Novel pre-targeting antibodies and uses thereof

Abstract

The present invention relates to novel antibodies that bind to an antigen on a target cell, such as a tumor cell, and target radionuclides to said cell, uses of these antibodies, and methods of making them.

Description

Technical Field

[0001] This invention relates to antibodies that bind to antigens on target cells and target said cells with radionuclides, and to methods of using a group of antibodies. Background Technology

[0002] Selective destruction of individual cells or specific cell types is often desired in a variety of clinical settings. For example, a primary goal of cancer therapy is to specifically destroy tumor cells while leaving healthy cells and tissues intact.

[0003] In this regard, bispecific antibodies have been designed, which use one "arm" to bind to surface antigens on target cells and a second "arm" to bind to effector moieties such as drugs. Various bispecific forms have been developed, but the task of developing bispecific antibodies is by no means easy.

[0004] In pre-targeted radioimmunotherapy (PRIT), the antibody construct used has affinity for both tumor-associated antigens and radiolabeled compounds. In the first step, the antibody is administered and localized to the tumor. Subsequently, the radiolabeled compound is administered. Because the radiolabeled compound is small, it can be rapidly delivered to the tumor, and unbound compounds are rapidly cleared, thereby reducing radiation exposure outside the tumor (Goldenberg et al., Theranostics 2012, 2(5), 523-540). A similar procedure can also be used for imaging. Pre-targeting can utilize bispecific antibodies or a vividin-biotin system, although the latter has the disadvantage of vividin / streptavidin being immunogenic.

[0005] Pre-targeted radioimmunotherapy or imaging methods typically utilize scavengers or blockers, administered between the steps of antibody administration and the administration of a radiolabeled compound. The aim is to remove antibodies from the blood and / or block the binding sites of circulating antibodies to the radiolabeled compound (see, for example, Karacay et al., Bioconj. Chem., 13(5), 1054-1070 (2002)). The use of scavengers or blockers allows for the delivery of sufficient levels of radiation for effective treatment while limiting adverse toxicities, but the timing and dosage must be carefully chosen, and there is a risk of introducing adverse reactions such as immune responses with scavengers. Therefore, the use of the scavenging phase is a complex aspect of pre-targeted methods. Such methods are disclosed, for example, in WO2019 / 201959 and WO2019 / 202399.

[0006] Therefore, recent approaches to pre-targeted radioimmunotherapy aim to avoid the use of scavengers. Such approaches are disclosed in WO2021 / 009047, WO2022 / 152656, and WO2022 / 008688. However, there remains a need to provide a set of antibodies for use in such approaches that possess modified properties to allow for large-scale manufacturing while providing the desired antitumor effect and an acceptable safety profile. Attached Figure Description

[0007] Figure 1: Experimental design of scheme 1819

[0008] Figure 2: Pre-targeting with SPLIT antibody injection 212 24 hours after Pb-DOTAM administration, SCID mice carrying SC BxPC3 tumors showed pre-targeted [therapeutic effects]. 212 Biodistribution of Pb-DOTAM (periods 1–3). Radioactivity in organs and tissues is expressed as mean % IA / g ± standard deviation (SD; n = 3 or 5).

[0009] Figure 3: Mean tumor volume ± standard deviation (SD; n = 10) in the AD group of the SC BxPC3 model. According to the study design, the vertical dashed line indicates that all PRIT treatment groups received [treatment name missing]. 212 Pb-DOTAM (20 µCi).

[0010] Figure 4: Tumor growth curves of individual mice in the AD group of the SC BxPC3 model (n=10). According to the study design, the vertical dashed line indicates that all PRIT treatment groups received [treatment / treatment]. 212 Pb-DOTAM (20 µCi).

[0011] Figure 5: Kaplan-Meier curves show the survival rates in groups A through D of the SC BxPC3 model (n=10). According to the study design, the vertical dashed line indicates that all PRIT treatment groups received [the treatment / medication]. 212 Pb-DOTAM(20 µCi).

[0012] Figure 6: Mean change in body size (BW) after various treatments, expressed as a percentage of initial BW ± SD. According to the study design, the vertical dashed line indicates that all PRIT treatment groups received [treatment name missing]. 212 Pb-DOTAM (20 µCi).

[0013] Figure 7: Experimental design of scheme 1917

[0014] Figure 8: Pre-targeting with SPLIT antibody injection 212 24 hours after Pb-DOTAM administration, tumor-bearing SCID mice showed [significant changes / infections]. 212 Biodistribution of Pb (periods 1-3). Radioactivity levels in organs and tissues are expressed as mean % IA / g ± standard deviation (SD; n = 3).

[0015] Figure 9: Mean tumor volume ± standard deviation (SD; n = 10) in groups A and B of the SC BxPC3 model. According to the study design, the vertical dashed line indicates the PRIT treatment group. 212 Pb-DOTAM (20 µCi).

[0016] Figure 10: Tumor growth curves of individual mice in groups A and B of the SC BxPC3 model (n=10). According to the study design, the vertical dashed line indicates the PRIT treatment group. 212 Pb-DOTAM (20 µCi)

[0017] Figure 11: Kaplan-Meyer curves show survival rates in groups A and B of the SC BxPC3 model (n=10). According to the study design, the vertical dashed line indicates the PRIT treatment group. 212 Pb-DOTAM (20 µCi).

[0018] Figure 12: Mean change in body size (BW) after various treatments, expressed as a percentage of initial BW ± SD. According to the study design, the vertical dashed line indicates the PRIT treatment group. 212 Pb-DOTAM (20 µCi).

[0019] Figure 13A / B: Schematic diagram of antibody forms and names used herein. Full-length antibodies (“proteins”) are named with “P1A” (also referred to herein as “protein ID”), while the names of different antibody sub-parts, such as heavy and light chains (HC, LC), begin with “D1A”. For example, the antibody of the present invention with protein ID “P1AI0446” consists of a first heavy chain called D1AC4023, a second heavy chain called D1AK6882, and a light chain called D1AD3421. Terms used in this figure (e.g., “target”, “effecton”) have the meanings defined as in the specification. In particular, the term “effecton” encompasses antibodies chelated by DOTAM. 212 Pb. Detailed Implementation

[0020] This invention provides a group of antibodies comprising:

[0021] i) A first antibody that binds to an antigen expressed on the surface of a target cell, and further comprises a VH domain for an antigen-binding site of a radiolabeled compound, but does not comprise a VL domain for an antigen-binding site of a radiolabeled compound, or comprises a VL domain that does not effectively form an antigen-binding site of the radiolabeled compound together with the VH domain; and

[0022] ii) A second antibody that binds to the antigen expressed on the surface of target cells, and further comprises a VL domain for an antigen-binding site for a radiolabeled compound, but does not comprise a VH domain for an antigen-binding site for a radiolabeled compound, or comprises a VH domain that does not effectively form an antigen-binding site for the radiolabeled compound together with the VL domain.

[0023] The VH domain of the first antibody and the VL domain of the second antibody together form a functional antigen-binding site against a radiolabeled compound, wherein each of the VH domains or the VL domains is connected to the C-terminus of one of the two heavy chains of the first antibody or the second antibody via its N-terminal region through a peptide linker selected from GGSGGGGSGGGGSGGGSGG (SEQ ID NO: 35) or GGGGSGGGGSGGGSGGSGG (SEQ ID NO: 36).

[0024] I. Definition

[0025] Unless otherwise defined below, the terminology used herein is generally as it is used in the art.

[0026] 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 said antigen with sufficient affinity, such that the antibody can be used as a diagnostic and / or therapeutic agent targeting said antigen. In one aspect, for example, as measured by surface plasmon resonance (SPR), the degree to which the antibody binds to unrelated non-antigen proteins is less than about 10% of the degree to which the antibody binds to the antigen. In other aspects, the dissociation constant (Ki) of the antibody binding to the antigen expressed on the surface of the target cell... D ) is ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM or ≤ 0.001 nM (e.g., 10 -8 M or smaller, such as 10 -8 M to 10 -13 M, for example, 10-9 M to 10 -13 M). When the antibody K D When the concentration is 1 μM or less, the antibody is said to have "specifically bound" to the antigen expressed on the surface of the target cell. In some respects, the antibody binds to an epitope of the antigen that is conserved across said antigens from different species.

[0027] In some respects, unless otherwise indicated, “antigen expressed on the surface of target cells” (or simply “target”) is carcinoembryonic antigen-associated cell adhesion molecule 5 (“CEACAM5”, sometimes simply referred to as carcinoembryonic antigen or “CEA”), and refers to any naturally occurring CEACAM5 derived from any vertebrate, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). The term covers “full-length”, unprocessed CEACAM5, as well as any form of CEACAM5 produced by intracellular processing. The term also covers naturally occurring variants of CEACAM5, such as splice variants or allelic variants. In a particular respect, CEACAM5 is membrane-bound CEACAM5. In a further particular respect, CEACAM5 is human CEACAM5. See UniProt (www.uniprot.org) accession number P06731 (version 195) or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_004354.2. In a preferred embodiment, the antibody binds to human CEACAM5. In a further preferred embodiment, the antibody binds to membrane-bound CEACAM5.

[0028] The term "antigen-binding site of a radiolabeled compound" or "functional antigen-binding site of a radiolabeled compound" refers to an antigen-binding site containing both VH and VL domains, capable of binding a radiolabeled compound with sufficient affinity, allowing the antibody to be used as a diagnostic and / or therapeutic agent to associate the radiolabeled compound with the antibody. In one aspect, for example, as measured by surface plasmon resonance (SPR), the degree of binding of the antigen-binding site to unrelated non-antigen compounds is less than approximately 10% of the binding of the antibody to the radiolabeled compound. In some aspects, the dissociation constant (KD) of the antigen-binding site binding to the radiolabeled compound is ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM (e.g., 10 μM). -8 M or smaller, for example, 10 -8 M to 10 -13M, for example, 10 -9 M to 10 -13 The KD (kD) of the antigen binding site is preferably 100 pM, 50 pM, 20 pM, 10 pM, 5 pM, 1 pM or less, for example, 0.9 pM or less, 0.8 pM or less, 0.7 pM or less, 0.6 pM or less or 0.5 pM or less. For example, the functional binding site can bind the radiolabeled compound with a KD of about 1 pM to 1 nM, for example, about 1-10 pM, 1-100 pM, 5-50 pM, 100-500 pM or 500 pM to 1 nM. When the KD of the antigen binding site is 1 μM or less, the antigen binding site is referred to as "specifically binding" to the radiolabeled compound.

[0029] The terms "effecton" or "effect agent" mean a radiolabeled compound. The term "radiolabeled compound" means a chelated radioactive nucleus (or radioactive isotope), preferably an emitter of alpha or beta radiation, such as... 212 Pb, 212 Bi、 213 Bi、 90 Y、 177 Lu、 225 Ac、 211 At、 227 Th、 223 Ra. In one respect, chelated radioactive isotopes are chelated. 212 Pb. On the other hand, chelated radioactive isotopes mean those chelated by DOTAM. 212 Pb. The term "DOTAM" means 1,4,7,10-tetra(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane, which is a compound of the following formula:

[0030]

[0031] The term "antibody" encompasses a variety of antibody structures exhibiting the desired antigen-binding activity, including but not limited to monoclonal antibodies, polyclonal antibodies, and antibody fragments. Sometimes, the antibodies of this invention are specified as "SPLIT constructs," "SPLIT antibodies," or "CEA-Split-DOTAM VH / VL antibodies." As used herein, the term "SPLIT" means a split V domain bonding technique. In some aspects, the antibodies of this invention are monoclonal antibodies (mAbs). In some aspects, the antibodies of this invention belong to the IgG antibody class, more particularly to the IgG1, IgG2, IgG3, or IgG4 subclasses (isotypes). In some aspects, the antibodies of this invention are human or humanized monoclonal antibodies. In some aspects, the antibodies of this invention are human or humanized monoclonal IgG1 antibodies.

[0032] An "antibody fragment" is a molecule other than a complete antibody that contains a portion of the complete antibody and binds to the antigen bound by the complete antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; bisomatic antibodies; linear antibodies; single-chain antibody molecules (e.g., scFv and scFab); single-domain antibodies; and multispecific antibodies formed from antibody fragments. For a review of some antibody fragments, see Hollinger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0033] The terms “full-length antibody,” “intact antibody,” and “all antibody” are used interchangeably in this document and refer to antibodies with a structure substantially similar to that of natural antibodies.

[0034] As used herein, the term "monoclonal antibody" refers to an antibody derived from a substantially homologous population of antibodies, meaning that the individual antibodies contained in this population are identical and / or bind to the same epitopes, but exclude possible variant antibodies, such as those containing naturally occurring mutations or generated during the production of the monoclonal antibody formulation, which are generally present in small quantities. In contrast to polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen. Therefore, the modifier "monoclonal" indicates that the antibody is derived from a substantially homogeneous population of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies can be prepared using 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 the human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0035] "Isolated" antibodies are antibodies that have been separated from components of their natural environment. In some aspects, antibodies are purified to a purity greater than 95% or 99%, determined by methods such as electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC, affinity chromatography, size exclusion chromatography). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007). In some aspects, the antibodies provided by this invention are isolated antibodies.

[0036] "Humanized" antibodies are those comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In some aspects, humanized antibodies will contain substantially all at least one (and usually two) variable domains, wherein all or substantially all CDRs correspond to those of the non-human antibody ("parental" antibody), and all or substantially all FRs correspond to those of the human antibody. Such variable domains are referred to herein as "humanized variable regions." Humanized antibodies may optionally contain at least a portion of the antibody constant region derived from a human antibody. In some aspects, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., an antibody from which CDR residues are derived, i.e., the parental antibody), for example to restore or improve antibody specificity or affinity. The term "humanized" antibody also covers antibodies that contain certain mutations (e.g., amino acid substitutions) in their CDRs compared to those of the parental antibody. In some respects, compared to parental antibodies, humanized antibodies contain up to six (i.e., no, one, two, three, four, five, or six) amino acid substitutions in one or more of their CDRs. In some respects, humanized antibodies contain at least one CDR identical to the corresponding CDR of the parent antibody (i.e., without any amino acid substitutions compared to the parent antibody). In some respects, humanized antibodies contain at least three CDRs identical to the corresponding CDR of the parent antibody (particularly at least one heavy chain CDR (more particularly at least HCDR3) and at least one light chain CDR). The “humanized form” of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.

[0037] A "human antibody" is an antibody whose amino acid sequence corresponds to that of antibodies produced by humans or human cells, or to a non-human antibody derived from a complete human antibody library or other human antibody-coding sequences. This definition of a human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues. In some respects, human antibodies are derived from non-human transgenic mammals, such as mice, rats, or rabbits. In some respects, human antibodies are derived from hybridoma cell lines. Antibodies or antibody fragments isolated from human antibody libraries in this paper are also considered human antibodies or human antibody fragments.

[0038] The term "antigen-binding domain" refers to a portion of an antibody that contains a region that binds partially or completely to and is complementary to an antigen. The antigen-binding domain can be provided, for example, by one or more antibody variable domains (also called antibody variable regions). In a preferred aspect, the antigen-binding domain comprises an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).

[0039] The term "variable region" or "variable domain" refers to a domain of the antibody heavy or light chain involved in antibody-antigen binding. The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved frame regions (FRs) and complementarity-determining regions (CDRs). See, for example, Kindt et al., Kuby Immunology, p. 6. See, WH Freeman & Co., p. 91 (2007). A single VH or VL domain may be sufficient to confer antigen binding specificity. Furthermore, antibodies binding to a specific antigen can be separated using either the VH or VL domain from the antibody binding that antigen, to screen libraries containing complementary VL or VH domains. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0040] Glutamine or glutamate residues at the N-terminus of the antibody heavy or light chain can spontaneously convert to pyroglutamic acid (see, for example, Liu et al., Journal of Pharmaceutical Sciences 97, 2426-2447 (2008); Rehder et al., Journal of Chromatography A 1102, 164-175 (2006); Chelius et al., Anal Chem 78, 2370-2376 (2006)). Therefore, the variable regions or variable domains disclosed herein that contain glutamine (Q) or glutamate (E) amino acid residues at the N-terminus of the antibody heavy or light chain may contain N-terminal pyroglutamic acid (pyroE) residues instead of N-terminal Q or E residues. Similarly, antibody heavy or light chains disclosed herein that contain glutamine (Q) or glutamate (E) amino acid residues at the N-terminus may contain an N-terminal pyroglutamic acid (pyroE) residue instead of an N-terminal Q or E residue. Therefore, for each antibody heavy chain, light chain, or variable domain or region sequence disclosed herein containing an N-terminal Q or E residue, the corresponding sequence having an N-terminal pyroE residue is also covered.

[0041] As used herein, the term "complementarity-determining region" or "CDR" refers to each of the regions of an antibody's variable structural domains that are highly variable in sequence and determine antigen-binding specificity. Typically, an antibody contains six CDRs: three in the VH region (CDR-H1, CDR-H2, CDR-H3) and three in the VL region (CDR-L1, CDR-L2, CDR-L3). CDRs are defined by those skilled in the art using various methods / systems. These systems and / or definitions have been developed and refined over many years and include Kabat, Chothia, IMGT, AbM, and Contact.

[0042] "Frame" or "FR" refers to the variable domain residues other than the complementarity-determining region (CDR). A variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the CDR and FR sequences usually appear in the VH (or VL) as follows: FR1-CDR-H1(CDR-L1)-FR2-CDR-H2(CDR-L2)-FR3-CDR-H3(CDR-L3)-FR4.

[0043] The term "Fc region" used herein is used to define the C-terminal region of an immunoglobulin heavy chain that comprises at least a portion of a constant region. This term includes both native sequence Fc regions and variant Fc regions. In one aspect, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the C-terminus of the heavy chain. However, antibodies produced by host cells can undergo post-translational cleavage of one or more (particularly one or two) amino acids from the C-terminus of the heavy chain. Therefore, antibodies produced by host cells by expressing a specific nucleic acid molecule encoding the full-length heavy chain can comprise the full-length heavy chain, or the antibody can comprise a cleaved variant of the full-length heavy chain. This is particularly likely in cases where the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, Kabat EU number). Therefore, the C-terminal lysine (Lys447) or the C-terminal glycine (Gly446) and lysine (Lys447) of the Fc region may or may not be present.

[0044] Unless otherwise indicated, CDR residues and other residues (e.g., FR residues) in the variable domains are numbered in this paper according to the method described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0045] The "percentage of amino acid sequence identity (%)" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to those in the reference polypeptide sequence after aligning the candidate sequence with the reference polypeptide sequence and introducing vacancies (if necessary) to achieve the maximum percentage of sequence identity, without considering any conserved substitutions as part of the sequence identity. Alignment used to determine the percentage of amino acid sequence identity can be performed in various ways within the scope of the art, such as using publicly available computer software, such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software, or the FASTA package. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment across the full length of the sequences being compared. Alternatively, the sequence comparison computer program ALIGN-2 can be used to generate the percentage of identity values. The ALIGN-2 sequence comparison computer program was developed by Genentech, and the source code has been submitted with the user documentation to the US Copyright Office, Washington DC, 20559, registered under US Copyright Registry No. TXU510087 and described in WO 2001 / 007611. Unless otherwise stated, for the purposes of this document, amino acid sequence identity % values ​​were generated using the BLOSUM50 comparison matrix and the ggsearch program in FASTA package version 36.3.8c or later. The FASTA package was created by the following authors: WR Pearson and DJ Lipman (“Improved Tools for Biological Sequence Analysis”, PNAS 85 (1988) 2444-2448); WRPearson (“Effective protein sequence comparison” Meth. Enzymol. 266 (1996) 227-258); and Pearson et al. (Genomics 46 (1997) 24-36), and is publicly available at www.fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml or www.ebi.ac.uk / Tools / sss / fasta.Alternatively, sequences can be compared using a public server accessible via fasta.bioch.virginia.edu / fasta_www2 / index.cgi, using the ggsearch (global protein:protein) program with default options (BLOSUM50; open: -10; ext: -2; Ktup = 2) to ensure a global rather than local alignment. The percentage of amino acid identity is given in the output alignment header.

[0046] An "antigen binding site" refers to a site on an antigen-binding molecule that provides for interaction with an antigen, namely one or more amino acid residues. For example, the antigen binding site of an antibody contains amino acid residues from the complementarity-determining region (CDR). A "functional antigen binding site" is a binding site that performs its desired function. A binding site may become nonfunctional (or ineffective), for example, by modifying its amino acid residues. According to the present invention, a nonfunctional (or ineffective) binding site can be used to protect a portion of a functional binding site (see, for example, Figure 13B).

[0047] The terms "polynucleotide" or "nucleic acid molecule" include any compound and / or substance comprising a nucleotide polymer. 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 ester group. Typically, nucleic acid molecules are described by a base sequence, where the bases represent the primary structure (linear structure) of the nucleic acid molecule. Base sequences are typically represented from 5' to 3'. In this document, 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. Furthermore, the term nucleic acid molecule includes both sense and antisense strands, as well as 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 having derivatized sugar or phosphate backbone bonds or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for direct expression of antibodies used in this 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 may be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule, thereby injecting mRNA into a subject to generate antibodies in vivo (see, for example, Stadler et al. (2017) Nature Medicine 23:815-817, or EP 2101823 B1).

[0048] "Isolated" nucleic acid molecules refer to nucleic acid molecules that have been isolated from components of their natural environment. Isolated nucleic acid molecules include those contained in cells that normally contain said nucleic acid molecules, but which are located outside chromosomes or at chromosomal locations different from their natural chromosomal locations.

[0049] "Isolated polynucleotides (or nucleic acids) encoding antibodies" refers to one or more polynucleotide molecules that encode the heavy and light chains (or fragments thereof) of an antibody, including such polynucleotide molecules in a single or different vectors, and such polynucleotide molecules present at one or more locations in the host cell.

[0050] As used herein, the term "vector" refers to a nucleic acid molecule capable of carrying another nucleic acid linked to it. This term includes vectors that function as self-replicating nucleic acid structures, as well as vectors incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of the nucleic acid to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0051] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells in which exogenous nucleic acids have been introduced, including progeny of such cells. Host cells include “transformers” and “transformed cells,” which include primary transformed cells and progeny derived from those primary transformed cells, regardless of passage number. Progeny may not be identical to the nucleic acid contents of the parent cells and may contain mutations. This includes mutant progeny with the same function or biological activity as screened or selected in the original transformed cells. Host cells are any type of cell system that can be used to produce the antibodies of the present invention. Host cells include cultured cells, such as mammalian cultured cells, such as, to name just a few, HEK cells, CHO cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, or hybridoma cells, yeast cells, insect cells, and plant cells, and also include cells contained in transgenic animals, transgenic plants, or cultured plant or animal tissues. In one aspect, the host cell of the present invention is a eukaryotic cell, particularly a mammalian cell. In another aspect, the host cell is an isolated host cell. In yet another aspect, the host cell is not a cell found in the human body.

[0052] The terms “pharmaceutical composition” or “pharmaceutical formulation” refer to preparations in a form that allows the bioactivity of the active ingredient contained therein to be effective and that do not contain any other components that would have unacceptable toxicity to a subject to whom the pharmaceutical composition will be administered.

[0053] "Pharmaceutically acceptable carriers" refer to components in a pharmaceutical composition or formulation other than the active ingredient that are non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffer solutions, excipients, stabilizers, or preservatives.

[0054] As used herein, “treatment” (and its grammatical variations such as treat or treating) refers to an attempt to alter the natural course of a disease in the treated individual and can be performed for prevention or may be performed during a clinicopathological process. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or mitigating the disease state, and alleviating or improving prognosis. In some aspects, the antibodies of this invention are used to delay the development of disease or slow its progression.

[0055] The “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (such as cows, sheep, cats, dogs, and horses), primates (such as humans and non-human primates, such as monkeys), rabbits, and rodents (such as mice and rats). In some respects, the individual or subject is a human.

[0056] The “effective amount” of a pharmaceutical agent (e.g., a pharmaceutical composition) refers to the amount that is sufficient to effectively achieve the desired therapeutic or preventative outcome at the necessary dose for the necessary period of time.

[0057] The term "cancer" includes hematologic malignancies or solid tumors, preferably solid tumors. Solid tumors include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, stomach cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer.

[0058] As used herein, the term "Pb" or "lead" includes its ions, such as Pb(II). References to other metals also include their ions. Therefore, those skilled in the art will understand that, for example, the terms lead, Pb, 212 Pb or 203 Pb is intended to cover the ionic form of the element, particularly Pb(II).

[0059] As used in this article, the term "PRIT" means pre-targeted radioimmunotherapy.

[0060] II. Compositions and Methods

[0061] This invention is based on a group of antibodies comprising a first antibody and a second antibody, each of which can bind to an antigen on a target cell, but wherein a functional antigen-binding site for an effector is formed only when the first and second antibodies associate with each other. More specifically, this invention provides antibodies that bind to CEA (CEACAM5) and, when combined, to a radiolabeled compound as defined herein (such as, for example, an α-emitter chelated by DOTAM). Schematic diagrams and the names of the antibodies or antibody pairs used herein are shown in Figures 13A and 13B.

[0062] The inventors have discovered that modifications to the sequence of the CEA and / or DOTAM conjugates improve the properties of the antibodies of the present invention, resulting in better developability or manufacturability. In one aspect, such modifications are present in the receptor framework of the CEA and / or DOTAM conjugates and are reflected in the specific sequences disclosed herein. Improvements in the developability or manufacturability of the antibodies of the present invention are generally characterized by improved yield and / or stability. In one aspect, such improvements are characterized by higher aggregation temperatures (T0). agg The modifications may include a lower percentage of high molecular weight (“HMW”) products or improved hydrophilicity of the antibodies of the present invention, or a combination of several or all of these parameters. In one aspect, such modifications provide more hydrophilic DOTAM conjugates and / or improved CEA conjugates (T84.66v, or “new” T84.66). Therefore, the antibodies of the present invention exhibit improved developability and / or manufacturability, combined with other properties advantageous for therapeutic applications, such as those relating to immunogenicity, affinity, specificity, efficacy, and safety / tolerability. More specifically, in one aspect, any improvement to the antibodies of the present invention relates to antibodies disclosed in this art, such as those disclosed in WO2019 / 201959 and WO2021 / 009047.

[0063] In one aspect, the present invention provides a set of antibodies comprising:

[0064] i) A first antibody that binds to an antigen expressed on the surface of a target cell, and further comprises a VH domain for an antigen-binding site of a radiolabeled compound, but does not comprise a VL domain for an antigen-binding site of a radiolabeled compound, or comprises a VL domain that does not effectively form an antigen-binding site of the radiolabeled compound together with the VH domain; and

[0065] ii) A second antibody that binds to the antigen expressed on the surface of target cells, and further comprises a VL domain for an antigen-binding site for a radiolabeled compound, but does not comprise a VH domain for an antigen-binding site for a radiolabeled compound, or comprises a VH domain that does not effectively form an antigen-binding site for the radiolabeled compound together with the VL domain.

[0066] The VH domain of the first antibody and the VL domain of the second antibody together form a functional antigen-binding site against a radiolabeled compound, wherein each of the VH domains or the VL domains is connected to the C-terminus of one of the two heavy chains of the first antibody or the second antibody via its N-terminal region through a peptide linker selected from GGSGGGGSGGGGSGGGSGG (SEQ ID NO: 35) or GGGGSGGGGSGGGSGGSGG (SEQ ID NO: 36).

[0067] In another aspect, the present invention provides a set of antibodies as defined herein, wherein each of the VH or VL domains is connected via its N-terminal region to the C-terminus of one of the two heavy chains of a first or second antibody through a peptide linker GGSGGGGSGGGGSGGGG (SEQ ID NO:35).

[0068] In another aspect, the present invention provides a set of antibodies as defined herein, wherein each of the VH domain or VL domain is connected via its N-terminal region to the C-terminus of one of the two heavy chains of a first antibody or a second antibody through a peptide linker GGGGSGGGGSGGGSGGSGG (SEQ ID NO:36).

[0069] On the other hand, the present invention provides a group of antibodies as defined herein, wherein the radiolabeled compound comprises Pb-DOTAM (or 212 Pb-DOTAM).

[0070] In another aspect, the present invention provides a set of antibodies as defined herein, wherein the functional binding site for Pb-DOTAM binds with a binding affinity Kd value of 100 pM, 50 pM, 20 pM, 10 pM, 5 pM, 1 pM or less, for example, 0.9 pM or less, 0.8 pM or less, 0.7 pM or less, 0.6 pM or less, or 0.5 pM or less.

[0071] In another aspect, the present invention provides a set of antibodies as defined herein, wherein a functional binding site for Pb-DOTAM binds to both Pb-DOTAM and Bi-DOTAM.

[0072] In another aspect, the present invention provides a set of antibodies as defined herein, wherein the VH domain of the antigen-binding site for a radiolabeled compound comprises:

[0073] a) Heavy chain CDR2, having the amino acid sequence FIGRGDTYYASWAKG (SEQ ID NO: 42), or a variant thereof having at most 1, 2 or 3 substitutions in SEQ ID NO: 42, wherein such substitutions do not include Phe50, Asp56 and / or Tyr58, and optionally also do not include Gly52 and / or Arg54; or wherein the variant is the amino acid sequence AIGSRGDTAYASWAKG (SEQ ID NO: 47).

[0074] b) Heavy chain CDR3, having the amino acid sequence ERDPYGGGAYPPHL (SEQ ID NO:43), or a variant thereof having at most 1, 2, or 3 substitutions in SEQ ID NO:43, wherein these substitutions do not include Glu95, Arg96, Asp97, Pro98, and optionally also exclude Ala100C, Tyr100D, and / or Pro100E, and / or optionally also exclude Tyr99; and

[0075] c) Heavy chain CDR1, which is the amino acid sequence TYSMS (SEQ ID NO:41);

[0076] In another aspect, the present invention provides a set of antibodies as defined herein, wherein the VH domain of the antigen-binding site for the radiolabeled compound comprises: (a) VH CDR1, which comprises the amino acid sequence TYSMS (SEQ ID NO:41); (b) VH CDR2, which comprises the amino acid sequence FIGSRGDTYYASWAKG (SEQ ID NO:42); and (c) VH CDR3, which comprises the amino acid sequence ERDPYGGGAYPPHL (SEQ ID NO:43).

[0077] In another aspect, the present invention provides a set of antibodies as defined herein, 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: 44 and SEQ ID NO: 48, or a variant thereof comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 44 or SEQ ID NO: 48.

[0078] In another aspect, the present invention provides a group of antibodies as defined herein, wherein the VH domain of the antigen-binding site for a radiolabeled compound consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 44 or SEQ ID NO: 48.

[0079] In another aspect, the present invention provides a set of antibodies as defined herein, wherein residue A or AST is added to the C-terminus of the VH domain of the antigen-binding site of a radiolabeled compound.

[0080] On the other hand, the present invention provides a set of antibodies as defined herein, wherein the VL domain of the antigen-binding site for a radiolabeled compound comprises

[0081] a) The light chain CDR1, comprising the amino acid sequence QSSHSVYSDNDLA (SEQ ID NO:37) or a variant thereof having up to 1, 2 or 3 substitutions in SEQ ID NO:37, wherein such substitutions do not include Tyr28 and Asp32; or the amino acid sequence QSSHSVASDNRLA (SEQ ID NO:45).

[0082] b) The light chain CDR3, comprising the amino acid sequence LGGYDDESDTYG (SEQ ID NO:39) or a variant thereof having at most one, two, or three substitutions in SEQ ID NO:39, wherein these substitutions do not include Gly91, Tyr92, Asp93, Thr95c, and Tyr96.

[0083] c) Light chain CDR 2, comprising the amino acid sequence QASKLAS (SEQ ID NO: 38) or a variant thereof having at least 1, 2 or 3 substitutions in SEQ ID NO: 5, optionally excluding Gln50.

[0084] In another aspect, the present invention provides a set of antibodies as defined herein, wherein the VL domain of the antigen-binding site for the radiolabeled compound comprises: (a) LC CDR1 of the amino acid sequence QSSHSVYSDNDLA (SEQ ID NO:37); (b) LC CDR2 of the amino acid sequence QASKLAS (SEQ ID NO:38); and (c) LC CDR3 of the amino acid sequence LGGYDDESDTYG (SEQ ID NO:39).

[0085] In another aspect, the present invention provides a set of antibodies as defined herein, wherein the VL domain of the antigen-binding site of the radiolabeled compound comprises the amino acid sequence of SEQ ID NO: 40 or 46, or a variant thereof comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 40 or 46.

[0086] In another aspect, the present invention provides a set of antibodies as defined herein, wherein the VL domain of the antigen-binding site for a radiolabeled compound consists of the amino acid sequence of SEQ ID NO: 40 or 46.

[0087] In another aspect, the present invention provides a set of antibodies as defined herein, wherein a first antibody and a second antibody bind to the same epitope of an antigen expressed on the surface of a target cell.

[0088] In another aspect, the present invention provides a set of antibodies as defined herein, wherein a first antibody and a second antibody bind to different epitopes of an antigen.

[0089] In another aspect, the present invention provides a group of antibodies as defined herein, wherein the antigen expressed on the surface of target cells is a tumor-associated antigen.

[0090] In another aspect, the present invention provides a group of antibodies as defined herein, wherein the antigens expressed on the surface of target cells are 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.

[0091] In another respect, the present invention provides a group of antibodies as defined herein, wherein the antigen expressed on the surface of target cells is carcinoembryonic antigen (CEA).

[0092] In another aspect, the present invention provides a set of antibodies as defined herein, wherein one or both of the first antibody and the second antibody contain an antigen-binding site that binds to CEA, the antigen-binding site comprising...

[0093] The light chain variable region comprises: (a) LCCDR1 containing the amino acid sequence KASAAVGTYVA (SEQ ID NO:1); (b) LCCDR2 containing the amino acid sequence SASYRKR (SEQ ID NO:2); and (c) LCCDR3 containing the amino acid sequence HQYYTYPLFT (SEQ ID NO:3); and

[0094] The heavy chain variable region comprises: (d) HC CDR1 containing the amino acid sequence EFGMN (SEQ ID NO:6); (e) HC CDR2 containing the amino acid sequence WINTKTGEATYVEEFKG (SEQ ID NO:7); and (f) HC CDR3 containing the amino acid sequence WDFAYYVEAMDY (SEQ ID NO:8).

[0095] In another aspect, the present invention provides a set of antibodies as defined herein, 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: 9, or a variant thereof comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 9.

[0096] In another aspect, the present invention provides a set of antibodies as defined herein, wherein the antigen-binding site against CEA comprises a VH sequence having the amino acid sequence of SEQ ID NO: 9.

[0097] In another aspect, the present invention provides a set of antibodies as defined herein, wherein the first antibody comprises an antigen-binding site against CEA comprising a VL sequence, the VH sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4, or a variant thereof comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 4.

[0098] In another aspect, the present invention provides a set of antibodies as defined herein, wherein the antigen-binding site against CEA comprises a VL sequence having the amino acid sequence of SEQ ID NO: 4.

[0099] In another aspect, the present invention provides a set of antibodies as defined herein, wherein one or both of the first antibody and the second antibody contain an antigen-binding site that binds to CEA, the antigen-binding site comprising...

[0100] The light chain variable region comprises: (a) LC CDR1 containing the amino acid sequence RAGESVDIFGVGFLH (SEQ ID NO:11); (b) LC CDR2 containing the amino acid sequence RGSNRAT (SEQ ID NO:12); and (c) LC CDR3 containing the amino acid sequence QQTSEYPYT (SEQ ID NO:13); and

[0101] The heavy chain variable region comprises: (d) HC CDR1 containing the amino acid sequence DTYMH (SEQ ID NO:16); (e) HC CDR2 containing the amino acid sequence RIDPANGNSKYADSVKG (SEQ ID NO:17); and (f) HC CDR3 containing the amino acid sequence FGYYVSDYAMAY (SEQ ID NO:18).

[0102] In another aspect, the present invention provides a set of antibodies as defined herein, 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: 19, or a variant thereof comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 19.

[0103] In another aspect, the present invention provides a set of antibodies as defined herein, wherein the antigen-binding site against CEA comprises a VH sequence having the amino acid sequence of SEQ ID NO: 19.

[0104] In another aspect, the present invention provides a set of antibodies as defined herein, wherein the first antibody comprises an antigen-binding site against CEA comprising a VL sequence, the VH sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 14, or a variant thereof comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 14.

[0105] In another aspect, the present invention provides a set of antibodies as defined herein, wherein the antigen-binding site against CEA comprises a VL sequence having the amino acid sequence of SEQ ID NO: 14.

[0106] In another aspect, the present invention provides a set of antibodies as defined herein, wherein

[0107] The first antibody (“P1AI0446”) comprises or consists of the following: the first heavy chain of SEQ ID NO: 21, the second heavy chain of SEQ ID NO: 23, and the light chain of SEQ ID NO: 5; and

[0108] The second antibody (“P1AI0084”) comprises or consists of the following: the first heavy chain of SEQ ID NO: 22, the second heavy chain of SEQ ID NO: 24, and the light chain of SEQ ID NO: 5.

[0109] In another aspect, the present invention provides a set of antibodies as defined herein, wherein

[0110] The first antibody (“P1AJ6059”) comprises or consists of the following: a first heavy chain of SEQ ID NO: 25, a second heavy chain of SEQ ID NO: 26, and a light chain of SEQ ID NO: 15; and

[0111] The second antibody (“P1AJ6062”) comprises or consists of the following: the first heavy chain of SEQ ID NO: 27, the second heavy chain of SEQ ID NO: 28, and the light chain of SEQ ID NO: 15.

[0112] In another aspect, the present invention provides a set of antibodies as defined herein, wherein

[0113] The first antibody (“P1AI0034”) comprises or consists of the following: a first heavy chain of SEQ ID NO: 29, a second heavy chain of SEQ ID NO: 23, and a light chain of SEQ ID NO: 5; and

[0114] The second antibody (“P1AI1622”) comprises or consists of the following: the first heavy chain of SEQ ID NO: 30, the second heavy chain of SEQ ID NO: 31, and the light chain of SEQ ID NO: 5.

[0115] In another aspect, the present invention provides a set of antibodies as defined herein, wherein

[0116] The first antibody (“P1AJ6063”) comprises or consists of the following: a first heavy chain of SEQ ID NO: 32, a second heavy chain of SEQ ID NO: 26, and a light chain of SEQ ID NO: 15; and

[0117] The second antibody (“P1AJ6064”) comprises or consists of the following: the first heavy chain of SEQ ID NO: 33, the second heavy chain of SEQ ID NO: 34, and the light chain of SEQ ID NO: 15.

[0118] In some respects, the antibodies presented herein are humanized antibodies. Typically, non-human antibodies are humanized to reduce immunogenicity in humans while retaining the specificity and affinity of the parent non-human antibody. Generally, humanized antibodies contain one or more variable domains, wherein the CDR (or a portion thereof) is derived from the non-human antibody, and the FR (or a portion thereof) is derived from the human antibody sequence. Humanized antibodies may also optionally contain at least a portion of the human constant region. In some respects, some FR residues in the humanized antibody are substituted with corresponding residues from the non-human antibody (e.g., the antibody from which the CDR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0119] Humanized antibodies and their preparation methods have been reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described, for example, in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Natl. Acad. Sci. USA 86:10029-10033 (1989); US patents 5,821,337, 7,527,791, 6,982,321 and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity-determining region (SDR) transplantation); Padlan, Mol. Immunol. 28:489-498 (1991) (describing “surface reshaping”); Dall'Acqua Methods 36:43-60 (2005) (describes “FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describes a “guided selection” approach for FR shuffling).

[0120] Human frame regions that can be used for humanization include, but are not limited to: frame regions selected using a “best-fit” method (see, for example, Sims et al., J. Immunol. 151:2296 (1993)); frame regions derived from the common sequences of human antibodies from specific subgroups of light or heavy chain variable regions (see, for example, Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al., J. Immunol., 151:2623 (1993)); human maturation (somatic mutation) frame regions or human germline frame regions (see, for example, Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and frame regions derived from screening FR libraries (see, for example, Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok). et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0121] Polynucleotide and Recombinant Methods

[0122] The present invention further provides a set of nucleic acids that express a set of antibodies as defined herein.

[0123] In one aspect, the present invention provides an isolated polynucleotide encoding the antibody of the present invention. The isolated polynucleotide may be a single polynucleotide or multiple polynucleotides.

[0124] The polynucleotide encoding the antibody of the present invention can be expressed as a single polynucleotide encoding the entire antibody or as multiple (e.g., two or more) polynucleotides co-expressed. The polypeptides encoded by the co-expressed polynucleotides can associate via, for example, disulfide bonds or other means to form a functional antibody. For example, the light chain portion of the antibody can be encoded by a polynucleotide separate from the portion of the antibody comprising the heavy chain. When co-expressed, the heavy chain polypeptide will associate with the light chain polypeptide to form the antibody. In another example, an antibody comprising one subunit of two Fc domain subunits and optionally a portion of one or more Fab molecules can be encoded by a polynucleotide separate from the antibody comprising the other subunit of the two Fc domain subunits and optionally a portion of a Fab molecule. When co-expressed, the Fc domain subunits will associate to form the Fc domain.

[0125] In some respects, the isolated polynucleotides encode the complete antibody molecule according to the invention as described herein. In other respects, the isolated polynucleotides encode polypeptides contained in antibodies according to the invention as described herein.

[0126] In some respects, the polynucleotide or nucleic acid is DNA. In other respects, the polynucleotide of the present invention is RNA, for example, in the form of messenger RNA (mRNA). The RNA of the present invention can be single-stranded or double-stranded.

[0127] In another aspect, the present invention provides an expression vector or a set of expression vectors comprising the set of nucleic acids described above. In yet another aspect, the present invention provides a host cell or a set of host cells comprising the expression vector or a set of expression vectors.

[0128] For recombinant production, one or more polynucleotides encoding antibodies, such as those described above, are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such polynucleotides can be readily isolated and sequenced using conventional methods. In some aspects, a vector, particularly an expression vector, is provided that contains the polynucleotides of the present invention (i.e., a single polynucleotide or multiple polynucleotides). Expression vectors containing the coding sequence of an antibody and appropriate transcription / translation control signals can be constructed using methods well known to those skilled in the art. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination / genetic recombination. See, for example, the techniques described in the following literature: Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY (1989); and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, NY (1989). The expression vector can be a plasmid, part of a virus, or a fragment of nucleic acid. The expression vector includes an expression cassette into which the polynucleotide encoding the antibody (i.e., the coding region) is operatively associated with a promoter and / or other transcription or translation control elements. As used herein, a “coding region” is a portion of a nucleic acid that consists of codons that translate into amino acids. Although a “stop codon” (TAG, TGA, or TAA) is not translated into amino acids, it can be considered part of the coding region (if present), while any flanking sequences, such as promoters, ribosome binding sites, transcription terminators, introns, 5' and 3' untranslated regions, etc., are not part of the coding region. Two or more coding regions may be present in a single polynucleotide construct (e.g., on a single vector) or in separate polynucleotide constructs (e.g., on separate (different) vectors). Furthermore, any vector may contain a single coding region or may contain two or more coding regions; for example, the vectors of the present invention may encode one or more polypeptides that are cleaved into the final protein post-translation or during translation via proteolytic cleavage. Additionally, the vectors, polynucleotides, or nucleic acids of the present invention may encode heterologous coding regions that are fused or not fused with a polynucleotide or a variant or derivative thereof encoding an antibody of the present invention. Heterologous coding regions include, but are not limited to, specialized elements or motifs, such as secretory signal peptides or heterologous functional domains. Operable association occurs when the coding region of a gene product (e.g., a polypeptide) is associated in a certain way with one or more regulatory sequences so that the expression of the gene product is under the influence or control of the regulatory sequences.If the induction of promoter function leads to the transcription of mRNA encoding the desired gene product, and the nature of the connector between the two DNA segments does not interfere with the ability of the expression regulatory sequence to direct the expression of the gene product, nor with the ability of the DNA template to be transcribed, then the two DNA segments (e.g., a polypeptide coding region and a promoter associated with it) are “operably associated.” Therefore, if a promoter can influence the transcription of the nucleic acid, the promoter region will operably associate with the nucleic acid encoding the polypeptide. The promoter can be a cell-specific promoter that directs the substantial transcription of DNA only in a predetermined cell. In addition to promoters, other transcriptional control elements, such as enhancers, operons, repressors, and transcription termination signals, can operably associate with polynucleotides to direct cell-specific transcription. Suitable promoters and other transcriptional control regions are disclosed herein. Various transcriptional control regions are known to those skilled in the art. These transcriptional control regions include, but are not limited to, transcriptional control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (e.g., the immediate early promoter binding intron-A), simian virus 40 (e.g., the early promoter), and retroviruses (e.g., Raoult sarcoma virus). Other transcriptional control regions include those derived from vertebrate genes (such as actin, heat shock protein, bovine growth hormone, and rabbit β-globin), as well as other sequences capable of controlling gene expression in eukaryotic cells. Other suitable transcriptional control regions include tissue-specific promoters and enhancers, and inducible promoters (e.g., tetracycline-inducible promoters). Similarly, various translational control elements are known to those skilled in the art. These translational control elements include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from viral systems (particularly internal ribosome entry sites, or IRES, also known as CITE sequences). Expression cassettes may also include other features such as origin of replication and / or chromosomal integration elements, such as retroviral long terminal repeats (LTRs) or adeno-associated virus (AAV) inverted terminal repeats (ITRs).

[0129] The polynucleotide and nucleic acid coding regions of the present invention can associate with additional coding regions encoding secretory peptides or signal peptides, which in turn direct the secretion of polypeptides encoded by the polynucleotides of the present invention. For example, if antibody secretion is desired, DNA encoding a signal sequence can be placed upstream of the nucleic acid of an antibody or fragment thereof of the present invention. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence that is cleaved from the mature protein once the protein chain has begun to export across the rough endoplasmic reticulum. Those skilled in the art know that polypeptides secreted by vertebrate cells typically have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the translated polypeptide to produce the secretory or “mature” form of the polypeptide. In some aspects, natural signal peptides (e.g., immunoglobulin heavy or light chain signal peptides) or functional derivatives of the sequence that retain the ability to direct the secretion of polypeptides with which they operatively associate are used. Alternatively, heterologous mammalian signal peptides or functional derivatives thereof can be used. For example, the wild-type leader sequence can be replaced by the leader sequence of human tissue plasminogen activator (TPA) or mouse β-glucuronidase.

[0130] DNA encoding short protein sequences (which can be used to facilitate subsequent purification (e.g., histidine tagging) or to help label antibodies) can be contained inside or at the end of the antibody (fragment) encoding a polynucleotide.

[0131] In another aspect, host cells comprising the polynucleotides (i.e., a single polynucleotide or multiple polynucleotides) of the present invention are provided. In some aspects, host cells comprising the vectors of the present invention are provided. The polynucleotides and vectors may be incorporated, alone or in combination, into any features described herein with respect to the polynucleotides and vectors, respectively. In some such aspects, the host cell comprises one or more vectors (e.g., transformed or transfected therein) that contain one or more polynucleotides encoding (a portion of) an antibody of the present invention. As used herein, the term “host cell” refers to any kind of cellular system that can be engineered to produce the antibody or fragment thereof of the present invention. Host cells suitable for replicating and supporting antibody expression are well known in the art. Such cells can be appropriately transfected or transduced with specific expression vectors, and large quantities of cells containing the vectors can be grown for inoculation of large-scale fermenters to obtain sufficient quantities of antibody for clinical application. Suitable host cells include prokaryotic microorganisms, such as *Escherichia coli*, or various eukaryotic cells, such as Chinese hamster ovary cells (CHO), insect cells, etc. For example, peptides can be produced in bacteria, particularly when glycosylation is not required. After expression, peptides can be separated from bacterial cell paste in a soluble fraction and can be further purified. Besides prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are also suitable cloning or expression hosts for vectors encoding peptides, including fungal and yeast strains whose glycosylation pathways have been "humanized," resulting in the production of peptides with partially or fully human glycosylation patterns. See Gerngross, Nat Biotech 22, 1409-1414 (2004) and Li et al., Nat Biotech 24, 210-215 (2006). Suitable host cells for expressing (glycosylated) peptides also originate from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains have been identified that can be used with insect cells, particularly for transfecting Spodoptera frugiperda cells. Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLATNIBODIES for producing antibodies in transgenic plants). TM(Technology). Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension may be useful. Other examples of useful mammalian host cell lines include the monkey kidney CV1 line (COS-7) transformed from SV40; human embryonic kidney lines (293 or 293T cells, as described, for example, in Graham et al., J GenVirol 36, 59 (1977)); young hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells, as described, for example, in Mather, Biol Reprod 23, 243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); Buffalo rat hepatocytes (BRL3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor cells (MMT 060562); and TRI cells (as described, for example, in Mather et al., Annals NYAcad Sci 383, 44-68 (1982)). (As described above), MRC5 cells, and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including dhfr - CHO cells (Urlaub et al., Proc Natl Acad Sci USA 77, 4216 (1980)); and myeloma cell lines such as YO, NSO, P3X63, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for protein production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (edited by BKCLo, Humana Press, Totowa, NJ), pp. 255–268 (2003). Host cells include cultured cells, such as mammalian cultured cells, yeast cells, insect cells, bacterial cells, and plant cells, to name just a few, as well as cells contained in transgenic animals, transgenic plants, or cultured plant or animal tissues. In some respects, host cells are eukaryotic cells, particularly mammalian cells, such as Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, or lymphoid cells (e.g., YO, NSO, Sp20 cells). In some respects, the host cell is a separate host cell. In other respects, the host cell is not a cell within the human body.

[0132] Standard techniques for expressing exogenous genes in these systems are known in the art. Cells expressing polypeptides containing heavy or light chains of antibodies, such as antigen-binding domains, can be engineered to also express another antibody chain, such that the expressed product is an antibody having both heavy and light chains.

[0133] In one aspect, a method for producing an antibody according to the invention is provided, wherein the method comprises culturing a host cell containing a polynucleotide encoding an antibody as provided herein under conditions suitable for antibody expression, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0134] The components of the (multispecific) antibody of the present invention can be genetically fused together. The (multispecific) antibody can be engineered such that its components fuse directly or indirectly with each other via adapter sequences. The composition and length of the adapter can be determined according to methods well known in the art, and the efficacy of the adapter can be tested. Examples of adapter sequences between different components of the (multispecific) antibody are provided herein. Additional sequences (e.g., endopeptidase recognition sequences) may be included, if desired, to incorporate cleavage sites to separate the fused components.

[0135] The antibodies prepared as described herein can be purified using techniques known in the art, such as high-performance liquid chromatography (HPLC), ion-exchange chromatography (IEC), gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The specific conditions used for purifying a particular protein will depend in part on factors such as net charge, hydrophobicity, and hydrophilicity, and will be apparent to those skilled in the art. For affinity chromatography purification, antibodies, ligands, receptors, or antigens that bind to the antibody can be used. For example, for affinity chromatography purification of the antibodies of the present invention, a matrix having protein A or protein G can be used. Essentially as described in the examples, antibodies can be separated using sequential protein A or G affinity chromatography and size exclusion chromatography. The purity of the antibody can be determined by any of a variety of well-known analytical methods, including gel electrophoresis, high-performance liquid chromatography, etc.

[0136] Composition, Formulation and Administration

[0137] In one aspect, the present invention provides a pharmaceutical composition comprising a group of antibodies as defined herein and pharmaceutically acceptable excipients.

[0138] In another aspect, the present invention provides a pharmaceutical composition comprising a first or second antibody as defined herein and a pharmaceutically acceptable excipient.

[0139] In another aspect, the present invention provides a pharmaceutical product for administration to a patient with cancer in combination, concurrently or sequentially, the product comprising: A) a pharmaceutical composition as a first component comprising a first antibody; and B) a pharmaceutical composition as a second component comprising a second antibody, wherein the antibody is as defined herein.

[0140] In another aspect, the present invention provides a pharmaceutical product for administration to a patient with cancer in combination, concurrently or sequentially, the product comprising: A) a pharmaceutical composition as a first component comprising a first antibody as defined herein; and B) a pharmaceutical composition as a second component comprising a second antibody as defined herein.

[0141] The pharmaceutical compositions of the present invention comprise an effective amount of antibody lysed or dispersed in a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" means that the molecular entity and composition are generally non-toxic to the recipient at the doses and concentrations employed, i.e., do not produce adverse, allergic, or other adverse reactions when administered to animals (e.g., humans) as appropriate. Preparation of pharmaceutical compositions containing antibodies and optionally additional active ingredients will be known to those skilled in the art according to this disclosure, as illustrated in Remington's Pharmaceutical Sciences, 18th edition, Mack Printing Company, 1990, which is incorporated herein by reference. Furthermore, for animal (e.g., human) administration, it should be understood that the preparations should meet the sterility, pyrogenicity, general safety, and purity standards required by the FDA Office of Biostandards or other relevant national authorities. Preferred compositions are lyophilized formulations or aqueous solutions. As used herein, "pharmaceuticalally acceptable carriers" include any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delay agents, salts, preservatives, antioxidants, proteins, pharmaceuticals, pharmaceutical stabilizers, polymers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, and similar substances, as well as combinations thereof, as should be known to a person skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th edition, Mack Printing Company, 1990, pp. 1289-1329, which is incorporated herein by reference). The use of such carriers in pharmaceutical compositions is contemplated, except in cases where any conventional carrier is incompatible with the active ingredient.

[0142] The antibodies (and any other therapeutic agents) of the present invention can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal administration, and, if desired, for local treatment or intralesional application. Parenteral administration includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Administration can be carried out by any suitable route, such as by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term.

[0143] Parenteral compositions include those designed for administration by injection (e.g., subcutaneous, intradermal, intralesional, intravenous, intra-arterial, intramuscular, intrathecal, or intraperitoneal injection). For injection, the antibodies of the present invention can be formulated in aqueous solutions, particularly in physiologically compatible buffers (e.g., Hanks' solution, Ringer's solution, or saline buffer). The solution may contain a formulatory agent, such as a suspending agent, stabilizer, and / or dispersant. Alternatively, the antibody may be in powder form for use prior to preparation with a suitable solvent (e.g., sterile pyrogen-free water). Sterile injectable solutions are prepared by incorporating the antibodies of the present invention in the desired amount with various other components listed below into a suitable solvent as needed. For example, sterility can be readily achieved by filtration through a sterile filter membrane. Typically, dispersions are prepared by incorporating various sterilized active ingredients into a sterile solvent containing a base dispersion medium and / or other components. In the case of sterile powders used to prepare sterile injectable solutions, suspensions, or emulsions, the preferred preparation method is vacuum drying or lyophilization, which produces a powder of the active ingredient from a previously sterile filtered liquid medium, plus any additional desired components. If necessary, the liquid medium should be appropriately buffered, and the liquid diluent should be isotonicized with sufficient saline or glucose before injection. The composition must be stable under the manufacturing and storage conditions and preserved against contamination by microorganisms such as bacteria and fungi. It should be understood that endotoxin contamination should be kept to a safe level, for example, below 0.5 ng / mg protein. Suitable pharmaceutically acceptable carriers include, but are not limited to: buffers such as histidine, phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethyl ammonium chloride; benzalkonium chloride; benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol, and m-cresol); and low molecular weight (less than about 10). (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 dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming ions, such as sodium; metal complexes (e.g., zinc protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG). Aqueous injection suspensions may contain compounds that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, dextran, etc. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of high-concentration solutions.In addition, suspensions of the active compounds can be prepared into suitable oily injectable suspensions. Suitable lipophilic solvents or media include fatty oils, such as sesame oil; or synthetic fatty acid esters, such as ethyl oleate or triglycerides; or liposomes.

[0144] Pharmaceutical compositions containing the antibodies of the present invention can be prepared by conventional methods of mixing, dissolving, emulsifying, encapsulating, embedding, or lyophilizing. The pharmaceutical compositions can be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants that facilitate the processing of proteins into pharmaceutically usable formulations. Appropriate formulations depend on the chosen route of administration.

[0145] Antibodies can be formulated into compositions in the form of free acids or bases, neutral or salts. Pharmaceutically acceptable salts are those that essentially retain the biological activity of the free acid or free base. These pharmaceutically acceptable salts include acid addition salts, such as those formed with the free amino group of a protein composition, or those formed with inorganic acids (such as hydrochloric acid or phosphoric acid) or organic acids (such as acetic acid, oxalic acid, tartaric acid, or mandelic acid). Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide; or organic bases such as isopropylamine, trimethylamine, histidine, or procaine. Pharmaceutically acceptable salts tend to be more soluble in aqueous and other protic solvents compared to their corresponding free base forms.

[0146] Treatment methods and uses

[0147] The antibodies provided herein, or any of a group of antibodies or antibody pairs, can be used in therapeutic applications. The antibodies of this invention can be used as radiotherapy agents, for example, for treating cancers, particularly cancers characterized by the expression of CEACAM5, such as colorectal cancer.

[0148] Therefore, in one aspect, the present invention provides a method for pre-targeted radioimmunotherapy, the method comprising:

[0149] i) administering to a subject a group of antibodies according to any one of claims 1 to 38, wherein the first antibody and the second antibody are administered simultaneously or sequentially, wherein these antibodies bind to the target antigen and are localized on the surface of cells expressing the target antigen; and wherein the association of the first antibody and the second antibody forms a functional binding site against the radiolabeled compound; and

[0150] ii) Subsequently, a radiolabeled compound is applied, wherein the radiolabeled compound binds to a functional binding site for the radiolabeled compound.

[0151] In another aspect, the present invention provides a method as described above, wherein the radiolabeled compound is led-DOTAM (Pb-DOTAM or 212 Pb-DOTAM).

[0152] For use in therapeutic applications, the antibodies of this invention will be formulated, administered, and applied in accordance with good medical practice. Factors considered in this context include the specific disease being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the disease, the site of delivery of the drug, the method of administration, the timing of administration, and other factors known to medical practitioners.

[0153] In some aspects, antibodies of the present invention are provided for use as medicines. In other aspects, antibodies of the present invention are provided for treating diseases. In some aspects, antibodies of the present invention are provided for treatment methods. In some aspects, the present invention provides an antibody of the present invention for treating a disease in an individual in need. In some aspects, the present invention provides a method of using an antibody to treat an individual suffering from a disease, the method comprising administering an effective amount of the antibody to the individual. In some aspects, the disease is a proliferative disorder. In some aspects, the disease is cancer, particularly cancer expressing CEACAM5, such as colorectal cancer. In some aspects, if the disease to be treated is cancer, the method further comprises administering an effective amount of at least one other therapeutic agent, such as an anticancer agent, to the individual. The "individual" according to any of the foregoing aspects can be a mammal, preferably a human.

[0154] In another aspect, the present invention provides the use of the antibody of the present invention in the manufacture or preparation of a medicament. In some aspects, the medicament is used to treat a disease in an individual in need. In another aspect, the medicament is used in a method of treating a disease, the method comprising administering an effective amount of the medicament to an individual suffering from the disease. In some aspects, the disease is a proliferative disorder. In some aspects, the disease is cancer, particularly cancer expressing CEACAM5, such as colorectal cancer. In some aspects, the method further comprises administering an effective amount of at least one other therapeutic agent, such as an anticancer agent, to the individual (if the disease to be treated is cancer). The “individual” according to any of the foregoing aspects can be a mammal, preferably a human.

[0155] In a further aspect, the present invention provides a medicament suitable for treating a disease, comprising the antibody of the present invention. In some aspects, the medicament is suitable for treating a disease in an individual in need. In still other aspects, a method of treating a disease with the medicament comprises administering an effective amount of the medicament to an individual suffering from the disease. In some aspects, the disease is a proliferative disorder. In some aspects, the disease is cancer, particularly cancer expressing CEACAM5, such as colorectal cancer. In some aspects, the treatment or method of treatment further comprises administering an effective amount of at least one other therapeutic agent, such as an anticancer agent, to the individual (if the disease to be treated is cancer). The “individual” according to any of the foregoing aspects can be a mammal, preferably a human.

[0156] In a further aspect, the present invention provides a method for treating a disease. In some aspects, the method comprises administering a therapeutically effective amount of an antibody of the present invention to an individual suffering from such a disease. In some aspects, the individual is administered a composition comprising an antibody of the present invention in a pharmaceutically acceptable form. In some aspects, the disease is a proliferative disorder. In some aspects, the disease is cancer, particularly cancer expressing CEACAM5, such as colorectal cancer. In some aspects, if the disease to be treated is cancer, the method further comprises administering an effective amount of at least one other therapeutic agent, such as an anticancer agent, to the individual. The “individual” according to any of the foregoing aspects can be a mammal, preferably a human.

[0157] In some respects, the diseases requiring treatment are proliferative disorders, such as cancer, preferably solid tumors. Non-limiting examples of cancer include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, stomach cancer, prostate cancer, leukemia, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer. Other proliferative disorders include, but are not limited to, tumors located in the following sites: abdomen, bones, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal glands, parathyroid glands, pituitary gland, testes, ovaries, thymus, thyroid gland), eyes, head and neck, nervous system (central and peripheral nervous systems), lymphatic system, pelvis, skin, soft tissue, spleen, chest, and genitourinary system. Precancerous conditions or lesions and metastases are also included. In some respects, cancer is cancer expressing CEACAM5. In some respects, cancers are categorized into groups consisting of: colorectal cancer, stomach cancer, pancreatic cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), and breast cancer. Technicians readily recognize that in many cases, antibodies may not provide a cure, but may only offer partial benefit. In some respects, physiological changes that offer certain benefits are also considered to have a therapeutic benefit. Therefore, in some respects, the amount of antibody providing physiological changes is considered an "effective amount." Subjects, patients, or individuals requiring treatment are typically mammals, and more specifically, humans.

[0158] In some respects, an effective amount of the antibodies of the present invention will be administered to an individual to treat a disease.

[0159] For the prevention or treatment of disease, the appropriate dosage of the antibody of the present invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the route of administration, the patient's weight, the type of antibody, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous or concurrent therapeutic interventions, the patient's clinical history and response to the antibody, and the judgment of the attending physician. In any case, the practitioner responsible for administration will determine the concentration and appropriate dosage of the active ingredient in the composition for the individual subject. Various dosing schedules are considered herein, including but not limited to single or multiple administrations at various time points, bolus administration, and pulse infusion.

[0160] Antibodies are administered to patients either once or as part of a series of treatments. Depending on the type and severity of the disease, an antibody of approximately 1 µg / kg to 15 mg / kg (e.g., 0.1 mg / kg - 10 mg / kg) may be an initial candidate dose administered to the patient, for example, by a single or multiple administrations alone or by continuous infusion. Depending on the factors mentioned above, a typical daily dose range may be from approximately 1 µg / kg to 100 mg / kg or more. For repeated administration over several days or longer, treatment will generally continue until the desired suppression of disease symptoms is achieved, depending on the condition. Progression of this therapy can be easily monitored using routine techniques and assays.

[0161] The antibodies of the present invention will generally be used in an amount that effectively achieves the intended purpose. For the treatment or prevention of a condition, the antibodies of the present invention or their pharmaceutical compositions will be administered or applied in an effective amount.

[0162] For systemic administration, the effective dose can be initially estimated based on in vitro assays, such as cell culture assays. The dose can then be formulated in animal models to achieve the IC50, as determined in cell culture. 50 The circulating concentration range, including [specific concentration range]. This type of information can be used to more accurately determine the useful dose for humans.

[0163] The initial dose can also be estimated using techniques well known in the art, based on in vivo data (e.g., animal models).

[0164] The dosage and interval can be individually adjusted to provide sufficient plasma antibody levels to maintain therapeutic efficacy. Therapeuticly effective plasma levels can be achieved by administering multiple doses daily. Plasma levels can be measured, for example, by HPLC.

[0165] Effective doses of the antibodies of this invention will generally provide therapeutic benefit without causing significant toxicity. The toxicity and therapeutic efficacy of the antibodies can be determined using standard pharmaceutical methods in cell culture or laboratory animals. Cell culture assays and animal studies can be used to determine the LD50. 50 (50% of the lethal dose) and ED 50 (The dose that is effective in 50% of the population). The dose ratio between toxicity and efficacy is the therapeutic index, which can be expressed as the ratio LD50. 50 / ED 50 Antibodies exhibiting a high therapeutic index are preferred. In some respects, the antibodies according to the invention exhibit a high therapeutic index. Data obtained from cell culture assays and animal studies can be used to formulate a range of doses suitable for humans. The doses are preferably within the range of ED (excessive toxicity) including those with little or no toxicity. 50The dosage can vary within this range depending on various factors, such as the dosage form used, the route of administration, and the patient's condition. The exact formulation, route of administration, and dosage can be selected by an individual physician based on the patient's condition (see, for example, Fingl et al., 1975, in: The Pharmacological Basis of Therapeutics, Chapter 1, page 1, the entire contents of which are incorporated herein by reference).

[0166] The attending physician of a patient treated with the antibody of this invention will know how and when to terminate, interrupt, or adjust administration due to toxicity, organ dysfunction, etc. Conversely, if the clinical response is inadequate (excluding toxicity), the attending physician will also know to adjust the treatment to a higher level. The dosage administered in the management of the target disease will vary depending on the severity of the disease being treated, the route of administration, etc. For example, the severity of the disease can be assessed in part by standard prognostic assessment methods. Furthermore, the dosage and possible dosing frequency will also vary based on the individual patient's age, weight, and response.

[0167] The antibodies of the present invention can be administered in combination with one or more other therapeutic agents. For example, the antibodies of the present invention can be administered together with at least one additional therapeutic agent. The term "therapeutic agent" includes any agent administered to treat symptoms or diseases of an individual requiring such treatment. Such additional therapeutic agents may contain any active ingredient suitable for the specific disease being treated, preferably active ingredients having complementary activities that do not adversely affect each other. In some aspects, the additional therapeutic agent is an immunomodulator, a cell growth inhibitor, a cell adhesion inhibitor, a cytotoxic agent, an apoptosis activator, or an agent that increases the sensitivity of cells to apoptosis inducers. In some aspects, the additional therapeutic agent is an anticancer agent, such as a microtubule disruptor, antimetabolite, topoisomerase inhibitor, DNA intercalating agent, alkylating agent, hormone therapy, kinase inhibitor, receptor antagonist, tumor cell apoptosis activator, or antiangiogenic agent.

[0168] Other such agents are appropriately available in combinations of amounts effective for the intended purpose. The effective amount of these other agents depends on the amount of antibody used, the type of disease or treatment, and other factors discussed above. Antibodies are typically used at the same dosage and route of administration as described herein, or at approximately 1% to 99% of the dosage described herein, or at any dosage and route of administration empirically / clinically determined to be appropriate.

[0169] Such combination therapies include combined administration (where two or more therapeutic agents are included in the same or different compositions) and single administration. In the case of single administration, the antibody of the present invention may be administered before, simultaneously with and / or after administration of additional therapeutic agents and / or adjuvants.

[0170] Products

[0171] In another aspect of the invention, an article is provided containing a substance that can be used to treat, prevent, and / or diagnose the aforementioned conditions. The article includes a container and a label or packaging insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, intravenous (IV) solution bags, etc. The container can be formed from a variety of materials such as glass or plastic. The container contains a composition that, on its own or in combination with another composition, is effective for treating, preventing, and / or diagnosing the condition, and the container may have a sterile inlet (e.g., the container may be an IV solution bag or vial with a stopper capable of being punctured by a hypodermic needle). At least one active agent in the composition is an antibody of the present invention. The label or packaging insert indicates that the composition is for treating the selected condition. Furthermore, the article may include (a) a first container containing a composition comprising the antibody of the present invention; and (b) a second container containing a composition comprising additional cytotoxic agents or other therapeutic agents. The article of this aspect of the invention may also include a packaging insert indicating that the composition is for treating a specific condition. Alternatively or additionally, the article may further comprise a second (or third) container containing pharmaceutically acceptable buffer solutions, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and glucose solution. It may further include other materials desired from a commercial and user perspective, including additional buffer solutions, diluents, filters, needles, and syringes.

[0172] The following set of terms defines the present invention and its preferred aspects and embodiments:

[0173] 1. A group of antibodies comprising:

[0174] i) A first antibody that binds to an antigen expressed on the surface of a target cell, and further comprises a VH domain for an antigen-binding site of a radiolabeled compound, but does not comprise a VL domain for an antigen-binding site of a radiolabeled compound, or comprises a VL domain that does not effectively form an antigen-binding site of the radiolabeled compound together with the VH domain; and

[0175] ii) A second antibody that binds to the antigen expressed on the surface of target cells, and further comprises a VL domain for an antigen-binding site for a radiolabeled compound, but does not comprise a VH domain for an antigen-binding site for a radiolabeled compound, or comprises a VH domain that does not effectively form an antigen-binding site for the radiolabeled compound together with the VL domain.

[0176] The VH domain of the first antibody and the VL domain of the second antibody together form a functional antigen-binding site against a radiolabeled compound, wherein each of the VH domains or the VL domains is connected to the C-terminus of one of the two heavy chains of the first antibody or the second antibody via its N-terminal region through a peptide linker selected from GGSGGGGSGGGGSGGGSGG (SEQ ID NO: 35) or GGGGSGGGGSGGGSGGSGG (SEQ ID NO: 36).

[0177] 2. A group of antibodies according to claim 1, wherein each of the VH domain or the VL domain is connected via its N-terminal region to the C-terminus of one of the two heavy chains of the first antibody or the second antibody through the peptide linker GGSGGGGSGGGGSGGGG (SEQ ID NO: 35).

[0178] 3. A set of antibodies according to Clause 1, wherein each of the VH domain or the VL domain is connected via its N-terminal region to the C-terminus of one of the two heavy chains of the first antibody or the second antibody through the peptide linker GGGGSGGGGSGGGSGGSGG (SEQ ID NO: 36).

[0179] 4. A group of antibodies according to any one of clauses 1 to 3, wherein the radiolabeled compound comprises Pb-DOTAM.

[0180] 5. A group of antibodies according to Clause 4, wherein the functional binding site against Pb-DOTAM binds with a binding affinity Kd value of 100 pM, 50 pM, 20 pM, 10 pM, 5 pM, 1 pM or less, such as 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.

[0181] 6. A group of antibodies according to Clause 4 or Clause 5, wherein the said functional binding site against Pb-DOTAM binds to both Pb-DOTAM and Bi-DOTAM.

[0182] 7. A group of antibodies according to any one of clauses 4 to 7, wherein the VH domain of the antigen-binding site of the radiolabeled compound comprises:

[0183] a) Heavy chain CDR2, having the amino acid sequence FIGRGDTYYASWAKG (SEQ ID NO: 42), or a variant thereof having at most 1, 2 or 3 substitutions in SEQ ID NO: 42, wherein such substitutions do not include Phe50, Asp56 and / or Tyr58, and optionally also do not include Gly52 and / or Arg54; or wherein the variant is the amino acid sequence AIGSRGDTAYASWAKG (SEQ ID NO: 47).

[0184] b) Heavy chain CDR3, having the amino acid sequence ERDPYGGGAYPPHL (SEQ ID NO:43), or a variant thereof having at most 1, 2, or 3 substitutions in SEQ ID NO:43, wherein these substitutions do not include Glu95, Arg96, Asp97, Pro98, and optionally also exclude Ala100C, Tyr100D, and / or Pro100E, and / or optionally also exclude Tyr99; and

[0185] c) Heavy chain CDR1, which is the amino acid sequence TYSMS (SEQ ID NO:41);

[0186] 8. A group of antibodies according to Clause 6, wherein the VH domain at the antigen-binding site of the radiolabeled compound comprises (a) VH CDR1, which comprises the amino acid sequence TYSMS (SEQ ID NO: 41); (b) VH CDR2, which comprises the amino acid sequence FIGSRGDTYYASWAKG (SEQ ID NO: 42); and (c) VH CDR3, which comprises the amino acid sequence ERDPYGGGAYPPHL (SEQ ID NO: 43).

[0187] 9. A group of antibodies according to any one of claims 4 to 8, wherein the VH domain of the antigen-binding site against the radiolabeled compound comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 44 and SEQ ID NO: 48, or a variant thereof comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 44 or SEQ ID NO: 48.

[0188] 10. A group of antibodies according to any one of clauses 4 to 9, wherein the VH domain of the antigen-binding site against the radiolabeled compound consists of an amino acid sequence selected from SEQ ID NO: 44 or SEQ ID NO: 48.

[0189] 11. A group of antibodies according to Clause 10, wherein residue A or AST is added to the C-terminus of the VH domain of the antigen-binding site of the radiolabeled compound.

[0190] 12. A group of antibodies according to any one of clauses 4 to 11, wherein the VL domain of the antigen-binding site against the radiolabeled compound comprises

[0191] a) The light chain CDR1, comprising the amino acid sequence QSSHSVYSDNDLA (SEQ ID NO:37) or a variant thereof having up to 1, 2 or 3 substitutions in SEQ ID NO:37, wherein such substitutions do not include Tyr28 and Asp32; or the amino acid sequence QSSHSVASDNRLA (SEQ ID NO:45).

[0192] b) The light chain CDR3, comprising the amino acid sequence LGGYDDESDTYG (SEQ ID NO:39) or a variant thereof having at most one, two, or three substitutions in SEQ ID NO:39, wherein these substitutions do not include Gly91, Tyr92, Asp93, Thr95c, and Tyr96.

[0193] c) Light chain CDR 2, comprising the amino acid sequence QASKLAS (SEQ ID NO: 38) or a variant thereof having at least 1, 2 or 3 substitutions in SEQ ID NO: 5, optionally excluding Gln50.

[0194] 13. A group of antibodies according to Clause 12, wherein the VL domain of the antigen-binding site of the radiolabeled compound comprises (a) LC CDR1, which is the amino acid sequence QSSHSVYSDNDLA (SEQ ID NO: 37); (b) LC CDR2, which is the amino acid sequence QASKLAS (SEQ ID NO: 38); and (c) LC CDR3, which is the amino acid sequence LGGYDDESDTYG (SEQ ID NO: 39).

[0195] 14. A group of antibodies according to any one of claims 4 to 13, wherein the VL domain of the antigen-binding site against the radiolabeled compound comprises the amino acid sequence of SEQ ID NO: 40 or 46, or a variant thereof comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 40 or 46.

[0196] 15. A group of antibodies according to any one of clauses 4 to 13, wherein the VL domain of the antigen-binding site against the radiolabeled compound consists of the amino acid sequence of SEQ ID NO: 40 or 46.

[0197] 16. A group of antibodies according to any one of clauses 1 to 15, wherein the first antibody and the second antibody bind to the same epitope of the antigen expressed on the surface of a target cell.

[0198] 17. A group of antibodies according to any one of clauses 1 to 15, wherein the first antibody and the second antibody bind to the same epitope of the antigen expressed on the surface of a target cell.

[0199] 18. A group of antibodies according to any one of clauses 1 to 17, wherein the antigen expressed on the surface of the target cell is a tumor-associated antigen.

[0200] 19. A group of antibodies according to Clause 18, wherein the antigen expressed on the surface of 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.

[0201] 20. A group of antibodies according to Clause 19, wherein the antigen expressed on the surface of the target cell is carcinoembryonic antigen (CEA).

[0202] 21. A group of antibodies according to Clause 20, wherein one or both of the first antibody and the second antibody comprise an antigen-binding site for binding to CEA, the antigen-binding site comprising...

[0203] The light chain variable region comprises: (a) LCCDR1 containing the amino acid sequence KASAAVGTYVA (SEQ ID NO:1); (b) LCCDR2 containing the amino acid sequence SASYRKR (SEQ ID NO:2); and (c) LCCDR3 containing the amino acid sequence HQYYTYPLFT (SEQ ID NO:3); and

[0204] The heavy chain variable region comprises: (d) HC CDR1 containing the amino acid sequence EFGMN (SEQ ID NO:6); (e) HC CDR2 containing the amino acid sequence WINTKTGEATYVEEFKG (SEQ ID NO:7); and (f) HC CDR3 containing the amino acid sequence WDFAYYVEAMDY (SEQ ID NO:8).

[0205] 22. A set of antibodies according to claim 21, wherein the first antibody comprises the LC CDR and HC CDR disclosed in claim 21, and the second antibody binds to different CEA epitopes.

[0206] 23. A set of antibodies according to claim 21, wherein both the first antibody and the second antibody comprise the LC CDR and HC CDR disclosed in claim 21.

[0207] 24. A group of antibodies according to any one of clauses 20 to 23, wherein the antigen-binding site against CEA comprises a VH sequence, said VH sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 9, or a variant thereof comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 9.

[0208] 25. A group of antibodies according to any one of clauses 20 to 23, wherein the antigen-binding site against CEA comprises a VH sequence having the amino acid sequence of SEQ ID NO: 9.

[0209] 26. A group of antibodies according to any one of clauses 20 to 25, wherein the antigen-binding site against CEA comprises a VL sequence, the VL sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4, or a variant thereof comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 4.

[0210] 27. A group of antibodies according to any one of clauses 20 to 25, wherein the antigen-binding site against CEA comprises a VL sequence having the amino acid sequence of SEQ ID NO: 4.

[0211] 28. A group of antibodies according to Clause 20, wherein one or both of the first antibody and the second antibody comprise an antigen-binding site for binding to CEA, the antigen-binding site comprising...

[0212] The light chain variable region comprises: (a) LC CDR1 containing the amino acid sequence RAGESVDIFGVGFLH (SEQ ID NO:11); (b) LC CDR2 containing the amino acid sequence RGSNRAT (SEQ ID NO:12); and (c) LC CDR3 containing the amino acid sequence QQTSEYPYT (SEQ ID NO:13); and

[0213] The heavy chain variable region comprises: (d) HC CDR1 containing the amino acid sequence DTYMH (SEQ ID NO:16); (e) HC CDR2 containing the amino acid sequence RIDPANGNSKYADSVKG (SEQ ID NO:17); and (f) HC CDR3 containing the amino acid sequence FGYYVSDYAMAY (SEQ ID NO:18).

[0214] 29. A set of antibodies according to claim 28, wherein the first antibody comprises the LC CDR and HC CDR disclosed in claim 28, and the second antibody binds to different CEA epitopes.

[0215] 30. A set of antibodies according to claim 28, wherein both the first antibody and the second antibody comprise the LC CDR and HC CDR disclosed in claim 28.

[0216] 31. A group of antibodies according to any one of clauses 28 to 30, wherein the antigen-binding site against CEA comprises a VH sequence, the VH sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 19, or a variant thereof comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 19.

[0217] 32. A group of antibodies according to any one of clauses 28 to 30, wherein the antigen-binding site against CEA comprises a VH sequence having the amino acid sequence of SEQ ID NO: 19.

[0218] 33. A group of antibodies according to any one of clauses 28 to 32, wherein the antigen-binding site against CEA comprises a VL sequence, the VL sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 14, or a variant thereof comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 14.

[0219] 34. A group of antibodies according to any one of clauses 28 to 32, wherein the antigen-binding site against CEA comprises a VL sequence having the amino acid sequence of SEQ ID NO: 14.

[0220] 35. A group of antibodies according to clause 1 or 4, wherein

[0221] The first antibody comprises or consists of the following: a first heavy chain of SEQ ID NO: 21, a second heavy chain of SEQ ID NO: 23, and a light chain of SEQ ID NO: 5; and

[0222] The second antibody comprises or consists of the following: the first heavy chain of SEQ ID NO: 22, the second heavy chain of SEQ ID NO: 24, and the light chain of SEQ ID NO: 5.

[0223] 36. A group of antibodies according to clause 1 or 4, wherein

[0224] The first antibody comprises or consists of the following: a first heavy chain of SEQ ID NO: 25, a second heavy chain of SEQ ID NO: 26, and a light chain of SEQ ID NO: 15; and

[0225] The second antibody comprises or consists of the following: the first heavy chain of SEQ ID NO: 27, the second heavy chain of SEQ ID NO: 28, and the light chain of SEQ ID NO: 15.

[0226] 37. A group of antibodies according to clause 1 or 4, wherein

[0227] The first antibody comprises or consists of the following: a first heavy chain of SEQ ID NO: 29, a second heavy chain of SEQ ID NO: 23, and a light chain of SEQ ID NO: 5; and

[0228] The second antibody comprises or consists of the following: the first heavy chain of SEQ ID NO: 30, the second heavy chain of SEQ ID NO: 31, and the light chain of SEQ ID NO: 5.

[0229] 38. A group of antibodies according to clause 1 or 4, wherein

[0230] The first antibody comprises or consists of the following: a first heavy chain of SEQ ID NO: 32, a second heavy chain of SEQ ID NO: 26, and a light chain of SEQ ID NO: 15; and

[0231] The second antibody comprises or consists of the following: the first heavy chain of SEQ ID NO: 33, the second heavy chain of SEQ ID NO: 34, and the light chain of SEQ ID NO: 15.

[0232] 39. A pharmaceutical composition comprising a group of antibodies according to any one of clauses 1 to 38, and a pharmaceutically acceptable excipient.

[0233] 40. A pharmaceutical composition comprising a first or second antibody according to any one of clauses 1 to 38, and a pharmaceutically acceptable excipient.

[0234] 41. A pharmaceutical product for administration to a cancer patient in combination, concurrently or sequentially, the product comprising A) a pharmaceutical composition comprising a first antibody as a first component; and B) a pharmaceutical composition comprising a second antibody as a second component.

[0235] 42. A pharmaceutical product for administration to a cancer patient in combination, concurrently or sequentially, the product comprising A) a pharmaceutical composition comprising a first antibody as a first component, comprising a first antibody as described in clauses 35 to 38; and B) a pharmaceutical composition comprising a second antibody as a second component, comprising a second antibody as described in clauses 35 to 38.

[0236] 43. A group of nucleic acids that express a group of antibodies according to any one of clauses 1 to 38.

[0237] 44. An expression vector or a set of expression vectors comprising a set of nucleic acids in accordance with clause 43.

[0238] 45. A host cell or a group of host cells comprising an expression vector or a group of expression vectors as described in Clause 44.

[0239] 46. ​​A method for pre-targeted radioimmunotherapy, comprising:

[0240] i) Administering to a subject a group of antibodies according to any one of clauses 1 to 38, wherein the first antibody and the second antibody are administered simultaneously or sequentially in any order, wherein the antibodies bind to the target antigen and are localized on the surface of cells expressing the target antigen; and wherein the association of the first antibody and the second antibody forms a functional binding site against the radiolabeled compound; and

[0241] ii) Subsequently, a radiolabeled compound is applied, wherein the radiolabeled compound binds to a functional binding site for the radiolabeled compound.

[0242] 47. The method according to Clause 46, wherein the radiolabeled compound is led-DOTAM (Pb-DOTAM).

[0243] 48. The method according to clause 46 or 47, wherein the subject is a person.

[0244] 49. The method according to any one of clauses 46 to 48, wherein the target antigen is a cancer-associated or tumor-associated antigen and the method is a method for radioimmunotherapy of a tumor or cancer.

[0245] 50. A group of antibodies according to any one of Clauses 1 to 38, used in a method of pre-targeted radioimmunotherapy according to any one of Clauses 46 to 49.

[0246] Example

[0247] I. Efficacy and Biodistribution

[0248] Materials and methods

[0249] conventional

[0250] Health monitoring and termination criteria

[0251] All experimental protocols were reviewed and approved by the local authorities (Comité Régional d'Ethique de l'Expérimentation Animale du Limousin [CREEAL], Laboratoire Départemental d'Analyses et de Recherches de la Haute-Vienne). In accordance with ethical guidelines, female severely combined immunodeficient (SCID) mice (Charles River) were maintained in a SOPF-free environment with daily light-dark cycles (12 h / 12 ​​h). No manipulation was performed for the first 5 days after arrival to allow the animals to acclimatize to the new environment. Clinical symptoms and adverse events in the animals were monitored daily.

[0252] Solid xenografts were established by subcutaneous (SC) injection of CEA-expressing tumor cells into a cell culture medium mixed 1:1 with Corning® Matrigel® basement membrane matrix (growth factor reduced; catalog number 354230). Tumor volume was estimated three times weekly using manual calipers and calculated using the following formula: Volume = 0.5 × Length × Width 2 Additional tumor measurements may be performed as needed, depending on the tumor growth rate.

[0253] To minimize the reingestion of radioactive urine / feces, during administration... 212 Following Pb-DOTAM administration, mice were placed in cages with wire mesh flooring for 4 hours, after which they were transferred to new cages with standard bedding. All cages were then changed 24 hours after the injection (pi). Nutrients and hydration solutions (SAFE® gel diet energy) were provided to all mice starting the day after the radioactive injection for 7 days or until all individuals had fully recovered from any acute BW relief.

[0254] Mice exhibiting unbearable suffering or signs of pain due to tumor burden, injection side effects, or other reasons should be euthanized before the predetermined endpoint. Signs of pain, suffering, or discomfort include, but are not limited to, acute weight loss (BW), unkempt coat, diarrhea, hunchback posture, and lethargy. General guidelines for immediate euthanasia are a reduction in BW exceeding 20% ​​of the initial BW or a tumor volume reaching 2000 mm². 3 BW of treated animals was measured three times a week, with additional measurements as needed based on health condition. Other factors for euthanasia for ethical reasons included tumor status (e.g., ulcers, necrotic areas, blood / fluid leakage, signs of self-mutilation) and the animal’s overall appearance (e.g., fur, posture, movement).

[0255] Organizational gains

[0256] After each PRIT cycle, injection 212 The scout mice were euthanized and autopsied 24 hours after Pb-DOTAM administration.

[0257] Blood was collected from the venous sinus via retroorbital hemorrhage in anesthetized mice at termination, followed by cervical dislocation. Additional tissue was then harvested for radiometric measurements and / or histological analysis. Any unexpected or abnormal events were documented. A 2470 WIZARD was used. 2 The PerkinElmer automated gamma counter weighs and measures the radioactivity of organs and tissues collected for biodistribution purposes, and then calculates the percentage of injected activity per gram of tissue (%IA / g), including decay and background correction.

[0258] Test compounds

[0259] The compounds used in the described studies are listed in Table 1 (SPLIT antibodies) and Table 2 (radiolabeled chelates).

[0260] All antibody constructs were stored at -80°C until the day of injection, after which they were thawed and diluted in standard solvent buffer (20 mM histidine, 140 mM NaCl; pH 6.0) and mixed together to form a single injection solution for intraperitoneal (IP) administration (5 mg / kg of each antibody, 200 µL in total).

[0261] The DOTAM chelate used for radiolabeling was provided by Macrocyclics and stored at -20°C, and then radiolabeled by Orano Med (Razès, France). 212Pb-DOTAM (RO7205834) is produced by elution of DOTAM from a thorium generator, followed by quenching with Cu or Ca after marking. 212 The Pb-DOTAM solution was diluted with 0.9% NaCl to obtain the required amount for IV injection. 212 Pb active concentration.

[0262] Mice in the solvent control group received multiple injections of solvent buffer, rather than the SPLIT antibody or... 212 Pb-DOTAM.

[0263] Table 1: SPLIT Antibody

[0264]

[0265] Table 2: Radiolabeled chelates

[0266]

[0267] Tumor model

[0268] The tumor cells used for inoculation in mice are described in Table 3. BxPC3 is a human primary pancreatic adenocarcinoma cell line that naturally expresses CEA. Cells were cultured in RPMI 1640 medium enriched with 10% fetal bovine serum (GE Healthcare Hyclone SH30088.03), supplemented with GlutaMAX™, and HEPES (Gibco, reference number 72400-021). On day 0 of the study, solid xenografts were established in each SCID mouse by subcutaneous injection of cells from RPMI medium mixed 1:1 with Corning® Matrigel® basement membrane matrix (growth factor reduced; catalog number 354230) into the right abdomen.

[0269] Table 3: Tumor cell lines

[0270]

[0271] *European Certified Cell Culture Collection (Salisbury, UK)

[0272] Example 1 (Solution 1819): 212 Distribution and efficacy of Pb in vivo

[0273] The purpose of this example is to evaluate 212 The in vivo distribution of Pb and the efficacy of SC xenografts expressing CEA after three cycles in the treatment of immunodeficient mice with the antibody of the present invention.

[0274] SCID mice were injected with BxPC3 tumor cells, and tumors developed, followed by manual diameter measurement. The mice were then co-injected with SPLIT antibody, followed by radioactive injection 7 days later. 212 Pb-DOTAM was used for a two-step PRIT process. Biodistribution was assessed in scout mice after all three treatment cycles to confirm... 212 Targeting and clearance of Pb-DOTAM. Treatment efficacy was assessed based on tumor growth inhibition (TGI) and survival. A summary of the study is shown in Figure 1.

[0275] The timeline and design of Scheme 1819 are shown in Tables 5 and 6.

[0276] Table 4: Timeline of Scheme 1819

[0277]

[0278] Table 5: Research Groups in Scheme 1819*

[0279]

[0280] On day 0 of the study, primary solid xenografts (decreased growth factors; catalog number 354230) were established in SCID mice (8 weeks old) by injecting BxPC3 cells (generation 21) into RPMI medium mixed with Corning® Matrigel® basement membrane matrix at a 1:1 ratio. Thirteen days after tumor cell injection, mice were divided into four efficacy groups with comparable average tumor size (approximately 180 mm). 3 (n=10) and ten biological distribution reconnaissance groups (n=3).

[0281] Mice in the AD group were tracked to assess treatment efficacy until the end of the study or until one or more termination criteria were met.

[0282] In the 1st Second, 2nd The third or second Second-rate 212 Twenty-four hours after Pb-DOTAM injection, mice in groups E to N were sacrificed and necropsy was performed to confirm tumor uptake and normal tissue distribution. 212 Pb retention. The following organs and tissues were harvested and their radioactivity measured: blood, spleen, pancreas, kidney, liver, muscle, tail, and tumor.

[0283] Biological distribution

[0284] Figure 2 shows the cycles 1-3. 212 In all tissues collected 24 hours after Pb-DOTAM injection 212 Mean accumulation and retention of Pb. Tumor uptake was specific for all tested SPLIT antibody pairs. After pre-targeting with P1AI0084 + P1AI0446 in cycles 1, 2, and 3, the mean uptake in tumors was 20.5%, 25.5%, and 22.5% IA / g, respectively; for P1AI1622 + P1AI0034, the corresponding mean uptake in tumors was 7.4%, 14.3%, and 13.5% IA / g; for P1AJ6062 + P1AJ6059, the corresponding mean uptake in tumors was 17.2%, 28.3%, and 22.5% IA / g; P1AJ6063 + P1AJ6064 was administered for only one cycle, and the mean tumor uptake was 11.7% IA / g.

[0285] Tumor development and survival

[0286] The mean tumor volume after CEA-PRIT and control treatments is shown in Figure 3, and the corresponding individual curves are shown in Figure 4. All treatment groups showed tumor growth inhibition compared to the solvent. Overall survival is shown in Figure 5, based on tumor volume ≥ 2000 mm. 3 The termination criteria were as follows. The study was terminated on day 158 after cell injection, at which point 1 / 10 of the mice in both groups B and D were alive and tumors had developed.

[0287] Adverse events and toxicity

[0288] Figure 6 shows the mean body width (BW) development across all treatment groups. Injection 212 Pb-DOTAM caused transient weight loss in irradiated mice, slightly more pronounced in group D, until approximately day 100 post-injection. No mice in any group were euthanized due to acute reduction in body weight (BW) after injection.

[0289] Of all groups, 4 mice were euthanized due to tumor ulceration, 1 mouse was euthanized due to inactivity, and 1 mouse was euthanized due to a spontaneous tumor in the thymus; 1 solvent mouse was found dead in its cage. All adverse events are described in Table 6.

[0290] Table 6: Adverse Events in Plan 1819

[0291]

[0292] The study was conducted on days 21, 35, and 49. 212 Pb irradiation (20 µCi).

[0293] Example 2 (Solution 1917): 212 In vivo distribution of Pb and the efficacy of antibodies against P1AJ6063 and P1AJ6064

[0294] The purpose of this example is to evaluate 212 In vivo distribution of Pb and efficacy of Split-PRIT in treating CEA-expressing SC xenografts in immunodeficient mice after three cycles. SCID mice were injected with BxPC3 tumor cells, and tumors developed, followed by manual diameter measurement. The tumors were co-injected with the Split-PRIT antibody, followed by radioactive injection 7 days later. 212 Pb-DOTAM was used for a two-step PRIT process. Biodistribution was assessed in scout mice after all three treatment cycles to confirm... 212 Targeting and clearance of Pb-DOTAM. Treatment efficacy was assessed based on tumor growth inhibition (TGI) and survival. A summary of the study is shown in Figure 7.

[0295] The timeline and design of Scheme 1917 are shown in Tables 7 and 8, respectively.

[0296] Table 7: Timeline of Scheme 1917

[0297]

[0298] Table 8: Research Groups in Scheme 1917*

[0299]

[0300] On day 0 of the study, primary solid xenografts (decreased growth factors; catalog number 354230) were established in SCID mice (11 weeks old) by injecting BxPC3 cells (passage 19) into RPMI medium mixed with Corning® Matrigel® basement membrane matrix at a 1:1 ratio. Fourteen days after tumor cell injection, mice were divided into two efficacy groups (approximately 180 mm). 3 (n=10) and three biological distribution reconnaissance groups (n=3).

[0301] Mice in groups A and B will be tracked to assess therapeutic efficacy until the study ends or until one or more termination criteria are met.

[0302] In the 1st Second, 2nd The third or second Second-rate 212 Twenty-four hours after Pb-DOTAM injection, mice in groups C through E were sacrificed and necropsy was performed to confirm tumor uptake and normal tissue infiltration. 212 Pb retention. The following organs and tissues were harvested and their radioactivity measured: blood, spleen, pancreas, kidney, liver, muscle, tail, and tumor.

[0303] Biological distribution

[0304] Figure 8 shows the cycles 1-3. 212 In all tissues collected 24 hours after Pb-DOTAM injection 212 Mean accumulation and retention of Pb. Tumor uptake was specific in all cycles, with mean uptake in tumors of 21.1%, 16.9%, and 15.4% IA / g after pre-targeting with P1AJ6063 + P1AJ6064 in cycles 1, 2, and 3, respectively.

[0305] Tumor development and survival

[0306] The mean tumor volume after CEA-PRIT and control treatments is shown in Figure 9, and the corresponding individual curves are as follows: Figure 10 As shown in Figure 11, all treatment groups exhibited tumor growth inhibition compared to the solvent. Overall survival, based on tumor volume ≥ 2000 mm², is shown in Figure 11. 3 Termination criteria.

[0307] Adverse events and toxicity

[0308] Figure 12 shows the mean BW development across all treatment groups. Injection 212 Pb-DOTAM caused transient weight loss in irradiated mice. No mice in any group were euthanized due to acute reduction in body weight after injection.

[0309] Of all groups, six mice were euthanized due to tumor ulceration, and one mouse was euthanized due to a wound in the peripheral area accompanied by reduced BW. All adverse events are described in Table 9.

[0310] Table 9: Adverse Events in Plan 1917

[0311]

[0312] The study was conducted on days 21, 35, and 49. 212Pb irradiation (20 µCi).

[0313] Example 3: Production of CEA-split-DOTAM VH / VL antibodies

[0314] Generation of plasmids for recombinant expression of antibody heavy or light chains

[0315] The desired protein was expressed by transient transfection of human embryonic kidney cells (HEK 293). The transcription unit containing the following functional elements was used to express the desired gene / protein (e.g., full-length antibody heavy chain, full-length antibody light chain, or full-length antibody heavy chain containing additional domains, such as immunoglobulin heavy chain or light chain variable domain at its C-terminus):

[0316] Immediate early enhancers and promoters from human cytomegalovirus (P-CMV), including intron A,

[0317] - Human heavy chain immunoglobulin 5'-untranslated region (5'UTR),

[0318] - Mouse immunoglobulin heavy chain signal sequence (SS),

[0319] - The gene / protein to be expressed, and

[0320] - Bovine growth hormone polyadenylation sequence (BGH pA).

[0321] In addition to the expression unit / cassette containing the desired gene to be expressed, the basic / standard mammalian expression plasmid also contains

[0322] The origin of replication is derived from the vector pUC18, which allows the plasmid to replicate in E. coli, and

[0323] The β-lactamase gene confers ampicillin resistance in Escherichia coli.

[0324] Transient expression of SPLIT antibody molecules

[0325] Using the transfection reagent Expifectamine TM 293 (Life Technologies) in the suspension-adapted HEKExpi293F TM (Life Technologies) enables transient expression of the SPLIT molecule in cells.

[0326] Cells were thawed in 125 ml shake flasks and then passaged at least four times (30 ml volume) via dilution (incubated / shaked at 37°C, 7% CO2, 85% humidity, and 135 rpm). Cells were expanded to 0.5–2.4 x 10⁻⁶ cells / mL in 250 ml volumes. 6 Cells / ml. Then divide the cells and mix them in a 1-liter shake flask at 6 x 10⁻⁶ cells / ml. 5 Seeds were generated at a density of 10 cells / ml in a 250 ml volume. After 24 hours, the cells were cultured at a density of approximately 2.2–2.8 x 10⁻⁶ cells / ml. 6 Transfection was performed at a cell density of 1 cell / ml.

[0327] Prior to transfection, 250 µg of plasmid-DNA was diluted to a final volume of 12.5 ml using preheated (water bath; 37°C) Opti-MEM (LifeTechnologies). Simultaneously, ExpiFectamine transfection reagent was prepared in 12.5 ml of Opti-MEM. Both solutions were incubated at room temperature for no more than 5 min, then combined by gentle mixing, followed by incubation at room temperature for 15–20 min. The entire volume of the mixture was added dropwise to a 1 L shake flask containing 250 ml of HEK cell culture.

[0328] Incubate / shake at 37°C, 7% CO2, 85% humidity, and 135 rpm for 6 or 7 days. After 16–24 h, add 1.25 ml of enhancer 1 (Life Technologies) and 12.5 ml of enhancer 2 (Life Technologies) to each 250 ml culture.

[0329] The supernatant was harvested by filtration through 10g of Sartoclear Dynamics Lab Filter Aid (product number SDLKG-10.0-----2 (Sartorius Stedim Biotech)) diatomaceous earth, followed by filtration through a 0.22 µm top-mounted filter and storage in a refrigerator (-20°C).

[0330] Purification of SPLIT antibody constructs

[0331] Culture supernatant containing antigen-binding molecules was filtered and purified using two or three chromatographic steps. Antibodies were captured by affinity chromatography using HiTrapMabSelectSuRe (GE Healthcare) equilibrated with PBS (1 mM KH2PO4, 10 mM Na2HPO4, 137 mM NaCl, 2.7 mM KCl), pH 7.4. Unbound proteins were removed by washing with equilibration buffer, and antigen-binding molecules were recovered with 100 mM sodium acetate buffer, pH 3.0, and immediately neutralized to pH 6.0 with 1 M Tris-base (pH 9.0) after elution. TM Ion exchange chromatography on a 50 XS (Thermo Fisher) was used as an intermediate purification step. Ion exchange chromatography was performed using 40 mM sodium acetate buffer (pH 5.5) as loading and washing buffer. Elution was carried out in a gradient of 750 mM sodium acetate (pH 5.5). Fractions were collected and analyzed by CE-SDS and size exclusion chromatography. The fractions containing the desired product were collected and purified in the final chromatographic step. Superdex 200 TM Size exclusion chromatography (SUC) on a GE Healthcare scale was used as the final purification step. SUC was performed in 20 mM histidine buffer, 0.14 M NaCl, pH 6.0. The solution containing the bispecific antigen-binding molecules was concentrated using an Amicon Ultra centrifuge-filtration unit (Millipore, Billerica, MA) equipped with a cellulose membrane and stored at -80°C.

[0332] Example 4: Overview of Byproducts of PRIT Molecular Candidates

[0333] Analysis of the byproduct profile of candidate molecules

[0334] The exemplary antibodies of this invention, namely P1AJ6062 and P1AJ6059, underwent a two-step purification process to highlight resistant byproducts and support CHO pool selection and single-cell colony selection. Both molecules were purified by protein A chromatography followed by cation exchange gradient elution with a salt gradient. The fractions with the highest monomer content were combined, and byproducts present in the other fractions were analyzed. Both molecules exhibited complex byproduct profiles, requiring complex purification processes to achieve the desired product quality, such as, for example, good bioprocessing yield and sufficient protein purity.

[0335] Table 10: Overview of byproducts after chromatographic analysis of protein A (SE-HPLC purity):

[0336]

[0337] Table 11: Overview of byproducts after cation exchange chromatography (SE-HPLC purity):

[0338]

[0339] Example 5: Developability assessment of the antibody of the present invention

[0340] To evaluate the developability of the antibody according to the present invention, the following tests were performed:

[0341] Apparent hydrophobicity – Apparent hydrophobicity was evaluated using an appropriate hydrophobic interaction chromatography (HIC) HPLC method. The relative retention time (RRT) of each molecule was calculated after normalizing its absolute retention time to the absolute retention times of standards with known low and high hydrophobicity. The analyzed molecules were ranked based on their RRTs.

[0342] Surface charge distribution and pharmacokinetic prediction – by HPLC using a TSKgel heparin-5PW column (Tosoh) and a buffer gradient of increasing salt concentrations (A = 50 mM Tris pH 7.4, and B = 1 M sodium chloride, 50 mM Tris pH 7.4, 0.8 ml / min, 32 min) in a standard HPLC system (UltiMate 3000, Thermo Fisher Scientific) and taking into account the manufacturer’s instructions, and as previously described (doi: 10.1080 / 19420862.2019.1683432). The relative retention time (RRT) of each molecule was calculated after normalizing its absolute retention time to the absolute retention time of a standard with a known high heparin affinity (RRT = 1.0).

[0343] Thermal stability and aggregation tendency - To determine the thermal stability and aggregation tendency of molecules, they were exposed to temperatures increased under a controlled gradient using UNCLE instruments (UnchainedLabs). Structure unfolding, as measured by the increase in intrinsic fluorescence, indicates the melting temperature (T0). m Furthermore, the aggregation tendency, as measured by the increase in static light scattering (SLS), indicates the aggregation temperature (T). aggThe instrument was operated under standard conditions according to the manufacturer's instructions, measuring samples in triplicate at a concentration of 1 mg / ml. Alternatively, thermal stability was assessed using dynamic light scattering (DLS) readings on a DynaPro plate reader II (Wyatt Technology). Samples at concentrations of approximately 1 mg / ml in 20 mM His pH 6.0 and 140 mM NaCl were subjected to temperature gradients from 25 °C to 80 °C in increments of 0.05 °C / min. Aggregation and unfolding temperatures were determined by an experienced / skilled analyst based on evaluation of the resulting DLS curves and observed signal increases.

[0344] Small-scale stability assessment – ​​To assess the stability of the molecules, they were stored at concentrations of 1 mg / ml or 10 mg / ml under simulated physiological conditions (37°C, 1x phosphate-buffered saline, pH 7.4) and storage lifetime indicator conditions (40°C, 140 mM sodium chloride, 20 mM histidine buffer, pH 6.0) for two weeks. Subsequent analyses focused on changes observed in the aggregates of stored samples compared to an untreated control group stored at -80°C. Molecular aggregation was assessed by analyzing samples using size exclusion chromatography (SEC) methods suitable for IgG antibodies, specifically using a TSKgel UP-SW3000 or TSKgel G3000PWxl column (Tosoh) with isocratic elution (200 mM potassium phosphate buffer, pH 6.2, using 250 mM potassium chloride, 0.3 ml / min or 0.5 ml / min) in a standard HPLC system (UltiMate 3000, Thermo Fisher Scientific) and following the manufacturer's instructions. High molecular weight (HMW) substances with shorter residence times than the major substance were monitored using a UV A 280 nm signal. Peak values ​​were integrated using a custom baseline, and the area under the curve (AUC) was calculated using Chromeleon (Thermo Fisher Scientific).

[0345] result

[0346] For both heparin and HIC, the antibodies were ranked using their relative retention time compared to reference molecules with known properties. A high RRT value indicates stronger binding / retention, therefore a lower RRT is a desirable property for both methods.

[0347] Strong binding in heparin chromatography indicates the antibody's affinity for a strongly negatively charged matrix. This is used as a surrogate indicator of the potential rapid clearance of antibodies from the bloodstream. Therefore, antibodies with lower heparin binding are expected to have better pharmacokinetic (PK) properties (MAbs 2020 Jan-Dec; 12(1):1683432. doi: 10.1080 / 19420862.2019.1683432). Here, the novel CEA conjugate (T84.66v) used in the antibody of this invention is likely a contributor to the improvements demonstrated by these data.

[0348] Hydrophobic interaction chromatography (HIC) was used to determine and rank the hydrophobicity of monoclonal antibodies (mAbs). Stronger retention is a function of higher hydrophobicity and indicates a higher tendency to aggregate, lower stability, and potentially worse PK properties. (MAbs. Jan-Dec 2020; 12(1): 1743053 doi: 10.1080 / 19420862.2020.1743053) Based on this, antibodies with relatively higher retention rates can be expected to have poorer CMC properties (e.g., in terms of downstream processing, formulation, concentration, and overall stability). The data in Table 12 demonstrate the improvement from the reference antibody (e.g., PRIT1 split construct VL 0.82 and VH 0.97) to the antibodies of this invention (e.g., VL 0.55 and VH 0.85 for P1AJ6059 and P1AJ6062).

[0349] Thermal stability: High thermal stability is the ability of a protein to maintain its integrity at elevated temperatures. Thermal stability indicates a lower tendency for partial unfolding and long-term aggregation resistance. Furthermore, antibodies with lower thermal stability show lower expression (mAbs, 11:2, 239-264, DOI: 10.1080 / 19420862.2018.1553476). In particular, the data for the present invention's antibodies containing the DOTAM conjugate VH (60.8°C and 63.8°C) are significantly improved compared to the reference compound (50.7°C). See Table 12.

[0350] Table 12: Results of the Developmentability Assessment

[0351]

[0352] *Measurements were performed using DLS-based readings as described in this document.

[0353] +PRIT-0213 (P1AA7386) is an antibody that binds to CEA (“old” T84.66) and contains functional VH and VL DOTAM conjugates within the same antibody. PRIT-0213 is disclosed, for example, in WO2019 / 201959.

[0354] ++ P1AD8592 and P1AD8749 are antibody pairs that bind to CEA (CH1A1A), with one antibody containing a functional VL DOTAM conjugate (P1AD8592) and the other containing a functional VH DOTAM conjugate (P1AD8749). These antibodies are disclosed, for example, in WO2021 / 009047. P1AF0171 used herein is identical to P1AD8749, differing only in that for P1AF0171, an extension (AST) is added at the C-terminus of the DOTAM-VH domain. This extension reduces the pre-existing ADA response and has no material effect on the data shown in this table.

[0355] Forced stress studies are typically used to expose subject antibodies to harsh conditions in order to identify potential downstream reliability. Prolonged storage at high temperatures under different buffer conditions simulates physiological stress (37°C, pH 7.4) as well as manufacturing, transport, and storage stress (40°C, pH 6). Under these conditions, as measured by size exclusion chromatography (SEC), high percentages of aggregation and fragmentation lead to high (HMW) and low (LMW) molecular weight substances, respectively, and indicate lower long-term stability. Specifically, measuring aggregation at high temperature (40°C) can predict the long-term stability of antibodies at 5°C or 25°C (mAbs, 11:2, 239-264, DOI: 10.1080 / 19420862.2018.1553476) (mAbs, 12:1, DOI:10.1080 / 19420862.2020.1743053). Therefore, the higher the percentage of HMW, the higher the aggregation and thus the lower the stability. The data in Table 13 demonstrate that the HMW and LMW values ​​of the DOTAM-VH of the present invention containing the antibody are improved when compared to the corresponding reference compounds (i.e., for HMW, from up to 42.4% to up to 9.9%). Table 13 also shows that the HMW percentage of the antibodies of the present invention containing the DOTAM-VL conjugate is higher (e.g., up to 19.7%) compared to the reference compound (e.g., up to 8.8%). However, it is important to consider that the antibodies of the present invention are subjected to stress at much higher concentrations (10 mg / ml), which in itself constitutes a more stringent stress condition. Therefore, the higher HMW percentage of DOTAM-VL containing the antibodies of the present invention can be interpreted as a function of the higher concentration during stress.

[0356] Table 13:

[0357]

[0358] *See Table 12

[0359] -80℃ Ref.: Reference sample stored at -80℃

[0360] 37℃, pH 7.4: Physiological pressure 1x PBS pH 7.4, 37℃

[0361] 40℃, pH 6.0: CMC, or storage life indicator pressure 140 mM sodium chloride, 20 mM histidine buffer at 40℃

[0362] Abbreviation Glossary

[0363]

[0364] sequence

[0365]

Claims

1. A group of antibodies comprising: i) A first antibody that binds to an antigen expressed on the surface of a target cell, and further comprises a VH domain for an antigen-binding site of a radiolabeled compound, but does not comprise a VL domain for an antigen-binding site of the radiolabeled compound, or comprises a VL domain that does not effectively form an antigen-binding site of the radiolabeled compound together with the VH domain; and ii) A second antibody that binds to the antigen expressed on the surface of the target cells, and further comprises a VL domain for an antigen-binding site of the radiolabeled compound, but does not comprise a VH domain for the antigen-binding site of the radiolabeled compound, or comprises a VH domain that does not effectively form an antigen-binding site of the radiolabeled compound together with the VL domain. The VH domain of the first antibody and the VL domain of the second antibody together form a functional antigen-binding site against the radiolabeled compound, wherein each of the VH domain or the VL domain is connected to the C-terminus of one of the two heavy chains of the first antibody or the second antibody via its N-terminal region through a peptide linker selected from GGSGGGGSGGGGSGGGSGG (SEQ ID NO:35) or GGGGSGGGGSGGGSGGSGG (SEQ ID NO:36).

2. The group of antibodies according to claim 1, wherein each of the VH domain or the VL domain is connected to the C-terminus of one of the two heavy chains of the first antibody or the second antibody via the peptide linker GGSGGGGSGGGGSGGGGG (SEQ ID NO: 35) through its N-terminal region.

3. The group of antibodies according to claim 1, wherein each of the VH domain or the VL domain is connected to the C-terminus of one of the two heavy chains of the first antibody or the second antibody via the peptide linker GGGGSGGGGSGGGSGGSGG (SEQ ID NO: 36) through its N-terminal region.

4. A group of antibodies according to any one of claims 1 to 3, wherein the radiolabeled compound comprises Pb-DOTAM.

5. The group of antibodies according to claim 4, wherein the functional binding site against Pb-DOTAM binds with a binding affinity Kd value of 100 pM, 50 pM, 20 pM, 10 pM, 5 pM, 1 pM or less, for example 0.9 pM or less, 0.8 pM or less, 0.7 pM or less, 0.6 pM or less or 0.5 pM or less.

6. The group of antibodies according to claim 4 or claim 5, wherein the functional binding site against Pb-DOTAM binds to both Pb-DOTAM and Bi-DOTAM.

7. A group of antibodies according to any one of claims 4 to 7, wherein the VH domain of the antigen-binding site of the radiolabeled compound comprises: a) Heavy chain CDR2, having the amino acid sequence FIGRGDTYYASWAKG (SEQ ID NO: 42), or a variant thereof having at most 1, 2 or 3 substitutions in SEQ ID NO: 42, wherein such substitutions do not include Phe50, Asp56 and / or Tyr58, and optionally also do not include Gly52 and / or Arg54; or said variant having the amino acid sequence AIGSRGDTAYASWAKG (SEQ ID NO: 47); b) Heavy chain CDR3, having the amino acid sequence ERDPYGGGAYPPHL (SEQ ID NO:43), or a variant thereof having at most 1, 2, or 3 substitutions in SEQ ID NO:43, wherein these substitutions do not include Glu95, Arg96, Asp97, Pro98, and optionally also exclude Ala100C, Tyr100D, and / or Pro100E, and / or optionally also exclude Tyr99; and c) Heavy chain CDR1, which is the amino acid sequence TYSMS (SEQ ID NO:41).

8. The group of antibodies according to claim 6, wherein the VH domain at the antigen-binding site of the radiolabeled compound comprises (a) VH CDR1, which comprises the amino acid sequence TYSMS (SEQ ID NO: 41); (b) VH CDR2, which comprises the amino acid sequence FIGSRGDTYYASWAKG (SEQ ID NO: 42); and (c) VH CDR3, which comprises the amino acid sequence ERDPYGGGAYPPHL (SEQ ID NO: 43).

9. A group of antibodies according to any one of claims 4 to 8, wherein the VH domain of the antigen-binding site against the radiolabeled compound comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 44 and SEQ ID NO: 48, or a variant thereof comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 44 or SEQ ID NO:

48.

10. A group of antibodies according to any one of claims 4 to 9, wherein the VH domain of the antigen-binding site of the radiolabeled compound is composed of an amino acid sequence selected from SEQ ID NO: 44 or SEQ ID NO:

48.

11. The group of antibodies according to claim 10, wherein residue A or AST is added to the C-terminus of the VH domain of the antigen-binding site of the radiolabeled compound.

12. A group of antibodies according to any one of claims 4 to 11, wherein the VL domain of the antigen-binding site of the radiolabeled compound comprises a) The light chain CDR1 comprising the amino acid sequence QSSHSVYSDNDLA (SEQ ID NO: 37) or a variant thereof having at most one, two or three substitutions in SEQ ID NO: 37, wherein such substitutions do not include Tyr28 and Asp32; or the amino acid sequence QSSHSVASDNRLA (SEQ ID NO: 45). b) The light chain CDR3, comprising the amino acid sequence LGGYDDESDTYG (SEQ ID NO: 39) or a variant thereof having at most one, two, or three substitutions in SEQ ID NO: 39, wherein these substitutions do not include Gly91, Tyr92, Asp93, Thr95c, and Tyr96. c) Light chain CDR 2, comprising the amino acid sequence QASKLAS (SEQ ID NO: 38) or a variant thereof having at least 1, 2 or 3 substitutions in SEQ ID NO: 5, optionally excluding Gln50.

13. The group of antibodies according to claim 12, wherein the VL domain of the antigen-binding site of the radiolabeled compound comprises (a) LC CDR1, which is the amino acid sequence QSSHSVYSDNDLA (SEQ ID NO: 37); (b) LC CDR2, which is the amino acid sequence QASKLAS (SEQ ID NO: 38); and (c) LC CDR3, which is the amino acid sequence LGGYDDESDTYG (SEQ ID NO: 39).

14. A group of antibodies according to any one of claims 4 to 13, wherein the VL domain of the antigen-binding site against the radiolabeled compound comprises the amino acid sequence of SEQ ID NO: 40 or 46, or a variant thereof comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 40 or 46.

15. A group of antibodies according to any one of claims 4 to 13, wherein the VL domain of the antigen-binding site of the radiolabeled compound consists of the amino acid sequence of SEQ ID NO: 40 or 46.

16. A group of antibodies according to any one of claims 1 to 15, wherein the first antibody and the second antibody bind to the same epitope of the antigen expressed on the surface of a target cell.

17. A group of antibodies according to any one of claims 1 to 15, wherein the first antibody and the second antibody bind to different epitopes of the antigen.

18. A group of antibodies according to any one of claims 1 to 17, wherein the antigen expressed on the surface of the target cell is a tumor-associated antigen.

19. The group of antibodies according to claim 18, wherein the antigen expressed on the surface of the target cell 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.

20. The group of antibodies according to claim 19, wherein the antigen expressed on the surface of the target cell is carcinoembryonic antigen (CEA).

21. The group of antibodies according to claim 20, wherein one or both of the first antibody and the second antibody comprise an antigen-binding site for binding to CEA, the antigen-binding site comprising... The light chain variable region comprises: (a) LCCDR1 containing the amino acid sequence KASAAVGTYVA (SEQ ID NO:1); (b) LCCDR2 containing the amino acid sequence SASYRKR (SEQ ID NO:2); and (c) LCCDR3 containing the amino acid sequence HQYYTYPLFT (SEQ ID NO:3); and The heavy chain variable region comprises: (d) HC CDR1 containing the amino acid sequence EFGMN (SEQ ID NO:6); (e) HC CDR2 containing the amino acid sequence WINTKTGEATYVEEFKG (SEQ ID NO:7); and (f) HC CDR3 containing the amino acid sequence WDFAYYVEAMDY (SEQ ID NO:8).

22. The group of antibodies according to claim 21, wherein the first antibody comprises the LC CDR and HC CDR disclosed in claim 21, and the second antibody binds to different CEA epitopes.

23. The group of antibodies according to claim 21, wherein both the first antibody and the second antibody comprise the LC CDR and HC CDR disclosed in claim 21.

24. A group of antibodies according to any one of claims 20 to 23, wherein the antigen-binding site against CEA comprises a VH sequence, the VH sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 9, or a variant thereof comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:

9.

25. A group of antibodies according to any one of claims 20 to 23, wherein the antigen-binding site against CEA comprises a VH sequence having the amino acid sequence of SEQ ID NO:

9.

26. A group of antibodies according to any one of claims 20 to 25, wherein the antigen-binding site against CEA comprises a VL sequence, the VL sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4, or a variant thereof comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:

4.

27. A group of antibodies according to any one of claims 20 to 25, wherein the antigen-binding site against CEA comprises a VL sequence having the amino acid sequence of SEQ ID NO:

4.

28. The group of antibodies according to claim 20, wherein one or both of the first antibody and the second antibody comprise an antigen-binding site for binding to CEA, the antigen-binding site comprising... The light chain variable region comprises: (a) LCCDR1 containing the amino acid sequence RAGESVDIFGVGFLH (SEQ ID NO:11); (b) LCCDR2 containing the amino acid sequence RGSNRAT (SEQ ID NO:12); and (c) LCCDR3 containing the amino acid sequence QQTSEYPYT (SEQ ID NO:13); and The heavy chain variable region comprises: (d) HC CDR1 containing the amino acid sequence DTYMH (SEQ ID NO:16); (e) HC CDR2 containing the amino acid sequence RIDPANGNSKYADSVKG (SEQ ID NO:17); and (f) HC CDR3 containing the amino acid sequence FGYYVSDYAMAY (SEQ ID NO:18).

29. The group of antibodies according to claim 28, wherein the first antibody comprises the LC CDR and HC CDR disclosed in claim 28, and the second antibody binds to different CEA epitopes.

30. The group of antibodies according to claim 28, wherein both the first antibody and the second antibody comprise the LC CDR and HC CDR disclosed in claim 28.

31. A group of antibodies according to any one of claims 28 to 30, wherein the antigen-binding site against CEA comprises a VH sequence, the VH sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 19, or a variant thereof comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:

19.

32. A group of antibodies according to any one of claims 28 to 30, wherein the antigen-binding site against CEA comprises a VH sequence having the amino acid sequence of SEQ ID NO:

19.

33. A group of antibodies according to any one of claims 28 to 32, wherein the antigen-binding site against CEA comprises a VL sequence, the VL sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 14, or a variant thereof comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:

14.

34. A group of antibodies according to any one of claims 28 to 32, wherein the antigen-binding site against CEA comprises a VL sequence having the amino acid sequence of SEQ ID NO:

14.

35. A group of antibodies according to claim 1 or 4, wherein The first antibody comprises or is composed of the following: The first heavy chain of SEQ ID NO: 21, the second heavy chain of SEQ ID NO: 23, and the light chain of SEQ ID NO: 5; and The second antibody comprises or consists of the following: the first heavy chain of SEQ ID NO: 22, the second heavy chain of SEQ ID NO: 24, and the light chain of SEQ ID NO:

5.

36. A group of antibodies according to claim 1 or 4, wherein The first antibody comprises or is composed of the following: The first heavy chain of SEQ ID NO: 25, the second heavy chain of SEQ ID NO: 26, and the light chain of SEQ ID NO: 15; and The second antibody comprises or consists of the following: the first heavy chain of SEQ ID NO: 27, the second heavy chain of SEQ ID NO: 28, and the light chain of SEQ ID NO:

15.

37. A group of antibodies according to claim 1 or 4, wherein The first antibody comprises or is composed of the following: The first heavy chain of SEQ ID NO: 29, the second heavy chain of SEQ ID NO: 23, and the light chain of SEQ ID NO: 5; and The second antibody comprises or consists of the following: the first heavy chain of SEQ ID NO: 30, the second heavy chain of SEQ ID NO: 31, and the light chain of SEQ ID NO:

5.

38. A group of antibodies according to claim 1 or 4, wherein The first antibody comprises or is composed of the following: The first heavy chain of SEQ ID NO: 32, the second heavy chain of SEQ ID NO: 26, and the light chain of SEQ ID NO: 15; and The second antibody comprises or consists of the following: a first heavy chain of SEQ ID NO: 33, a second heavy chain of SEQ ID NO: 34, and a light chain of SEQ ID NO:

15.

39. A pharmaceutical composition comprising a group of antibodies according to any one of claims 1 to 38, and a pharmaceutically acceptable excipient.

40. A pharmaceutical composition comprising a first antibody or a second antibody according to any one of claims 1 to 38, and a pharmaceutically acceptable excipient.

41. A pharmaceutical product for administration to a cancer patient in combination, simultaneously or sequentially, the product comprising A) a pharmaceutical composition comprising a first antibody according to claims 1 to 38 as a first component; and B) a pharmaceutical composition comprising a second antibody according to claims 1 to 38 as a second component.

42. A pharmaceutical product for administration to a cancer patient in combination, simultaneously or sequentially, the product comprising A) a pharmaceutical composition comprising a first antibody according to claims 35 to 38 as a first component; and B) a pharmaceutical composition comprising a second antibody according to claims 35 to 38 as a second component.

43. A set of nucleic acids expressing an antibody according to any one of claims 1 to 38.

44. An expression vector or a set of expression vectors comprising a set of nucleic acids according to claim 43.

45. A host cell or a group of host cells comprising the expression vector or a group of expression vectors according to claim 44.

46. ​​A method for pre-targeted radioimmunotherapy, the method comprising: i) administering to a subject a group of antibodies according to any one of claims 1 to 38, wherein the first antibody and the second antibody are administered simultaneously or sequentially in any order, wherein the antibodies bind to the target antigen and are localized on the surface of cells expressing the target antigen; and wherein the association of the first antibody and the second antibody forms a functional binding site against the radiolabeled compound; and ii) Subsequently, a radiolabeled compound is applied, wherein the radiolabeled compound binds to a functional binding site for the radiolabeled compound.

47. The method of claim 46, wherein the radiolabeled compound is led-DOTAM (Pb-DOTAM).

48. The method of claim 46 or 47, wherein the subject is a human being.

49. The method according to any one of claims 46 to 48, wherein the target antigen is a cancer-associated or tumor-associated antigen and the method is a method for radioimmunotherapy of tumors or cancer.

50. A group of antibodies according to any one of claims 1 to 38, used in a method of pre-targeted radioimmunotherapy according to any one of claims 46 to 49.

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