fab fragment. In another embodiment, the C-terminus of the light chain fragment of the first Fab fragment is connected to the N-terminus of the heavy chain or light chain fragment of the second Fab fragment. Thus, in some embodiments, the Fab tandem of the first antibody and / or the second antibody comprises three chains as follows: 1) a light chain fragment of a first Fab fragment ((VLCL)1), a heavy chain fragment of a first Fab fragment connected via a peptide tether to a heavy chain fragment of a second Fab fragment ((VHCH1)1-tether-(VHCH1)2), and a light chain fragment of a second Fab fragment ((VLCL)2); or 2) a light chain fragment of a first Fab fragment ((VLCL)1), a heavy chain fragment of a first Fab fragment connected via a peptide tether to a light chain fragment of a second Fab fragment ((VHCH1)1-tether-(VLCL)2), and a heavy chain fragment of a second Fab fragment ((VH-CH1)2); or 3) a heavy chain fragment of a first Fab fragment (VHCH1), a light chain fragment of a first Fab fragment connected via a peptide tether to a light chain fragment of a second Fab fragment ((VLCL)1-tether-(VLCL)2), and a heavy chain fragment of a second Fab fragment; or 4) a heavy chain fragment of a first Fab fragment (VHCH1), a light chain fragment of a first Fab fragment connected via a peptide tether to a heavy chain fragment of a second Fab fragment ((VLCL)1-tether-(VHCH1)2), and a light chain fragment of a second Fab fragment ((VLCL)2). may include:

[0277] In another embodiment, the first antibody and / or the second antibody can each bind to more than one different epitope, optionally two different epitopes, of the target antigen. Thus, one or both of the antibodies can be biparatopic with respect to the target antigen. In some embodiments, the first antibody and the second antibody each comprise: i) an antibody fragment comprising an antigen-binding site specific for a first epitope of the target antigen A; ii) an antibody fragment comprising an antigen-binding site for a second epitope of the target antigen A; iii) a VL domain or a VH domain (but not both) of an antigen-binding site for a radiolabeled compound; and iv) optionally an Fc region.

[0278] In such embodiments, proper assembly of light chains with their respective heavy chains can be aided by using cross-mab technology. For example, in one embodiment, each antibody can comprise a tandem Fab, comprising one Fab and one cross-Fab, where one fragment selected from the Fab and cross-Fab is specific for a first epitope and the other is specific for a second epitope.

[0279] In one particular example, the first antibody is a) a tandem Fab comprising a first fragment and a second fragment, wherein the first fragment is connected by its C-terminus to the N-terminus of the second fragment via a peptide tether, the first fragment binds to a first epitope of a target antigen A, and the second fragment binds to a second epitope of the target antigen A, and one of the fragments selected from the first fragment and the second fragment is a Fab and the other is a cross-Fab; b) i) an antibody heavy chain variable domain (VH); or ii) an antibody heavy chain variable domain (VH) and an antibody heavy chain constant domain (CH1), wherein the C-terminus of the VH domain is fused to the N-terminus of the CH1 domain; A polypeptide comprising or consisting of: a polypeptide fused by the N-terminus of the VH domain, preferably via a peptide linker, to the C-terminus of one of the chains of a second fragment may include:

[0280] The second antibody is c) a tandem Fab comprising a first fragment and a second fragment, wherein the first fragment is connected by its C-terminus to the N-terminus of the second fragment, the first fragment binds to a first epitope of target antigen A, and the second fragment binds to a second epitope of target antigen A, wherein one of the fragments selected from the first fragment and the second fragment is a Fab and the other is a cross-Fab; and d) i) an antibody light chain variable domain (VL); or ii) an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL) in which the C-terminus of the VL domain is fused to the N-terminus of the light chain constant domain; A polypeptide comprising or consisting of: a polypeptide fused by the N-terminus of the VL domain, preferably via a peptide linker, to the C-terminus of one of the chains of a second fragment may include:

[0281] The antibody heavy chain variable domain (VH) of the first antibody and the antibody light chain variable domain (VL) of the second antibody together form a functional antigen binding site for a radiolabeled compound.

[0282] Optionally, the polypeptide of portion b(i) can additionally comprise one or more residues, optionally one or more alanine residues, optionally a single alanine residue, at the C-terminus of the VH domain. Optionally, the additional residues can be 1 to 10 residues from the N-terminal portion of the CH1 domain described above, e.g., the CH1 domain of human IgG1, e.g., from the N-terminus of the CH1 domain. For example, the additional residue can be AST.

[0283] The first fragment or the second fragment can be a crossover Fab, so long as the tandem Fab comprises one conventional Fab and one crossover Fab.

[0284] In any of the above-described embodiments for tandem Fabs (including those involving cross-Fabs), optionally, the first antibody can consist essentially of or consist of components (a) and (b), and the second antibody can consist essentially of or consist of components (c) and (d). In either case, the first antibody does not comprise an antibody light chain variable domain (VL) capable of associating with component (b) of the first antibody to form a functional antigen-binding site for a radiolabeled compound; and the second antibody does not comprise an antibody heavy chain variable domain (VH) capable of associating with component (d) of the second antibody to form a functional antigen-binding site for a radiolabeled compound.

[0285] In any of the tandem Fab embodiments (including those involving crossed Fabs), the peptide tether connecting the Fab fragment of the first antibody to the Fab fragment of the second antibody can be a peptide with an amino acid sequence at least 5 amino acids in length, preferably 5-100, more preferably 10-50 amino acids in length. 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.

[0286] As mentioned above, in some embodiments, the first antibody and the second antibody may each comprise an Fc domain, optionally engineered to reduce or eliminate effector function.

[0287] In one embodiment, each of the first antibody and the second antibody can comprise: i) an Fc domain; ii) at least one antibody fragment, such as an scFv, Fv, Fab, or cross-Fab fragment, comprising an antigen-binding site specific for a target antigen; and iii) a VL domain or a VH domain (but not both) of an antigen-binding site for a radiolabeled compound.

[0288] Optionally, an antibody comprising an Fc domain may be monovalent with respect to binding to a target antigen. In other embodiments, an antibody comprising an Fc domain may be multivalent, e.g., bivalent. The first antibody and the second antibody may each be multivalent and monospecific for the same epitope of the target antigen. In yet other embodiments, the first antibody and the second antibody may each have binding sites for different epitopes of the target antigen (e.g., the first antibody and the second antibody may be biparatopic).

[0289] The antibody fragment may be an scFv. Thus, in one embodiment, the first antibody comprises: a) an scFv fragment that binds to a target antigen; b) an Fc domain; and c) i) an antibody heavy chain variable domain (VH); or ii) an antibody heavy chain variable domain (VH) and an antibody heavy chain constant domain (CH1), wherein the C-terminus of the VH domain is fused to the N-terminus of the constant domain; A polypeptide comprising or consisting of and The scFv in (a) is fused to the N-terminus of the Fc domain, and the polypeptide in c) is fused to the C-terminus of the Fc domain via the N-terminus of the VH domain, preferably via a peptide linker.

[0290] Optionally, the polypeptide of portion c(i) can additionally comprise one or more residues, optionally one or more alanine residues, optionally a single alanine residue, at the C-terminus of the VH domain. Optionally, the additional residues can be 1 to 10 residues from the N-terminal portion of the CH1 domain described above, e.g., the CH1 domain of human IgG1, e.g., from the N-terminus of the CH1 domain. For example, the additional residue can be AST.

[0291] The second antibody is d) a second scFv that binds to the target antigen; e) an Fc domain; and f) i) an antibody light chain variable domain (VL); or ii) an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL) in which the C-terminus of the VL domain is fused to the N-terminus of the constant domain; A polypeptide comprising or consisting of and The scFv in (d) is fused to the N-terminus of the Fc domain, and the polypeptide in (f) is fused to the C-terminus of the Fc domain via the N-terminus of the VH domain, preferably via a peptide linker.

[0292] In another embodiment, the first antibody and the second antibody can each be a single-arm IgG and Fc domain comprising a Fab against the target antigen (e.g., a single Fab against the target antigen). i) complete light chain fragment; ii) complete heavy chain; iii) an additional Fc chain lacking Fd; and iv) A polypeptide comprising or consisting of a VH domain of an antigen-binding site for a radiolabeled compound. and The light chain of (i) and the heavy chain of (ii) together provide an antigen-binding site for the target antigen; a polypeptide comprising or consisting of a VH domain of the antigen-binding site for the radiolabeled compound is fused by its N-terminus to the C-terminus of (ii) or (iii), preferably via a linker.

[0293] The second antibody is v) complete light chain fragment; vi) complete heavy chain; vii) an additional Fc chain lacking Fd; and viii) a polypeptide comprising or consisting of a VL domain of an antigen-binding site for a radiolabeled compound. and The light chain of (v) and the heavy chain of (vi) together provide an antigen-binding site for the target antigen; a polypeptide comprising or consisting of the VL domain of the antigen-binding site for the radiolabeled compound is fused by its N-terminus to the C-terminus of (vi) or (vii), preferably via a linker.

[0294] The polypeptide comprising or consisting of a VH domain of an antigen-binding site for a radiolabeled compound is i) an antibody heavy chain variable domain (VH), wherein the polypeptide may additionally comprise one or more residues at the C-terminus of the VH domain, optionally one or more alanine residues, optionally a single alanine residue, or optionally the N-terminal portion of a CH1 domain as described above; or ii) an antibody heavy chain variable domain (VH) and an antibody heavy chain constant domain (CH1), wherein the C-terminus of the VH domain is fused to the N-terminus of the CH1 domain; A polypeptide comprising or consisting of It is possible.

[0295] The polypeptide comprising or consisting of the VL domain of the antigen-binding site for the radiolabeled compound is i) an antibody heavy chain variable domain (VL); or ii) an antibody heavy chain variable domain (VL) and an antibody light chain constant domain, wherein the C-terminus of the VL domain is fused to the N-terminus of the constant domain; A polypeptide comprising or consisting of It is possible.

[0296] When the first and second antibodies are heterodimers, e.g., for single-arm IgG, assembly of the first and second antibodies can be assisted by the use of knob-into-hole technology, as described further below.

[0297] In another embodiment, the antibodies may each comprise a tandem Fab described above (e.g., comprising two Fab fragments, where both the first and second Fab fragments bind to the same epitope on target antigen A; or comprising an Fab and a cross-Fab, where one of the Fab fragments binds to a first epitope on target antigen A and the other binds to a second epitope on target antigen A), wherein the Fab tandems are fused (e.g., via their C-terminus) to the N-terminus of an Fc domain, and a peptide comprising or consisting of a VH domain or VL domain of an antigen-binding site for a radiolabeled compound is fused (e.g., via its N-terminus) to the C-terminus of the Fc domain.

[0298] Thus, the first antibody a) i) a tandem Fab comprising two Fab fragments, wherein a first Fab fragment and a second Fab fragment bind to target antigen A, the epitope to which the first Fab fragment binds is the same as the epitope to which the second Fab fragment binds, and the first Fab fragment and the second Fab fragment are connected via a peptide tether, and the first Fab is connected via its C-terminus to the N-terminus of the second Fab; and ii) a tandem Fab comprising a first fragment and a second fragment, wherein the first fragment is connected by its C-terminus to the N-terminus of the second fragment via a peptide tether, the first fragment binds to a first epitope of a target antigen A, and the second fragment binds to a second epitope of the target antigen A, and one of the fragments selected from the first fragment and the second fragment is a Fab and the other is a cross-Fab. A tandem Fab selected from: b) an Fc domain; and c) i) an antibody heavy chain variable domain (VH); or ii) an antibody heavy chain variable domain (VH) and an antibody heavy chain constant domain (CH1), wherein the C-terminus of the VH domain is fused to the N-terminus of the CH1 domain; A polypeptide comprising or consisting of and The tandem Fab is fused to the N-terminus of one of the chains of the Fc domain, and the polypeptide of c) is fused to the C-terminus of one of the chains of the Fc domain by the N-terminus of the VH domain, preferably via a peptide linker.

[0299] Optionally, the polypeptide of portion c(i) can additionally comprise one or more residues, optionally one or more alanine residues, optionally a single alanine residue, at the C-terminus of the VH domain. Optionally, the additional residues can be 1 to 10 residues from the N-terminal portion of the CH1 domain described above, e.g., the CH1 domain of human IgG1, e.g., from the N-terminus of the CH1 domain. For example, the additional residue can be AST.

[0300] The second antibody is d) i) a tandem Fab comprising two Fab fragments, wherein a first Fab fragment and a second Fab fragment bind to target antigen A, the epitope to which the first Fab fragment binds is the same as the epitope to which the second Fab fragment binds, and the first Fab fragment and the second Fab fragment are connected via a peptide tether, and the first Fab is connected via its C-terminus to the N-terminus of the second Fab; and ii) a tandem Fab comprising a first fragment and a second fragment, wherein the first fragment is connected by its C-terminus to the N-terminus of the second fragment via a peptide tether, the first fragment binds to a first epitope of a target antigen A, and the second fragment binds to a second epitope of the target antigen A, and one of the fragments selected from the first fragment and the second fragment is a Fab and the other is a cross-Fab. A tandem Fab selected from: e) an Fc domain; and f) i) an antibody heavy chain variable domain (VL); or ii) an antibody heavy chain variable domain (VL) and an antibody light chain constant domain, wherein the C-terminus of the VL domain is fused to the N-terminus of the light chain constant domain; A polypeptide comprising or consisting of and The tandem Fab of (d) is fused to the N-terminus of one of the chains of the Fc domain, and the polypeptide of (f) is fused to the C-terminus of one of the chains of the Fc domain by the N-terminus of the VL domain, preferably via a peptide linker.

[0301] The VH domain of the first antibody and the VL domain of the second antibody together, ie, when the two antibodies associate, form an antigen-binding site for the radiolabeled compound.

[0302] If the first antibody comprises a tandem Fab according to (a)(i), it will generally be the case that the second antibody comprises a tandem Fab according to d(i); if the first antibody comprises a tandem Fab according to (a)(ii), it will generally be the case that the second antibody comprises a tandem Fab according to d(ii).

[0303] The tandem Fab may generally be as described above. For example, the tethered linkage of the two fragments of the tandem Fab may be as described above. The tandem Fab may be composed of any of the chain sets defined above. Generally, the heavy chain fragment of the second Fab (which may be a cross-Fab) may be linked to an Fc domain.

[0304] In a further embodiment, each of the first and second antibodies can comprise: a) an Fc domain; b) at least one antibody fragment, such as an scFv, Fv, Fab, or cross-Fab fragment, containing an antigen-binding site for a target antigen; and c) a polypeptide containing a VL or VH domain (but not both) of an antigen-binding site for a radiolabeled compound, wherein the C-terminus of the antibody fragment (b) is fused to the N-terminus of one chain of the Fc domain, and the C-terminus of the polypeptide (c) is fused to the N-terminus of the other chain of the Fc domain. Fusion of the antibody fragment (b) is preferably via the hinge region. Fusion of the polypeptide (c) can also be via a linker located 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-terminal residue according to the EU numbering index). In one embodiment, the antibody fragment (b) can be a Fab fragment. In one embodiment, in the first antibody, the polypeptide (c) consists of a VH domain of an antigen-binding site for a radiolabeled compound; and in the second antibody, the polypeptide (c) consists of a VL domain of an antigen-binding site for a radiolabeled compound.

[0305] Thus, in one embodiment, the first antibody is i) intact light chain; ii) complete heavy chain; iii) an additional Fc chain; and iv) A polypeptide comprising or consisting of a VH domain of an antigen-binding site for a radiolabeled compound. and The light chain of (i) and the heavy chain of (ii) together provide an antigen-binding site for the target antigen; a polypeptide comprising or consisting of a VH domain of an antigen-binding site for a radiolabeled compound is fused by its C-terminus to the N-terminus of (iii), preferably via a linker.

[0306] The second antibody is v) intact light chain; vi) complete heavy chain; vii) an additional Fc chain; and viii) a polypeptide comprising or consisting of a VL domain of an antigen-binding site for a radiolabeled compound. and The light chain of (v) and the heavy chain of (vi) together provide an antigen-binding site for the target antigen; a polypeptide comprising or consisting of the VL domain of the antigen-binding site for the radiolabeled compound is fused by its C-terminus to the N-terminus of (vii), preferably via a linker.

[0307] The linker can comprise any flexible linker known to one of skill in the art, for example, the linker GGGGSGGGGSGGGGSGGSGG (SEQ ID NO: 152). The linker can further include all of the top of the hinge region, for example, extending from Asp221 to the beginning of the Fc chain (e.g., at Cys226).

[0308] In still further embodiments, the first antibody and / or the second antibody each comprise a full-length antibody having an antigen-binding site for a target antigen, and further comprise a VL domain or a VH domain of an antigen-binding site for a radiolabeled compound.

[0309] In one particular embodiment, the first antibody is a) a first full-length antibody consisting of two antibody heavy chains and two antibody light chains, wherein at least one arm of the full-length antibody binds to target antigen A; and b) i) an additional antibody heavy chain variable domain (VH); or ii) a further antibody heavy chain variable domain (VH) and a further antibody constant domain (CH1), wherein the C-terminus of the VH domain is fused to the N-terminus of the CH1 domain; A polypeptide comprising or consisting of: a polypeptide fused by the N-terminus of the VH domain to the C-terminus of one of the two heavy chains of said first full-length antibody, preferably via a peptide linker; may include:

[0310] The second antibody is c) a second full-length antibody consisting of two antibody heavy chains and two antibody light chains, wherein at least one arm of the full-length antibody binds to target antigen A; and d) i) an additional antibody light chain variable domain (VL); or ii) a further antibody light chain variable domain (VL), and a further antibody light chain constant domain (CL), wherein the C-terminus of the VL domain is fused to the N-terminus of the CL domain. A polypeptide comprising or consisting of: a polypeptide fused by the N-terminus of the VL domain to the C-terminus of one of the two heavy chains of said second full-length antibody, preferably via a peptide linker; may include:

[0311] The antibody heavy chain variable domain (VH) of the first antibody and the antibody light chain variable domain (VL) of the second antibody together, i.e., when the two antibodies associate, form a functional antigen-binding site for a radiolabeled compound.

[0312] Optionally, the polypeptide of portion b(i) can additionally comprise one or more residues, optionally one or more alanine residues, optionally a single alanine residue, at the C-terminus of the VH domain. Optionally, the additional residues can be 1 to 10 residues from the N-terminal portion of the CH1 domain described above, e.g., the CH1 domain of human IgG1, e.g., from the N-terminus of the CH1 domain. For example, the additional residue can be AST.

[0313] Optionally, the first antibody can consist essentially of or consist of the above-listed components (a) and (b), and the second antibody can consist essentially of or consist of the above-listed components (c) and (d). In either case, the first antibody does not contain an antibody light chain variable domain (VL) that can associate with component (b) of the first antibody to form a functional antigen-binding site for a radiolabeled compound; and the second antibody does not contain an antibody heavy chain variable domain (VH) that can associate with component (b) of the second antibody to form a functional antigen-binding site for a radiolabeled compound.

[0314] It may be preferable that both arms of a full-length antibody have binding specificity for target antigen A. If the antibody is bivalent for the target antigen, both arms of the full-length antibody may bind to the same epitope of target antigen A.

[0315] In another embodiment, the antibody may be biparatopic with respect to the target antigen; for example, one arm of a full-length antibody may bind to a first epitope on target antigen A, and one arm may bind to a second epitope on target antigen A. In such an embodiment, one arm of the antibody may comprise a Fab, and one arm may comprise a cross-Fab to assist in proper assembly of light chains with their respective heavy chains. Thus, in one embodiment, the first heavy chain of the full-length antibody may comprise a VL domain (e.g., VL-CH1-hinge-CH2-CH3) in place of a VH domain, and the first light chain may comprise a VH domain (e.g., VH-CL) swapped with a VL domain, or the first heavy chain may comprise a CL domain (e.g., VH-CL-hinge-CH2-CH3) in place of an HC1 domain, and the first light chain may comprise a CH1 domain (e.g., VL-CH1) in place of a CL domain. In this embodiment, the second heavy chain and the second light chain have conventional domain structures (e.g., VH-CH1-hinge-CH2-CH3 and VL-CL, respectively). In an alternative embodiment, the second heavy chain of the full-length antibody can comprise a VL domain (e.g., VL-CH1-hinge-CH2-CH3) instead of a VH domain, and the second light chain can comprise a VH domain (e.g., VH-CL) swapped with a VL domain, or the second heavy chain can comprise a CL domain (e.g., VH-CL-hinge-CH2-CH3) instead of an HC1 domain, and the second light chain can comprise a CH1 domain (e.g., VL-CH1) instead of a CL domain. In this embodiment, the first heavy chain and the first light chain have conventional domain structures.

[0316] Additionally or alternatively, in some embodiments, proper assembly of light chains with their respective heavy chains may be aided by the use of charge modifications, discussed further below.

[0317] The correct assembly of heterodimeric heavy chains can be assisted by knob-into-hole technology.

[0318] As used herein, the term "full-length antibody" refers to an antibody consisting of two "full-length antibody heavy chains" and two "full-length antibody light chains." A "full-length antibody heavy chain" may be a polypeptide composed of, from N- to C-terminus, an antibody heavy chain variable domain (VH), antibody constant heavy chain domain 1 (CH1), antibody hinge region (HR), antibody heavy chain constant domain 2 (CH2), and antibody heavy chain constant domain 3 (CH3), abbreviated as VH-CH1-HR-CH2-CH3; and, optionally, in the case of antibodies of subclass IgE, antibody heavy chain constant domain 4 (CH4). Preferably, a "full-length antibody heavy chain" is a polypeptide composed, from N- to C-terminus, of VH, CH1, HR, CH2, and CH3. Reference to "full length" is not intended to exclude the possibility of cross-Mab formation (thus, a heavy chain may have a VH domain swapped for a VL domain or a CH1 domain swapped for a CL domain). A "full-length antibody light chain" can be a polypeptide composed of, from N- to C-terminal, an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL), abbreviated as VL-CL. Alternatively, in the case of cross-mAbs, the VL domain may be swapped with a VH domain, and the CL domain may be swapped with a CH1 domain. The antibody light chain constant domain (CL) can be a κ (kappa) domain or a λ (lambda) domain. Two full-length antibody chains are linked together via interpolypeptide disulfide bonds between the CL domain and the CH1 domain and between the hinge region of the full-length antibody heavy chain. Typical examples of full-length antibodies are natural antibody-like IgG (e.g., IgG1 and IgG2), IgM, IgA, IgD, and IgE). Full-length antibodies according to the present invention can be derived from a single species, e.g., human, or can be chimeric or humanized. The full-length antibodies described herein, in some embodiments, comprise two antigen-binding sites, each formed by a pair of a VH and a VL, both of which may specifically bind to the same antigen or different antigens. The C-terminus of the heavy or light chain of the full-length antibody refers to the last amino acid at the C-terminus of the heavy or light chain.

[0319] b) The N-terminus of an antibody heavy chain variable domain (VH) of a polypeptide below, and d) the N-terminus of an antibody light chain variable domain (VL) of a polypeptide below, refers to the last amino acid at the N-terminus of the VH domain or VL domain.

[0320] Known techniques for making multispecific antibodies can also be used to make any of the heterodimers described herein, including, but not limited to, recombinant coexpression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature, 305:537 (1983)), and "knob-into-hole" engineering (see, e.g., U.S. Patent No. 5,731,168; and Atwell et al., J. Mol. Biol., 270:26 (1997)). Other methods include the manipulation of electrostatic steering effects to create antibody Fc heterodimeric molecules (see, e.g., WO2009 / 089004); cross-linking of two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980; and Brennan et al., Science, 229:81 (1985)); the use of leucine zippers (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992) and WO2011 / 034605); and the use of general light chain technology to circumvent the light chain mispairing problem (see, e.g., WO98 / 50431).

[0321] The CH3 domains of the full-length antibodies described above can be modified by the "knob-into-hole" technique, which is described in detail, with some examples, in, for example, WO 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 surfaces of the two CH3 domains are 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," while the other is a "hole." For example, one contains a so-called "knob mutation" (T366W and, optionally, one of S354C or Y349C) and the other contains a so-called "hole mutation" (T366S, L368A, and Y407V and, optionally, Y349C or S354C) according to the numbering according to the EU index (see, e.g., Carter, P. et al., Immunotechnol. 2 (1996) 73).

[0322] Additionally or alternatively, the introduction of disulfide bridges can be used 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.

[0323] Thus, in some embodiments, the first antibody and / or the second antibody 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 each meet at an interface comprising the interface between the CH3 domains of the original antibodies, wherein said interface has been modified to promote antibody formation, the modification comprising: a) where the CH3 domain of one heavy chain meets the CH3 domain of the other heavy chain in the antibody, amino acid residues are replaced with amino acid residues with larger side chain volumes, thereby creating a convex portion in the interface of the CH3 domain of one heavy chain, which is then altered to be positioned within a concave portion in the interface of the CH3 domain of the other heavy chain; b) In the original interface of the second CH3 domain where the CH3 domain of the other heavy chain meets the original interface of the first CH3 domain in the antibody, amino acid residues are replaced with amino acid residues with smaller side chain volumes, thereby creating a recess in the interface of the second CH3 domain, into which the protrusion in the interface of the first CH3 domain is located. It is characterized in that

[0324] The amino acid residue having a large side chain capacity may optionally be selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). The amino acid residue having a small side chain capacity may optionally be selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V).

[0325] Optionally, in some embodiments, both CH3 domains are further modified by the introduction of 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.

[0326] Multispecific (e.g., biparatopic) antibodies of the present invention can contain amino acid substitutions within the Fab molecules (including cross-Fab molecules) contained therein that are particularly effective in reducing mispairing of light chains with unmatched heavy chains (Bence-Jones by-products) that can occur in the generation of Fab-based bi / multispecific antigen-binding molecules involving VH / VL exchange in one of their binding arms (or more than one in the case of molecules comprising more than two antigen-binding Fab molecules) (see also PCT Publication No. 2015 / 150447, in particular the Examples therein, which is incorporated herein by reference in its entirety). The ratio of desired multispecific antibodies to undesired by-products, in particular Bence-Jones by-products occurring in one of their binding arms, can be improved by the introduction of charged amino acids with opposite charges (sometimes referred to herein as "charge modifications") at specific amino acid positions within the CH1 and CL domains of the Fab molecules.

[0327] Thus, in some embodiments, antibodies of the invention, including Fab molecules, comprise at least one Fab with a heavy chain constant domain, a CH1 domain, that contains the charge modifications described herein, and a light chain constant domain, a CL domain, that contains the charge modifications described herein.

[0328] Charge modifications may be made in the conventional Fab molecule(s) comprised in an antibody of the invention, or in the crossover Fab molecule(s) comprised in an antibody of the invention, but not in both. In certain embodiments, charge modifications are made in the conventional Fab molecule(s) comprised in an antibody of the invention.

[0329] In some embodiments, in a Fab or cross-Fab comprising a light chain constant domain, CL, that comprises a charge modification, and a heavy chain constant domain, CHI, that comprises a charge modification, the charge modification in the light chain constant domain, CL, is at position 124, optionally at position 123 (numbering according to Kabat), and the charge modification in the heavy chain constant domain, CHI, is at position 147 and / or 213 (numbering according to Kabat EU index). In some embodiments, the amino acid at position 124 of the light chain constant domain, CL, is independently substituted with lysine (K), arginine (R), or histidine (H) (numbering according to Kabat) (in a preferred embodiment, independently with lysine (K)), and the amino acid at position 147 and / or the amino acid at position 213 of the heavy chain constant domain, CHI, is independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

[0330] H. Exemplary Antibodies In some embodiments, aspects and embodiments relating to target binding (e.g., binding to CEA, binding to FAP, or binding to GPRC5D) may be combined with aspects and embodiments relating to binding to DOTA. That is, it may be preferred that a first antibody and a second antibody each comprise a binding site for CEA, FAP, or GPRC5D, e.g., comprising any 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 any of the sequences described above. It is also expressly contemplated that aspects and embodiments relating to binding to CEA, FAP, or GPRC5D and / or binding to DOTA may be combined with the preferred formats for antibodies described above (i.e., in any of the preferred formats, the moiety that binds to the target antigen may comprise the CDR or variable region sequences described above, and / or the moiety that binds to the radionuclide-labeled compound may be a DOTA binder having the CDR and / or variable region sequences described above).

[0331] In one particular embodiment, the first antibody is a) a first full-length antibody that specifically binds to CEA and consists of two antibody heavy chains and two antibody light chains; and b) A polypeptide comprising or consisting of an antibody heavy chain variable domain (VH) comprising the heavy chain CDRs of SEQ ID NOs: 35 to 37 (or wherein CDR-H1 has the sequence GFSLTDYGVH (SEQ ID NO: 148)) and / or having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 41, a polypeptide fused by the N-terminus of the VH domain to the C-terminus of one of the two heavy chains of said first full-length antibody, preferably via a peptide linker; may include:

[0332] The first antibody does not include a light chain domain that associates with the polypeptide of (b) to form a functional binding domain for a radiolabeled compound.

[0333] It may be preferred that the polypeptide of (b) further comprises one or more residues, e.g., 1 to 10 residues, at the C-terminus of the VH domain. Optionally, these may be one or more alanine residues, optionally a single alanine residue. In another embodiment, the further residues may be 1 to 10 residues derived from the N-terminal portion of the CH1 domain described above, e.g., the CH1 domain of human IgG1, e.g., from the N-terminus of the CH1 domain. For example, the further residue may be AST.

[0334] In some embodiments, the two antibody heavy chains in portion (a) have identical variable domains, optionally identical variable CH1 and / or variable CH2 domains. The two antibody heavy chains in portion (a) can optionally differ only in their CH3 domains, for example, by creating knob-into-hole mutations and other mutations intended to promote proper assembly of the heterodimer.

[0335] The second antibody is c) a second full-length antibody that specifically binds to CEA and consists of two antibody heavy chains and two antibody light chains; and d) A polypeptide comprising or consisting of an antibody light chain variable domain (VL) comprising the CDRs of SEQ ID NOs: 38-40 and / or having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 42. and The polypeptide is fused by the N-terminus of the VL domain to the C-terminus of one of the two heavy chains of the second full-length antibody, preferably via a peptide linker, and the second antibody does not contain a heavy chain domain that associates with the polypeptide of (d) to form a functional binding domain for a radiolabeled compound.

[0336] In some embodiments, the two antibody heavy chains in portion (c) have identical variable domains, optionally identical variable CH1 and / or variable CH2 domains, to each other. The two antibody heavy chains in portion (c) can optionally differ only in their CH3 domains, for example, by creating knob-into-hole mutations and other mutations intended to promote proper assembly of the heterodimer.

[0337] The CEA binding site / sequence can be any of the CEA binding sites / sequences described above.

[0338] In one particular embodiment, the first antibody may have a CEA binding sequence (ie, CDRs or VH / VL domains) derived from antibody CH1A1A.

[0339] For example, the two light chains in (a) can comprise the CDRs of SEQ ID NOs: 22-24 and / or can comprise a light chain variable domain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 26. In some embodiments, the two light chains in (a) can have at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 103. In some embodiments, it may be preferable that the two light chains in (a) are identical to each other.

[0340] The two antibody heavy chains in portion (a) can comprise the CDRs of SEQ ID NOs: 19-21, and / or the two antibody heavy chains in portion (a) comprise variable domains having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 25. In one embodiment, one heavy chain in portion (a) has the sequence of SEQ ID NO: 100, and the other heavy chain has the sequence of SEQ ID NO: 102.

[0341] In one specific embodiment, the first antibody may comprise a first heavy chain of SEQ ID NO: 100, and a second heavy chain of SEQ ID NO: 101 (in which case the C-terminal AST is optional and may be absent or replaced with another C-terminal extension described herein), and a light chain of SEQ ID NO: 103.

[0342] The second antibody can also have CEA binding sequences (ie, CDRs or VH / VL domains) derived from antibody CH1A1A.

[0343] For example, the two light chains in (c) can comprise the CDRs of SEQ ID NOs: 22-24 and / or can comprise a light chain variable domain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 26. In some embodiments, the two light chains in (c) can have at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 103. In some embodiments, it may be preferred that the two light chains in (c) are identical to each other. In some embodiments, it may be preferred that the two light chains in (c) have the same sequence as the light chains in (a) of the first antibody, e.g., all of the light chains in portions (a) and (c) have the same sequence.

[0344] In some embodiments, the two antibody heavy chains in portion (c) comprise the CDRs of SEQ ID NOs: 19-21 and / or the two antibody heavy chains in portion (c) comprise variable domains having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 25. In one embodiment, one heavy chain in portion (c) has the sequence of SEQ ID NO: 97 and the other heavy chain has the sequence of SEQ ID NO: 99.

[0345] In one specific embodiment, the second antibody may comprise a first heavy chain of SEQ ID NO:97, and a second heavy chain of SEQ ID NO:98, and a light chain of SEQ ID NO:103.

[0346] Similarly, in some embodiments, aspects and embodiments relating to target binding (e.g., binding to CEA, binding to FAP, or binding to GPRC5D) may be combined with aspects and embodiments relating to binding to Pb-DOTAM, i.e., it may be preferred that a first antibody and a second antibody each comprise a binding site for CEA, FAP, or GPRC5D, e.g., comprising any of the sequences described above, and that the first antibody and second antibody associate to form a binding site for a Pb-DOTAM chelate having any of the sequences described above. It is also expressly contemplated that aspects and embodiments relating to binding to CEA, FAP, or GPRC5D, and / or binding to Pb-DOTAM, may be combined with the preferred formats for antibodies described above (i.e., in any of the preferred formats, the moiety that binds to the target antigen may comprise the CDR or variable region sequences described above, and / or the moiety that binds to the radionuclide-labeled compound may be a Pb-DOTAM binder having the CDR and / or variable region sequences described above).

[0347] In one particular embodiment, the first antibody is a) a first full-length antibody that specifically binds to CEA and consists of two antibody heavy chains and two antibody light chains; and b) comprising or consisting of an antibody heavy chain variable domain (VH) comprising the heavy chain CDRs of SEQ ID NOs: 1-3 and / or having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 7; a polypeptide fused by the N-terminus of the VH domain to the C-terminus of one of the two heavy chains of said first full-length antibody, preferably via a peptide linker; may include:

[0348] The first antibody does not include a light chain domain that associates with the polypeptide of (b) to form a functional binding domain for a radiolabeled compound.

[0349] It may be preferred that the polypeptide of (b) further comprises one or more residues, optionally one or more alanine residues, optionally a single alanine residue, at the C-terminus of the VH domain. For example, the polypeptide of (b) may comprise SEQ ID NO: 7 with a C-terminal alanine extension, i.e., the sequence VTLKESGPVLVKPTETLTLTCTVSGFSLSTYSMSWIRQPPGKALEWLGFIGSRGDTYYASWAKGRLTISKDTSKSQVVLTMTNMDPVDTATYYCARERDPYGGGAYPPHLWGRGTLVTVSSA (SEQ ID NO: 150) It may comprise or consist of:

[0350] In another embodiment, the additional residues can be 1 to 10 residues from the N-terminal portion of the CH1 domain described above, e.g., the CH1 domain of human IgG1, e.g., from the N-terminus of the CH1 domain. For example, the additional residues can be AST.

[0351] In some embodiments, the two antibody heavy chains in portion (a) have identical variable domains, optionally identical variable CH1 and / or variable CH2 domains. The two antibody heavy chains in portion (a) can optionally differ only in their CH3 domains, for example, by creating knob-into-hole mutations and other mutations intended to promote proper assembly of the heterodimer.

[0352] The second antibody is c) a second full-length antibody that specifically binds to CEA and consists of two antibody heavy chains and two antibody light chains; and d) A polypeptide comprising or consisting of an antibody light chain variable domain (VL) comprising the CDRs of SEQ ID NOs: 4 to 6 and / or having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 8. and The polypeptide is fused by the N-terminus of the VL domain to the C-terminus of one of the two heavy chains of the second full-length antibody, preferably via a peptide linker, and the second antibody does not contain a heavy chain domain that associates with the polypeptide of (d) to form a functional binding domain for a radiolabeled compound.

[0353] In some embodiments, the two antibody heavy chains in portion (c) have identical variable domains, optionally identical variable CH1 and / or variable CH2 domains, to each other. The two antibody heavy chains in portion (c) can optionally differ only in their CH3 domains, for example, by creating knob-into-hole mutations and other mutations intended to promote proper assembly of the heterodimer.

[0354] In certain embodiments, the first antibody may have a CEA-binding sequence (ie, CDRs or VH / VL domains) derived from antibody CH1A1A.

[0355] For example, the two light chains in (a) can comprise the CDRs of SEQ ID NOs: 22-24 and / or can comprise a light chain variable domain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 26. In some embodiments, the two light chains in (a) can have at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 34. In some embodiments, it may be preferred that the two light chains in (a) are identical to each other.

[0356] The two antibody heavy chains in portion (a) can comprise the CDRs of SEQ ID NOs: 19-21, and / or the two antibody heavy chains in portion (a) comprise variable domains having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 25. In one embodiment, one heavy chain in portion (a) has the sequence of SEQ ID NO: 27, and the other heavy chain has the sequence of SEQ ID NO: 28.

[0357] In one specific embodiment, the first antibody may comprise a first heavy chain of SEQ ID NO: 28 and a second heavy chain of SEQ ID NO: 32 (or variants thereof comprising other C-terminal extensions described herein, such as an extension with an additional C-terminal alanine, or AST), and a light chain of SEQ ID NO: 34. Variants of SEQ ID NO: 32 with C-terminal alanine extensions are shown below: (SEQ ID NO: 153) Shown below.

[0358] In another specific embodiment, the first antibody may have CEA binding sequences (ie, CDRs or VH / VL domains) derived from antibody A5B7 (including humanized versions thereof).

[0359] For example, the two light chains in (a) can comprise the CDRs of SEQ ID NOs: 46-48 and / or can comprise a light chain variable domain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 50. In some embodiments, the two light chains in (a) can have at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 54. In some embodiments, it may be preferred that the two light chains in (a) are identical to each other.

[0360] In some embodiments, the two antibody heavy chains in portion (a) can comprise the CDRs of SEQ ID NOs: 43-45 and / or the two antibody heavy chains in portion (a) comprise variable domains having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 49. In one embodiment, one heavy chain in portion (a) has the sequence of SEQ ID NO: 51 and the other heavy chain has the sequence of SEQ ID NO: 53.

[0361] In one specific embodiment, the first antibody may comprise a first heavy chain of SEQ ID NO: 51, and a second heavy chain of SEQ ID NO: 52 (or a variant thereof with other C-terminal extensions described herein, such as a C-terminal alanine extension, or an extension with AST), and a light chain of SEQ ID NO: 54.

[0362] In another specific embodiment, the first antibody may have CEA binding sequences (ie, CDRs or VH / VL domains) derived from antibody T84.66 (including humanized versions thereof).

[0363] For example, the two light chains in (a) can comprise the CDRs of SEQ ID NOs: 14-16 and / or can comprise a light chain variable domain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 18. In some embodiments, the two light chains in (a) can have at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 89. In some embodiments, it may be preferred that the two light chains in (a) are identical to each other.

[0364] In some embodiments, the two antibody heavy chains in portion (a) can comprise the CDRs of SEQ ID NOs: 11-13, and / or the two antibody heavy chains in portion (a) comprise variable domains having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 17. In one embodiment, one heavy chain in portion (a) has the sequence of SEQ ID NO: 86, and the other heavy chain has the sequence of SEQ ID NO: 88.

[0365] In one specific embodiment, the first antibody may comprise a first heavy chain of SEQ ID NO: 86, and a second heavy chain of SEQ ID NO: 87 (or a variant thereof in which the C-terminal "AST" is absent or replaced with a different C-terminal extension as disclosed herein), and a light chain of SEQ ID NO: 89.

[0366] In another specific embodiment, the first antibody may have CEA binding sequences (ie, CDRs or VH / VL domains) derived from antibody 28A9 (including humanized versions thereof).

[0367] For example, the two light chains in (a) can comprise the CDRs of SEQ ID NOs: 62-64 and / or can comprise a light chain variable domain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 66. In some embodiments, the two light chains in (a) can have at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 96. In some embodiments, it may be preferred that the two light chains in (a) are identical to each other.

[0368] In some embodiments, the two antibody heavy chains in portion (a) can comprise the CDRs of SEQ ID NOs: 59-61, and / or the two antibody heavy chains in portion (a) comprise variable domains having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 65. In one embodiment, one heavy chain in portion (a) has the sequence of SEQ ID NO: 93, and the other heavy chain has the sequence of SEQ ID NO: 95.

[0369] In one specific embodiment, the first antibody may comprise a first heavy chain of SEQ ID NO: 93, and a second heavy chain of SEQ ID NO: 94 (or a variant thereof without the C-terminal "AST" or substituted with a different C-terminal extension as described herein), and a light chain of SEQ ID NO: 96.

[0370] In some embodiments, the second antibody may have a CEA-binding sequence (ie, CDRs or VH / VL domains) derived from antibody CH1A1A.

[0371] For example, the two light chains in (c) can comprise the CDRs of SEQ ID NOs: 22-24 and / or can comprise a light chain variable domain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 26. In some embodiments, the two light chains in (c) can have at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 34. In some embodiments, it may be preferred that the two light chains in (c) are identical to each other. In some embodiments, it may be preferred that the two light chains in (c) have the same sequence as the light chains in (a) of the first antibody, e.g., all of the light chains in portions (a) and (c) have the same sequence.

[0372] In some embodiments, the two antibody heavy chains in portion (c) comprise the CDRs of SEQ ID NOs: 19-21 and / or the two antibody heavy chains in portion (c) comprise variable domains having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 25. In one embodiment, one heavy chain in portion (c) has the sequence of SEQ ID NO: 29 and the other heavy chain has the sequence of SEQ ID NO: 30.

[0373] In one specific embodiment, the second antibody may comprise a first heavy chain of SEQ ID NO:30, and a second heavy chain of SEQ ID NO:33, and a light chain of SEQ ID NO:34.

[0374] In another specific embodiment, the second antibody may have CEA binding sequences (ie, CDRs or VH / VL domains) derived from A5B7 (including humanized versions thereof).

[0375] For example, the two light chains in (c) can comprise the CDRs of SEQ ID NOs: 46-48 and / or can comprise a light chain variable domain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 50. In some embodiments, the two light chains in (c) can have at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 58. In some embodiments, it may be preferred that the two light chains in (c) are identical to each other. In some embodiments, it may be preferred that the two light chains in (c) have the same sequence as the light chains in (a) of the first antibody, e.g., all of the light chains in portions (a) and (c) have the same sequence.

[0376] In some embodiments, the two antibody heavy chains in portion (c) comprise the CDRs of SEQ ID NOs: 43-45 and / or the two antibody heavy chains in portion (c) comprise variable domains having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 49. In one embodiment, one heavy chain in portion (c) has the sequence of SEQ ID NO: 55 and the other heavy chain has the sequence of SEQ ID NO: 57.

[0377] In one specific embodiment, the second antibody may comprise a first heavy chain of SEQ ID NO:55, and a second heavy chain of SEQ ID NO:56, and a light chain of SEQ ID NO:58.

[0378] In another specific embodiment, the second antibody may have CEA binding sequences (ie, CDRs or VH / VL domains) derived from antibody T84.66 (including humanized versions thereof).

[0379] For example, the two light chains in (c) can comprise the CDRs of SEQ ID NOs: 14-16 and / or can comprise a light chain variable domain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 18. In some embodiments, the two light chains in (c) can have at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 89. In some embodiments, it may be preferred that the two light chains in (c) are identical to each other.

[0380] In some embodiments, the two antibody heavy chains in portion (c) can comprise the CDRs of SEQ ID NOs: 11-13 and / or the two antibody heavy chains in portion (c) comprise variable domains having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 17. In one embodiment, one heavy chain in portion (c) has the sequence of SEQ ID NO: 83 and the other heavy chain has the sequence of SEQ ID NO: 85.

[0381] In one specific embodiment, the second antibody may comprise a first heavy chain of SEQ ID NO:83, and a second heavy chain of SEQ ID NO:84, and a light chain of SEQ ID NO:89.

[0382] In another specific embodiment, the second antibody may have CEA binding sequences (ie, CDRs or VH / VL domains) derived from antibody 28A9 (including humanized versions thereof).

[0383] For example, the two light chains in (c) can comprise the CDRs of SEQ ID NOs: 62-64 and / or can comprise a light chain variable domain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 66. In some embodiments, the two light chains in (c) can have at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 96. In some embodiments, it may be preferred that the two light chains in (c) are identical to each other.

[0384] In some embodiments, the two antibody heavy chains in portion (c) can comprise the CDRs of SEQ ID NOs: 59-61, and / or the two antibody heavy chains in portion (a) comprise variable domains having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 65. In one embodiment, one heavy chain in portion (c) has the sequence of SEQ ID NO: 90 and the other heavy chain has the sequence of SEQ ID NO: 92.

[0385] In one specific embodiment, the second antibody may comprise a first heavy chain of SEQ ID NO:90, and a second heavy chain of SEQ ID NO:91, and a light chain of SEQ ID NO:96.

[0386] In some embodiments, the first antibody and the second antibody bind to the same epitope of CEA.Therefore, for example, both the first antibody and the second antibody can have the CEA binding sequence derived from antibody CH1A1A; both the first antibody and the second antibody can have the CEA binding sequence derived from A5B7 (including its humanized version); both the first antibody and the second antibody can have the CEA binding sequence derived from T84.66 (including its humanized version); both the first antibody and the second antibody can have the CEA binding sequence derived from 28A9 (including its humanized version); both the first antibody and the second antibody can have the CEA binding sequence derived from MFE23 (including its humanized version).

[0387] So, for example, i) the first antibody may comprise a first heavy chain of SEQ ID NO: 28, a second heavy chain of SEQ ID NO: 32 (optionally with a C-terminal extension described herein, e.g., AST), and a light chain of SEQ ID NO: 34; the second antibody may comprise a first heavy chain of SEQ ID NO: 30, a second heavy chain of SEQ ID NO: 33, and a light chain of SEQ ID NO: 34; ii) the first antibody may comprise a first heavy chain of SEQ ID NO: 51, a second heavy chain of SEQ ID NO: 52 (optionally with a C-terminal extension described herein, e.g., AST), and a light chain of SEQ ID NO: 54; the second antibody may comprise a first heavy chain of SEQ ID NO: 55, a second heavy chain of SEQ ID NO: 56, and a light chain of SEQ ID NO: 58; iii) the first antibody may comprise a first heavy chain of SEQ ID NO: 86, a second heavy chain of SEQ ID NO: 87 (in which case the C-terminal AST residue is optional and may be absent or replaced by an alternative C-terminal extension), and a light chain of SEQ ID NO: 89; the second antibody may comprise a first heavy chain of SEQ ID NO: 83, a second heavy chain of SEQ ID NO: 84, and a light chain of SEQ ID NO: 89; iv) The first antibody may comprise a first heavy chain of SEQ ID NO: 93, a second heavy chain of SEQ ID NO: 94 (in which case the C-terminal AST residue is optional and may be absent or replaced by an alternative C-terminal extension), and a light chain of SEQ ID NO: 96; the second antibody may comprise a first heavy chain of SEQ ID NO: 90, a second heavy chain of SEQ ID NO: 91, and a light chain of SEQ ID NO: 96.

[0388] In other embodiments, the first antibody and the second antibody bind to different epitopes of CEA, as discussed above.Therefore, for example, the first antibody can have the CEA binding sequence derived from antibody CH1A1A, and the second antibody can have the CEA binding sequence derived from A5B7; the first antibody can have the CEA binding sequence derived from antibody A5B7, and the second antibody can have the CEA binding sequence derived from CH1A1A.The example of the use of a double paratope (CH1A1A and A5B7) pair is described in Example 6c.

[0389] In still further specific embodiments, the target antigen can be GPRC5D or FAP, and the format can be as shown in Figure 25B. Optionally, the first antibody and the second antibody associate to form a functional antigen-binding site chelate to Pb-DOTAM (Pb-DOTAM).

[0390] Thus, in one embodiment (where the target antigen is GPRC5D): i) the first antibody comprises a first heavy chain of SEQ ID NO: 104, a second heavy chain of SEQ ID NO: 106 (in which the C-terminal alanine is optional and may be absent or replaced by an alternative C-terminal extension described herein), and a light chain of SEQ ID NO: 107; ii) The second antibody comprises a first heavy chain of SEQ ID NO: 104, a second heavy chain of SEQ ID NO: 105, and a light chain of SEQ ID NO: 107.

[0391] In another embodiment (wherein the target antigen is a FAP), i) the first antibody comprises a first heavy chain of SEQ ID NO: 108, a second heavy chain of SEQ ID NO: 110 (in which the C-terminal alanine is optional and may be absent or replaced by an alternative C-terminal extension described herein), and a light chain of SEQ ID NO: 111; ii) The second antibody comprises a first heavy chain of SEQ ID NO: 108, a second heavy chain of SEQ ID NO: 109, and a light chain of SEQ ID NO: 111.

[0392] In yet a further specific embodiment, the target can be CEA, for example, the antibody has a CEA binding sequence derived from antibody CH1A1A, and the format can be as shown in Figure 25C. Optionally, the first antibody and the second antibody associate to form a functional antigen binding site chelate to Pb-DOTAM (Pb-DOTAM). Thus, in one specific embodiment, i) the first antibody comprises a first heavy chain of SEQ ID NO: 112, a second heavy chain of SEQ ID NO: 114, and a light chain of SEQ ID NO: 115; ii) The second antibody comprises a first heavy chain of SEQ ID NO:112, a second heavy chain of SEQ ID NO:113, and a light chain of SEQ ID NO:115.

[0393] I. Antibody Variants In certain embodiments, amino acid sequence variants of the antibodies presented herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or may be prepared by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of, residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions may be made to arrive at the final construct, provided that the final construct possesses the desired properties, e.g., antigen binding.

[0394] Substitution, insertion, and deletion mutants In certain embodiments, antibody variants with one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include HVRs (CDRs) and FRs. Conservative substitutions are shown in Table 1 under the heading of "Preferred Substitutions." More substantial changes are presented in Table 1 under the heading of "Exemplary Substitutions," as further described below with reference to amino acid side chain classes. Amino acid substitutions may be introduced into an antibody of interest, and the products may be screened for a desired activity, e.g., retained / improved binding to antigen, reduced immunogenicity, or reduced or eliminated ADCC or CDC. TIFF2026000913000009.tif189170

[0395] Amino acids have common side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe They can be grouped according to:

[0396] Non-conservative substitutions would involve exchanging a member of one of these classes for a member of another class.

[0397] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have altered (e.g., improved) certain biological properties (e.g., increased affinity, reduced immunogenicity) compared to the parent antibody and / or will also substantially retain certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity-matured antibody, which can be conveniently generated using, for example, phage-display-based affinity maturation methods, such as those described herein. Briefly, one or more CDR residues are mutated, and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0398] Alterations (e.g., substitutions) can be made within the CDRs to, for example, improve antibody affinity. Such alterations can be made in CDR "hot spots," i.e., residues encoded by codons that frequently undergo mutation during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol., 207:179-196 (2008)), and / or antigen-contacting residues, and the resulting VH or VL variants are tested for binding affinity. Affinity maturation by construction and reselection from secondary libraries is described, for example, in Hoogenboom et al., Methods in Molecular Biology, 178:1-37 (O'Brien et al., eds., Humana Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves CDR assignment, 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 often particularly targeted.

[0399] In certain embodiments, substitutions, insertions, or deletions can be made within one or more CDRs, as long as such changes do not substantially reduce the antibody's ability to bind to the antigen. For example, conservative changes (e.g., conservative substitutions provided herein) that do not substantially reduce binding affinity can be made within a CDR. Such changes can be, for example, outside of antigen-contacting residues within the CDR. In certain variant VH and VL sequences provided above, each CDR contains either no change or no more than one, two, or three amino acid substitutions.

[0400] 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. In this method, a residue or target group of residues (e.g., charged residues such as arg, asp, his, lys, and glu) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the antibody's interaction with the antigen is affected. Further substitutions can be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitution. Alternatively, or in addition, a crystal structure of an antigen-antibody complex can be used to identify contact points between the antibody and antigen. Such contact residues and nearby residues can be targeted as candidate residues for substitution or eliminated. Mutants can be screened to determine whether they contain the desired properties.

[0401] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion includes an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules include the N- or C-terminal fusion of an antibody to an enzyme (e.g., for ADEPT (antibody directed enzyme prodrug therapy)) or a polypeptide which increases the serum half-life of the antibody.

[0402] Glycosylation variants In certain embodiments, the antibodies presented herein are altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0403] If the antibody comprises an Fc region, the oligosaccharides attached to the antibody can be altered. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides, generally N-linked to Asn297 in the CH2 domain of the Fc region. See, e.g., Wright et al., TIBTECH, 15:26-32 (1997). Oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the antibodies of the invention can be made to create antibody variants with improved specific properties.

[0404] In one embodiment, antibody variants are provided that have nonfucosylated oligosaccharides, i.e., oligosaccharide structures lacking fucose attached (directly or indirectly) to the Fc region. Such nonfucosylated oligosaccharides (also referred to as "afucosylated" oligosaccharides) are N-linked oligosaccharides that lack a fucose residue attached to the first GlcNAc, particularly in the stem of a biantennary oligosaccharide structure. In one embodiment, antibody variants are provided that have an increased proportion of nonfucosylated oligosaccharides in the Fc region compared to the native or parent antibody. For example, the proportion of nonfucosylated oligosaccharides can be at least about 20%, at least about 40%, at least about 60%, at least about 80%, or even about 100% (i.e., no fucosylated oligosaccharides are present). The percentage of nonfucosylated oligosaccharides is the (average) amount of oligosaccharides lacking a fucose residue compared to the sum of all oligosaccharides (e.g., complex mannose structures, hybrid mannose structures, and high mannose structures) conjugated to Asn297, as measured by MALDI-TOF mass spectrometry, for example, as described in WO2006 / 082515. Asn297 refers to an asparagine residue located at approximately position 297 (EU numbering for residues in the Fc region) within the Fc region; however, due to minor sequence variations within antibodies, Asn297 may also be located approximately ±3 amino acids upstream or downstream from position 297, i.e., between positions 294 and 300. Such antibodies with an increased proportion of nonfucosylated oligosaccharides within the Fc region may exhibit improved binding to the FcγRIIIa receptor and / or improved effector function, particularly ADCC function. See, for example, US2003 / 0157108; US2004 / 0093621.

[0405] Examples of cell lines capable of producing antibodies with reduced fucosylation include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al., Arch. Biochem. Biophys., 249:533-545 (1986); US 2003 / 0157108; and WO 2004 / 056312, inter alia, in Example 11), and CHO cells in which the alpha-1,6-fucosyltransferase gene, FUT8, has been knocked out (e.g., Yamane-Ohnuki et al., Biotech. Bioeng., 87:614-622 (2004); Kand and WO2003 / 085107), or cells in which the activity of a GDP-fucose synthesis protein or a GDP-fucose transporter protein is reduced or eliminated (see, e.g., US2004259150, US2005031613, US2004132140, US2004110282).

[0406] In a further aspect, there is provided an antibody variant having bisected oligosaccharides, e.g., biantennary oligosaccharides attached to the Fc region of the antibody are bisected 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); WO99 / 54342, WO2004 / 065540, and WO2003 / 011878.

[0407] Also provided are antibody variants in which at least one galactose residue in the oligosaccharide is attached to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO1997 / 30087; WO1998 / 58964; and WO1999 / 22764.

[0408] It may be preferable to modify the antibody to reduce the degree of glycosylation. In some embodiments, the antibody may be non-glycosylated or deglycosylated. The antibody may include a substitution at N297, for example, N297D / A.

[0409] Fc region variants In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody presented herein, thereby creating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, human IgG2, human IgG3, or human IgG4 Fc region) that includes an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0410] In certain embodiments, the present invention contemplates antibody variants with reduced effector function, e.g., antibody variants with reduced or abolished CDC, ADCC, and / or binding to FcγR. In certain aspects, the present invention contemplates antibody variants that retain some, but not all, effector functions, making them desirable candidates for applications where in vivo antibody half-life is important but certain effector functions, such as complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC), are unnecessary or deleterious.

[0411] In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that an antibody lacks binding to FcγR (and thus likely lacks ADCC activity) but retains the ability to bind to FcRn. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol., 9:457-492 (1991). Non-limiting examples of in vitro assays to evaluate ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA, 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA, 82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med., 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be employed (see, e.g., the ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (Cell Technology, Inc., Mountain View, CA); and CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI). Useful effector cells for assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that 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 is unable to bind C1q and thus lacks CDC activity.See, for example, the C1q binding ELISA and C3c binding ELISA in WO2006 / 029879 and WO2005 / 100402. To assess complement activation, CDC assays 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)). Determination of binding to FcRn and in vivo clearance / half-life can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol., 18(12):1759-1769 (2006); WO2013 / 120929 A1).

[0412] Antibodies with reduced effector function include antibodies with substitutions at one or more of residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region (U.S. Patent No. 6,737,056), e.g., P329G. Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0413] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that attenuate binding to FcγR, e.g., substitutions at positions 234 and 235 of the Fc region (EU numbering of residues). In one embodiment, the substitutions are L234A and L235A (LALA). In certain embodiments, the antibody variant further comprises D265A and / or P329G in the Fc region, which are derived from the Fc region of human IgG1. In one embodiment, the substitutions are L234A, L235A, and P329G in the Fc region, which are derived from the Fc region of human IgG1 (LALA-PG) (see, e.g., WO2012 / 130831). In another embodiment, the substitutions are L234A, L235A, and D265A in the Fc region, which are derived from the Fc region of human IgG1 (LALA-DA).

[0414] In other embodiments, it may be possible to use IgG subtypes with reduced effector function, such as IgG4 or IgG2.

[0415] Certain antibody variants with improved or diminished binding to FcRs have been described (see, e.g., U.S. Pat. No. 6,737,056; WO 2004 / 056312; and Shields et al., J. Biol. Chem., 9(2):6591-6604 (2001)).

[0416] In some embodiments, alterations are made in the Fc region that result in altered (i.e., improved or attenuated, preferably attenuated) binding to C1q and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551; WO 99 / 51642; and Idusogie et al., J. Immunol., 164:4178-4184 (2000).

[0417] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that reduce binding to FcRn, e.g., substitutions at positions 253 and / or 310 and / or 435 (EU numbering of residues) of the Fc region. In certain embodiments, the antibody variant comprises an Fc region with amino acid substitutions at positions 253, 310, and 435. In one embodiment, the substitutions are I253A, H310A, and H435A in the Fc region, which are derived from the Fc region of human IgG1. See, e.g., Grevys, A. et al., J. Immunol., 194 (2015) 5497-5508.

[0418] In certain aspects, the antibody mutant comprises an Fc region with one or more amino acid substitutions that reduce binding to FcRn, for example, substitutions at positions 310 and / or 433 and / or 436 (EU numbering of residues) of the Fc region. In certain aspects, the antibody mutant comprises an Fc region with amino acid substitutions at positions 310, 433, and 436. In one aspect, the substitutions are H310A, H433A, and Y436A in the Fc region, which are derived from the Fc region of human IgG1. (See, e.g., WO2014 / 177460 A1). For example, in some embodiments, normal binding to FcRn may be used.

[0419] See also Duncan and Winter, Nature, 322:738-40 (1988); US Pat. No. 5,648,260; US Pat. No. 5,624,821; and WO 94 / 29351 for other examples of Fc region variants.

[0420] The heavy chain C-terminus of a full-length antibody reported herein can be a complete C-terminus, terminating at amino acid residue PGK. The C-terminus of a heavy chain can be a truncated C-terminus, in which one or two of the C-terminal amino acid residues are removed. The C-terminus of a heavy chain can be a truncated C-terminus, terminating at PG. In one aspect of all embodiments reported herein, an antibody comprising a heavy chain comprising a C-terminal CH3 domain as specified herein comprises a C-terminal glycine residue (G446; amino acid positions numbered according to the EU index). This is also explicitly encompassed by the term "full-length antibody" or "full-length heavy chain" as used herein.

[0421] antibody derivative In certain embodiments, the antibodies provided herein can be further modified to contain additional nonproteinaceous moieties known in the art and readily available. Moieties suitable for derivatizing antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone), polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer can be of any molecular weight and can be branched or unbranched. The number of polymers conjugated to an antibody can vary, and when more than one polymer is conjugated, they can be the same molecule or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used therapeutically under defined conditions, etc.

[0422] J. Recombinant Methods and Compositions Antibodies can be made using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid or set of isolated nucleic acids is provided that encodes the set of antibodies described herein.

[0423] For example, the set of nucleic acids may include the following nucleic acid encoding a first antibody: i) a nucleic acid encoding a first heavy chain of a first antibody, said first heavy chain comprising the heavy chain of a full-length antibody that specifically binds to a target antigen, said first heavy chain being fused via its C-terminus to a polypeptide comprising a VH domain of an antigen-binding site for a radiolabeled compound; ii) a nucleic acid encoding a second heavy chain of the first antibody, said second heavy chain comprising a heavy chain of a full-length antibody that specifically binds to a target antigen and not comprising a VL domain of an antigen-binding site for a radiolabeled compound (optionally, the second heavy chain consists of a heavy chain of a full-length antibody that specifically binds to a target antigen); iii) a nucleic acid encoding the light chain of the first antibody may include:

[0424] Additionally or alternatively, the set of nucleic acids according to the invention may comprise the following nucleic acid encoding the second antibody: iv) a nucleic acid encoding a first heavy chain of a second antibody, said first heavy chain comprising the heavy chain of a full-length antibody that specifically binds to a target antigen, said first heavy chain being fused via its C-terminus to a polypeptide comprising a VL domain of an antigen-binding site for a radiolabeled compound; v) a nucleic acid encoding a second heavy chain of a second antibody, said second heavy chain comprising a heavy chain of a full-length antibody that specifically binds to a target antigen and not comprising a VH domain of an antigen-binding site for a radiolabeled compound (optionally, the second heavy chain consists of a heavy chain of a full-length antibody that specifically binds to a target antigen); vi) nucleic acid encoding the light chain of the second antibody may include:

[0425] In some embodiments, certain of these nucleic acids may be identical to each other, for example, the nucleic acid in (iii) may be identical to the nucleic acid in (vi) such that the entire set contains only five significantly different nucleic acid sequences.

[0426] The nucleic acids can be incorporated into one or more nucleic acid molecules or expression vectors.

[0427] Thus, in a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acid(s) are provided. In one embodiment, each of the respective heavy and light chains is expressed from an individual plasmid.

[0428] In further embodiments, a host cell or set of host cells comprising such nucleic acid(s) or vector(s) is provided. In one embodiment, a first host cell is provided that expresses a first antibody, and a second host cell is provided that expresses a second antibody.

[0429] In one such embodiment, the first host cell comprises (e.g., has been transformed with) (1) a vector comprising the above nucleic acids (i) to (iii), or (2) a first vector comprising nucleic acid (i), a second vector comprising nucleic acid (ii), and a third vector comprising nucleic acid (iii), or (3) two vectors comprising the above nucleic acids (i) to (iii) together. The second host cell comprises (e.g., has been transformed with) (1) a vector comprising the above nucleic acids (iv) to (vi), or (2) a first vector comprising nucleic acid (iv), a second vector comprising nucleic acid (v), and a third vector comprising nucleic acid (vi), or (3) two vectors comprising the above nucleic acids (iv) to (vi) together.

[0430] In one embodiment, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphocyte (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of making an antibody in accordance with the invention is provided, comprising culturing a host cell containing nucleic acid encoding an antibody as set forth above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell medium).

[0431] For recombinant antibody production, for example, nucleic acids encoding the antibodies described above 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 (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody).

[0432] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., US 5,648,237, US 5,789,199, and US 5,840,523 (see also Charlton, KA, Methods in Molecular Biology, Vol. 248, Lo, BKC (eds.), Humana Press, Totowa, NJ (2003), pp. 245-254, which describes the expression of antibody fragments in E. coli). After expression, the antibody can be isolated from the bacterial cell paste in a soluble fraction and further purified.

[0433] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast, including fungal and yeast strains whose glycosylation pathways have been "humanized," resulting in the production of antibodies with partially or fully human glycosylation patterns, are also suitable cloning or expression hosts for antibody-encoding vectors. See Gerngross, TU, Nat. Biotech., 22 (2004), 1409-1414; and Li, H. et al., Nat. Biotech., 24 (2006), 210-215.

[0434] Suitable host cells for the expression of (glycosylated) antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. In particular, numerous baculovirus strains have been identified that can be used with insect cells for the transfection of Spodoptera frugiperda cells.

[0435] Plant cell cultures may also be utilized as hosts. See, e.g., US 5,959,177, US 6,040,498, US 6,420,548, US 7,125,978, and US 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0436] Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include SV40 (COS-7) transformed monkey kidney CV1 cell lines; human embryonic kidney cell lines (e.g., 293 or 293T cells, described in Graham, FL et al., J. Gen Virol., 36 (1977), 59-74); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells, described in Mather, JP, Biol. Reprod., 23 (1980), 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells (e.g., Mather, JP et al., Annals NY Acad. Sci., 383 (1982), 44-68); MRC5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA, 77 (1980), 4216-4220); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki, P. and Wu, AM, "Methods in Molecular Biology," Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0437] In one embodiment, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphocyte (e.g., a Y0, NS0, Sp20 cell).

[0438] K. Assay The antibodies presented herein may be identified, screened for, or characterized for their physical / chemical properties and / or biological activity by a variety of assays known in the art.

[0439] In one embodiment, the antibodies of the invention are examined for their antigen-binding activity by known methods, such as ELISA or Western blot.

[0440] Antibody affinity In certain embodiments, the antibodies provided herein have an activity against a target antigen of ≦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, e.g., 10 -8 M~10 -13 M, e.g., 10 -9 M~10 -13 M), or a dissociation constant (KD) as stated elsewhere herein.

[0441] In certain embodiments, the antigen binding site for the radiolabeled compound has an affinity of ≦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, e.g., 10 -8 M~10 -13 M, e.g., 10 -9 M~10 -13 In some embodiments, the Kd 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 stated elsewhere herein. For example, a functional binding site may bind a radiolabeled compound / metal chelate with a Kd of about 1 pM to 1 nM, e.g., about 1-10 pM, 1-100 pM, 5-50 pM, 100-500 pM, or 500 pM to 1 nM.

[0442] In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA). In one embodiment, the RIA is performed with a Fab variant of the antibody of interest and its antigen. For example, the binding affinity of a Fab to an antigen in solution is determined by binding the Fab to a minimum concentration of ( 125 The activity of the antibody is measured by equilibrating the plate with a 1-I labeled (I-labeled) antigen and then capturing the bound antigen with an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol., 293:865-881 (1999)). To establish the conditions for the assay, MICROTITER® multiwell plates (Thermo Scientific) are coated overnight with 5 μg / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), followed by blocking with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23°C). For non-adsorbent plates (Nunc; product number 269620), 100 pM or 26 pM [ 125 [I]-antigen is mixed with serial dilutions of the Fab of interest (e.g., based on the evaluation of Fab-12, an anti-VEGF antibody, in Presta et al., Cancer Res., 57:4593-4599 (1997)). The Fab of interest is then incubated overnight, although incubation can be continued for longer periods (e.g., approximately 65 hours) to ensure equilibrium is reached. The mixture is then transferred to a capture plate for incubation at room temperature (e.g., for 1 hour). The solution is then removed, and the plate is washed eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate has dried, 150 μl of scintillant (MICROSCINT-20™; Packard) is added per well, and the plate is counted for 10 minutes on a TOPCOUNT™ gamma counter (Packard). Concentrations of each Fab that exhibit 20% or less of maximal binding are selected for use in competitive binding assays.

[0443] According to another embodiment, Kd is measured using a BIACORE® surface plasmon resonance assay. For example, assays using a BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) are performed at 25°C with an immobilized antigen CM5 chip of approximately 10 response units (RU). In one embodiment, a carboxymethylated dextran biosensor chip (CM5; BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / ml (approximately 0.2 μM) in 10 mM sodium acetate, pH 4.8, and then injected at a flow rate of 5 μl / min to achieve protein coupling of approximately 10 response units (RU). After antigen injection, 1 M ethanolamine is injected to block unreactive groups. To measure the reaction rate, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20 (Tween-20™) surfactant (PBST) at 25°C and a flow rate of approximately 25 μl / min. on ) and dissociation rate (k off The equilibrium dissociation constant (Kd) is calculated by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2). off / k on See, for example, Chen et al., J. Mol. Biol., 293:865-881 (1999). If the association rate by the surface plasmon resonance assay described above is 10 6 M -1 seconds -1above, the association rate can be determined by using a fluorescence quenching technique to measure the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, with a 16 nm bandpass) at 25°C of 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen, as measured by a spectrometer such as a spectrophotometer equipped with a stopped flow (Aviv Instruments) or an 8000 series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with a stirred cuvette.

[0444] In another embodiment, Kd is measured using a solution equilibration titration (SET) assay. According to 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 by a labeled / tagged anti-species antibody, typically using electrochemiluminescence (e.g., as described in Haenel et al., Analytical Biochemistry, 339 (2005), 182-184).

[0445] For example, in one embodiment, a 384-well streptavidin plate (Nunc, Microcoat; model number: 11974998001) is incubated overnight at 4°C with 25 μl per well of antigen-biotin-isomer mix at a concentration of 20 ng / ml in PBS buffer. To equilibrate the antibody sample with free antigen, 0.01 nM to 1 nM antibody is titrated with the antigen of interest in serial dilutions of 1:3, 1:2, or 1:1.7, starting at concentrations of 2500 nM, 500 nM, or 100 nM. The samples are incubated overnight at 4°C in a sealed REMP Storage polypropylene microplate (Brooks). After overnight incubation, the streptavidin plate is washed three times with 90 μl per well of PBST. 15 μl of each sample was transferred from the equilibrated plate to the assay plate and incubated for 15 minutes at room temperature, followed by three washing steps with 90 μl of PBST buffer. Detection was carried out by adding 25 μl of goat anti-human IgG antibody-POD conjugate (Jackson, 109-036-088, 1:4000 in OSEP), followed by six washing steps with 90 μl of PBST buffer. 25 μl of TMB substrate (Roche Diagnostics GmbH; Cat. No. 11835033001) was added to each well. Measurements were performed at 370 / 492 nm on a Safire 2 reader (Tecan).

[0446] In another embodiment, Kd is measured using a Kinetic Exclusion (KinExA) assay. According to this assay, typically, antigen is titrated to a fixed concentration of antibody binding sites, the sample is allowed to equilibrate, and then rapidly removed through a flow cell. Free antibody binding sites are captured on antigen-coated beads, while the antigen-saturated antibody complexes are washed away. The bead-captured antibody is then detected by a labeled anti-species antibody, e.g., a fluorescent label (Bee et al., PloS One, 2012, 7(4):e36261). For example, in one embodiment, KinExA experiments are performed at room temperature (RT) using PBS pH 7.4 as the running buffer. Samples are prepared in a running buffer ("sample buffer") supplemented with 1 mg / ml BSA. A flow rate of 0.25 ml / min is used. A fixed amount of antibody, with a binding site concentration of 5 pM, is titrated with antigen via two-fold serial dilutions starting at 100 pM (concentration range: 0.049 pM to 100 pM). One antibody sample without antigen is used as 100% signal (i.e., signal without inhibition). The antigen-antibody complex is incubated at RT for at least 24 hours to allow equilibrium to be reached. The equilibrated mixture is then passed through a column of antigen-coupled beads in a volume of 5 ml in the KinExA system, allowing unbound antibody to be captured by the beads without disturbing the solution equilibrium. The captured antibody is detected using 250 ng / ml Dylight 650 (©)-conjugated anti-human Fc fragment-specific secondary antibody in sample buffer. Each sample is measured in duplicate for every equilibration experiment. KD is obtained from nonlinear regression analysis of the data using a one-site homogeneous binding model contained within the KinExA software (Version 4.0.11) using the "standard analysis" method.

[0447] L. Therapeutic Methods and Compositions The set of antibodies described herein can be used in therapeutic methods. In one aspect, a set of antibodies described herein is provided for use as a medicament. In certain aspects, a set of antibodies is provided for use in treatment methods.

[0448] As discussed above, in some aspects, the antibody set according to the present invention is suitable for any treatment in which it is desired to deliver a radionuclide to target cells in a subject. For example, the antibody set described herein is provided for use in pretargeting radioimmunotherapy, for example, for the treatment of cancer.

[0449] In certain embodiments, the present invention provides a set of antibodies for use in pretargeting radioimmunotherapy in an individual, comprising administering to the individual an effective amount of the set of antibodies. The "individual" according to any of the above embodiments is preferably a human.

[0450] As mentioned above, the treatment can be any condition treatable by cytotoxic activity targeted to the patient's affected 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 can also be the treatment of viral infection or infection with another pathogenic organism, such as a prokaryotic organism. Optionally, the targeting can also be the targeting of T cells for the treatment of T cell-driven autoimmune diseases or T cell-mediated blood cancers. Thus, the conditions to be treated can include viral infections such as HIV, rabies, EBV, and Kaposi's sarcoma-associated herpesvirus, as well as autoimmune diseases such as multiple sclerosis and graft-versus-host disease.

[0451] The term "cancer" as used herein may refer to lymphoma, lymphocytic leukemia, lung cancer, non-small cell lung (NSCL) cancer, bronchioloalveolar cell lung cancer, bone cancer, pancreatic cancer, including pancreatic ductal adenocarcinoma (PDAC), skin cancer, head or neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, anal region cancer, stomach (gastric) cancer, colon cancer and / or rectal cancer, including refractory forms of any of the following cancers, checkpoint inhibitor-treated forms of any of the above cancers, or a combination of one or more of the following cancers: This includes both solid and hematologic cancers, such as colorectal 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 system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvis cancer, mesothelioma, hepatocellular carcinoma, bile duct cancer, central nervous system (CNS) neoplasms, spinal axis tumors, brain stem glioma, glioblastoma multiforme, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, and Ewing's sarcoma.

[0452] Methods for targeting radioisotopes to cells, tissues, or organs for therapeutic purposes include: i) administering to a subject (in either order, simultaneously or sequentially) a first antibody and a second antibody described herein, wherein the antibodies bind to a target antigen and localize to the surface of a cell expressing the target antigen; and wherein the association of the first antibody with the second antibody forms a functional binding site for a radiolabeled compound; and ii) subsequently administering a radiolabeled compound that binds to the functional binding site for the radiolabeled compound. may include:

[0453] Radiolabeled compounds are labeled with a radioisotope that is cytotoxic to cells. Suitable radioisotopes include the alpha-emitters and beta-emitters discussed above.

[0454] In pretargeting radioimmunotherapy using bispecific antibodies (i.e., antibodies that are not "split" antibodies according to the present invention), it is common practice to administer a clearing agent or blocking agent between the administration of the antibody and the administration of the radiolabeled compound. Clearing agents bind to antibodies and enhance their clearance rate from the body. Clearing agents include anti-idiotypic antibodies. Blocking agents are typically agents that bind to the antigen binding site of a radiolabeled compound but are not themselves radiolabeled. For example, if a radiolabeled compound contains a chelating agent loaded with a radioisotope of a certain chemical element (e.g., metal), the blocking agent can include the same chelating agent loaded with a non-radioactive isotope of the same element (e.g., metal), or can include an unloaded chelating agent or a chelating agent loaded with a different non-radioactive moiety (e.g., a non-radioactive isotope of a different element), provided that it can still be bound by the antigen binding site. In some cases, the blocking agent can additionally contain a moiety that increases the size and / or hydrodynamic radius of the molecule. These hinder the molecule's ability to access the tumor without interfering with its ability to bind to circulating antibodies. Exemplary moieties include hydrophilic polymers. The moiety can be, for example, a polymer or copolymer of dextran, dextrin, PEG, polysialic acid (PSA), hyaluronic acid, hydroxyethyl starch (HES), or poly(2-ethyl-2-oxazoline) (PEOZ). In other embodiments, the moiety can be an unstructured peptide or protein, such as an XTEN polypeptide (an unstructured hydrophilic protein polymer), homo-amino acid polymer (HAP), proline-alanine-serine polymer (PAS), elastin-like peptide (ELP), or gelatin-like protein (GLK). Further exemplary moieties include proteins such as albumin, e.g., bovine serum albumin, or IgG. Suitable molecular weights for the moiety / polymer can be, for example, at least 50 kDa, e.g., in the range of 50 kDa to 2000 kDa.For example, the molecular weight can be between 200 and 800 kDa, optionally greater than 300, 350, 400, or 450 kDa, and optionally less than 700, 650, 600, or 550 kDa, optionally about 500 kDa.

[0455] According to certain aspects of the present invention, there is no step of administering a clearing agent or blocking agent to the subject. In certain aspects, there is no step of administering any agent that binds to the first antibody or the second antibody between the administration of the antibody and the administration of the radiolabeled compound. In certain aspects, there is no step of administering any agent between the administration of the antibody and the administration of the radiolabeled compound, except, optionally, a compound selected from a chemotherapeutic agent, an immunotherapeutic agent, and a radiosensitizer. 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.

[0456] In some embodiments, the method can be a two-step pretargeting radioimmunotherapy, which consists of or essentially consists of: i) administering a set of antibodies (wherein the first antibody and the second antibody can be administered simultaneously or sequentially in any order); and ii) subsequently administering a radiolabeled compound. Treatment can involve multiple cycles of such treatment, i.e., multiple cycles of these two steps. The duration of an exemplary treatment cycle is 28 days, where the set of antibodies is administered on the first day of the cycle, and the radiolabeled compound is optionally administered on the first, second, third, fourth, fifth, sixth, seventh, or eighth day of the cycle, for example, on the seventh day. The number of treatment cycles can vary. In one embodiment, 4, 5, or 6 treatment cycles can be administered.

[0457] Surprisingly, the inventors have determined that, using antibodies according to the invention, it is possible to obtain tumor uptake of therapeutically effective radiolabeled compounds while avoiding the accumulation of excess radioactivity in normal tissues. Indeed, in the examples, it was found that the level of accumulation of radioactivity in non-target tissues was lower than in the case of the three-step PRIT method, which uses bispecific antibodies and a clearing step and also results in the use of a simpler procedure.

[0458] In some embodiments, the radiolabeled compound can be administered to a subject after allowing time for the first and second antibodies to localize to target cells. For example, in some embodiments, the radiolabeled compound can be administered to a 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 can be administered no more than 3 days, 5 days, or 7 days after the first and second antibodies. In one specific embodiment, the radiolabeled compound can be administered to a subject 2 to 7 days after the first and second antibodies.

[0459] In some embodiments, the antibodies described herein may be administered as part of a combination therapy. For example, the antibodies described herein may be administered in combination with one or more chemotherapeutic agents; the chemotherapeutic agents and antibodies may be administered simultaneously or sequentially in any order. Additionally, or alternatively, the antibodies described herein may be administered in combination with one or more immunotherapeutic agents; the immunotherapeutic agents and antibodies may be administered simultaneously or sequentially in any order.

[0460] In some embodiments, in addition or alternatively, the antibodies described herein may be administered in combination with a radiosensitizer. The radiosensitizer and antibody may be administered simultaneously or sequentially, in any order.

[0461] The antibodies of the invention (and any additional therapeutic agents, e.g., radiolabeled compounds) may be administered by any suitable means, including parenteral, intrapulmonary, and intranasal administration, and, if desired for localized treatment, by intralesional administration. Parenteral administration includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration can be by any suitable route, e.g., by injection, such as intravenous or subcutaneous injection.

[0462] In some embodiments, one or more dosimetry cycles may be used prior to one or more treatment cycles described above. A dosimetry cycle may include i) administering a set of antibodies (wherein the first and second antibodies may be administered simultaneously or sequentially in either order), followed by ii) administering a compound suitable for imaging radiolabeling with a gamma emitter (wherein the radiolabeled compound binds to a functional binding site for the radiolabeled compound). The compound may be the same as the compound used in the subsequent treatment cycle, 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 The radiolabeled compound used in the treatment cycle can be Pb-DOTAM. 212 The compound may be Pb-DOTAM. The patient may be subjected to imaging to determine tumor uptake of the compound and / or 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.

[0463] M. Pharmaceutical Preparations The first antibody and the second antibody described herein may be formulated in a single pharmaceutical composition or in separate pharmaceutical compositions. Thus, in a further aspect, the present invention provides a pharmaceutical composition comprising the first antibody and the second antibody of the present invention, or a first 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 of the therapeutic or diagnostic methods 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 additional therapeutic agent, for example, as described below.

[0464] Pharmaceutical formulations of the antibodies described herein may be prepared by mixing such antibodies having the desired purity with one or more optional pharmaceutically acceptable carriers ("Remington's Pharmaceutical Sciences", 16th Edition, Osol, A., ed. (1980)) in the form of a lyophilized formulation or an aqueous solution.

[0465] Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed and include buffers such as histidine, phosphate, citrate, acetate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); small molecule Examples of suitable pharmaceutically acceptable carriers include, but are not limited to, small (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; 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 non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersion agents, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Halozyme, Inc.). Certain exemplary sHASEGPs, including rHuPH20, and their methods of use are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one embodiment, a sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0466] Exemplary lyophilized antibody compositions are described in U.S. Patent No. 6,267,958. Aqueous antibody compositions include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter compositions including a histidine-acetate buffer.

[0467] The formulations herein may also contain more than one active ingredient as needed for the specific indication being treated, preferably active ingredients with complementary activities that do not adversely affect each other. For example, it may be desirable to further provide chemotherapeutic agents, immunotherapeutic agents, and / or radiosensitizers, as discussed above. Suitably, such active ingredients are present in combination in amounts effective for the intended purpose.

[0468] The active ingredient can be encapsulated in, for example, microcapsules prepared by coacervation or interfacial polymerization, for example, in hydroxymethylcellulose microcapsules, or gelatin microcapsules and poly(methyl methacrylate) microcapsules, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions, respectively. Such techniques are disclosed in "Remington's Pharmaceutical Sciences," 16th edition, edited by Osol, A. (1980).

[0469] Sustained-release preparations may also be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

[0470] Preparations to be used for in vivo administration are generally sterile. Sterility may be readily achieved, for example, by filtration through sterile filtration membranes.

[0471] N. Methods and Compositions for Diagnostics and Detection The antibody set described herein can also be used in d...

Claims

1. i) a first antibody that binds to an antigen expressed on the surface of a target cell, the first antibody further comprising a VH domain of an antigen-binding site for a radiolabeled compound, but not comprising a VL domain of an antigen-binding site for a radiolabeled compound; and ii) a second antibody that binds to the antigen expressed on the surface of a target cell, the second antibody further comprising a VL domain of an antigen-binding site for a radiolabeled compound, but not comprising a VH domain of an antigen-binding site for a radiolabeled compound. Including; the VH domain of the first antibody and the VL domain of the second antibody are capable of together forming a functional antigen-binding site for a radiolabeled compound; Antibody set.

2. The set of antibodies described in claim 1, wherein the first antibody and the second antibody each comprise: i) an antibody fragment comprising an antigen-binding site specific for the antigen expressed on the surface of a target cell, and ii) a VL domain or a VH domain of an antigen-binding site for a radiolabeled compound.

3. The set of antibodies according to claim 2, wherein the antibody fragments are selected from at least one Fv fragment, scFv fragment, or Fab fragment, or cross-Fab fragment.

4. The first antibody is a) a Fab fragment that binds to the antigen; and b) i) an antibody heavy chain variable domain (VH) of the antigen-binding site for a radiolabeled compound, or ii) an antibody heavy chain variable domain (VH) and an antibody heavy chain constant domain of an antigen-binding site for a radiolabeled compound, with the C-terminus of the VH domain fused to the N-terminus of the constant domain; A polypeptide comprising or consisting of: A polypeptide fused by the N-terminus of a VH domain to the C-terminus of the CL domain or CH1 domain of a Fab fragment. comprising or consisting of The second antibody c) a Fab fragment that binds to the antigen; and d) iii) an antibody light chain variable domain (VL) of the antigen-binding site for a radiolabeled compound; or iv) an antibody light chain variable domain (VL) and an antibody light chain constant domain of an antigen-binding site for a radiolabeled compound, wherein the C-terminus of the VL domain is fused to the N-terminus of the constant domain; A polypeptide comprising or consisting of: A polypeptide fused by the N-terminus of the VL domain to the C-terminus of the CL domain or CH1 domain of a Fab fragment. comprising or consisting of the antibody heavy chain variable domain (VH) of the polypeptide of (b) and the antibody light chain variable domain (VL) of the polypeptide of (d) are capable of together forming a functional antigen-binding site for a radiolabeled compound; The set of antibodies described in claim 3.

5. The set of antibodies described in claim 4, wherein the polypeptide (b) is fused to the C-terminus of the CL domain or CH1 domain of the Fab fragment of (a) via a peptide linker at the N-terminus of the VH domain; and the polypeptide (d) is fused to the C-terminus of the CL domain or CH1 domain of the Fab fragment of (c) via a peptide linker at the N-terminus of the VL domain.

6. The first antibody is a) a tandem Fab comprising two Fab fragments, wherein a first Fab fragment and a second Fab fragment each bind to the same epitope of the antigen, and wherein the first Fab fragment and the second Fab fragment are connected via a peptide tether, and wherein the first Fab is connected via its C-terminus to the N-terminus of the second Fab; and b) i) an antibody heavy chain variable domain (VH); or ii) an antibody heavy chain variable domain (VH) and an antibody constant domain (CH1), wherein the C-terminus of the VH domain is fused to the N-terminus of the CH1 domain; A polypeptide comprising or consisting of: a polypeptide fused by the N-terminus of the VH domain to the C-terminus of the CL domain or CH1 domain of a second Fab fragment Including; The second antibody c) a tandem Fab comprising two Fab fragments, wherein a first Fab fragment and a second Fab fragment each bind to the same epitope of the antigen, and wherein the first Fab fragment and the second Fab fragment are connected via a peptide tether, and wherein the first Fab is connected via its C-terminus to the N-terminus of the second Fab; and d) i) an antibody light chain variable domain (VL); or ii) an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL) in which the C-terminus of the VH domain is fused to the N-terminus of the constant domain; A polypeptide comprising or consisting of: a polypeptide fused by the N-terminus of the VL domain to the C-terminus of the CL domain or CH1 domain of a second Fab fragment Including, the antibody heavy chain variable domain (VH) of the polypeptide of (b) and the antibody light chain variable domain (VL) of the polypeptide of (d) are capable of together forming a functional antigen-binding site for a radiolabeled compound; The set of antibodies described in claim 3.

7. The set of antibodies described in claim 6, wherein the polypeptide (b) is fused to the C-terminus of the CL domain or CH1 domain of the second Fab fragment of (a) via a peptide linker at the N-terminus of the VH domain; and the polypeptide (d) is fused to the C-terminus of the CL domain or CH1 domain of the second Fab fragment of (c) via a peptide linker at the N-terminus of the VL domain.

8. The first antibody is a) a tandem Fab comprising a first fragment and a second fragment, wherein the first fragment is connected by its C-terminus to the N-terminus of the second fragment via a peptide tether, the first fragment binds to a first epitope of the antigen, and the second fragment binds to a second epitope of the antigen, and one of the fragments selected from the first fragment and the second fragment is a Fab and the other is a crossover Fab; b) i) an antibody heavy chain variable domain (VH); or ii) an antibody heavy chain variable domain (VH) and an antibody heavy chain constant domain (CH1), wherein the C-terminus of the VH domain is fused to the N-terminus of the CH1 domain; A polypeptide comprising or consisting of: a polypeptide fused by the N-terminus of the VH domain to the C-terminus of one of the chains of a second fragment Including; The second antibody c) a tandem Fab comprising a first fragment and a second fragment, wherein the first fragment is connected by its C-terminus to the N-terminus of the second fragment, the first fragment binds to a first epitope of the antigen, and the second fragment binds to a second epitope of the antigen, and one of the fragments selected from the first fragment and the second fragment is a Fab and the other is a crossover Fab; and d) i) an antibody light chain variable domain (VL); or ii) an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL), wherein the C-terminus of the VL domain is fused to the N-terminus of the light chain constant domain; A polypeptide comprising or consisting of: a polypeptide fused by the N-terminus of the VL domain to the C-terminus of one of the chains of a second fragment Including; the antibody heavy chain variable domain (VH) of the polypeptide of (b) and the antibody light chain variable domain (VL) of the polypeptide of (d) are capable of together forming a functional antigen-binding site for a radiolabeled compound; The set of antibodies described in claim 3.

9. 9. The set of antibodies described in claim 8, wherein the polypeptide (b) is fused to the C-terminus of one of the chains of the second fragment (a) via a peptide linker at the N-terminus of the VL domain; and the polypeptide (d) is fused to the C-terminus of one of the chains of the second fragment (c) via a peptide linker at the N-terminus of the VL domain.

10. The set of antibodies described in any one of claims 1 to 3, wherein the first antibody and the second antibody each further comprise an Fc domain.

11. The set of antibodies described in claim 10, wherein the first antibody and the second antibody each comprise: i) an antibody fragment comprising an antigen-binding site specific for the antigen expressed on the surface of a target cell; ii) an Fc region; and iii) a VL domain or VH domain of an antigen-binding site for a radiolabeled compound fused to the Fc region.

12. 12. The set of antibodies of claim 10 or 11, wherein the Fc domain is modified to reduce or eliminate effector function.

13. The first antibody is i) a complete light chain fragment; ii) complete heavy chain; iii) an additional Fc chain lacking Fd; and iv) a polypeptide comprising or consisting of a VH domain of an antigen-binding site for a radiolabeled compound. comprising or consisting of; the light chain of (i) and the heavy chain of (ii) together provide an antigen-binding site for said antigen; a polypeptide comprising or consisting of a VH domain of an antigen-binding site for a radiolabeled compound is fused by its N-terminus to the C-terminus of (ii) or (iii); The second antibody v) complete light chain fragment; vi) complete heavy chain; vii) an additional Fc chain lacking Fd; and viii) a polypeptide comprising or consisting of a VL domain of an antigen-binding site for a radiolabeled compound; comprising or consisting of; the light chain of (v) and the heavy chain of (vi) together provide an antigen-binding site for said antigen; a polypeptide comprising or consisting of a VL domain of an antigen-binding site for a radiolabeled compound is fused by its N-terminus to the C-terminus of (vi) or (vii); the antibody heavy chain variable domain (VH) of the polypeptide of (iv) and the antibody light chain variable domain (VL) of the polypeptide of (viii) are capable of together forming a functional antigen-binding site for a radiolabeled compound; A set of antibodies according to any one of claims 10 to 12.

14. A set of antibodies described in claim 13, wherein the polypeptide (iv) is fused by its N-terminus to the C-terminus of (ii) or (iii) via a linker; and the polypeptide (viii) is fused by its N-terminus to the C-terminus of (vi) or (vii) via a linker.

15. 13. The set of antibodies according to any one of claims 10 to 12, wherein each of the first antibody and the second antibody comprises: a) an Fc domain; b) at least one antibody fragment, such as an scFv, Fv, Fab, or cross-Fab fragment, comprising an antigen-binding site for a target antigen; and c) a polypeptide comprising a VL domain or VH domain of an antigen-binding site for a radiolabeled compound, wherein the C-terminus of the antibody fragment (b) is fused to the N-terminus of one chain of the Fc domain, and the C-terminus of the polypeptide (c) is fused to the N-terminus of the other chain of the Fc domain.

16. The first antibody is i) intact light chain; ii) complete heavy chain; iii) an additional Fc chain; and iv) a polypeptide comprising or consisting of a VH domain of an antigen-binding site for a radiolabeled compound. Including; the light chain of (i) and the heavy chain of (ii) together provide an antigen-binding site for a target antigen; a polypeptide comprising or consisting of a VH domain of an antigen-binding site for a radiolabeled compound is fused by its C-terminus to the N-terminus of (iii) via a linker; The second antibody v) intact light chain; vi) complete heavy chain; vii) an additional Fc chain; and viii) a polypeptide comprising or consisting of a VL domain of an antigen-binding site for a radiolabeled compound; Including; 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 of a VL domain of an antigen-binding site for a radiolabeled compound is fused by its C-terminus to the N-terminus of (vii) via a linker. A set of antibodies according to claim 15.

17. i) the first antibody comprises a first heavy chain of SEQ ID NO: 112, a second heavy chain of SEQ ID NO: 114, and a light chain of SEQ ID NO: 115; ii) the second antibody comprises a first heavy chain of SEQ ID NO: 112, a second heavy chain of SEQ ID NO: 113, and a light chain of SEQ ID NO: 115; A set of antibodies according to claim 16.

18. The first antibody is a) a first full-length antibody consisting of two antibody heavy chains and two antibody light chains, wherein at least one arm of the full-length antibody binds to the antigen; and b) i) an antibody heavy chain variable domain (VH); or ii) an antibody heavy chain variable domain (VH) and an antibody constant domain (CH1); A polypeptide consisting of a polypeptide fused by the N-terminus of the VH domain to the C-terminus of one of the two heavy chains of said first full-length antibody; Including, The second antibody c) a second full-length antibody consisting of two antibody heavy chains and two antibody light chains, wherein at least one arm of the antibody binds to the antigen; and d) i) an antibody light chain variable domain (VL); or ii) an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL) A polypeptide consisting of a polypeptide fused by the N-terminus of the VL domain to the C-terminus of one of the two heavy chains of the second full-length antibody; Including, the polypeptide of (b) and the polypeptide of (d) are capable of forming together a functional antigen-binding site for a radiolabeled compound; A set of antibodies according to any one of claims 10 to 12.

19. 19. The set of antibodies described in claim 18, wherein polypeptide (b) is fused to the C-terminus of one of the two heavy chains of the first full-length antibody via a peptide linker at the N-terminus of the VH domain; and polypeptide (d) is fused to the C-terminus of one of the two heavy chains of the second full-length antibody via a peptide linker at the N-terminus of the VL domain.

20. 20. The set of antibodies described in claim 18 or 19, wherein each of the first antibody and the second antibody is bivalent for the antigen.

21. The set of antibodies described in claim 20, wherein both arms of the full-length antibodies bind to the same epitope of the antigen.

22. 20. The set of antibodies described in claim 18 or 19, wherein each of the first antibody and the second antibody is dual paratopic with respect to the antigen.

23. The set of antibodies described in claim 22, wherein the two arms of the first antibody each bind to an epitope on the antigen that is different from each other; and the two arms of the second antibody each bind to an epitope on the antigen that is different from each other.

24. 24. The set of antibodies of any one of claims 1 to 23, wherein the radiolabeled compound comprises radiolabeled DOTA or a salt or functional variant thereof.

25. 25. The set of antibodies according to any one of claims 1 to 24, wherein the radiolabeled compound is DOTA radiolabeled with a radioactive isotope of Lu or Y, or a salt or functional variant thereof.

26. A set of antibodies described in claim 24 or 25, wherein the VH domain of the antigen-binding site for the radiolabeled compound comprises: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 35; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 36; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:

37.

27. A set of antibodies described in any one of claims 24 to 26, 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 comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:

41.

28. 28. The set of antibodies described in claim 27, wherein one or more alanine residues are added to the C-terminus of the VH domain of the antigen-binding site for the radiolabeled compound, or one or more residues derived from the N-terminus of the CH1 domain are added to the C-terminus of the VH domain of the antigen-binding site for the radiolabeled compound.

29. 29. The set of antibodies of claim 28, wherein residues AST are added to the C-terminus of the VH domain of the antigen binding site for the radiolabeled compound.

30. A set of antibodies described in any one of claims 24 to 29, wherein the VL domain of the antigen-binding site for the radiolabeled compound comprises: (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 38; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 39; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:

40.

31. A set of antibodies described in any one of claims 24 to 30, wherein the VL domain of the antigen binding site for the radiolabeled compound comprises the amino acid sequence of SEQ ID NO: 42 or a variant thereof comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:

42.

32. The set of antibodies according to any one of claims 1 to 23, wherein the radiolabeled compound comprises Pb-DOTAM.

33. 33. The set of antibodies of claim 32, wherein the functional binding sites for Pb-DOTAM bind with a Kd value for 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.

34. A set of antibodies according to claim 32 or claim 33, wherein the functional binding site for Pb-DOTAM binds to Pb-DOTAM and Bi-DOTAM.

35. The VH domain of the antigen-binding site for the radiolabeled compound is a) a heavy chain CDR2 comprising the amino acid sequence FIGSRGDTYYASWAKG (SEQ ID NO:2), or a variant thereof having up to one, two, or three substitutions within 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 of 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, and optionally also do not include Ala100C, Tyr100D, and / or Pro100E, and / or optionally also do not include Tyr99; and optionally, c) a heavy chain CDR1 comprising the amino acid sequence of GFSLSTYSMS (SEQ ID NO: 1), or a variant thereof having up to one, two, or three substitutions within SEQ ID NO: 1; 35. The set of antibodies of any one of claims 32 to 34, comprising:

36. The set of antibodies described in claim 35, wherein the VH domain of the antigen-binding site for the radiolabeled compound comprises (a) CDR-H1 having the amino acid sequence of GFSLSTYSMS (SEQ ID NO: 1); (b) CDR-H2 having the amino acid sequence of FIGSRGDTYYASWAKG (SEQ ID NO: 2); and (c) CDR-H3 having the amino acid sequence of ERDPYGGGAYPPHL (SEQ ID NO: 3).

37. A set of antibodies described in any one of claims 32 to 36, wherein the VH domain of the antigen binding site for the radiolabeled compound comprises an amino acid sequence selected from the group consisting of SEQ ID NO:7 and SEQ ID NO:9, or variants thereof comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:7 or SEQ ID NO:

9.

38. 38. The set of antibodies described in claim 37, wherein one or more alanine residues are added to the C-terminus of the VH domain of the antigen-binding site for the radiolabeled compound, or one or more residues derived from the N-terminus of the CH1 domain are added to the C-terminus of the VH domain of the antigen-binding site for the radiolabeled compound.

39. 39. The set of antibodies of claim 38, wherein residues AST are added to the C-terminus of the VH domain of the antigen binding site for the radiolabeled compound.

40. The VL domain of the antigen-binding site for the radiolabeled compound is d) a light chain CDR1 comprising the amino acid sequence of QSSHSVYSDNDLA (SEQ ID NO: 4), or a variant thereof having up to one, two, or three substitutions within 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 within SEQ ID NO: 6, wherein these substitutions do not include Gly91, Tyr92, Asp93, Thr95c, and Tyr96. and optionally, f) a light chain CDR2 comprising the amino acid sequence of QASKLAS (SEQ ID NO: 5), or a variant thereof having at least one, two, or three substitutions within SEQ ID NO: 5, and optionally excluding Gln50; 40. The set of antibodies of any one of claims 32 to 39, comprising:

41. The set of antibodies described in claim 40, wherein the VL domain of the antigen-binding site for the radiolabeled compound comprises: (d) CDR-L1 comprising the amino acid sequence QSSHSVYSDNDLA (SEQ ID NO: 4); (e) CDR-L2 comprising the amino acid sequence QASKLAS (SEQ ID NO: 5); and (f) CDR-L3 comprising the amino acid sequence LGGYDDESDTYG (SEQ ID NO: 6).

42. A set of antibodies described in any one of claims 32 to 41, 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 comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:

8.

43. 43. The set of antibodies of any one of claims 1 to 42, wherein the first antibody and the second antibody bind to the same epitope of an antigen expressed on the surface of a target cell.

44. 43. The set of antibodies of any one of claims 1 to 42, wherein a first antibody binds to a different epitope of an antigen than a second antibody.

45. 45. A set of antibodies according to any one of claims 1 to 44, wherein the antigen expressed on the surface of the target cell is a tumor-associated antigen.

46. 46. ​​The set of antibodies of any one of claims 1 to 45, wherein the antigen expressed on the surface of the target cells is selected from the group consisting of carcinoembryonic antigen (CEA), CD20, HER2, EGP-1 (epithelial glycoprotein 1 also known as trophoblast 2), colon-specific antigen p (CSAp), pancreatic mucin MUC1, GPRC5D, and FAP.

47. 47. The set of antibodies of any one of claims 1 to 46, wherein the antigen expressed on the surface of the target cell is selected from the group consisting of CEA, GPRC5D, and FAP.

48. i) the first antibody comprises a first heavy chain of SEQ ID NO: 104, a second heavy chain of SEQ ID NO: 106, wherein the C-terminal alanine of SEQ ID NO: 106 is optional and may be absent or replaced by an alternative C-terminal extension, and a light chain of SEQ ID NO: 107; ii) the second antibody comprises a first heavy chain of SEQ ID NO: 104, a second heavy chain of SEQ ID NO: 105, and a light chain of SEQ ID NO: 107; 48. A set of antibodies according to claim 47.

49. i) the first antibody comprises a first heavy chain of SEQ ID NO: 108, a second heavy chain of SEQ ID NO: 110, wherein the C-terminal alanine of SEQ ID NO: 110 is optional and may be absent or replaced by an alternative C-terminal extension, and a light chain of SEQ ID NO: 111; ii) the second antibody comprises a first heavy chain of SEQ ID NO: 108, a second heavy chain of SEQ ID NO: 109, and a light chain of SEQ ID NO: 111; 48. A set of antibodies according to claim 47.

50. 48. A set of antibodies according to any one of claims 1 to 47, wherein the antigen expressed on the surface of the target cell is CEA.

51. The first antibody is (a) a heavy chain variable region comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 19; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 20; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 21; and (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:22; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:23; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

24.

51. The set of antibodies of claim 50, comprising an antigen binding site that binds to CEA, comprising:

52. 52. The set of antibodies of any one of claims 50 to 51, wherein the first antibody comprises an antigen binding site against CEA comprising a VH sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO:25 or a variant thereof comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:

25.

53. 53. The set of antibodies of any one of claims 50 to 52, wherein the first antibody comprises an antigen binding site against CEA comprising a VL sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO:26 or a variant thereof comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:

26.

54. The set of antibodies of claim 50, wherein the first antibody comprises an antigen-binding site that binds to CEA, comprising a heavy chain variable region comprising: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 43; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 44; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 45; and a light chain variable region comprising: (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 46; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 47; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:

48.

55. 55. The set of antibodies described in claim 50 or 54, wherein the first antibody comprises an antigen binding site against CEA comprising a VH sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO:49 or a variant thereof comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:

49.

56. 56. The set of antibodies of any one of claims 50, 54, or 55, wherein the first antibody comprises an antigen binding site against CEA comprising a VL sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 50, or a variant thereof comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:

50.

57. The first antibody is (a) a heavy chain variable region comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 11; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 12; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 13; and (d) a light chain variable region comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 14; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 15; and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

16.

51. The set of antibodies of claim 50, comprising an antigen binding site that binds to CEA, comprising:

58. 58. The set of antibodies described in claim 50 or 57, wherein the first antibody comprises an antigen binding site against CEA comprising a VH sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 17 or a variant thereof comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:

17.

59. 59. The set of antibodies of any one of claims 50, 57, or 58, wherein the first antibody comprises an antigen binding site against CEA comprising a VL sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 18, or a variant thereof comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:

18.

60. The first antibody is (a) a heavy chain variable region comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 59; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 60; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 61; and (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 62; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 63; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

64.

51. The set of antibodies of claim 50, comprising an antigen binding site that binds to CEA, comprising:

61. 61. The set of antibodies described in claim 50 or claim 60, wherein the first antibody comprises an antigen binding site against CEA comprising a VH sequence comprising an amino acid sequence selected from the group consisting of 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 to SEQ ID NO:

65.

62. 62. The set of antibodies of any one of claims 50, 60, or 61, wherein the first antibody comprises an antigen binding site against CEA comprising a VL sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 66, or a variant thereof comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:

66.

63. 51. The set of antibodies of claim 50, wherein the first antibody comprises an antigen-binding site that binds to CEA, comprising a heavy chain variable region comprising: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 156; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 157 or 158; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 159; and a light chain variable region comprising: (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 160, 161, or 162; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 163, 164, or 165; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:

166.

64. 64. The set of antibodies of claim 50 or 63, wherein the first antibody comprises an antigen-binding site that binds to CEA, comprising: a heavy chain variable region (VH) comprising an amino acid sequence selected from SEQ ID NO: 169, 170, 171, 172, 173, or 174, or a sequence with 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity thereto; and a light chain variable region (VL) comprising an amino acid sequence selected from SEQ ID NO: 175, 176, 177, 178, 179, or 180, or a sequence with 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity thereto.

65. The first antibody is (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 169, and a VL domain comprising the amino acid sequence of SEQ ID NO: 179; or (b) a VH domain comprising the amino acid sequence of SEQ ID NO: 173 and a VL domain comprising the amino acid sequence of SEQ ID NO: 179; or (c) a VH domain comprising the amino acid sequence of SEQ ID NO: 170, and a VL domain comprising the amino acid sequence of SEQ ID NO: 179; or (d) a VH domain comprising the amino acid sequence of SEQ ID NO: 174, and a VL domain comprising the amino acid sequence of SEQ ID NO: 178; or (e) a VH domain comprising the amino acid sequence of SEQ ID NO: 173, and a VL domain comprising the amino acid sequence of SEQ ID NO: 178; or (f) a VH domain comprising the amino acid sequence of SEQ ID NO: 171, and a VL domain comprising the amino acid sequence of SEQ ID NO: 178; or (g) a VH domain comprising the amino acid sequence of SEQ ID NO: 169, and a VL domain comprising the amino acid sequence of SEQ ID NO: 178 65. The set of antibodies of any one of claims 50, 63, or 64, comprising an antigen binding site that binds to CEA, comprising:

66. The second antibody (a) a heavy chain variable region comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 19; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 20; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 21; and (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:22; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:23; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

24.

66. The set of antibodies of any one of claims 50 to 65, comprising an antigen binding site that binds to CEA, comprising:

67. 67. The set of antibodies of any one of claims 50 to 66, wherein the second antibody comprises an antigen binding site against CEA comprising a VH sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 25 or a variant thereof comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:

25.

68. 68. The set of antibodies of any one of claims 50 to 67, wherein the second antibody comprises an antigen binding site against CEA comprising a VL sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO:26 or a variant thereof comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:

26.

69. 66. The set of antibodies of any one of claims 50 to 65, wherein the second antibody comprises an antigen-binding site that binds to CEA, comprising a heavy chain variable region comprising: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 43; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 44; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 45; and a light chain variable region comprising: (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 46; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 47; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:

48.

70. 70. The set of antibodies of any one of claims 50 to 65 or 69, wherein the second antibody comprises an antigen binding site against CEA comprising a VH sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO:49 or a variant thereof comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:

49.

71. 71. The set of antibodies of any one of claims 50 to 65, 69, or 70, wherein the second antibody comprises an antigen binding site against CEA comprising a VL sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 50, or a variant thereof comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:

50.

72. The second antibody (a) a heavy chain variable region comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 11; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 12; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 13; and (d) a light chain variable region comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 14; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 15; and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

16.

66. The set of antibodies of any one of claims 50 to 65, comprising an antigen binding site that binds to CEA, comprising:

73. 73. The set of antibodies of any one of claims 50 to 65 or 72, wherein the second antibody comprises an antigen binding site against CEA comprising a VH sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 17 or a variant thereof comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:

17.

74. 74. The set of antibodies of any one of claims 50 to 65, 72, or 73, wherein the second antibody comprises an antigen binding site against CEA comprising a VL sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 18, or a variant thereof comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:

18.

75. The second antibody (a) a heavy chain variable region comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 59; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 60; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 61; and (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 62; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 63; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

64.

66. The set of antibodies of any one of claims 50 to 65, comprising an antigen binding site that binds to CEA, comprising:

76. 76. The set of antibodies of any one of claims 50 to 65 or 75, wherein the second antibody comprises an antigen binding site against CEA comprising a VH sequence comprising an amino acid sequence selected from the group consisting of 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 to SEQ ID NO:

65.

77. 80. The set of antibodies of any one of claims 50 to 65, 75, or 76, wherein the second antibody comprises an antigen binding site against CEA comprising a VL sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 66, or a variant thereof comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:

66.

78. 66. The set of antibodies of any one of claims 50 to 65, wherein the second antibody comprises an antigen-binding site that binds to CEA, comprising a heavy chain variable region comprising: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 156; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 157 or 158; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 159; and a light chain variable region comprising: (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 160, 161, or 162; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 163, 164, or 165; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:

166.

79. 80. The set of antibodies of any one of claims 50 to 65 or 78, wherein the second antibody comprises an antigen-binding site that binds to CEA, comprising: a heavy chain variable region (VH) comprising an amino acid sequence selected from SEQ ID NO: 169, 170, 171, 172, 173, or 174, or a sequence with 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity thereto; and a light chain variable region (VL) comprising an amino acid sequence selected from SEQ ID NO: 175, 176, 177, 178, 179, or 180, or a sequence with 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity thereto.

80. The second antibody (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 169, and a VL domain comprising the amino acid sequence of SEQ ID NO: 179; or (b) a VH domain comprising the amino acid sequence of SEQ ID NO: 173 and a VL domain comprising the amino acid sequence of SEQ ID NO: 179; or (c) a VH domain comprising the amino acid sequence of SEQ ID NO: 170, and a VL domain comprising the amino acid sequence of SEQ ID NO: 179; or (d) a VH domain comprising the amino acid sequence of SEQ ID NO: 174, and a VL domain comprising the amino acid sequence of SEQ ID NO: 178; or (e) a VH domain comprising the amino acid sequence of SEQ ID NO: 173, and a VL domain comprising the amino acid sequence of SEQ ID NO: 178; or (f) a VH domain comprising the amino acid sequence of SEQ ID NO: 171, and a VL domain comprising the amino acid sequence of SEQ ID NO: 178; or (g) a VH domain comprising the amino acid sequence of SEQ ID NO: 169, and a VL domain comprising the amino acid sequence of SEQ ID NO: 178 80. The set of antibodies of any one of claims 50 to 65, 78, or 79, comprising an antigen binding site that binds to CEA, comprising:

81. 51. A set of antibodies according to claim 50, wherein the first antibody is an antibody according to any one of claims 57 to 59 and the second antibody is an antibody according to any one of claims 66 to 68.

82. The set of antibodies of claim 1 , wherein the first antibody comprises a first heavy chain of SEQ ID NO: 28, a second heavy chain of SEQ ID NO: 32, and a light chain of SEQ ID NO:

34.

83. A set of antibodies described in claim 82, wherein one or more alanine residues are added to the C-terminus of SEQ ID NO: 32, or an AST sequence is added to the C-terminus of SEQ ID NO:

32.

84. The set of antibodies of claim 1 , wherein the first antibody comprises a first heavy chain of SEQ ID NO: 51, a second heavy chain of SEQ ID NO: 52, and a light chain of SEQ ID NO:

54.

85. A set of antibodies described in claim 84, wherein one or more alanine residues are added to the C-terminus of SEQ ID NO: 52, or the sequence of AST is added to the C-terminus of SEQ ID NO:

32.

86. 2. The set of antibodies of claim 1, wherein the first antibody comprises a first heavy chain of SEQ ID NO: 86, a second heavy chain of SEQ ID NO: 110, wherein the C-terminal AST residue is optional and may be absent or replaced with an alternative C-terminal extension, and a light chain of SEQ ID NO:

89.

87. 2. The set of antibodies of claim 1, wherein the first antibody comprises a first heavy chain of SEQ ID NO: 93, a second heavy chain of SEQ ID NO: 94, wherein the C-terminal AST residue is optional and may be absent or replaced with an alternative C-terminal extension, and a light chain of SEQ ID NO:

96.

88. 88. The set of antibodies of any one of claims 1 or 82 to 87, wherein the second antibody comprises a first heavy chain of SEQ ID NO: 30, a second heavy chain of SEQ ID NO: 33, and a light chain of SEQ ID NO:

34.

89. 88. The set of antibodies of any one of claims 1 or 82 to 87, wherein the second antibody comprises a first heavy chain of SEQ ID NO: 55, a second heavy chain of SEQ ID NO: 56, and a light chain of SEQ ID NO:

58.

90. 88. The set of antibodies of any one of claims 1 or 82 to 87, wherein the second antibody comprises a first heavy chain of SEQ ID NO: 83, a second heavy chain of SEQ ID NO: 84, and a light chain of SEQ ID NO:

89.

91. 88. The set of antibodies of any one of claims 1 or 82 to 87, wherein the second antibody comprises a first heavy chain of SEQ ID NO: 90, a second heavy chain of SEQ ID NO: 91, and a light chain of SEQ ID NO:

96.

92. 92. A set of nucleic acids expressing the set of antibodies of any one of claims 1 to 91.

93. 93. An expression vector or set of expression vectors comprising the set of nucleic acids of claim 92.

94. 94. A host cell or set of host cells comprising the expression vector or set of expression vectors of claim 93.

95. 1. A method of pretargeting radioimmunotherapy, comprising: i) administering to a subject a set of antibodies according to any one of claims 1 to 91, wherein the first antibody and the second antibody are administered simultaneously or sequentially in any order, and wherein 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 with the second antibody forms a functional binding site for a radiolabeled compound; and ii) subsequently administering a radiolabeled compound that binds to the functional binding site for the radiolabeled compound. A method comprising:

96. 96. The method of claim 95, wherein the method does not include administering a clearing or blocking agent.

97. 97. The method of claim 95 or 96, wherein the subject is a human.

98. 98. The method of any one of claims 95 to 97, wherein the target antigen is a cancer-associated antigen or a tumor-associated antigen and is radioimmunotherapy for tumors or cancer.

99. A set of antibodies according to any one of claims 1 to 91 for use in a method of pretargeting radioimmunotherapy according to any one of claims 95 to 98.

100. 1. A method for targeting a radioisotope to a tissue or organ for radiological imaging, comprising: i) administering to a subject a set of antibodies according to any one of claims 1 to 91, wherein the first antibody and the second antibody are administered simultaneously or sequentially in any order, and 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 with the second antibody forms a functional binding site for a radiolabeled compound; and ii) subsequently administering a radiolabeled compound that binds to the functional binding site for the radiolabeled compound. A method comprising:

101. 101. The method of claim 100, wherein the method does not include administering a clearing or blocking agent.

102. 102. The method of claim 100 or 101, further comprising an imaging step.

103. The method of claim 102, wherein the target antigen is a cancer-associated antigen or a tumor-associated antigen, and the method is for imaging tumors or cancer.

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