HK40063708A - Antibodies which bind to cancer cells and target radionuclides to said cells - Google Patents

Description

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

Technical Field

The present invention relates to antibodies that bind to antigens on target cells and target radionuclides to the cells and methods of use thereof.

Background

Monoclonal antibodies have been developed to target drugs to cancer cells. By conjugating a toxic agent to an antibody that binds to a tumor-associated antigen, there is the potential to provide more specific tumor kill and less damage to surrounding tissues.

In pre-targeted radioimmunotherapy (PRIT), antibody constructs are utilized which have an affinity on the one hand for tumor-associated antigens and on the other hand for compounds with a radioactive label. In the first step, the antibody is administered and localized to the tumor. Subsequently, a radiolabeled compound is administered. Because the radiolabeled compounds are small, they can be delivered to tumors quickly and cleared quickly, which reduces radiation exposure outside of the tumor (golden berg et al, Theranostics 2012,2(5), 523-540). Similar procedures can also be used for imaging. Pretargeting may utilize bispecific antibodies or systems using avidin-biotin, but the latter has the disadvantage that avidin/streptavidin is immunogenic.

Methods of pre-targeted radioimmunotherapy or imaging typically utilize a clearing or blocking agent that is administered between the step of administering the antibody and the step of administering the radiolabeled compound. The aim is to clear the antibodies from the blood and/or to block the binding sites of circulating antibodies of radiolabeled compounds (see, e.g., Karacay et al, Biocon j. chem.,13(5), 1054-. The use of a scavenger or blocking agent allows for a sufficient level of radioactivity to be administered for effective treatment while limiting undesirable toxicity, but the timing and dosage must be carefully selected. Thus, the use of a clearance phase in a pre-targeting approach is a complex aspect.

Disclosure of Invention

The present invention provides a panel of antibodies useful in pre-targeting methods and methods of use thereof.

In one aspect, the invention provides a set of antibodies comprising:

i) a first antibody that binds to an antigen expressed on the surface of a target cell and further comprises V of the antigen binding site of a radiolabeled compound_HV of domain, but not comprising the antigen binding site of the radiolabeled compound_LA domain; and

ii) a second antibody that binds to an antigen expressed on the surface of a target cell and further comprises V of the antigen binding site of a radiolabeled compound _LV of domain, but not comprising the antigen binding site of the radiolabeled compound_HThe domain(s) is (are),

wherein said V of the first antibody_HThe V of domain and second antibody_LThe domains can together form a functional antigen binding site for the radiolabeled compound.

Neither the first antibody nor the second antibody alone comprises a functional antigen binding site for the radiolabeled compound. The first antibody has only V from the functional binding site of the radiolabeled compound_HDomain and does not have V_LA domain. The second antibody has only V_LDomain and does not have V_HA domain.

When V of the first antibody and the second antibody_HAnd V_LWhen the domains associate, a functional antigen binding site for the radiolabeled compound is formed. This may occur, for example, when the first and second antibodies bind to the same individual target cell or to adjacent cells.

The first and second antibodies described herein may be referred to herein as "single domain split antibodies", "split antibodies", or "half antibodies" (demibodies). V together forming an antigen binding site capable of binding to a radiolabeled compound_HDomain and V_LThe domain is split between the two antibodies and does not exist as part of the same antibody.

Split-domain format refers to a radiolabeled compound that is not bound to a first antibody alone or to a second antibody alone. In blood, there is little or no stable association between the primary and secondary antibodies, and thus little or no stable binding of the radiolabeled compound.

The antigen expressed on the surface of a target cell may be referred to herein as a "target antigen" or "TA". According to the present invention, the first and second antibodies described above have binding sites for the same target antigen. (for the avoidance of doubt, where it is stated that an antibody binds to the same target antigen, this is meant to have a binding site capable of binding to the same target antigen and includes the possibility that the antibody may bind to two individual antigen molecules that are identical to each other). For example, in one embodiment, both the first and second antibodies bind to CEA.

In some embodiments, the first antibody and the second antibody can bind to the same epitope of the target antigen (have binding sites for the same epitope of the target antigen). In other embodiments, the first antibody may bind to an epitope of a target antigen that is different from the second antibody (having a binding site for an epitope of the target antigen that is different from the second antibody).

In some embodiments, the first antibody and the second antibody may comprise the same antigen binding site for the target antigen. That is, it may comprise an antigen binding site capable of binding to a target antigen, the antigen binding site comprising V_LSequence and V _HSequences in which, in the first antibody and in the second antibody, the V forming the antigen-binding site_LSequence and V_HThe sequences are identical.

In some embodiments, each of the first and second antibodies is bivalent to the target antigen. In some embodiments, for epitopes, each is bivalent and monospecific. In other embodiments, each of the first and second antibodies is biparatopic for the target antigen, i.e., the first and second antibodies each have a binding site for two different epitopes of the target antigen.

In some embodiments, it may be preferred that the first antibody and/or the second antibody comprise an Fc region. The presence of the Fc region is beneficial in the context of radioimmunotherapy and radioimaging, for example to prolong the circulating half-life of the protein and/or to result in higher tumor uptake than can be observed with smaller fragments. In this case, the "split-domain" format described herein may be particularly advantageous because it reduces the greater likelihood of association with the radiolabeled compound that would otherwise occur due to the prolonged presence of circulating antibodies.

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

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

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

In some embodiments, an antibody as described herein is used in a pre-targeted radioimmunotherapy (PRIT) method or in a pre-targeted radioimaging method.

In one aspect, the invention provides a method of pre-targeted radioimmunotherapy comprising:

i) administering to the individual a first antibody and a second antibody as described above; and

ii) subsequently administering to the individual a radiolabeled compound.

In another aspect, the invention provides the first and second antibodies described above for use in a method of treatment comprising administering the first and second antibodies to an individual and subsequently administering a radiolabeled compound to the individual. In another aspect, the invention provides a first antibody as described above for use in a method of treatment comprising administering to an individual a first antibody and a second antibody and subsequently administering to the individual a radiolabeled compound. In another aspect, the invention provides a second antibody as described above for use in a method of treatment comprising administering to an individual a first antibody and a second antibody and subsequently administering to the individual a radiolabeled compound.

In another aspect, the present invention provides a radiological imaging method, comprising:

i) administering to the individual a first antibody and a second antibody as described herein, wherein the antibodies bind to the target antigen and are localized to the surface of a cell expressing the target antigen;

ii) subsequent administration of a radiolabeled compound; and optionally

iii) imaging the tissue or organ in which the radionuclide is located.

In another aspect, the invention provides a first antibody and a second antibody as described herein for use in a method of diagnosis of a human or animal body, wherein the method comprises

i) Administering to the individual a first antibody and a second antibody as described herein, wherein the antibodies bind to the target antigen and are localized to the surface of a cell expressing the target antigen;

ii) subsequent administration of a radiolabeled compound; and optionally

iii) imaging the tissue or organ in which the radionuclide is located.

The imaging step may be followed by the step of forming a diagnosis and optionally delivering the diagnosis to the subject. In some embodiments, the method may further comprise determining an appropriate treatment and optionally administering the treatment to the individual.

In each of the above methods/uses, binding of the first antibody and the second antibody to the same or adjacent target cells causes V of the antigen binding site of the radiolabeled compound_HDomain and V_LThe association of domains and the formation of functional antigen binding sites for radiolabeled compounds. Thus, after administration of the radiolabeled compound, the radiolabeled compound binds to the target by association V _HAnd V_LForming a functional antigen binding site.

In any of the methods and uses described herein, the first and second antibodies may be administered simultaneously or sequentially in either order.

Generally in the art, PRIT or radiological imaging methods involve a cleanup step. The clearing step comprises administering an agent between the administration of the antibody and the administration of the radiolabeled compound, wherein the agent increases the rate of removal of the antibody from the blood and/or blocks binding of the radiolabeled compound to the antibody.

In one embodiment of the methods and uses described herein, the method does not comprise a cleanup step. That is, it does not include the step of administering a scavenger or blocking agent between (i.e., after administration of the antibody but before administration of the radiolabeled compound) the administration of the first and second antibodies and the administration of the radiolabeled compound. In another embodiment, no agent other than the optionally selected radiosensitizer, immunotherapeutic, and/or chemotherapeutic agent is administered between the administration of the first and second antibodies and the administration of the radiolabeled compound. In another embodiment, no agent is administered between the administration of the first and second antibodies and the administration of the radiolabeled compound.

In some embodiments, the antibodies described herein can be administered as part of a combination therapy. For example, it may be administered in combination with one or more radiosensitizers, immunotherapeutics, and/or chemotherapeutics: the radiosensitizer, immunotherapeutic, or chemotherapeutic agent and antibody can be administered simultaneously or sequentially in either order.

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

In another aspect, the invention provides a kit comprising:

i) a first antibody and a second antibody as described herein;

ii) a radiolabeled compound bound to an antigen-binding site formed by combining the first and second antibodies.

The kit may optionally exclude (i.e., not include) a scavenger or blocking agent as described herein.

The kit may optionally further comprise a radiosensitizer, an immunotherapeutic, or a chemotherapeutic.

In some embodiments, the first antibody and the second antibody may be present in the same pharmaceutical composition. In other embodiments, the first antibody and the second antibody may be present in separate pharmaceutical compositions. In some embodiments, the radiolabeled compound is present in the pharmaceutical composition separately from the antibody.

Drawings

FIG. 1 shows the schematic structures of a Target Antigen (TA) -DOTAM bispecific antibody (TA-DOTAM BsAb) belonging to the comparative example and an exemplary TA-split-DOTAM-VH/VL antibody of the invention.

FIG. 2 is a schematic showing the assembly of division-VH/VL DOTAM binders on tumor cells. Non-heavy binding of TA-division-DOTAM-VH/VL antibodies²¹²Pb-DOTAM unless bound to a Tumor Antigen (TA) on the targeted cell, where the two domains of the DOTAM binder are assembled.

FIG. 3 shows a schematic overview of an embodiment of the three-step TA-PRIT concept involving scavenger use.

FIG. 4 shows a schematic overview of an embodiment of the two-step TA-PRIT concept in which no scavenger is used.

Figure 5 shows the binding of a split antibody for demonstrating CEA binding ability to MKN45 cells. Antibody detection was performed using human IgG-specific secondary antibodies.

Figure 6 shows the binding of a split antibody to MKN45 cells for exhibiting DOTAM binding ability. Antibody detection was performed using Pb-DOTAM-FITC.

Figure 7A shows an exemplary scheme of two-step PRIT using CEA-split-DOTAM-VH/VL in SCID mice bearing SC BxPC3 tumors (h ═ h, d ═ day, w ═ week).

Fig. 7B shows an exemplary protocol for three-step PRIT control in SCID mice bearing SC BxPC3 tumor (h-hour, d-day, w-week).

FIG. 8 shows pre-targeting of CEA-division-DOTAM-VH alone, CEA-division-DOTAM-VL alone or in combination with two complementary antibodies or using a standard three-step PRIT²¹²Pretargeting 6 hours after Pb-DOTAM²¹²Biodistribution of Pb-DOTAM in SC BxPC3 tumor bearing SCID mice (% ID/g ± SD, n ═ 4).

FIG. 9 shows CEA-division-DOTAM-VH/VL pharmacokinetics after IV injection in SCID mice.

FIG. 10 shows the experimental design of protocol 158 comprising CEA-PRIT in 2-step (top) or 3-step (bottom) in SCID mice bearing SC BxPC3 tumor. CEA disruption DOTAM BsAb dose was adjusted to compensate for the hole/hole impurities in the 2/4 construct.

FIG. 11 shows pre-targeting²¹²Biodistribution of Pb-DOTAM in SCID mice harboring SC BxPC3 tumor (6h p.i).). Distribution is pre-targeted by injecting a double complementary site combination of CEA-DOTAM BsAb or CEA-split-DOTAM antibodies²¹²In SCID mice bearing tumors 6 hours after Pb-DOTAM²¹²And Pb. The radioactivity content in organs and tissues was expressed as mean% ID/g ± SD (n ═ 4).

FIG. 12 shows the experimental schedule of a one cycle protocol 160 comprising 3 steps CEA-PRIT (apical), 2 steps CEA-PRIT (intermediate), or 1 step CEA-RIT in SCID mice bearing SC BxPC3 tumor. Biodistribution (BD) scout mice were euthanized 24 hours after the radiation injection, while mice in the efficacy group were carefully maintained and monitored until termination criteria were reached.

FIG. 13 shows pretargeting²¹²Pb-DOTAM and²¹²biodistribution of Pb-DOTAM-CEA-DOTAM in SCID mice bearing SC BxPC3 tumor (24h p.i.). Distribution is pre-targeted by CEA-DOTAM injection²¹²Pb-DOTAM or pre-incubated²¹²Tumor-bearing SCID mice 24 hours after Pb-DOTAM-CEA-DOTAM²¹²And Pb. The radioactivity content in organs and tissues was expressed as mean% ID/g ± SD (n ═ 3).

Fig. 14 shows mean tumor growth + standard error (n-10) for PRIT treated and control (groups a-E) in BxPC3 model. The curve is truncated at n<5. The vertical lines indicating some or all of the groups designed according to the study²¹²Pb-DOTAM administration (20. mu. Ci).

Fig. 15 shows individual tumor growth curves (n-10) for PRIT treated group and control (groups a-E) in the BxPC3 model. Vertical line indication²¹²Pb labeled compound was applied (20 μ Ci).

Figure 16 shows the mean body weight loss of mice treated with CEA-PRIT and CEA-RIT in the BxPC3 model (group a-E, n ═ 10). The curve is truncated at n<5. The vertical lines indicating some or all of the groups designed according to the study²¹²Pb labeled compound is administered.

FIG. 17 shows the experimental design of protocol 175 comprising two-step CEA-PRIT in SCID mice bearing SC BxPC3 tumor, where injection was performed²¹²Sacrifice and necropsy 24 hours after Pb-DOTAM. The CEA-split-DOTAM-VH-AST dose is adjusted to compensate for the hole/hole impurities.

FIG. 18 shows pre-targeting by CEA-division-DOTAM-VH/VL antibody injection²¹²24 hours after Pb-DOTAM²¹²Distribution of Pb in tumor-bearing SCID mice (protocol 175). The radioactivity content in organs and tissues was expressed as mean% ID/g ± SD (n ═ 4).

FIG. 19 shows the experimental design of a protocol 185 comprising a two-step CEA-PRIT in SCID mice bearing SC BxPC3 tumor, in which injection was performed²¹²Sacrifice and necropsy were performed 6 hours after Pb-DOTAM. The CEA-split-DOTAM-VH-AST (CH1A1A) dose was adjusted to compensate for the pocket/pocket impurities.

FIG. 20 shows pre-targeting by CEA-division-DOTAM-VH/VL antibody injection²¹²6 hours after Pb-DOTAM²¹²Distribution of Pb in tumor-bearing SCID mice (scheme 185). The radioactivity content in organs and tissues was expressed as mean% ID/g ± SD (n ═ 5).

Figure 21 shows the distribution of CEA-division-DOTAM-VH/VL pairs (pooled VH and VL antibodies) in two selected SC BxPC3 tumors 7 days after injection. A and B show tumor sections from mouse A3 injected with CEA-division-DOTAM-VH/VL targeted to T84.66, where A shows CEA expression and B shows the corresponding CEA-division-DOTAM-VH/VL distribution. C and D show tumor sections from mouse C5 injected with CEA-division-DOTAM-VH/VL targeting CH1A 1A: c shows CEA expression and D shows the corresponding CEA-division-DOTAM-VH/VL profile.

FIG. 22 shows the experimental design of a protocol 189 comprising a two-step CEA-PRIT in SCID mice bearing SC BxPC3 tumor, in which injection was performed²¹²Sacrifice and necropsy were performed 6 hours after Pb-DOTAM. The CEA-split-DOTAM-VH-AST (CH1A1A) dose was adjusted to compensate for the pocket/pocket impurities.

FIG. 23 shows pretargeting in a biparatopic pair injected with CEA-division-DOTAM-VH/VL antibody (T84.66 and CH1A1A) compared to positive control (CH1A1A only)²¹²6 hours after Pb-DOTAM²¹²Distribution of Pb in tumor-bearing SCID mice. The radioactivity content in organs and tissues is expressed as mean% ID/g ± SD.

Figure 24 shows the Mean Fluorescence Intensity (MFI) of the SPLIT antibody as determined by FACS. Binding of Pb-DOTA-FITC as determined by FACS can be shown only for co-incubation of two SPLIT antibodies with Pb-DOTA-FITC. Single SPLIT antibodies do not produce a significant signal.

Fig. 25A-C show exemplary formats of antibodies as described herein.

Figure 26 shows the results of experiment 1 of example 11, which evaluated the binding of individual TA-division-DOTAM-VH and TA-division-DOTAM-VL antibodies to biotin-labeled DOTAM captured on chip.

FIG. 27 shows the results of experiment 2 of example 11, which evaluated the binding of DOTAM to individual TA-division-DOTAM-VH and TA-division-DOTAM-VL antibodies captured on-chip.

Figure 28 shows the results of experiment 3 of example 11, evaluating the binding of DOTAM to TA-split-DOTAM-VH/VL antibodies (antibody pair) captured on a chip.

Detailed Description

Definition of

For purposes herein, an "acceptor human framework" refers to a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework as defined below. An acceptor human framework "derived" from a human immunoglobulin framework or human consensus framework may comprise the same amino acid sequence of a human immunoglobulin framework or human consensus framework, or it may contain amino acid sequence variations. In some aspects, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some aspects, the VL acceptor human framework has sequence identity to a VL human immunoglobulin framework sequence or a human consensus framework sequence.

"affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and a binding partner (e.g., an antigen) of the molecule. As used herein, "binding affinity" refers to an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen), unless otherwise indicated. The affinity of a molecule X for its partner Y can generally be determined by the dissociation constant (K) _D) And (4) showing. Affinity can be determined by methods known in the artIncluding the methods described herein. Specific illustrative and exemplary methods for measuring binding affinity are described below.

An "affinity matured" antibody is one that has one or more alterations in one or more Complementarity Determining Regions (CDRs) compared to a parent antibody that does not have the alterations that result in improved affinity of the antibody for the antigen.

The term "antibody that binds to an antigen expressed on the surface of a target cell" refers to an antibody that is capable of binding to the antigen with sufficient affinity to allow the antibody to be used as a diagnostic and/or therapeutic agent in targeting the antigen. In one aspect, the extent of binding of the antibody to an unrelated, non-antigenic protein is less than about 10% of the binding of the antibody to the antigen, as measured, for example, by Surface Plasmon Resonance (SPR). In certain aspects, the dissociation constant (K) of an antibody that binds to an antigen expressed on the surface of a target cell_D) At less than 1 μ M, less than 100nM, less than 10nM, less than 1nM, less than 0.1nM, less than 0.01nM or less than 0.001nM (e.g., 10 nM)^-8M or less, e.g. 10^-8M to 10^-13M, e.g. 10^-9M to 10^-13M). Allegedly when the K of the antibody is _DAt 1 μ M or less, the antibody "specifically binds" to an antigen expressed on the surface of the target cell. In certain aspects, the antibody binds to an epitope of the antigen that is conserved among the antigens from different species.

The term "antigen-binding site of a radiolabeled compound" or "functional antigen-binding site of a radiolabeled compound" refers to an antigen-binding site comprising a VH domain and a VL domain that is capable of binding to a radiolabeled compound with sufficient affinity to allow the antibody to be used as a diagnostic and/or therapeutic agent in order to associate the radiolabeled compound with the antibody. In one aspect, the extent of binding of the antigen binding site to an unrelated, non-antigenic compound is less than about 10% of the binding of the antibody to the radiolabeled compound, as measured, for example, by Surface Plasmon Resonance (SPR). In certain aspects, the dissociation constant (KD) of the antigen-binding site bound to the radiolabeled compound is 1 μ M or less, 100nM or less, 10nM or less, 1nM or less, or less0.1nM, ≦ 0.01nM, or ≦ 0.001nM (e.g. 10)^-8M or less, e.g. 10^-8M to 10^-13M, e.g. 10^-9M to 10^-13M). It may be preferred that it has a KD of 100pM, 50pM, 20pM, 10pM, 5pM, 1pM or less, e.g. 0.9pM or less, 0.8pM or less, 0.7pM or less, 0.6pM or less or 0.5pM or less. For example, a functional binding site may bind a radiolabeled compound with a Kd of about 1pM to 1nM, e.g., about 1 to 10pM, 1 to 100pM, 5 to 50pM, 100pM or 500pM to 1 nM. The antigen binding site is said to "specifically bind" to the radiolabeled compound when the KD of the antigen binding site is 1 μ M or less.

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

An "antibody fragment" refers to a molecule that comprises, in addition to an intact antibody, a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, cross Fab, Fab '-SH, F (ab')₂(ii) a A bifunctional antibody; a linear antibody; single chain antibody molecules (e.g., scFv and scFab); single domain antibodies (dabs); and multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23: 1126-. Thus, the term "Fab fragment" refers to an antibody fragment comprising a light chain comprising a VL domain and a CL domain, and a heavy chain fragment comprising a VH domain and a CH1 domain. A "Fab' fragment" differs from a Fab fragment in that a residue is added at the carboxy terminus of the CH1 domain that includes one or more cysteines from the antibody hinge region. For Fab and F (ab') containing salvaged receptor binding epitope residues and having extended half-life in vivo ₂See U.S. Pat. No. 5,869,046 for a discussion of fragments. The term "crossover Fab fragment" or "xFab fragment" or "crossover Fab fragment" refers to a Fab fragment in which the variable or constant regions of the heavy and light chains are interchanged. Cross-Fab fragments comprising a variable light chainPolypeptide chains consisting of the region (VL) and the heavy chain constant region 1(CH1) and polypeptide chains consisting of the heavy chain variable region (VH) and the light chain constant region (CL). Asymmetric Fab arms can also be engineered by introducing charged or uncharged amino acid mutations into the domain interface to direct proper Fab pairing. See, for example, WO 2016/172485.

A "single chain variable fragment" or "scFv" is a fusion protein of the heavy (VH) and light (VL) chain variable domains of an antibody connected by a peptide linker. In particular, the linker is a short polypeptide of 10 to 25 amino acids and is generally glycine rich for flexibility and serine or threonine rich for solubility and can link the N-terminus of VH with the C-terminus of VL and vice versa. Regardless of constant region removal and linker introduction, this protein still retains the specificity of the original antibody. For a summary of scFv fragments see, for example, Pl ü ckthun, in The Pharmacology of Monoclonal Antibodies, Vol.113, compiled by Rosenburg and Moore, (Springer-Verlag, New York), p.269-315 (1994); see also WO 93/16185; and U.S. patent nos. 5,571,894 and 5,587,458.

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

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

The term "clearing step" or "clearing stage" as used herein encompasses the use of a blocking or clearing agent. Some agents may act as scavengers and as blocking agents.

The term "epitope" refers to a site on a protein or non-protein antigen to which an antibody binds. Epitopes can be formed by stretches of contiguous amino acids (linear epitopes) or comprise non-contiguous amino acids that are spatially adjacent, for example, as a result of antigen folding, i.e., as a result of tertiary folding of the protein antigen (conformational epitopes). Linear epitopes typically remain bound to the antibody after exposure of the protein antigen to the denaturant, whereas conformational epitopes are typically destroyed after treatment with the denaturant. An epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation.

Screening for Antibodies that bind to a particular epitope (i.e., Antibodies that bind to the same epitope) can be performed using methods conventional in the art, such as, but not limited to, alanine scanning, peptide dot method (see meth.mol.biol.248(2004)443-463), peptide cleavage analysis, epitope excision, epitope extraction, antigen chemical modification (see prot.sci.9(2000)487-496), and cross-blocking (see "Antibodies", Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY).

Antibody profiling based on Antigen Structure (ASAP), also known as Modified Assisted Profiling (MAP), allows binning of a population of monoclonal antibodies from a population that specifically binds to an antigen based on the binding profile of each antibody to a chemically or enzymatically modified antigen surface (see e.g. US 2004/0101920). The antibodies in each bin bind to the same epitope, which may be a unique epitope distinct or partially overlapping with the epitope represented by the other group.

In addition, competitive binding can be used to readily determine whether an antibody binds to the same epitope as a reference antibody, or competes for binding with a reference antibody. For example, "an antibody that binds to the same epitope as a reference antibody" refers to an antibody that blocks binding of the reference antibody to its antigen by 50% or more in a competition assay, and conversely, the reference antibody blocks binding of the antibody to its antigen by 50% or more in a competition assay. Further, for example, to determine whether an antibody binds to the same epitope as a reference antibody, the reference antibody is allowed to bind to the antigen under saturating conditions. After removal of excess reference antibody, the ability of the antibody in question to bind to antigen was assessed. If the antibody in question is capable of binding to an antigen after saturation binding of the reference antibody, it can be concluded that: the antibody in question binds to a different epitope than the reference antibody. However, if the antibody in question is unable to bind to the antigen after saturation binding by the reference antibody, the antibody in question may bind to the same epitope as the epitope bound by the reference antibody. To confirm that the antibodies in question are binding to the same epitope or binding is hindered only for steric reasons, routine experiments can be used (e.g. peptide mutation and binding assays using ELISA, RIA, surface plasmon resonance, flow cytometry or any other quantitative or qualitative antibody binding assay available in the art). This analysis should be performed in both settings, i.e. in case both antibodies are saturated antibodies. If, in both settings, only the first (saturating) antibody is able to bind to the antigen, it can be concluded that: the antibody in question and the reference antibody compete for binding to the antigen.

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

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

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

An antibody "class" refers to the type of constant domain or constant region that a heavy chain has. There are five main classes of antibodies: IgA, IgD, IgE, IgG and IgM, and several of these antibodies may be further divided into subclasses (isotypes), e.g. IgG ₁、IgG₂、IgG₃、IgG₄、IgA₁And IgA₂. In certain aspects, the antibody is an IgG₁Isoforms. In certain aspects, the antibody isIgG with mutations P329G, L234A and L235A for attenuating effector function of Fc region₁Isoforms. In other aspects, the antibody is an IgG₂Isoforms. In certain aspects, the antibodies are of the type used to improve IgG₄IgG with S228P mutation in hinge region for antibody stability₄Isoforms. The constant domains of the heavy chains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. Antibody light chains can be assigned to one of two types, called kappa (kappa) and lambda (lambda), based on the amino acid sequence of their constant domains.

"Effector function" refers to those biological activities attributable to the Fc region of an antibody that vary with antibody isotype. Examples of antibody effector functions include: c1q binding and Complement Dependent Cytotoxicity (CDC); fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

An "effective amount" of an agent (e.g., a pharmaceutical composition) refers to an amount effective to achieve a desired therapeutic or prophylactic result at the requisite dosage and for the requisite period of time.

The term "tandem Fab" refers to an antibody comprising two Fab fragments linked via a peptide linker/tether. In some embodiments, a tandem Fab may comprise one Fab fragment and one cross Fab fragment connected by a peptide linker/tether.

The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region. The term includes native sequence Fc regions as well as variant Fc regions. In one aspect, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxy-terminus of the heavy chain. However, the antibody produced by the host cell may undergo post-translational cleavage of one or more, in particular one or two, amino acids from the C-terminus of the heavy chain. Thus, an antibody produced by a host cell by expression of a particular nucleic acid molecule encoding a full-length heavy chain may comprise the full-length heavy chain, or it may comprise a cleaved variant of the full-length heavy chain. This may be the case where the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, according to the EU index numbering). Thus, the C-terminal lysine (Lys447) or C-terminal glycine (Gly446) and lysine (Lys447) of the Fc region may or may not be present. In one aspect, the heavy chain comprising the Fc region as defined herein comprised in the antibody of the invention comprises another C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to the EU index). In one aspect, the heavy chain comprising an Fc region as defined herein comprised in an antibody of the invention comprises another C-terminal glycine residue (G446, numbering according to the EU index). Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al, Sequences of Proteins of Immunological Interest, published Health Service 5 th edition, National Institutes of Health, Bethesda, MD, 1991.

"framework" or "FR" refers to variable domain residues other than Complementarity Determining Regions (CDRs). The FRs of a variable domain typically consist of the following four FR domains: FR1, FR2, FR3 and FR 4. Thus, in VH (or VL), the CDR and FR sequences are generally present in the form of: FR1-CDR-H1(CDR-L1) -FR2-CDR-H2(CDR-L2) -FR3-CDR-H3(CDR-L3) -FR 4.

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

The terms "host cell," "host cell strain," and "host cell culture" are used interchangeably and refer to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. The nucleic acid content of the progeny may not be identical to that of the maternal cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the original transformed cell are included herein.

"human antibody" refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by human or human cells or derived from a non-human source using a repertoire of human antibodies or other human antibody coding sequences. This human antibody definition specifically excludes humanized antibodies comprising non-human antigen binding residues.

"human consensus framework" refers to a framework representing the amino acid residues most frequently present in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selected human immunoglobulin VL or VH sequences are from a subset of variable domain sequences. Generally, this subset of Sequences is a subset as in Kabat et al, Sequences of Proteins of Immunological Interest, fifth edition, NIH Publication 91-3242, Bethesda MD (1991), volumes 1-3. In one aspect, for VL, this subgroup is as in Kabat et al, supra, subgroup kappa I. In one aspect, for the VH, this subgroup is subgroup III as in Kabat et al, supra.

"humanized" antibodies refer to chimeric antibodies comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain aspects, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs correspond to CDRs of a non-human antibody and all or substantially all of the FRs correspond to FRs of a human antibody. Optionally, the humanized antibody may comprise at least a portion of an antibody constant region derived from a human antibody. "humanized forms" of antibodies (e.g., non-human antibodies) refer to antibodies that have undergone humanization.

The term "hypervariable region" or "HVR" as used herein refers to a region of an antibody variable domain which has high sequence denaturation and determines antigen-binding specificity, e.g., "complementarity determining regions" ("CDRs").

Generally, an antibody comprises six CDRs: three CDRs in VH (CDR-H1, CDR-H2, CDR-H3) and three CDRs in VL (CDR-L1, CDR-L2, CDR-L3). Exemplary CDRs herein include:

(a) hypervariable loops present at amino acid residues 26-32(L1), 50-52(L2), 91-96(L3), 26-32(H1), 53-55(H2) and 96-101(H3) (Chothia and Lesk, J.mol.biol.196:901-917 (1987));

(b) CDRs present at amino acid residues 24-34(L1), 50-56(L2), 89-97(L3), 31-35b (H1), 50-65(H2) and 95-102(H3) (Kabat et al, Sequences of Proteins of Immunological Interest, 5 th edition Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and

(c) antigen junctions present at amino acid residues 27c-36(L1), 46-55(L2), 89-96(L3), 30-35b (H1), 47-58(H2) and 93-101(H3) (MacCallum et al.J.mol.biol.262:732-745 (1996)).

Unless otherwise indicated, CDRs are determined according to Kabat et al, supra. Those skilled in the art will appreciate that the CDR names may also be determined according to the following: chothia, supra; McCallum, supra; or any other scientifically accepted nomenclature system. Instead of the above, the sequence of CDR-H1 as described herein may extend from Kabat26 to Kabat35, e.g. for Pb-DOTAM binding variable domains.

In one aspect, the CDR residues comprise CDR residues identified in the sequence listing or elsewhere in this specification.

Unless otherwise indicated, HVR/CDR residues and other residues (e.g., FR residues) in the variable domains are numbered herein according to Kabat et al, supra.

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

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

A molecule as described herein may be "isolated". An "isolated" antibody is an antibody that has been separated from components of its natural environment. In some aspects, the antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) methods. For a review of methods for assessing antibody purity, see, e.g., Flatman et al, j.chromager.b 848:79-87 (2007).

The term "nucleic acid molecule" or "polynucleotide" includes any compound and/or substance comprising a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Nucleic acid molecules are often described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The base sequence is usually represented from 5 'to 3'. As used herein, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cdna) and genomic DNA; ribonucleic acid (RNA), particularly messenger RNA (mrna); synthetic forms of DNA or RNA; and mixed polymers comprising two or more of these molecules. The nucleic acid molecule may be linear or circular. In addition, the term nucleic acid molecule includes sense and antisense strands as well as single-stranded and double-stranded forms. In addition, the nucleic acid molecules described herein can contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases or chemically modified residues with derivatized sugar or phosphate backbone linkages. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for direct expression of the antibodies of the invention in vitro and/or in vivo, e.g., in a host or patient. The DNA (e.g., cDNA) or RNA (e.g., mRNA) vector may be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and/or the expression of the encoded molecule, such that the mRNA can be injected into a subject to generate antibodies in vivo (see, e.g., Stadler et al, Nature Medicine 2017,2017, published on-line 6.12.12.d., doi:10.1038/nm.4356 or EP 2101823B 1).

An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule that is contained in a cell that typically contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location that is different from its native chromosomal location.

"isolated nucleic acid encoding an antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains of an antibody (or fragments thereof), including such nucleic acid molecules in a single vector or in different vectors and such nucleic acid molecules present at one or more locations in a host cell.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population have identity and/or bind the same epitope, except for possibly variant antibodies (e.g., those containing naturally occurring mutations or produced during the production of a monoclonal antibody preparation, which variants are typically present in minor amounts). In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the character of being obtained from a population of substantially homogeneous antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies of the invention can be made by a variety of techniques including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, the methods described herein for making monoclonal antibodies, and other exemplary methods.

"naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabeled. Naked antibodies may be present in a pharmaceutical composition.

"Natural antibody" refers to a naturally occurring immunoglobulin molecule having a different structure. For example, a native IgG antibody is a heterotetrameric glycoprotein of about 150,000 daltons (dalton) composed of two identical light chains and two identical heavy chains that are disulfide-bonded. Each heavy chain has, from N-terminus to C-terminus, a variable domain (VH), also referred to as variable domain or heavy chain variable domain, followed by three constant heavy domains (CH1, CH2, and CH 3). Similarly, each light chain has a variable domain (VL), also referred to as a variable light domain or light chain variable domain, from N-terminus to C-terminus, followed by a Constant Light (CL) domain.

The term "package insert" is used to refer to a package insert that is typically included in a commercial package for a therapeutic product and that contains information regarding the indication, usage, dosage, administration, combination therapy, contraindications and/or warnings associated with the use of the therapeutic product.

"percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity and not considering any conservative substitutions as part of the sequence identity for the purposes of the alignment. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in various ways within the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software, or the FASTA package. One skilled in the art can determine parameters suitable for aligning sequences, including any algorithms required to achieve maximum alignment over the full length of the sequences being compared. Alternatively, the percent identity value may be generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech corporation and the source code has been submitted with the user file in the united states copyright Office of colombia, Washington (d.c., 20559), Washington, which is registered with us copyright registration number TXU510087 and described in WO 2001/007611.

For purposes herein, unless otherwise indicated, percent amino acid sequence identity values are generated using the BLOSUM50 comparison matrix using the ggsearch program, either edition c or later, of FASTA suite 36.3.8. The FASTA package is available from W.R. Pearson and D.J. Lipman (1988), "Improved Tools for Biological Sequence Analysis", PNAS 85: 2444-; W.R. Pearson (1996) "Effective protein sequence compliance" meth.Enzymol.266: 227-; and Pearson et al (1997) Genomics 46:24-36, and published from 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 common server accessible through fasta. bioch. virginia. edu/fasta _ www2/index. cgi, using the ggsearch program and default options (BLOSUM 50; open: -10; ext: -2; Ktup ═ 2) to ensure that a global alignment is performed rather than a local alignment. Percent amino acid identity is given in the output alignment heading.

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

"pharmaceutically acceptable carrier" refers to an ingredient of a pharmaceutical composition or formulation other than an active ingredient that is not toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

Unless otherwise indicated, reference to a target antigen as used herein refers to any native target antigen from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length," unprocessed target antigen, as well as any form of target antigen that results from processing in a cell. The term also encompasses naturally occurring variants of the target antigen, such as splice variants or allelic variants. For example, the target antigen CEA may have the amino acid sequence of human CEA, particularly carcinoembryonic antigen-associated cell adhesion molecule 5(CEACAM5), shown in UniProt (www.uniprot.org) accession number P06731 (model 119) or NCBI (www.ncbi.nlm.nih.gov /) RefSeq NP _ 004354.2. Another example of a target antigen is Fibroblast Activation Protein (FAP). The amino acid sequence of human FAP is shown in UniProt (www.uniprot.org) accession number Q12884 (model 149) or NCBI (www.ncbi.nlm.nih.gov /) RefSeq NP-004451.2. Another example of a target antigen is GPRC5D (for human sequences, see UniProt accession No. Q9NZD1 (model 115); NCBI RefSeq accession No. NP-061124.1).

The terms "split antibody (split antibodies/split antibodies)", "single domain split antibody" or "SPLIT PRIT" as referred to herein means that the VH and VL domains which together form an antigen binding site capable of binding to the radiolabeled compound are split between the two antibodies and are not present as part of the same antibody (prior to in vivo assembly). "CEA-targeting SPLIT PRIT" refers to a CEA-targeting split antibody. The term "SPLIT PRIT" may also be used interchangeably with the term "TA-division-DOTAM-VH/VL" (e.g., where "TA" or target antigen is CEA, FAP or GPRC 5D). The term "CEA-targeting SPLIT PRIT" is used interchangeably with the term "CEA-cleavage-DOTAM-VH/VL".

As used herein, "treatment" (and grammatical variations thereof, such as "treating") refers to a clinical intervention that attempts to alter the natural course of disease in the treated individual and may be performed either prophylactically or during the course of clinical pathology. Desirable therapeutic effects include, but are not limited to, preventing occurrence or recurrence of disease, alleviating symptoms, alleviating any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, ameliorating or palliating the disease state, and alleviating or improving prognosis. In some aspects, the antibodies of the invention are used to delay disease progression or to slow disease progression.

The term "variable region" or "variable domain" refers to a domain of an antibody heavy or light chain that is involved in binding of the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain comprising four conserved Framework Regions (FRs) and three Complementarity Determining Regions (CDRs). (see, e.g., kit et al. kuby Immunology, 6 th edition, w.h.freeman and co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. Furthermore, antibodies that bind a particular antigen can be isolated using the VH domain or VL domain from the antibody that binds the antigen to screen a repertoire of complementary VL domains or VH domains, respectively. See, e.g., Portolano et al, J.Immunol.150:880-887 (1993); clarkson et al, Nature 352: 624-.

The term "vector" as used herein refers to a nucleic acid molecule capable of transmitting another nucleic acid to which it is linked. The term includes vectors in the form of self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. The vector is referred to herein as an "expression vector".

The term "Pb" or "lead" as used herein includes its dissociation such as Pb (ii) And (5) performing secondary treatment. Other metals mentioned also include ions thereof. Thus, for example, the skilled reader understands the terms lead, Pb, B,²¹²Pb or²⁰³Pb is intended to encompass the ionic form of the element, in particular Pb (ii).

Compositions and methods

In one aspect, the invention is based, in part, on a panel of antibodies comprising a first antibody and a second antibody, wherein each antibody can bind to an antigen on a target cell, but wherein a functional antigen binding site for an effector agent is formed only when the first and second antibodies are associated with each other. The antibodies of the invention are useful, for example, in methods of pre-targeted immunotherapy and/or pre-targeted imaging. In a preferred aspect, the method eliminates the step of applying a scavenger or sealant.

A. Target antigens

Antigens expressed on the surface of target cells are also referred to herein as "target antigens".

Where the invention relates to a method of treatment and with respect to a product for use in said method of treatment, it is applicable to any condition treatable by cytotoxic activity targeted to a patient cell, for example a diseased cell. Thus, the target cell is any cell that needs to be targeted for cytotoxicity, e.g., any diseased cell. The treatment preferably belongs to a tumor or cancer. However, the applicability of the present invention is not limited to tumors and cancers. For example, treatment can also pertain to viral infection (by targeting infected cells) or T cell driven autoimmune disease (by targeting T cells). Immunotoxins directed against viral antigens expressed on the surface of infected cells have been studied against various viral infections such as HIV, rabies, and EBV. Cai and Berger 2011Antiviral Research 90(3):143-50 use immunotoxins containing PE38 to target cells infected with Kaposi's sarcoma-associated herpesvirus (Kaposi). In addition, the first and second substrates are, (A-dmDT390-bisFv (UCHT1)) selectively kills human malignant T cells and transiently depletes normal T cells, and is considered to be capable of treating diseases such as multiple sclerosis and graft-versus-host diseaseT cell driven autoimmune disease of the disease and the T cell leukemia to which it has undergone clinical trials. Likewise, the methods of the present invention may be applied to any cell type requiring radiographic imaging, including but not limited to cancer cells or tumor cells.

Thus, suitable target antigens may include cancer cell antigens, viral antigens, or microbial antigens.

The antigen is typically a normal cell surface antigen that is overexpressed or expressed at an abnormal time. Ideally, the target antigen is expressed only on diseased cells (such as tumor cells), however, this is rarely observed in practice. Thus, the target antigen is typically selected based on differential expression between diseased tissue and healthy tissue.

For example, the cell surface marker or target antigen can be a tumor-associated antigen.

The term "tumor-associated antigen" or "tumor-specific antigen" as used herein refers to any molecule (e.g., protein, peptide, lipid, carbohydrate, etc.) that is expressed or overexpressed solely or predominantly by tumor cells and/or cancer cells or by other tumor stromal cells such as cancer-associated fibroblasts, such that the antigen is associated with one or more tumors and/or one or more cancers. Tumor-associated antigens may additionally be expressed by normal, non-tumor or non-cancer cells. However, in such cases, tumor-associated antigen expression by normal, non-tumor or non-cancer cells is less robust than expression by tumor or cancer cells. In this regard, a tumor cell or cancer cell may overexpress or express an antigen at a significantly higher level than the expression of the antigen by normal, non-tumor or non-cancer cells. Furthermore, tumor-associated antigens may additionally be expressed by cells of different states of development or maturation. For example, tumor-associated antigens may additionally be expressed by cells at embryonic or fetal stages, which are not typically found in adult hosts. Alternatively, the tumor-associated antigen may additionally be expressed by stem cells or precursor cells, which are not normally found in adult hosts.

The tumor-associated antigen can be an antigen expressed by any cell of any cancer or tumor, including the cancers and tumors described herein. The tumor-associated antigen may be a tumor-associated antigen of only one type of cancer or tumor such that the tumor-associated antigen is associated with or is characteristic of only one type of cancer or tumor. Alternatively, the tumor-associated antigen may be (e.g., may be characteristic of) a tumor-associated antigen of more than one type of cancer or tumor. For example, tumor-associated antigens may be expressed by breast and prostate cancer cells, and not by normal, non-tumor or non-cancer cells at all.

Exemplary tumor-associated antigens to which the antibodies of the invention can bind include, but are not limited to, mucin 1 (MUCl; tumor-associated epithelial mucin), melanoma preferential expression antigen (PRAME), carcinoembryonic antigen (CEA), Prostate Specific Membrane Antigen (PSMA), PSCA, EpCAM, Trop2 (Proderm-2, also known as EGP-1), granulocyte-macrophage colony stimulating factor receptor (GM-CSFR), CD56, human epidermal growth factor receptor 2(HER2/neu) (also known as erbB-2), CDS, CD7, tyrosinase-related protein (TRP) I, and TRP 2. In another embodiment, the tumor antigen may be selected from the group consisting of: cluster of Differentiation (CD)19, CD20, CD21, CD22, CD25, CD30, CD33 (sialic acid binding Ig-like lectin 3, myeloid cell surface antigen), CD79b, CD123 (interleukin 3 receptor alpha), transferrin receptor, EGF receptor, mesothelin, cadherin, lewis y (lewis y), glypican-3, FAP (fibroblast activation protein alpha), GPRC5D (G protein coupled receptor class C group 5 member D), PSMA (prostate specific membrane antigen), CA9 CAIX (carbonic anhydrase IX), Ll (neuroadhesion molecule L1), endosialin, HER3 (activated conformation of epidermal growth factor receptor family member 3), Alkl differentiation/BMP 9 complex (non-lymphomatal kinase 1/bone morphogenic protein 9), TPBG ═ 5T4 (CAM), r1 (tyrosine receptor surface antigen-like kinase) HER1 (activated conformation of epidermal growth factor receptor) and CLL1 (C-type lectin domain family 12 member a). Mesothelin is expressed in, for example, ovarian cancer, mesothelioma, non-small cell lung cancer, lung adenocarcinoma, fallopian tube cancer, head and neck cancer, cervical cancer, and pancreatic cancer. CD22 is expressed in, for example, hairy cell leukemia, Chronic Lymphocytic Leukemia (CLL), prolymphocytic leukemia (PLL), non-Hodgkin's lymphoma, Small Lymphocytic Lymphoma (SLL), and Acute Lymphocytic Leukemia (ALL). CD25 is expressed in, for example, leukemias and lymphomas, including hairy cell leukemia and hodgkin's lymphoma. The lewis Y antigen is expressed in, for example, bladder cancer, breast cancer, ovarian cancer, colorectal cancer, esophageal cancer, gastric cancer, lung cancer, and pancreatic cancer. CD33 is expressed in, for example, Acute Myeloleukemia (AML), Chronic Myelomonocytic Leukemia (CML), and myeloproliferative disorders.

Exemplary antibodies that specifically bind to a tumor-associated antigen include, but are not limited to, anti-transferrin receptor antibodies (e.g., HB21 and variants thereof), anti-CD 22 antibodies (e.g., RFB4 and variants thereof), anti-CD 25 antibodies (e.g., Tac antibodies and variants thereof), anti-mesothelin antibodies (e.g., SS1, rabb-009, SS, HN1, HN2, MN, MB and variants thereof), and anti-lewis Y antigen antibodies (e.g., B3 and variants thereof). In this aspect, the targeting moiety (cell binding agent) can be an antibody and antigen binding portion thereof selected from the group consisting of B3, RFB4, SS1, MN, MB, HN1, HN2, HB21, and moab-009. Additional exemplary targeting moieties suitable for use in the chimeric molecules of the invention are disclosed, for example, in: us patent 5,242,824 (anti-transferrin receptor); us patent 5,846,535 (anti-CD 25); us patent 5,889,157 (liu yi Y); us patent 5,981,726 (liu yi Y); us patent 5,990,296 (liu yi Y); us patent 7,081,518 (anti-mesothelin); us patent 7,355,012 (anti-CD 22 and anti-CD 25); us patent 7,368,110 (anti-mesothelin); us patent 7,470,775 (anti-CD 30); us patent 7,521,054 (anti-CD 25); and U.S. Pat. No. 7,541,034 (anti-CD 22); U.S. patent application publication 2007/0189962 (anti-CD 22); frankel et al, Clin. cancer Res.,6: 326-; and Kreitman et al, AAPS Journal,8(3) E532-E551(2006), each of which is incorporated herein by reference.

Additional antibodies have been cultured that target specific tumor-associated antigens including: teratoma-derived growth factor (Cripto), CD30, CD19, CD33, glycoprotein NMB, CanAg, Her2(ErbB2/Neu), CD56(NCAM), CD22(Siglec2), CD33(Siglec3), CD79, CD138, PSCA, PSMA (prostate-specific membrane antigen), BCMA, CD20, CD70, E-selectin, EphB2, melanotransferrin, Muc16 and TMEFF 2. Any of the above antibodies, or antigen binding fragments thereof, may be suitable for use in the present invention, i.e., may be incorporated into the antibodies described herein.

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

CEA is advantageous in the context of the present invention because it internalizes relatively slowly and thus a high percentage of the antibody will remain available on the cell surface after initial treatment to bind to the radionuclide. Other hypointernalization targets/tumor associated antigens may also be preferred. Other examples of tumor-associated antigens include CD20 or HER 2. In yet another embodiment, the target may be EGP-1 (epithelial glycoprotein-1, also known as Zingiber dermatum-2), colon specific antigen-p (CSAP), or pancreatic mucin MUC 1. See, for example, golden berg et al 2012 (therapeutics 2(5)) which is incorporated herein by reference. This reference also describes antibodies such as: mu-9 bound to CSAP (see also Sharkey et al cancer Res.2003; 63:354-63), hPAM4 bound to MUC1 (see also Gold et al cancer Res.2008:68:4819-26), veltuzumab bound to CD20 (see also Sharkey et al cancer Res.2008; 68:5282-90), and hRS7 bound to EGP-1 (see also Cubas et al Biochim Biophys Acta 2009; 1796: 309-14). Any of the above antibodies, or antigen binding portions thereof, may be suitable for use in the present invention, i.e., may be incorporated into the antibodies described herein. An example of a cultured anti-CEA antibody is T84.66 (as shown in NCBI accession number: CAA36980 for the heavy chain and CAA36979 for the light chain, or as shown in SEQ ID NOs 317 and 318 of WO 2016/075278) and humanized and chimeric versions thereof, such as T84.66-LCHA as described in WO2016/075278A1 and/or WO 2017/055389. Another example is CH1A1a as an anti-CEA antibody as described in WO2012/117002 and WO 2014/131712; and CEA hMN-14 (see also US 6676924 and US 5874540). Another anti-CEA antibody is A5B7 as described in M.J.Banfield et al, Proteins 1997,29(2), 161-171. Humanized antibodies derived from the murine antibody A5B7 have been disclosed in WO 92/01059 and WO 2007/071422. See also co-pending application PCT/EP 2020/067582. An example of a humanized version of A5B7 is A5H1EL1 (G54A). Another exemplary anti-CEA antibody is MFE23 and humanized versions thereof described in US7626011 and/or co-pending application PCT/EP 2020/067582. Yet another example of an anti-CEA antibody is 28A 9. Any of the above antibodies or antigen binding fragments thereof may be used to form the CEA binding portion of the present invention.

In some embodiments, FAP (fibroblast activation protein α) or GPRC5D (G protein-coupled receptor class C group 5 member D) may also be preferred. FAP is an established target for imaging and therapy due to its widespread expression in the microenvironment of multiple tumor types, such as pancreatic, breast and lung cancer (Lindner, t., Loktev, a., Giesel, f.et al.targeting of activated fibroblastics for imaging and therapy. ejnmi radiopharm.4, 16 (2019)). Thus, SPLIT PRIT using FAP-division-DOTAM-VH/VL antibodies is expected to be produced on activated cancer-associated fibroblasts²¹²Specific accumulation of Pb-DOTAM. Thus, it is expected that in addition to a limited direct tumor killing effect on adjacent tumor cells, the emitted alpha radiation also adversely affects the immunosuppression of FAP-expressing malignancies. The G protein-coupled receptor family C, group 5 member D (GPRC5D), is overexpressed on multiple myeloma plasma cells (Atamaniuk J, Gleiss A, Porpaczy E, Kainz B, Grunt TW, Raderer M, et al. overexpression of G protein-coupled receptor 5D in the bone marrowed with protein inhibitors with polypeptide inhibitors with multiple polypeptide molecules Europe J Clin invest.2012; 42:953-60.) and SC (subcutaneous) in vivo models have been established that reflect expression of, for example, OPM-2 and NCI-H929 (Kodama T, Kochi Y, Nakai W, Mino H, Baba T, Hatmbu K, al et al. Ann. RC 5-28/CD 24-34-sample 869) found in multiple myeloma patients. Thus, we expected that SPLIT PRIT using GPRC 5D-division-DOTAM-VH/VL antibody produced tumor-specific accumulation of 212Pb-DOTAM, followed by radiation-induced tumor cell death.

In some embodiments, an antibody of the invention can specifically bind to a target antigen (e.g., a target as discussed herein)Any one of the antigens). In some embodiments, it can be ≦ 1 μ M ≦ 100nM, ≦ 10nM, ≦ 1nM, ≦ 0.1nM, ≦ 0.01nM, or ≦ 0.001nM (e.g., 10 nM)^-7M or less, e.g. 10^-7M to 10^-13M；10^-8M or less, e.g. 10^-8M to 10^-13M, e.g. 10^-9M to 10^-13M) dissociation constant (Kd) binding.

The first and second antibodies each bind to the same target antigen, which may be referred to as "antigen a" (i.e., the antibodies have binding specificity for the same target antigen). The antibodies may each have binding specificity for the same epitope on antigen a. Alternatively, the first antibody may bind to a first epitope on antigen a and the second antibody may bind to a second epitope on a different antigen a. For example, in one embodiment, one of the antibodies may bind to the T84.66 epitope of CEA and the other may bind to the A5B7 epitope of CEA.

In some embodiments, for antigen a, one or both of the first and/or second antibodies may be biparatopic-that is, each of the individual antibodies may bind to two different epitopes of antigen a. The first antibody can comprise a first binding site and a second binding site that bind to a first epitope and a second epitope, respectively, of antigen a, wherein the first epitope and the second epitope are different from each other. Alternatively, or additionally, the second antibody can comprise first and second binding sites that bind to first and second epitopes of antigen a, wherein the first and second epitopes are different from each other. In some embodiments, one or both of the epitopes bound by the first antibody may be different from one or both of the epitopes bound by the second antibody. In other embodiments, the two epitopes bound by the first antibody may be the same as the two epitopes bound by the second antibody.

B. Radiolabelled compounds

According to the invention, the combination of the first antibody and the second antibody forms a functional binding site for an effector molecule. The effector molecule of the present invention is a radiolabeled compound comprising a radioisotope, such as a radiolabeled hapten.

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

In some embodiments, a functional binding site for an effector molecule may bind to a chelate comprising a chelator and a radioisotope. In other embodiments, the antibody may be conjugated to a moiety conjugated to a chelated radioisotope, such as histamine-succinyl-glycine (HSG), digoxigenin (digoxigenin), biotin, or caffeine.

For example, the chelating agent may be a multidentate molecule such as an aminopolycarboxylic acid or an aminopolythiocarboxylic acid or a salt or functional variant thereof. For example, the chelating agent may be bidentate or tridentate or tetradentate. Examples of suitable metal chelators include molecules comprising: EDTA (ethylenediaminetetraacetic acid or salts such as CaNa)₂Salt forms of EDTA), DTPA (diethylenetriaminepentaacetic acid), DOTA (1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid), NOTA (2,2 '- (1,4, 7-triazacyclononane-1, 4, 7-triyl) triacetic acid), IDA (iminodiacetic acid), MIDA ((methyliminodiacetic) diacetic acid), TTHA (3,6,9, 12-tetrakis (carboxymethyl) -3,6,9, 12-tetraazatetradecanedioic acid), TETA (2,2' - (1,4,8, 11-tetraazacyclotetradecane-1, 4,8, 11-tetrayl) tetraacetic acid), DOTAM (1,4,7, 10-tetrakis (carbamoylmethyl) -1,4,7, 10-tetraazacyclododecane), HEHA (1,4,7,10,13, 16-hexaazacyclohexadecane-1, 4,7,10,13, 16-hexaacetic acid available from Macrocyclics, Plano, Texas), NTA (nitrilotriacetic acid), EDDHA (ethylenediamine-N, N' -bis (2-hydroxyphenylacetic acid), BAL (2,3, -dimercaptopropanol), DMSA (2, 3-dimercaptosuccinic acid), DMPS (2, 3-dimercapto-1-propanesulfonic acid), D-penicillamine (B-dimethylcysteine), MAG (MAG) ₃(mercaptoacetyltriglycine), Hynic (6-hydrazinopyridine-3-carboxylic acid), para-isothiocyanatobenzyl-deferoxamine (e.g. labelled with zirconium for imaging) and salts or functional variants/derivatives thereof capable of chelating metals. In some embodiments, it may be preferred that the chelating agent is DOTA or DOTAM or a salt or functional variant/derivative thereof capable of chelating a metal. Thus, the chelator may be or may comprise DOTA or DOTAM with a radioisotope chelated thereto.

The effector molecule may comprise or consist ofThe composition is as follows: functional variants or derivatives of the above chelators, and radionuclides. Suitable variants/derivatives have structures that differ to some limited extent and retain the ability to act as a chelator (i.e., retain sufficient activity for one or more of the purposes described herein). Functional variants/derivatives may also include chelators as described above, including small molecules, polypeptides, or carbohydrates, conjugated to one or more additional moieties or substituents. This attachment may occur, for example, via one of the constituent carbons in the backbone portion of the chelating agent. For example, suitable substituents may be hydrocarbyl groups such as alkyl, alkenyl, aryl or alkynyl groups; a hydroxyl group; an alcohol group; a halogen atom; a nitro group; a cyano group; a sulfonyl group; a thiol group; an amino group; an oxo group; a carboxyl group; a thiocarboxyl group; a carbonyl group; an amide group; an ester group; or a heterocyclic group, including heteroaryl. For example, the substituent may be as described below for the group "R ¹"one of the substituents defined. For example, the small molecule may be a dye (such as Alexa 647 or Alexa488), biotin or a biotin moiety, or a phenyl or benzyl moiety. For example, the polypeptide may be an oligopeptide, such as an oligopeptide with two or three amino acids. Exemplary carbohydrates include polydextrose, linear or branched polymers or copolymers (e.g., polyalkylene, poly (ethylene-lysine), polymethacrylate, polyamino acids, polysaccharides or oligosaccharides, dendrimers). Derivatives may also include polymers of the chelator compounds in which compounds as set out above are linked via a linker moiety. Derivatives may also include functional fragments of the supra-compounds that retain the ability to chelate metal ions.

Specific examples of derivatives include benzyl-EDTA and hydroxyethyl-thioureido-benzyl EDTA, DOTA-benzene (e.g., (S-2- (4-aminobenzyl) -1,4,7, 10-tetraazacyclododecane tetraacetic acid), DOTA-biotin and DOTA-TyrLys-DOTA.

In some embodiments of the invention, the functional binding site formed by the combination of the first antibody and the second antibody binds to a metal chelate comprising DOTAM and a metal such as lead (Pb). As mentioned above, "DOTAM" has the chemical name:

1,4,7, 10-tetrakis (carbamoylmethyl) -1,4,7, 10-tetraazacyclododecane,

which is a compound of the formula:

in certain aspects and embodiments, the invention can also utilize functional variants or derivatives of DOTAM that incorporate metal ions. Suitable variants/derivatives of DOTAM have a structure that differs to some limited extent from that of DOTAM and retain the ability to function (i.e., retain sufficient activity for one or more of the purposes described herein). In such aspects and embodiments, the DOTAM or functional variant/derivative of DOTAM may be one of the active variants disclosed in WO 2010/099536. Suitable functional variants/derivatives may be compounds of the formula:

or a pharmaceutically acceptable salt thereof; wherein

R^NIs H, C_1-6Alkyl radical, C_1-6Haloalkyl, C_2-6Alkenyl radical, C_2-6Alkynyl, C_3-7Cycloalkyl radical, C_3-7cycloalkyl-C_1-4Alkyl radical, C_2-7Heterocycloalkyl radical, C_2-7heterocycloalkyl-C_1-4Alkyl, phenyl-C_1-4Alkyl radical, C_1-7Heteroaryl and C_1-7heteroaryl-C_1-4An alkyl group; wherein C is_1-6Alkyl radical, C_1-6Haloalkyl, C_2-6Alkenyl and C_2-6Each alkynyl group is optionally substituted with 1, 2, 3 or 4 independently selected R^wSubstitution of radicals; and wherein the C_3-7Cycloalkyl, C_3-7cycloalkyl-C_1-4Alkyl radical, C_2-7Heterocycloalkyl, C _2-7heterocycloalkyl-C_1-4Alkyl, phenyl-C_1-4Alkyl radical, C_1-7Heteroaryl and C_1-7heteroaryl-C_1-4Alkyl radicals each independentlySelected from 1, 2, 3 or 4 independently selected R^xSubstituted by groups;

L¹independently is C_1-6Alkylene radical, C_1-6Alkenylene or C_1-6Alkynylene, each of which is optionally selected from R via 1, 2 or 3 independently¹Radical substitution of radicals;

L²is C_2-4Straight chain alkylene optionally substituted by independently selected R¹Substituted by groups; and which is optionally selected from C via 1, 2, 3 or 4 independently_1-4Alkyl and/or C_1-4Haloalkyl group substitution;

R¹independently selected from D¹-D²-D³Halogen, cyano, nitro, hydroxy, C_1-6Alkoxy radical, C_1-6Haloalkoxy, C_1-6Alkylthio radical, C_1-6Alkylsulfinyl radical, C_1-6Alkylsulfonyl, amino, C_1-6Alkylamino radical, di-C_1-6Alkylamino radical, C_1-4Alkylcarbonyl, carboxyl, C_1-6Alkoxycarbonyl group, C_1-6Alkylcarbonylamino, di-C_1-6Alkylcarbonylamino, C_1-6Alkoxycarbonylamino group, C_1-6Alkoxycarbonyl- (C)_1-6Alkyl) amino, carbamoyl, C_1-6Alkylcarbamoyl and di-C_1-6An alkylcarbamoyl group;

each D¹Is independently selected from C_6-10aryl-C_1-4Alkyl radical, C_1-9heteroaryl-C_1-4Alkyl radical, C_3-10cycloalkyl-C_1-4Alkyl radical, C_2-9heterocycloalkyl-C_1-4Alkyl radical, C_1-8Alkylene radical, C_1-8Alkenylene and C_1-8An alkynylene group; wherein the C_1-8Alkylene radical, C _1-8Alkenylene and C_1-8Alkynylene is optionally substituted with 1, 2, 3 or 4 independently selected R⁴Substitution of radicals; and wherein the C_6-10aryl-C_1-4Alkyl radical, C_1-9heteroaryl-C_1-4Alkyl radical, C_3-10cycloalkyl-C_1-4Alkyl radical, C_2-9heterocycloalkyl-C_1-4Each alkyl group optionally having 1, 2, 3 or 4 independently selected R⁵Substituted by groups;

each D²Independently of the other is absent or is C_1-20Straight chain alkylene, wherein the C_1-201 to 6 non-contiguous methylene groups of the linear alkylene group are each optionally independently selected-D⁴Module permutation with the proviso that C_1-20At least one methylene unit in the linear alkylene group not being optionally substituted by-D⁴-a module permutation; wherein the C_1-20The linear alkylene is optionally substituted with one or more groups independently selected from: halogen, cyano, nitro, hydroxy, C_1-4Alkyl radical, C_1-4Haloalkyl, C_1-4Alkoxy radical, C_1-4Haloalkoxy, amino, C_1-4Alkylamino radical, di-C_1-4Alkylamino radical, C_1-4Alkylcarbonyl, carboxyl, C_1-4Alkoxycarbonyl group, C_1-4Alkylcarbonylamino, di-C_1-4Alkylcarbonylamino, C_1-4Alkoxycarbonylamino group, C_1-4Alkoxycarbonyl- (C)_1-4Alkyl) amino, carbamoyl, C_1-4Alkylcarbamoyl and di-C_1-4An alkylcarbamoyl group;

each D³Independently selected from H, halogen, cyano, nitro, hydroxy, C_1-6Alkyl radical, C _1-6Haloalkyl, C_2-6Alkenyl radical, C_2-6Alkynyl, C_3-14Cycloalkyl radical, C_3-14cycloalkyl-C_1-4Alkyl radical, C_2-14Heterocycloalkyl radical, C_2-14heterocycloalkyl-C_1-4Alkyl radical, C_6-14Aryl radical, C_6-14aryl-C_1-4Alkyl radical, C_1-13Heteroaryl group, C_1-13heteroaryl-C_1-4An alkyl group; wherein the C is_1-6Alkyl radical, C_1-6Haloalkyl, C_2-6Alkenyl radical, C_2-6Each alkynyl group is optionally substituted with 1, 2, 3 or 4 independently selected R⁶Substituted by groups; and wherein the C_3-14Cycloalkyl radical, C_3-14cycloalkyl-C_1-4Alkyl radical, C_2-14Heterocycloalkyl radical, C_2-14heterocycloalkyl-C_1-4Alkyl radical, C_6-14Aryl radical, C_6-14aryl-C_1-4Alkyl radical, C_1-13Heteroaryl, C_1-13heteroaryl-C_1-4Alkyl radicals each independentlySelected from 1, 2, 3 or 4 independently selected R⁷Substitution of radicals;

each D⁴Independently selected from-O-, -S-, -NR^aC(＝O)-、-NR^aC(＝S)-、-NR^bC(＝O)NR^c-、-NR^bC(＝S)NR^c-、-S(＝O)-、-S(＝O)₂-、-S(＝O)NR^a-、-C(＝O)-、-C(＝S)-、-C(＝O)O-、-OC(＝O)NR^a-、-OC(＝S)NR^a-、-NR^a-、-NR^bS(＝O)NR^c-and NR^bS(＝O)₂NR^O-；

Each R⁴And R⁶Independently selected from halogen, cyano, nitro, hydroxy, C_1-4Alkoxy radical, C_1-4Haloalkoxy, C_1-4Alkylthio radical, C_1-4Alkylsulfinyl radical, C_1-4Alkylsulfonyl, amino, C_1-4Alkylamino radical, di-C_1-4Alkylamino radical, C_1-4Alkylcarbonyl, carboxyl, C_1-4Alkoxycarbonyl group, C_1-4Alkylcarbonylamino, di-C_1-4Alkylcarbonylamino, C_1-4Alkoxycarbonylamino group, C_1-4Alkoxycarbonyl- (C)_1-4Alkyl) amino, carbamoyl, C_1-4Alkylcarbamoyl and di-C_1-4An alkylcarbamoyl group;

each R⁵Independently selected from halogen, cyano, cyanate, isothiocyanate, nitro, hydroxy, C _1-4Alkyl radical, C_2-4Alkenyl radical, C_2-4Alkynyl, C_1-4Alkoxy radical, C_1-4Haloalkoxy, C_1-4Alkylthio radical, C_1-4Alkylsulfinyl radical, C_1-4Alkylsulfonyl, amino, C_1-4Alkylamino radical, di-C_1-4Alkylamino radical, C_1-4Alkylcarbonyl, carboxyl, C_1-4Alkoxycarbonyl group, C_1-4Alkylcarbonylamino, di-C_1-4Alkylcarbonylamino, C_1-4Alkoxycarbonylamino group, C_1-4Alkoxycarbonyl- (C)_1-4Alkyl) amino, carbamoyl, C_1-4Alkylcarbamoyl and di-C_1-4An alkylcarbamoyl group;

each R⁷Is independently selected fromHalogen, cyano, nitro, hydroxy, C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, C_3-7Cycloalkyl radical, C_3-7cycloalkyl-C_1-4Alkyl radical, C_2-7Heterocycloalkyl radical, C_2-7heterocycloalkyl-C_1-4Alkyl, phenyl-C_1-4Alkyl radical, C_1-7Heteroaryl group, C_1-7heteroaryl-C_1-4Alkyl, -OR^O、-SR^O、-S(＝O)R^P、-S(＝O)₂R^P、-S(＝O)NR^sR^t、-C(＝O)R^P、-C(＝O)OR^P、-C(＝O)NR^sR^t、-OC(＝O)R^P、-OC(＝O)NR^sR^t、-NR^sR^t、-NR^qC(＝O)R^r、-NR^qC(＝O)OR^r、-NR^qC(＝O)NR^r、-NR^qS(＝O)₂R^rand-NR^PS(＝O)₂NR^sR^t(ii) a Wherein the C is_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Each alkynyl group is optionally substituted with 1, 2, 3 or 4 independently selected R' groups; and wherein the C_3-7Cycloalkyl, C_3-7cycloalkyl-C_1-4Alkyl radical, C_2-7Heterocycloalkyl, C_2-7heterocycloalkyl-C_1-4Alkyl, phenyl-C_1-4Alkyl radical, C_1-7Heteroaryl, C_1-7heteroaryl-C_1-4Each alkyl group is optionally substituted with 1, 2, 3, or 4 independently selected R "groups;

each R^a、R^bAnd R^cIndependently selected from H, C_1-6Alkyl radical, C_1-6Haloalkyl, C_2-6Alkenyl radical, C_2-6Alkynyl, C _3-7Cycloalkyl, C_3-7cycloalkyl-C_1-4Alkyl radical, C_2-7Heterocycloalkyl radical, C_2-7heterocycloalkyl-C_1-4Alkyl, phenyl-C_1-4Alkyl radical, C_1-7Heteroaryl group, C_1-7heteroaryl-C_1-4An alkyl group; wherein the C is_1-6Alkyl radical, C_1-6Haloalkyl, C_2-6Alkenyl and C_2-6Each alkynyl group is optionally independently selected from 1, 2, 3 or 4R of (A) to (B)^wSubstituted by groups; and wherein the C_3-7Cycloalkyl, C_3-7cycloalkyl-C_1-4Alkyl radical, C_2-7Heterocycloalkyl, C_2-7heterocycloalkyl-C_1-4Alkyl, phenyl-C_1-4Alkyl radical, C_1-7Heteroaryl group, C_1-7heteroaryl-C_1-4Each alkyl group optionally having 1, 2, 3 or 4 independently selected R^xSubstitution of radicals;

each R^o、R^p、R^q、R^r、R^sAnd R^tIs independently selected from H, C_1-6Alkyl radical, C_1-6Haloalkyl, C_2-6Alkenyl radical, C_2-6Alkynyl, C_3-7Cycloalkyl, C_3-7cycloalkyl-C_1-4Alkyl radical, C_2-7Heterocycloalkyl, C_2-7heterocycloalkyl-C_1-4Alkyl, phenyl-C_1-4Alkyl radical, C_1-7Heteroaryl group, C_1-7heteroaryl-C_1-4An alkyl group; wherein the C_1-6Alkyl radical, C_1-6Haloalkyl, C_2-6Alkenyl radical, C_2-6Each alkynyl group is optionally substituted with 1, 2, 3 or 4 independently selected R^ySubstitution of radicals; and wherein the C_3-7Cycloalkyl, C_3-7cycloalkyl-C_1-4Alkyl radical, C_2-7Heterocycloalkyl radical, C_2-7heterocycloalkyl-C_1-4Alkyl, phenyl-C_1-4Alkyl radical, C_1-7Heteroaryl, C_1-7heteroaryl-C_1-4Each alkyl group optionally having 1, 2, 3 or 4 independently selected R ^zSubstitution of radicals;

each R', R^wAnd R^yIndependently selected from hydroxy, cyano, nitro, C_1-4Alkoxy radical, C_1-4Haloalkoxy, amino, C_1-4Alkylamino and di-C_1-4An alkylamino group; and

each R', R^xAnd R^zIndependently selected from hydroxy, halogen, cyano, nitro, C_1-4Alkyl radical, C_1-4Haloalkyl, C_1-4Alkoxy radical, C_1-4Haloalkoxy, amino, C_1-4Alkylamino and di-C_1-4An alkylamino group;

provided that the valency of each atom in the optionally substituted moiety is not exceeded.

Suitably, the affinity of the functional variant/derivative of the above formula for the antibody of the invention is comparable to or exceeds the affinity of DOTAM for the antibody of the invention, and the binding strength for Pb is comparable to or exceeds the binding strength of DOTAM for Pb ("affinity" is measured by dissociation constant as described above). For example, the dissociation constant of a functional variant/derivative and an antibody of the invention/Pb can be 1.1-fold or less, 1.2-fold or less, 1.3-fold or less, 1.4-fold or less, 1.5-fold or less, or 2-fold or less of the dissociation constant of DOTAM and the same antibody/Pb.

Each R^NCan be H, C_1-6Alkyl or C_1-6A haloalkyl group; preferably H, C_1-4Alkyl or C_1-4A haloalkyl group. Most preferably, each R ^NIs H.

For DOTAM variants, it is preferred that 1, 2, 3 or most preferably each L²Is C₂An alkylene group. Advantageously, C of DOTAM₂The alkylene variants may have a particularly high affinity for Pb. L is²The optional substituent of (A) may be R¹、C_1-4Alkyl or C_1-4A haloalkyl group. Suitably, L²The optional substituent of (A) may be C_1-4Alkyl or C_1-4A haloalkyl group.

Optionally, each L²May be unsubstituted C₂alkylene-CH₂CH₂-。

Each L¹Preferably C_1-4Alkylene, more preferably C₁Alkylene radicals, such as-CH₂-。

The functional variant/derivative of DOTAM may be a compound of the formula:

wherein each Z is independently R as defined above¹(ii) a p, q, r and s are 0, 1 or 2; and p + q + r + s is 1 or more. Youyou (an instant noodle)Optionally, p, q, r and s are 0 or 1 and/or p + q + r + s is 1. For example, the compound may have p + q + r + s ═ 1, where Z is p-SCN-benzyl module-such compounds are commercially available from Macrocyclics, Inc.

Radionuclides useful in the present invention may include radioisotopes of metals such as lead (Pb), lutetium (Lu), or yttrium (Y).

Radionuclides particularly useful in imaging applications may be radionuclides that are gamma emitters. For example, it may be selected from²⁰³Pb or²⁰⁵Bi。

Radionuclides that are particularly useful in therapeutic applications are those that act as alpha or beta emitters. For example, it may be selected from ²¹²Pb、²¹²Bi、²¹³Bi、⁹⁰Y、¹⁷⁷Lu、²²⁵Ac、²¹¹At、²²⁷Th、²²³Ra。

In some embodiments, it may be preferred that the DOTAM (or salt or functional variant thereof) is chelated with Pb or Bi, such as one of the Pb or Bi radioisotopes listed above. In other embodiments, it may be preferred that the DOTA (or salt or functional variant thereof) is chelated to Lu or Y, such as one of the Lu or Y radioisotopes listed above.

In some embodiments, methods and uses may include combination therapy and imaging methods utilizing radioisotope mixtures, such as radioisotopes suitable for therapy and radioisotopes suitable for imaging. For example, the above radioisotopes may be different radioisotopes of the same metal chelated by the same chelating agent. In one embodiment, the method may comprise administering the composition in the form of a mixture²⁰³Pb-DOTAM and²¹²Pb-DOTAM. In another embodiment, the method may comprise the use of a catalyst such as²⁰³Pb or²⁰⁵The first cycle of dosimetry using gamma emitters of Bi, followed by a second cycle using a second material such as²¹²Pb、²¹²Bi、²¹³Bi、⁹⁰Y、¹⁷⁷Lu、²²⁵Ac、²¹¹At、²²⁷Th or²²³Alpha or beta emitters of RaOne or more cycles of treatment. The method is further described below.

In some embodiments, a functional binding site formed by combining the first antibody and the second antibody can bind to the Pb-DOTAM chelate.

In some embodiments, a functional binding site formed by combining the first antibody and the second antibody can specifically bind to the radiolabeled compound. In some embodiments, it can bind to a radiolabeled compound, such as a Pb-DOTAM chelate complex, with a dissociation constant (Kd) for Pb-DOTAM and/or the target of 1 μ M or less, 100nM or less, 10nM or less, 1nM or less, 0.1nM or less, 0.01nM or less, or 0.001nM or less (e.g., 10nM or less)^-7M or less, e.g. 10^-7To 10^-13；10^-8M or less, e.g. 10^-8M to 10-13M, e.g. 10^-9M to 10^-13M). In some embodiments, it may be preferred that it binds with a binding affinity Kd value of 100pM, 50pM, 20pM, 10pM, 5pM, 1pM or less, e.g. 0.9pM or less, 0.8pM or less, 0.7pM or less, 0.6pM or less or 0.5pM or less. For example, a functional binding site may bind a metal chelate with a Kd of about 1pM to 1nM, such as about 1 to 10pM, 1 to 100pM, 5 to 50pM, 100pM or 500pM to 1 nM.

C. Exemplary antigen binding sites for DOTA

In a particular embodiment of the invention, the first and second antibodies are combined to form a conjugate for DOTA (or a functional derivative or variant thereof), e.g. with Lu or Y (e.g. with Lu or Y)¹⁷⁷Lu or ⁹⁰Y) a functional binding site for chelated DOTA. For example, a functional binding site may bind a radiolabeled compound with a Kd of about 1pM to 1nM, such as about 1 to 10pM, 1 to 100pM, 5 to 50pM, 100-500pM, or 500pM to 1 nM.

C825 is a known pair such as¹⁷⁷Lu and⁹⁰radiometal complexed DOTA-Bn (S-2- (4-aminobenzyl) -1,4,7, 10-tetraazacyclododecane tetraacetic acid) of Y has a high affinity scFv (see, e.g., Cheal et al 2018, theoratics 2018 and WO2010099536, herein incorporated by reference). Provided herein are the CDR sequences and VL and VH sequences of C825. In a 1In one embodiment, the heavy chain variable region forming part of the antigen binding site of the radiolabeled compound may comprise at least one, two or all three CDRs selected from: (a) CDR-H1 comprising the amino acid sequence of 35; (b) CDR-H2 comprising the amino acid sequence of 36; (c) CDR-H3 comprising the amino acid sequence of 37. In an alternative embodiment, the CDR-H1 can have the sequence GFSLTDYGVH (SEQ ID No.: 148). The variable region of the light chain forming part of the binding site of the radiolabeled compound may comprise at least one, two or all three CDRs selected from: (d) CDR-L1 comprising the amino acid sequence of 38; (e) CDR-L2 comprising the amino acid sequence of 39; and (f) CDR-L3 comprising the amino acid sequence of 40.

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

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

Combinations of embodiments relating to heavy chain variable regions and light chain variable regions are explicitly contemplated. Thus, a functional antigen binding site may be formed on the first and second antibodies by a heavy chain variable region as defined above and a light chain variable region as defined above, respectively.

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

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

D. Exemplary antigen binding sites for DOTAM

In another specific embodiment of the invention, the first antibody and the second antibody combine to form a functional antigen binding site for a Pb-DOTAM chelate (Pb-DOTAM).

In certain embodiments, the functional antigen-binding site that binds to Pb-DOTAM may have one or more of the following properties:

Specific binding to Pb-DOTAM and specific binding to Bi-DOTAM;

selectivity for Pb-DOTAM (and optionally Bi-DOTAM) as compared to other chelated metals such as Cu-DOTAM;

binding to Pb-DOTAM with very high affinity;

binds to the same epitope on Pb-DOTAM as an antibody described herein, e.g., PRIT-0213 or PRIT-0214, and/or has the same contact residues as the antibody.

Radioisotopes of Pb are useful in diagnostic and therapeutic methods. Specific radioisotopes of lead that may be used in the present invention include²¹²Pb and²⁰³Pb。

because of the combination of short path length and high linear energy transfer, radionuclides that are alpha-particle emitters are able to kill tumor cells more specifically with less damage to surrounding tissue than beta-emitters.²¹²Bi is an alpha-particle emitter, but its short half-life prevents its direct use.²¹²Pb is²¹²A parent radionuclide of Bi and can act as²¹²In vivo generation of Bi, thereby effectively overcoming²¹²The short half-life of Bi (Yong and Brechbiel, Dalton Trans.2001, 6.21; 40(23) 6068-6076).

²⁰³Pb can be used as an imaging isotope. Thus, is bonded to²⁰³Antibodies to Pb-DOTAM can be used in Radioimmunoassay (RII).

Generally, the radiometal is used in chelated form. In certain aspects of the invention, DOTAM is used as the chelating agent. Stable chelators with a DOTAM of Pb (II) (Yong and Brechbiel, Dalton Trans.2001, 6.21 days; 40(23)6068-Science, volume 27, pages 93-100, 2000). Thus, DOTAM and²¹²pb and²⁰³combinations of isotopes of lead as discussed above for Pb are particularly useful.

In some embodiments, it may be preferred that the antibody binds Pb-DOTAM with a binding affinity Kd value of 100pM, 50pM, 20pM, 10pM, 5pM, 1pM or less, e.g., 0.9pM or less, 0.8pM or less, 0.7pM or less, 0.6pM or less, or 0.5pM or less. For example, a functional binding site may bind a radiolabeled compound with a Kd of about 1pM to 1nM, e.g., about 1 to 10pM, 1 to 100pM, 5 to 50pM, 100pM or 500pM to 1 nM.

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

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

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

In some embodiments, the antibody may comprise one or more of CDR-H1, CDR-H2 and/or CDR-H3, or one or more of CDR-L1, CDR-L2 and/or CDR-L3 having substitutions, e.g., 1, 2 or 3 substitutions, as compared to the amino acid sequences of SEQ ID Nos. 1-6, respectively.

In some embodiments, the antibody may share the same contact residues as described herein: for example, these residues may be invariant. These residues may include the following:

a) in heavy chain CDR 2: phe50, Asp56 and/or Tyr58 and optionally in addition Gly52 and/or Arg 54;

b) in heavy chain CDR 3: glu95, Arg96, Asp97, Pro98, Tyr99, Ala100C and/or Tyr100D and optionally in addition Pro 100E;

c) in the light chain CDR 1: tyr28 and/or Asp 32;

d) in the light chain CDR 3: gly91, Tyr92, Asp93, Thr95c and/or Tyr 96;

e) in the light chain CDR 2: optionally Gln 50;

all according to Kabat numbering.

For example, in some embodiments, CDR-H2 may comprise amino acid sequence FIGSRGDTYYASWAKG (SEQ ID NO:2) or variants thereof having up to 1, 2, or 3 substitutions in SEQ ID NO:2, wherein the substitutions do not include Phe50, Asp56 and/or Tyr58, and optionally also do not include Gly52 and/or Arg 54, all according to Kabat numbering.

In some embodiments, CDR-H2 may be substituted at one or more positions as shown below. Here and in the following alternatives, substitutions are based on germline residues (underlined) or by amino acids that are theoretically matched in space and also present at sites in the crystallized repertoire. In some embodiments, residues as mentioned above may be fixed and other residues may be substituted according to the following table: in other embodiments, substitution of any residue may be made according to the following table.

Optionally, the CDR-H3 may comprise the amino acid sequence ERDPYGGGAYPPHL (SEQ ID NO:3) or variants thereof having up to 1, 2 or 3 substitutions in SEQ ID NO:3, wherein the substitutions do not include Glu95, Arg96, Asp97, Pro98, and optionally also do not include Ala100C, Tyr100D and/or Pro100E, and/or optionally also do not include Tyr 99. For example, in some embodiments, substitutions do not include Glu95, Arg96, Asp97, Pro98, Tyr99, Ala100C, and Tyr 100D.

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

WolfGuy	Kabat	AA	Substitution
				351	95	E
352	96	R	K、E
				353	97	D
354	98	P
				355	99	Y	F、G、S、T、D
356	100	G
				392	100A	G
393	100B	G
				394	100C	A	S、T
395	100D	Y	F
				396	100E	P
397	100F	P
				398	101	H	A、T、V、D
399	102	L	Y、V、I、H、F

Optionally, CDR-L1 may comprise the amino acid sequence QSSHSVYSDNDLA (SEQ ID NO:4) or variants thereof having up to 1, 2 or 3 substitutions in SEQ ID NO:4, wherein the substitutions do not include Tyr28 and/or Asp32(Kabat numbering).

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

Optionally, CDR-L3 may comprise amino acid sequence LGGYDDESDTYG (SEQ ID NO:6) or variants thereof having up to 1, 2 or 3 substitutions in SEQ ID NO:6, wherein the substitutions do not include Gly91, Tyr92, Asp93, Thr95c and/or Tyr96 (Kabat).

In certain embodiments, CDR-L3 may be substituted at the following positions as shown below. (since most residues are solvent exposed and have no antigenic junctions, many alternatives are conceivable). Furthermore, in some embodiments, residues as mentioned above may be fixed and other residues may be substituted according to the following table: in other embodiments, substitution of any residue may be made according to the following table.

WolfGuy	Kabat	AA	Substitution
				751	89	L	A,V,Q
752	90	G	A
				753	91	G
754	92	Y	A,D,E,F,G,H,I,K,L,N,Q,R,S,T,V
				755	93	D	A,E,F,G,H,I,K,L,M,N,Q,R,S,T,V,W,Y
756	94	D	A,E,F,G,H,I,K,L,M,N,Q,R,S,T,V,W,Y
				794	95	E	A,D,F,G,H,I,K,L,M,N,Q,R,S,T,V,W,Y
795	95A	S	A,F,G,H,I,K,L,M,N,Q,R,T,V,W,Y
				796	95B	D	A,E,F,G,H,I,L,M,N,Q,S,T,V,W,Y
797	95C	T	S
				798	96	Y	F,H,R
799	97	G	A,E,I,K,L,M,N,Q,S,T,V

The antibody may further comprise CDR-H1 or CDR-L2 optionally having the sequence of SEQ ID No. 1 or SEQ ID No. 5, respectively, or variants thereof having at least 1, 2 or 3 substitutions, optionally conservative substitutions, relative thereto.

Thus, the heavy chain variable domain forming part of the antigen binding site for Pb-DOTAM may comprise at least:

a) a heavy chain CDR2 comprising amino acid sequence FIGSRGDTYYASWAKG (SEQ ID NO:2) or a variant thereof having up to 1, 2 or 3 substitutions in SEQ ID NO:2, wherein the substitutions do not comprise Phe50, Asp56 and/or Tyr58, and optionally also Gly52 and/or Arg 54;

b) A heavy chain CDR3 comprising amino acid sequence ERDPYGGGAYPPHL (SEQ ID NO:3) or a variant thereof having up to 1, 2 or 3 substitutions in SEQ ID NO:3, wherein the substitutions do not comprise Glu95, Arg96, Asp97, Pro98, and optionally also do not comprise Ala100C, Tyr100D and/or Pro100E, and/or optionally also do not comprise Tyr 99.

In some embodiments, the heavy chain variable domain further comprises a heavy chain CDR1 selected from:

c) a heavy chain CDR1 comprising amino acid sequence GFSLSTYSMS (SEQ ID NO:1) or a variant thereof having up to 1, 2 or 3 substitutions in SEQ ID NO: 1.

In some embodiments, the heavy chain variable domain further comprises a C-terminal alanine (e.g., Ala114 according to the Kabat numbering system) to avoid binding of pre-existing antibodies recognizing free VH regions. As reported in Holland MC et al j. clin Immunol (2013), the free C-terminus appears to be critical for the binding of HAVH (human anti-VH domain) autoantibodies to VH domain antibodies, since HAVH autoantibodies do not bind to intact IgG or IgG fragments (fAb or modified VH molecules) containing the same VH framework sequence or to VK domain antibodies. Clinical and Experimental Immunology (2015) indicated the presence of a cryptic epitope at the C-terminal epitope of the VH dAb, which is not naturally accessible to HAVH antibodies in whole IgG molecules.

Thus, where the antibody comprises a free VH region (not fused to any other domain at its C-terminus), the sequence may be extended by one or more C-terminal residues. Extension may prevent binding of antibodies recognizing free VH regions. For example, extension may be by 1-10 residues, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues. In one embodiment, the VH sequence may be extended by one or more C-terminal alanine residues. The VH sequence may also be extended by the N-terminal part of the CH1 domain, for example by 1-10 residues from the N-terminus of the CH1 domain, for example the human IgG1 CH1 domain. (the first ten residues of the human IgG1 CH1 domain are ASTKGPSVFP (SEQ ID No.:149) and thus in one embodiment, 1-10 residues may be taken from the N-terminus of this sequence). For example, in one embodiment, the peptide sequence AST (corresponding to the first 3 residues of the IgG1 CH1 domain) is added into the C-terminus of the VH region.

In another embodiment, the light chain variable domain forming part of the antigen binding site for Pb-DOTAM comprises at least:

d) a light chain CDR1 comprising amino acid sequence QSSHSVYSDNDLA (SEQ ID NO:4) or a variant thereof having up to 1, 2 or 3 substitutions in SEQ ID NO:4, wherein the substitutions do not include Tyr28 and Asp 32;

e) A light chain CDR3 comprising amino acid sequence LGGYDDESDTYG (SEQ ID NO:6) or a variant thereof having up to 1, 2 or 3 substitutions in SEQ ID NO:6, wherein the substitutions do not include Gly91, Tyr92, Asp93, Thr95c and Tyr 96.

In some embodiments, the light chain variable domain further comprises a light chain CDR2 selected from the group consisting of:

f) light chain CDR2 comprising the amino acid sequence QASKLA (SEQ ID NO:5) or variants thereof having at least 1, 2 or 3 substitutions in SEQ ID NO:5, said substitutions optionally not comprising Gln 50.

In any embodiment of the invention that includes sequence variants comprising CDRs (e.g., with variable domains) as set forth above, the protein may be invariant in one or more of the CDR residues as set forth above.

Optionally, the heavy chain variable domain (on the first antibody) forming part of a functional antigen binding site for Pb-DOTAM comprises an amino acid sequence selected from the group consisting of SEQ ID NO 7 and 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 to SEQ ID NO 7 or SEQ ID NO 9. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity relative to a reference sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions, but the binding site comprising the sequence retains the ability to bind to Pb-DOTAM, preferably with affinity as described herein. The VH sequence may retain invariant residues as set out above. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO 7 or SEQ ID NO 9. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the CDR (i.e., in the FR). Optionally, the antibody comprises the VH sequence of SEQ ID No. 7 or SEQ ID No. 9, including post-translational modifications of that sequence, optionally with a C-terminal Ala. In a particular embodiment, the VH comprises one, two or three CDRs selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO. 1; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO. 2; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO. 3.

In some embodiments, as mentioned above, in some variants, SEQ ID NO 7 or 9 may be extended by one or more additional C-terminal residues, for example by one or more alanine residues, optionally by a single alanine residue. Thus, for example, in one specific variant, the sequence of SEQ ID NO. 7 can be extended to:

VTLKESGPVLVKPTETLTLTCTVSGFSLSTYSMSWIRQPPGKALEWLGFIGSRGDTYYASWAKGRLTISKDTSKSQVVLTMTNMDPVDTATYYCARERDPYGGGAYPPHLWGRGTLVTVSSA(SEQ ID NO.:150)

in other embodiments, extension may be through the N-terminal portion of the CH1 domain as described above, e.g., through 1-10 residues from the N-terminus of the CH1 domain, e.g., the human IgG1 CH1 domain. For example, the extension may be performed by the peptide sequence AST.

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

Combinations of embodiments relating to heavy chain variable regions and light chain variable regions are explicitly contemplated. Thus, a functional antigen binding site for Pb-DOTAM may be formed on the first and second antibodies by the heavy chain variable region as defined above and the light chain variable region as defined above, respectively.

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

In any of the above embodiments, the light chain variable region and the heavy chain variable region that form the anti-Pb-DOTAM binding site may be humanized. In one embodiment, the light chain variable region and the heavy chain variable region comprise CDRs as in any one of the above embodiments, and further comprise an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework. In another embodiment, the light chain variable region and/or the heavy chain variable region comprises CDRs as in any one of the above embodiments and further comprises framework regions derived from vk 139 and/or vh 226. In some embodiments, there may be no back-mutations for vk 139. For vh 226, germline Ala49 residues may be back-mutated to Gly 49.

E. Antigen binding site of exemplary CEA

In another particular embodiment of the invention, which may be combined with the embodiments discussed above, the target antigen to which the first and second antibodies bind may be CEA (carcinoembryonic antigen). Cultured anti-CEA antibodies include T84.66 and humanized and chimeric versions thereof, such as T84.66-LCHA as described in WO2016/075278A1 and/or WO2017/055389, CH1A1a, anti-CEA antibodies as described in WO2012/117002 and WO2014/131712, and CEAhMN-14 or labetazumab (labetuzumab) (e.g., as described in US 6676924 and US 5874540). Another exemplary anti-CEA antibody is A5B7 (e.g., as described in m.j.banfield et al, Proteins 1997,29(2), 161-171) or a humanized antibody derived from murine A5B7 as described in WO 92/01059 and WO 2007/071422. See also co-pending application PCT/EP 2020/067582. An example of a humanized version of A5B7 is A5H1EL1 (G54A). Another exemplary anti-CEA antibody is MFE23 and humanized versions thereof described in US 7626011 and/or co-pending application PCT/EP 2020/067582. Yet another example of an anti-CEA antibody is 28A 9. Any of the above antibodies or antigen binding fragments thereof may be used to form the CEA binding portion of the present invention.

Optionally, for monovalent binding, the antigen binding site that binds to CEA can bind with a Kd value of 1nM or less, 500pM or less, 200pM or less, or 100pM or less.

In some embodiments, the first antibody and/or the second antibody can bind to the CH1A1a epitope, A5B7 epitope, MFE23 epitope, T84.66 epitope, or 28a9 epitope of CEA.

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

Epitopes for CH1A1a and its parent murine antibody PR1A3 are described in WO2012/117002A1 and Durbin H.et al, Proc.Natl.Scad.Sci.USA,91: 4313-. The antibody that binds to the CH1A1a epitope binds to the B3 domain of the CEA molecule and a conformational epitope within the GPI anchor. In one aspect, the antibody binds to the same epitope as a CH1A1a antibody having a VH with SEQ ID No. 25 and a VL with SEQ ID No. 26 herein. The A5B7 epitope is described in co-pending application PCT/EP 2020/067582. The antibody that binds to the A5B7 epitope binds to the a2 domain of CEA, that is, to the domain comprising amino acids having SEQ ID NO: 154:

PKPFITSNNSNPVEDEDAVALTCEPEIQNTTYLWWVNNQSLPVSPRLQ LSNDNRTLTLLSVTRNDVGP YECGIQNKLSVDHSDPVILN(SEQ ID NO:154)。

In one aspect, the antibody binds to the same epitope as an A5B7 antibody having a VH with SEQ ID No. 49 and a VL with SEQ ID No. 50 herein.

In one aspect, the antibody binds to the same epitope as T84.66 described in WO 2016/075278. The antibody can bind to the same epitope as an antibody having a VH having SEQ ID NO 17 and a VL having SEQ ID NO 18 herein.

The epitope of MFE23 is described in co-pending application PCT/EP 2020/067582. The antibody that binds to the epitope of MFE23 binds to the a1 domain of CEA, that is, to the domain comprising amino acids having SEQ ID NO: 155:

PKPSISSNNSKPVEDKDAVAFTCEPETQDATYLWWVNNQSLPVSPRLQLSNGNRTLTLFNVTRNDTAS YKCETQNPVSARRSDSVILN(SEQ ID NO:155)。

in one aspect, the antibody can bind to the same epitope as an antibody having a VH domain of SEQ ID No. 167 and a VL domain of SEQ ID No. 168 herein.

In some embodiments, the first and/or second antibody may bind to the same CEA-epitope as an antibody provided herein, e.g., P1AD8749, P1AD8592, P1AE4956, P1AE4957, P1AF0709, P1AF0298, P1AF0710, or P1AF 0711.

In some embodiments, the first and second antibodies bind the same epitope of CEA as each other. Thus, for example, both the first and second antibodies can bind to the CH1A1a epitope, the A5B7 epitope, the MFE23 epitope, the T84.66 epitope, or the 28a9 epitope.

In some embodiments, both the first and second antibodies may have CEA binding sequences (i.e., CDR and/or VH/VL domains) from CH1 A1A; or both the first and second antibodies may have CEA binding sequences from A5B7 or a humanized version thereof; or both the first and second antibodies may have a CEA binding sequence from T84.66 or a humanized version thereof; or both the first and second antibodies may have CEA binding sequences from MFE23 or a humanized version thereof; or both the first and second antibodies may have CEA binding sequences from 28a9 or a humanized version thereof. Exemplary sequences are disclosed herein.

In other embodiments, the first and second antibodies bind to different epitopes of CEA. Thus, for example, i) one antibody can bind the CH1A1A epitope and another antibody can bind the A5B7 epitope, the T84.66 epitope, the MFE23 epitope, or the 28a9 epitope; ii) one antibody can bind to the A5B7 epitope and the other antibody can bind to the CH1A1A epitope, the T84.66 epitope, the MFE23 epitope, or the 28a9 epitope; iii) one antibody can bind to the MFE23 epitope and the other antibody can bind to the CH1A1A epitope, the A5B7 epitope, the T84.66 epitope, or the 28a9 epitope; iv) one antibody can bind to the T84.66 epitope and another antibody can bind to the CH1A1A epitope, the A5B7 epitope, the MFE23 epitope, or the 28a9 epitope; or v) one antibody can bind the 28a9 epitope and another antibody can bind the CH1A1a epitope, the A5B7 epitope, the MFE23 epitope, or the T84.66 epitope.

In some embodiments, i) one antibody may have CEA binding sequences (i.e., CDR or VH/VL domain) from CH1A1A and the other antibody may have CEA binding sequences from A5B7 or a humanized version thereof, from T84.66 or a humanized version thereof, from MFE23 or a humanized version thereof, or from 28a9 or a humanized version thereof; ii) one antibody may have a CEA binding sequence from A5B7 or a humanized version thereof and the other antibody may have a CEA binding sequence from CH1A1A, from T84.66 or a humanized version thereof, from MFE23 or a humanized version thereof, or from 28a9 or a humanized version thereof; iii) one antibody may have a CEA binding sequence from MFE23 or a humanized version thereof and the other antibody may have a CEA binding sequence from CH1A1A, from A5B7 or a humanized version thereof, from T84.66 or a humanized version thereof, or from 28a9 or a humanized version thereof; iv) one antibody may have a CEA binding sequence from T84.66 or a humanized version thereof and the other antibody may have a CEA binding sequence from CH1A1A, from A5B7 or a humanized version thereof, from MFE23 or a humanized version thereof, or from 28a9 or a humanized version thereof; v) one antibody may have a CEA binding sequence from 28a9 or a humanized version thereof and the other antibody may have a CEA binding sequence from CH1A1A, from A5B7 or a humanized version thereof, from T84.66 or a humanized version thereof, or from MFE23 or a humanized version thereof.

In a particular embodiment, one antibody may bind the CH1A1A epitope and the other antibody may bind the A5B7 epitope. The first antibody may have a CEA binding sequence from antibody CH1A1A and the second antibody may have a CEA binding sequence from A5B7 (including humanized versions thereof); or the first antibody may have CEA binding sequences from antibody A5B7 (including humanized versions thereof) and the second antibody may have CEA binding sequences from CH1 A1A.

In another particular embodiment, one antibody may bind the CH1A1A epitope and the other antibody may bind the T84.66 epitope. The first antibody may have a CEA binding sequence from antibody CH1A1A and the second antibody may have a CEA binding sequence from T84.66 (including humanized versions thereof); or the first antibody may have a CEA binding sequence from antibody T84.66 (including humanized versions thereof) and the second antibody may have a CEA binding sequence from CH1 A1A. In some embodiments, the first antibody may bind to a T84.66 epitope and/or have an antigen binding site as described in (i) below, and the second antibody may bind to a CH1A1A epitope and/or have an antigen binding site as described in (ii) below.

Exemplary CEA binding sequences i) -v) are disclosed below. The exemplary CEA binding sequences i) -v) provide examples of CEA binding sequences from i) T84.66, ii) CH1A1A, iii) A5B7, iv)28a9, and v) MFE23 (or from humanized versions thereof).

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

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

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

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

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

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

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

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

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

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

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

Optionally, the antigen binding site that binds to CEA comprises at least one, at least two, or all three VLCDR sequences selected from: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO. 22; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO. 23; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 24.

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

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

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

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

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

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

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

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

Optionally, the antigen binding site that binds to CEA comprises at least one, at least two, or all three VL CDR sequences selected from: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO. 46; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO. 47; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO. 48.

Optionally, the antigen binding site that binds to CEA comprises (a) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:43, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:44 and (iii) CDR-H3 comprising an amino acid sequence selected from SEQ ID NO: 45; and (b) a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from: (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO. 46, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO. 47, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO. 48. In some embodiments, the CDR-H1 may have the sequence GFTFTDYYMN (SEQ ID No.: 151).

In another aspect, the antigen binding site that binds to CEA comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 43; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO. 44; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO 45; (d) 46 comprising CDR-L1 of the amino acid sequence of SEQ ID NO; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO. 47; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO 48. In some embodiments, the CDR-H1 may have the sequence GFTFTDYYMN (SEQ ID No.: 151).

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

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

In another embodiment, the antigen binding site that binds to CEA comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO. 50. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity relative to a reference sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions, but the antigen-binding site comprising that sequence retains the ability to bind to CEA, preferably with affinity as set forth above. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in SEQ ID NO: 50. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the HVR (i.e., in the FR). Optionally, the antigen binding site of CEA comprises the VL sequence of SEQ ID NO. 50, including post-translational modifications of this sequence. In a particular embodiment, the VL comprises one, two or three CDRs selected from: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO. 46; (b) 47 or a CDR-L2 comprising the amino acid sequence of SEQ ID NO; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO 48.

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

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

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

Optionally, the antigen binding site that binds to CEA comprises at least one, at least two, or all three VL CDR sequences selected from: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO. 62; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO. 63; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 64.

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

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

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

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

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

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

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

Optionally, the antigen binding site that binds to CEA can comprise:

a VH CDR sequence that (a) comprises CDR-H1 of the amino acid sequence of SEQ ID NO: 156; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO 157 or 158; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 159; and/or

A VLCDR sequence that (a) includes CDR-L1 of the amino acid sequence of SEQ ID NO 160, 161, or 162; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO 163, 164 or 165; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 166.

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

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

In a particular aspect, the antigen binding domain capable of binding to CEA comprises:

(a) a VH domain comprising the amino acid sequence of SEQ ID NO. 169 and a VL domain comprising the amino acid sequence of SEQ ID NO. 179, or

(b) A VH domain comprising the amino acid sequence of SEQ ID NO 173 and a VL domain comprising the amino acid sequence of SEQ ID NO 179, or

(c) A VH domain comprising the amino acid sequence of SEQ ID NO:170 and a VL domain comprising the amino acid sequence of SEQ ID NO:179, or

(d) A VH domain comprising the amino acid sequence of SEQ ID NO. 174 and a VL domain comprising the amino acid sequence of SEQ ID NO. 178, or

(e) A VH domain comprising the amino acid sequence of SEQ ID NO 173 and a VL domain comprising the amino acid sequence of SEQ ID NO 178, or

(f) A VH domain comprising the amino acid sequence of SEQ ID NO. 171 and a VL domain comprising the amino acid sequence of SEQ ID NO. 178, or

(g) A VH domain comprising the amino acid sequence of SEQ ID NO 169 and a VL domain comprising the amino acid sequence of SEQ ID NO 178.

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

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

In another embodiment, the antigen binding site that binds to CEA comprises a VH as in any one of the embodiments provided above and a VL as in any one of the embodiments provided above.

F. Exemplary antigen binding sites for other targets

In another particular embodiment of the invention that may be combined with the embodiments discussed above (e.g., binding sites for DOTA or DOTAM), the target antigen to which the first and second antibodies bind may be GPRC5D or FAP.

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

Exemplary GPRC5D binding sequences are described below.

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

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

Optionally, the antigen binding site that binds to GPRC5D comprises at least one, at least two, or all three VL CDR sequences selected from: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO 70; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO 71; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 72.

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

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

In any of the above embodiments, the multispecific antibody may be humanized. In one embodiment, the anti-GPRC 5D antigen binding site comprises CDRs as in any one of the above embodiments and further comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.

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

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

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

An exemplary FAP binding sequence is described below.

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

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

Optionally, the antigen binding site that binds to FAP comprises at least one, at least two, or all three VL CDR sequences selected from: (a) 78, CDR-L1 comprising the amino acid sequence of SEQ ID NO; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO. 79; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 80.

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

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

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

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

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

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

G. Antibody formats

As described above, the present invention relates to a set 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 of the antigen binding site of the radiolabeled compound, but does not comprise a VL domain of the antigen binding site of the radiolabeled compound; and

ii) a second antibody that binds to an antigen expressed on the surface of a target cell and further comprises a VL domain of the antigen-binding site of the radiolabeled compound but does not comprise a VH domain of the antigen-binding site of the radiolabeled compound,

wherein the VH domain of the first antibody and the VL domain of the second antibody are capable of together forming a functional antigen-binding site for a radiolabeled compound.

In some embodiments, the first and second antibodies may each comprise an Fc domain. The presence of an Fc region in the context of radioimmunotherapy and radioimaging has benefits, such as extending the circulating half-life of the protein and/or causing tumor uptake above that which can be observed with smaller fragments.

In some embodiments, where an Fc region is present, it may be preferred that the Fc region is engineered to reduce or eliminate effector function. Such engineering may include substitution of one or more of residues 234, 235, 238, 265, 269, 270, 297, 327 and/or 329 of the Fc region, for example one or more of residues 234, 235 and/or 329. In some embodiments, the Fc region may be engineered to include substitutions Pro329 to Gly, Leu 234 to Ala, and/or Leu 235 to Ala (numbered according to the EU index).

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

In some embodiments, it may be preferred that when the first antibody and the second antibody are combined, they form an antibody complex that is monovalent for the radiolabeled compound. Thus, the first antibody may comprise only one VH domain of the antigen binding site of the radiolabeled compound and the second antibody may comprise only one VL domain of the antigen binding site of the radiolabeled compound such that together they form only one complete functional binding site of the radiolabeled compound.

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

The fusion may be direct or indirect. In some embodiments, fusion may be via a linker. For example, the Fc region may be fused to the antibody fragment via a hinge region or another suitable linker. Similarly, the linkage of the VL or VH domain of the antigen binding site of the radiolabeled compound to the rest of the antibody structure may be via a linker. The linker may be a peptide having at least 5 amino acids, preferably 5 to 100, more preferably 10 to 50 or 25 to 50 amino acids. The joint may be a rigid joint or a flexible joint. In some embodiments, it is a flexible linker comprising or consisting of: thr, Ser, Gly and/or Ala residues. For example, it may comprise or consist of: gly and Ser residues. In some embodiments, it may have repeating motifs such as (Gly-Ser) n, where n is, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the peptide linker is (GxS) n or (GxS) nGm, wherein G ═ glycine, S ═ serine, and (x ═ 3, n ═ 3, 4, 5 or 6, and m ═ 0, 1, 2 or 3) or (x ═ 4, n ═ 2, 3, 4 or 5, and m ═ 0, 1, 2 or 3), for example x ═ 4 and n ═ 2 or 3, for example where x ═ 4 and n ═ 2. In some embodiments, the linker may be or may comprise the sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID No.: 31). Other linkers can be used and can be identified by the skilled person.

In a particular embodiment, the first antibody may comprise or consist of:

a) a scFv fragment, wherein the scFv fragment binds a target antigen; and

b) a polypeptide comprising or consisting of:

i) antibody heavy chain variable domains (VH); or

ii) an antibody heavy chain variable domain (VH) and an antibody heavy chain constant domain, wherein the C-terminus of the VH domain is fused to the N-terminus of the constant domain;

wherein the polypeptide is fused to the C-terminus of the scFv fragment via the N-terminus of the VH domain, preferably via a peptide linker.

The second antibody may comprise or consist of:

c) a second scFv that binds a target antigen; and

d) a polypeptide comprising or consisting of:

i) an antibody light chain variable domain (VL); or

ii) an antibody light chain variable domain (VL) and an antibody light chain constant domain, wherein the C-terminus of the VL domain is fused to the N-terminus of the constant domain;

wherein the polypeptide is fused to the C-terminus of the scFv fragment through the N-terminus of the VL domain, preferably via a peptide linker.

The antibody heavy chain variable domain (VH) of the first antibody and the antibody light chain variable domain (VL) of the second antibody together form a functional antigen binding site for the radiolabeled compound when the two antibodies are combined.

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

The variable domains of the heavy and light chains of the scFv that recognize the target antigen may be linked by a peptide tether. Such peptide tethers may comprise from 1 to 25 amino acids, preferably from 12 to 20 amino acids, preferably from 12 to 16 or from 15 to 20 amino acids. Tethers described above may comprise one or more (G3S) and/or (G4S) motifs, in particular 1, 2, 3, 4, 5 or 6 (G3S) and/or (G4S) motifs, preferably 3 or 4 (G3S) and/or (G4S) motifs, more preferably 3 or 4 (G4S) motifs.

Optionally, the first antibody may consist essentially of or consist of components (a) and (b) listed above, and the second antibody may consist essentially of or consist of components (c) and (d) listed above. In any case, the first antibody does not comprise an antibody light chain variable domain (VL) capable of forming a functional antigen binding site for a radiolabeled compound in association with component (b) of the first antibody; and the second antibody does not comprise an antibody heavy chain Variable (VH) domain capable of forming a functional antigen binding site for a radiolabelled compound in association with component (d) of the second antibody.

In another particular embodiment, the first antibody may comprise or consist of:

a) Fab fragments that bind to a target antigen, and

b) a polypeptide comprising or consisting of:

i) the antibody heavy chain variable domain (VH) of the antigen binding site of the radiolabeled compound, or

ii) an antibody heavy chain variable domain (VH) and an antibody heavy chain constant domain of the antigen binding site of the radiolabeled compound, wherein the C-terminus of the VH domain is fused to the N-terminus of the constant domain;

wherein the polypeptide is fused to the CL domain or the C-terminus of the CH1 domain of the Fab fragment via the N-terminus of the VH domain, preferably via a peptide linker.

The second antibody may comprise or consist of:

c) fab fragments that bind to a target antigen, and

d) a polypeptide comprising or consisting of:

iii) the antibody light chain variable domain (VL) of the antigen binding site of the radiolabeled compound, or

iv) an antibody light chain variable domain (VL) and an antibody light chain constant domain of the antigen binding site of the radiolabeled compound, wherein the C-terminus of the VL domain is fused to the N-terminus of the constant domain;

wherein the polypeptide is fused to the CL domain or the C-terminus of the CH1 domain of the Fab fragment via the N-terminus of the VL domain, preferably via a peptide linker.

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

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

Optionally, the first antibody may consist essentially of or consist of components (a) and (b) listed above, and the second antibody may consist essentially of or consist of components (c) and (d) listed above. In any case, the first antibody does not comprise an antibody light chain variable domain (VL) capable of forming a functional antigen binding site for a radiolabeled compound in association with component (b) of the first antibody; and the second antibody does not comprise an antibody heavy chain Variable (VH) domain capable of forming a functional antigen binding site for a radiolabelled compound in association with component (d) of the second antibody.

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

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

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

In one embodiment, the first antibody comprises

a) A tandem Fab comprising two Fab fragments, wherein the first and second Fab fragments bind to the same target antigen ("target antigen a") and the epitope bound by the first Fab fragment is the same as the epitope bound by the second Fab fragment, and wherein the first and second Fab fragments are connected via a peptide tether, wherein the first Fab is connected via its C-terminus to the N-terminus of the second Fab; and

b) a polypeptide comprising or consisting of:

i) antibody heavy chain variable domains (VH); or

ii) an antibody heavy chain variable domain (VH) and an antibody constant domain (CH1), wherein the C-terminus of the VH domain is fused to the N-terminus of the CH1 domain;

wherein the polypeptide is fused to the CL domain of the second Fab fragment or the C-terminus of the CH1 domain via the N-terminus of the VH domain, preferably via a peptide linker;

and the second antibody comprises

c) A tandem Fab comprising two Fab fragments, wherein the first and second Fab fragments bind to the target antigen a and the epitope bound by the first Fab fragment is the same as the epitope bound by the second Fab fragment, and wherein the first and second Fab fragments are linked via a peptide tether, wherein the first Fab is linked via its C-terminus to the N-terminus of the second Fab; and

d) A polypeptide comprising or consisting of:

i) an antibody light chain variable domain (VL); or

ii) an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL), wherein the C-terminus of the VL domain is fused to the N-terminus of the constant domain;

wherein the polypeptide is fused to the CL domain of the second Fab fragment or the C-terminus of the CH1 domain via the N-terminus of the VL domain, preferably via a peptide linker.

The antibody heavy chain variable domain (VH) of part b (in the first antibody) and the antibody light chain variable domain (VL) of part (d) (in the second antibody) together form a functional antigen binding site for the radiolabelled compound, i.e. when the two antibodies are combined.

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

The chain of the tandem Fab fused to the polypeptide (i.e., whether or not the polypeptide is fused to the CL domain or the CH1 domain of the second Fab fragment) can be independently selected for the first and second antibodies.

As described above, the first Fab fragment of the tandem Fab is linked to the N-terminus of the second Fab fragment. In one embodiment, the C-terminus of the heavy chain fragment of the first Fab fragment is linked to the N-terminus of the heavy chain fragment or the light chain fragment of the second Fab fragment. In another embodiment, the C-terminus of the light chain fragment of the first Fab fragment is linked to the N-terminus of the heavy or light chain fragment of the second Fab fragment. Thus, in some embodiments, a tandem Fab of a first antibody and/or a second antibody may comprise three chains as follows:

1) A light chain fragment of the first Fab fragment ((VLCL)1), a heavy chain fragment of the first Fab fragment connected to a heavy chain fragment of the second Fab fragment via a peptide tether ((VHCH1) 1-tether- (VHCH1)2) and a light chain fragment of the second Fab fragment ((VLCL) 2); or

2) A light chain fragment of the first Fab fragment ((VLCL)1), a heavy chain fragment of the first Fab fragment connected to a light chain fragment of the second Fab fragment via a peptide tether ((VHCH1) 1-tether- (VLCL)2) and a heavy chain fragment of the second Fab fragment ((VH-CH1) 2); or

3) A heavy chain fragment of the first Fab fragment (VHCH1), a light chain fragment of the first Fab fragment linked to a light chain fragment of the second Fab fragment via a peptide tether ((VLCL) 1-tether- (VLCL)2), and a heavy chain fragment of the second Fab fragment; or

4) A heavy chain fragment of the first Fab fragment (VHCH1), a light chain fragment of the first Fab fragment ((VLCL) 1-tether- (VHCH1)2) linked to a heavy chain fragment of the second Fab fragment via a peptide tether, and a light chain fragment of the second Fab fragment ((VLCL) 2).

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

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

In one particular embodiment, the first antibody may comprise:

a) a tandem Fab comprising a first fragment and a second fragment, wherein the first fragment is connected via a peptide tether through its C-terminus to the N-terminus of the second fragment, wherein the first fragment binds a first epitope of target antigen A and the second fragment binds a second epitope of target antigen A, and wherein one of the fragments selected from the first fragment and the second fragment is a Fab and the other is a cross Fab,

b) a polypeptide comprising or consisting of:

i) antibody heavy chain variable domains (VH); or

ii) an antibody heavy chain variable domain (VH) and an antibody heavy chain constant domain (CH1), wherein the C-terminus of the VH domain is fused to the N-terminus of the CH1 domain;

wherein the polypeptide is fused to the C-terminus of one of the chains of the second fragment via the N-terminus of the VH domain, preferably via a peptide linker.

The second antibody may comprise

c) A tandem Fab comprising a first fragment and a second fragment, wherein the first fragment is connected by its C-terminus to the N-terminus of the second fragment, wherein the first fragment binds to a first epitope of target antigen a and the second fragment binds to a second epitope of target antigen a, and wherein one of the fragments selected from the first fragment and the second fragment is a Fab and the other is a cross Fab; and

d) a polypeptide comprising or consisting of:

i) an antibody light chain variable domain (VL); or

ii) an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL), wherein the C-terminus of the VL domain is fused to the N-terminus of the light chain constant domain

Wherein the polypeptide is fused to the C-terminus of one of the chains of the second fragment via the N-terminus of the VL domain, preferably via a peptide linker.

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

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

The first or second fragment may be a crossover Fab, as long as the tandem Fab comprises one conventional Fab and one crossover Fab.

In any of the tandem Fab embodiments described above, including those involving a crossover Fab, optionally, the first antibody may consist essentially of or consist of components (a) and (b) and the second antibody may consist essentially of or consist of components (c) and (d). In any case, the first antibody does not comprise an antibody light chain variable domain (VL) capable of forming a functional antigen binding site of the radiolabeled compound in association with component (b) of the first antibody; and the second antibody does not comprise an antibody heavy chain Variable (VH) domain capable of forming a functional antigen binding site for a radiolabelled compound in association with component (d) of the second antibody.

In any of the tandem Fab embodiments (including tandem Fab embodiments involving crossed fabs), the first antibody and the second antibodyThe peptide tether connecting the Fab fragment in the antibody may be a peptide having an amino acid sequence of at least 5 amino acids in length, preferably 5 to 100, more preferably 10 to 50 amino acids in length. In one embodiment, the peptide linker is (GxS) n or (GxS) nGm, wherein G ═ glycine, S ═ serine, and (x ═ 3, n ═ 3, 4, 5, or 6, and m ═ 0, 1, 2, or 3) or (x ═ 4, n ═ 2, 3, 4, or 5, and m ═ 0, 1, 2, or 3), preferably x ═ 4 and n ═ 2 or 3, more preferably wherein x ═ 4, n ═ 2. In one embodiment, the peptide tether is (G4S) ₂。

As mentioned above, in some embodiments, the first and second antibodies may each comprise an Fc domain that is optionally engineered to reduce or eliminate effector function.

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

Optionally, the antibody comprising the Fc domain may be monovalent with respect to binding to a target antigen. In other embodiments, it may be multivalent, for example bivalent. The first antibody and the second antibody can each be multivalent and monospecific for the same epitope of the target antigen. In still other embodiments, the first and second antibodies may each have a binding site for a different epitope of the target antigen-e.g., they may be biparatopic.

The antibody fragment may be an scFv. Thus, in one embodiment, the first antibody may comprise or consist of:

a) a scFv fragment, wherein the scFv fragment binds a target antigen;

b) an Fc domain; and

c) A polypeptide comprising or consisting of:

i) an antibody heavy chain variable domain (VH); or

ii) an antibody heavy chain variable domain (VH) and an antibody heavy chain constant domain (CH1), wherein the C-terminus of the VH domain is fused to the N-terminus of the constant domain;

wherein the scFv of (a) is fused to the N-terminus of the Fc domain, and wherein the polypeptide of C) is fused to the C-terminus of the Fc domain through the N-terminus of the VH domain, preferably via a peptide linker.

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

The second antibody may comprise or consist of:

d) a second scFv that binds a target antigen;

e) an Fc domain; and

f) a polypeptide comprising or consisting of:

i) an antibody light chain variable domain (VL); or

ii) an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL), wherein the C-terminus of the VL domain is fused to the N-terminus of the constant domain;

wherein the scFv of (d) is fused to the N-terminus of the Fc domain, and wherein the polypeptide of (f) is fused to the C-terminus of the Fc domain through the N-terminus of the VH domain, preferably via a peptide linker.

In another embodiment, the first and second antibodies may each be a one-armed IgG comprising Fab (e.g., a single Fab for the target antigen) and Fc domains for the target antigen. Thus, the first antibody may comprise or consist of:

i) a complete light chain fragment;

ii) an intact heavy chain;

iii) another Fc chain lacking Fd; and

iv) a VH domain comprising or consisting of the antigen-binding site of a radiolabeled compound;

wherein the light chain of (i) and the heavy chain of (ii) together provide an antigen binding site for a target antigen; and wherein the VH domain comprising or consisting of the antigen-binding site of the radiolabelled compound is fused via its N-terminus, preferably via a linker, to the C-terminus of (ii) or (iii).

The second antibody may comprise or consist of:

v) a complete light chain fragment;

vi) an intact heavy chain;

vii) another Fc chain lacking Fd; and

viii) a VL domain comprising or consisting of the antigen binding site of a radiolabeled compound;

wherein the light chain of (v) and the heavy chain of (vi) together provide an antigen binding site for a target antigen; and wherein the VL domain comprising or consisting of the antigen binding site of the radiolabeled compound is fused via its N-terminus, preferably via a linker, to the C-terminus of (vi) or (vii).

The VH domain comprising or consisting of the antigen binding site of the radiolabelled compound may be a polypeptide comprising or consisting of:

i) an antibody heavy chain variable domain (VH), in which case the polypeptide may additionally comprise one or more residues at the C-terminus of the VH domain, optionally one or more alanine residues, optionally a single alanine residue; or optionally the N-terminal portion of the CH1 domain as described above; or

ii) an antibody heavy chain variable domain (VH) and an antibody heavy chain constant domain (CH1), wherein the C-terminus of the VH domain is fused to the N-terminus of the CH1 domain.

The polypeptide comprising or consisting of the VL domain of the antigen binding site of the radiolabeled compound may be a polypeptide comprising or consisting of:

i) antibody heavy chain variable domains (VH); or

ii) an antibody heavy chain variable domain (VH) and an antibody light chain constant domain, wherein the C-terminus of the VH domain is fused to the N-terminus of the constant domain.

When the primary and secondary antibodies are heterodimers, for example in the case of a one-armed IgG, their assembly can be aided by the use of knob-in-hole (knob-hole) techniques as described further below.

In another embodiment, the antibodies may each comprise a tandem Fab as described above (e.g., comprising two Fab fragments, wherein the first and second Fab fragments both bind to the same epitope of target antigen a; or comprising a Fab and a cross Fab, one of which binds to a first epitope of target antigen a and the other of which binds to a second epitope of target antigen a), wherein the tandem Fab is fused (e.g., via its C-terminus) to the N-terminus of the Fc domain, and wherein a VH domain or a VL domain comprising or consisting of the antigen binding site of the radiolabelled compound is fused (e.g., via its N-terminus) to the C-terminus of the Fc domain.

Thus, the first antibody may comprise or consist of:

a) a tandem Fab selected from:

i) a tandem Fab comprising two Fab fragments, wherein the first and second Fab fragments bind to the target antigen a and the epitope bound by the first Fab fragment is the same as the epitope bound by the second Fab fragment, and wherein the first and second Fab fragments are connected via a peptide tether, wherein the first Fab is connected via its C-terminus to the N-terminus of the second Fab; and

ii) a tandem Fab comprising a first fragment and a second fragment, wherein the first fragment is linked by its C-terminus to the N-terminus of the second fragment via a peptide tether, wherein the first fragment binds to a first epitope of target antigen a and the second fragment binds to a second epitope of target antigen a, and wherein one of the fragments selected from the first fragment and the second fragment is a Fab and the other is a cross Fab;

b) an Fc domain; and

c) a polypeptide comprising or consisting of:

i) antibody heavy chain variable domains (VH); or

ii) an antibody heavy chain variable domain (VH) and an antibody heavy chain constant domain (CH1), wherein the C-terminus of the VH domain is fused to the N-terminus of the CH1 domain,

wherein the tandem Fab is fused to the N-terminus of one of the chains of the Fc domain and the polypeptide of C) is fused to the C-terminus of one of the chains of the Fc domain through the N-terminus of the VH domain, preferably via a peptide linker.

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

The second antibody may comprise or consist of:

d) a tandem Fab selected from:

i) a tandem Fab comprising two Fab fragments, wherein the first and second Fab fragments bind to the target antigen a and the epitope bound by the first Fab fragment is the same as the epitope bound by the second Fab fragment, and wherein the first and second Fab fragments are linked via a peptide tether, wherein the first Fab is linked via its C-terminus to the N-terminus of the second Fab; and

ii) a tandem Fab comprising a first fragment and a second fragment, wherein the first fragment is connected by its C-terminus to the N-terminus of the second fragment via a peptide tether, wherein the first fragment binds to a first epitope of target antigen a and the second fragment binds to a second epitope of target antigen a, and wherein one of the fragments selected from the first fragment and the second fragment is a Fab and the other is a cross Fab;

e) An Fc domain; and

f) a polypeptide comprising or consisting of:

i) an antibody heavy chain variable domain (VH); or

ii) an antibody heavy chain variable domain (VH) and an antibody light chain constant domain, wherein the C-terminus of the VH domain is fused to the N-terminus of the light chain constant domain,

wherein the tandem Fab of (d) is fused to the N-terminus of one of the chains of the Fc domain and the polypeptide of (f) is fused to the C-terminus of one of the chains of the Fc domain through the N-terminus of the VL domain, preferably via a peptide linker.

The VH domain of the first antibody and the VL domain of the second antibody together form the antigen binding site of the radiolabelled compound, i.e. when the two antibodies are combined.

If the first antibody comprises a tandem Fab according to (a) (i), the following is generally the case: the second antibody comprises a tandem Fab according to d (i); if the first antibody comprises a tandem Fab according to (a) (ii), the following is generally the case: the second antibody comprises a tandem Fab according to d (ii).

Tandem fabs can be generally as described above. For example, a tether linking two fragments of a tandem Fab may be as described above. A tandem Fab may be composed of any of the set of chains set forth above. In general, the heavy chain fragment of the second Fab (which may be a crossover Fab) may be linked to an Fc domain.

In another embodiment, each of the first and second antibodies may comprise a) an Fc domain; b) at least one antibody fragment comprising an antigen binding site for a target antigen, such as an scFv, Fv, Fab or cross Fab fragment; and C) a polypeptide comprising a VL domain or a VH domain (but not both) of the antigen binding site of the radiolabeled compound, wherein the C-terminus of the antibody fragment of (b) is fused to the N-terminus of one chain of the Fc domain and the C-terminus of the polypeptide of (C) is fused to the N-terminus of the other chain of the Fc domain. (b) The fusion of the antibody fragment of (a) is preferably performed via a hinge region. (c) The fusion of the polypeptide of (a) may be via a linker located between the C-terminus of the polypeptide and the N-terminus of the Fc region and/or via some or all of the upper hinge region (e.g., Asp221 and its C-terminal residue according to EU numbering index). In one embodiment, the antibody fragment of (b) may be a Fab fragment. In one embodiment, in the first antibody, the polypeptide of (c) consists of a VH domain of the antigen binding site of the radiolabeled compound; and in the second antibody, the polypeptide of (c) consists of the VL domain of the antigen-binding site of the radiolabeled compound.

Thus, in one embodiment, the first antibody may comprise or consist of:

i) a complete light chain;

ii) an intact heavy chain;

iii) another Fc chain; and

iv) a VH domain comprising or consisting of the antigen binding site of a radiolabeled compound;

wherein the light chain of (i) and the heavy chain of (ii) together provide an antigen binding site for a target antigen; and wherein the VH domain comprising or consisting of the antigen binding site of the radiolabelled compound is fused via its C-terminus, preferably via a linker, to the N-terminus of (iii).

The second antibody may comprise or consist of:

v) a complete light chain;

vi) an intact heavy chain;

vii) another Fc chain; and

viii) a VL domain comprising or consisting of the antigen binding site of a radiolabeled compound;

wherein the light chain of (v) and the heavy chain of (vi) together provide an antigen binding site for a target antigen; and wherein the VL domain comprising or consisting of the antigen binding site of the radiolabelled compound is fused via its C-terminus, preferably via a linker, to the N-terminus of (vii).

The linker may comprise any flexible linker as known to those of skill in the art, such as linker GGGGSGGGGSGGGGSGGSGG (SEQ ID No.: 152). The linker may further comprise a portion of the entire upper hinge region, e.g., may extend from Asp221 to the beginning of the Fc chain (e.g., at Cys 226).

In yet another embodiment, the first antibody and/or the second antibody each comprises a full length antibody having an antigen binding site for a target antigen, and further comprises a VL domain or a VH domain of the antigen binding site of the radiolabeled compound.

In a particular embodiment, the first antibody may comprise:

a) a first full-length antibody consisting of two antibody heavy chains and two antibody light chains, wherein at least one arm of the full-length antibody binds to target antigen a; and

b) a polypeptide comprising or consisting of:

i) another antibody heavy chain variable domain (VH); or

ii) a further antibody heavy chain variable domain (VH) and a further antibody constant domain (CH1), wherein the C-terminus of the VH domain is fused to the N-terminus of the CH1 domain,

wherein the polypeptide is fused to the C-terminus of one of the two heavy chains of the first full length antibody by the N-terminus of the VH domain, preferably via a peptide linker.

The second antibody may comprise

c) A second full-length antibody consisting of two antibody heavy chains and two antibody light chains, wherein at least one arm of the full-length antibody binds to target antigen a; and

d) a polypeptide comprising or consisting of:

i) another antibody light chain variable domain (VL); or

ii) another antibody light chain variable domain (VL) and another antibody light chain constant domain (CL), wherein the C-terminus of the VL domain is fused to the N-terminus of the CL domain,

Wherein the polypeptide is fused to the C-terminus of one of the two heavy chains of the second full length antibody via the N-terminus of the VL domain, preferably via a peptide linker.

The antibody heavy chain variable domain (VH) of the first antibody and the antibody light chain variable domain (VL) of the second antibody together form a functional antigen binding site for the radiolabelled compound, i.e. when the two antibodies are combined.

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

Optionally, the first antibody may consist essentially of or consist of components (a) and (b) listed above, and the second antibody may consist essentially of or consist of components (c) and (d) listed above. In any case, the first antibody does not comprise an antibody light chain variable domain (VL) capable of forming a functional antigen binding site for a radiolabeled compound in association with component (b) of the first antibody; and the second antibody does not comprise an antibody heavy chain Variable (VH) domain capable of forming a functional antigen binding site for a radiolabeled compound, in association with component (b) of the second antibody.

It may be preferred that both arms of the full-length antibody have binding specificity for target antigen a. In the case where the antibody is a bivalent antibody to the target antigen, both arms of the full length antibody may bind to the same epitope of target antigen a.

In another embodiment, the antibody may be biparatopic for the target antigen; for example, one arm of a full-length antibody can bind to a first epitope of target antigen a and one arm can bind to a second epitope of target antigen a. In such embodiments, one arm of the antibody may comprise a Fab and one arm may comprise a cross Fab to aid in the correct assembly of the light chain with its respective heavy chain. Thus, in one embodiment, the first heavy chain of the full length antibody may comprise a VL domain in place of a VH domain (e.g. VL-CH 1-hinge-CH 2-CH3) and the first light chain may comprise a VH domain (e.g. VH-CL) exchanged for a VL domain, or the first heavy chain may comprise a CL domain in place of an HC1 domain (e.g. VH-CL-hinge-CH 2-CH3) and the first light chain may comprise a CH1 domain in place of a CL domain (e.g. VL-CH 1). In this embodiment, the second heavy and light chains have conventional domain structures (e.g., VH-CH 1-hinge-CH 2-CH3 and VL-CL, respectively). In an alternative embodiment, the second heavy chain of the full length antibody may comprise a VL domain in place of a VH domain (e.g. VL-CH 1-hinge-CH 2-CH3) and the second light chain may comprise a VH domain (e.g. VH-CL) exchanged for a VL domain, or the second heavy chain may comprise a CL domain (e.g. VH-CL-hinge-CH 2-CH3) in place of an HC1 domain and the second light chain may comprise a CH1 domain (e.g. VL-CH1) in place of a CL domain. In this embodiment, the first heavy chain and the first light chain have conventional domain structures.

In some embodiments, proper assembly of the light chains with their respective heavy chains can be aided additionally and/or by using charge modifications as discussed further below.

Proper assembly of the heterodimeric heavy chain can be aided by segment-into-hole techniques.

The term "full-length antibody" as used herein means an antibody consisting of two "full-length antibody heavy chains" and two "full-length antibody light chains". The "full-length antibody heavy chain" may be a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody constant heavy chain domain 1(CH1), an antibody Hinge Region (HR), an antibody heavy chain constant domain 2(CH2), and an antibody heavy chain constant domain 3(CH3) (abbreviated as VH-CH1-HR-CH2-CH3) in the N-terminal to C-terminal direction, and an antibody heavy chain constant domain 4(CH4) optionally selected in the case of an antibody of subclass IgE. Preferably, a "full length antibody heavy chain" is a polypeptide consisting of VH, CH1, HR, CH2 and CH3 in the N-terminal to C-terminal direction. The possibility of cross-Mab formation is not intended to be excluded by the reference to "full-length" -thus, the heavy chain may have a VH domain transposed to a VL domain or a CH1 domain transposed to a CL domain. A "full-length antibody light chain" may be a polypeptide consisting of an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL), abbreviated VL-CL, in the N-terminal to C-terminal direction. Alternatively, in the case of a crossover Mab, the VL domain may be swapped for the VH domain, or the CL domain may be swapped for the CH1 domain. The antibody light chain constant domain (CL) may be kappa or lambda. Two full-length antibody chains are linked together via interpoly disulfide bonds between the CL domain and the CH1 domain and between the hinge region of the full-length antibody heavy chain. Examples of typical full-length antibodies are natural antibodies such as IgG (e.g., IgG1 and IgG2), IgM, IgA, IgD and IgE. The full length antibody of the invention may be from a single species, e.g., human, or it may be a chimeric or humanized antibody. The full-length antibodies described herein comprise two antigen-binding sites, each formed by a pair of VH and VL, which in some embodiments can both specifically bind to the same antigen or can bind to different antigens. The C-terminus of the heavy or light chain of the full-length antibody represents the last amino acid at the C-terminus of the heavy or light chain.

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

Techniques known for making multispecific antibodies may also be used to make any of the heterodimers described herein. Such techniques include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537(1983)) and "section-into-pocket" engineering (see, e.g., U.S. Pat. No. 5,731,168 and Atwell et al, j.mol. biol.270:26 (1997)). Other methods include engineering electrostatic manipulation effects for the manufacture of antibody Fc-heterodimer molecules (see, e.g., WO 2009/089004); crosslinking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980 and Brennan et al, Science,229:81 (1985)); using leucine zippers (see, e.g., Kostelny et al, j. immunol.,148(5): 1547-; and using light chain technology commonly used to circumvent the light chain mismatch problem (see, e.g., WO 98/50431).

The CH3 domain of full length antibodies as described above may be altered by the "node-in-hole" technique which is described in detail in, for example, WO 96/027011, Ridgway, J.B., et al, Protein Eng 9(1996) 617-681 and Merchant, A.M., et al, Nat Biotechnol 16(1998)677-681 in several examples. In this method, the interaction surface of the two CH3 domains is altered to increase heterodimerization of the two heavy chains containing these two CH3 domains. Each of the two CH3 domains (of the two heavy chains) may be a "knob" while the other is a "hole". For example, one CH3 domain comprises a so-called "knob mutation" (T366W and optionally one of S354C or Y349C) and the other CH3 domain comprises a so-called "hole mutation" (T366S, L368A and Y407V and optionally Y349C or S354C) according to EU index numbering (see e.g. Carter, p.et al, immunolotechnol.2 (1996) 73).

Additionally/or alternatively, the introduction of disulfide bridges can be used to stabilize the heterodimer (Merchant, A.M., et al., Nature Biotech 16(1998) 677-681; Atwell, S., et al., J.mol.biol.270(1997)26-35) and increase yield.

Thus, in some embodiments, the first antibody and/or the second antibody is further characterized by: the CH3 domain of one heavy chain of the full-length antibody and the CH3 domain of the other heavy chain of the full-length antibody each meet at an interface comprising the original interface between antibody CH3 domains (meet); wherein the interface is altered to promote antibody formation, wherein the alteration is characterized by:

a) the CH3 domain of one heavy chain is altered such that within the original interface where the CH3 domain of one heavy chain meets the original interface of the CH3 domain of the other heavy chain within the trivalent bispecific antibody, the amino acid residues are replaced with amino acid residues having a larger side chain volume, thereby generating a protuberance within the CH3 domain interface of one heavy chain that can be located in a cavity within the CH3 domain interface of the other heavy chain

And

b) the CH3 domain of the other heavy chain was altered such that the second CH3 domain encountered the original interface of the first CH3 domain within the trivalent bispecific antibody, the amino acid residues were replaced with amino acid residues having a smaller side chain volume, thereby generating a cavity within the second CH3 domain interface within which the protuberance within the first CH3 domain interface could be positioned.

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

Optionally, in some embodiments, the two CH3 domains are further altered by introducing cysteine (C) as an amino acid in the corresponding position of each CH3 domain such that a disulfide bridge may be formed between the two CH3 domains.

Multispecific (e.g. biparatopic) antibodies of the invention may comprise amino acid substitutions in Fab molecules (including crossed Fab molecules) contained therein which are particularly effective in reducing light chain and mismatch to unmatched heavy chains (Bence-Jones type by-products) which may occur in the generation of Fab-based bi/multispecific antigen-binding molecules with VH/VL exchange in one (or more, in the case of molecules comprising more than two antigen-binding Fab molecules) of their binding arms (see also PCT publication No. WO 2015/150447, particularly the examples therein, incorporated herein by reference in their entirety). The ratio of desired multispecific antibodies to undesired by-products, in particular the Bence Jones type by-products present in one of its binding arms, can be improved by introducing charged amino acids of opposite charge (sometimes referred to herein as "charge modifications") at specific amino acid positions in the CH1 domain and CL domain of the Fab molecule.

Thus, in some embodiments, an antibody of the invention comprising a Fab molecule comprises at least one Fab with a heavy chain constant domain CH1 domain comprising charge modification as described herein and a light chain constant CL domain comprising charge modification as described herein.

Charge modification may be performed in one or more conventional Fab molecules comprised in the antibody of the invention or in one or more exchanged Fab molecules comprised in the antibody of the invention (but not in both). In a particular embodiment, the charge modification is performed in one or more conventional Fab molecules comprised in the antibody of the invention.

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

H. Exemplary antibodies

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

In a particular embodiment, the first antibody may comprise:

a) a first full-length antibody that specifically binds to CEA and consists of two antibody heavy chains and two antibody light chains; and

b) A polypeptide comprising or consisting of an antibody heavy chain variable domain (VH), wherein the heavy chain variable domain comprises heavy chain CDRs having SEQ ID NOs 35-37 (or wherein CDR-H1 has sequence GFSLTDYGVH (SEQ ID No.:148)), and/or wherein the heavy chain variable domain has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO 41;

wherein the polypeptide is fused to the C-terminus of one of the two heavy chains of the first full length antibody using the N-terminus of the VH domain, preferably via a peptide linker.

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

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

In some embodiments, the two antibody heavy chains in part (a) have identical variable domains, optionally identical variable CH1 and/or CH2 domains. Optionally, it may differ only in its CH3 domain, e.g., by the generation of knob-in-hole mutations and other mutations intended to promote proper association of heterodimers.

The second antibody may comprise:

c) a second full length antibody that specifically binds CEA and consists of two antibody heavy chains and two antibody light chains; and

d) a polypeptide comprising or consisting of an antibody light chain variable domain (VL), wherein the light chain variable domain comprises CDRs having SEQ ID NOS 38-40 and/or wherein the light chain variable domain has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO 42;

wherein the polypeptide is fused to the C-terminus of one of the two heavy chains of the second full length antibody using the N-terminus of the VL domain, preferably via a peptide linker, and wherein the second antibody does not comprise a heavy chain domain that associates with the polypeptide of (d) to form a functional binding domain of a radiolabeled compound.

In some embodiments, the two antibody heavy chains in part (c) have variable domains identical to each other, optionally variable CH1 and/or CH2 domains identical. Optionally, it may differ only in its CH3 domain, e.g., by the generation of knob-in-hole mutations and other mutations intended to promote proper association of heterodimers.

The CEA binding site/sequence may be any of the CEA binding sites/sequences described above.

In a particular embodiment, the first antibody may have a CEA binding sequence (i.e., a CDR or VH/VL domain) from antibody CH1 A1A.

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

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

In one particular embodiment, the first antibody may comprise a first heavy chain having SEQ ID NO 100 and a second heavy chain having SEQ ID NO 101 (wherein the C-terminal AST is optional and may be absent or replaced with another C-terminal extension as described herein) and a light chain having SEQ ID NO 103.

The second antibody may also have CEA binding sequences (i.e., CDRs or VH/VL domains) from antibody CH1 A1A.

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

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

In a particular embodiment, the second antibody may comprise a first heavy chain having SEQ ID NO 97 and a second heavy chain having SEQ ID NO 98 and a light chain having SEQ ID NO 103.

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

In a particular embodiment, the first antibody may comprise:

a) a first full-length antibody that specifically binds to CEA and consists of two antibody heavy chains and two antibody light chains; and

b) a polypeptide comprising or consisting of an antibody heavy chain variable domain (VH), wherein the heavy chain variable domain comprises heavy chain CDRs having SEQ ID NOs 1-3, and/or wherein the heavy chain variable domain has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO 7;

wherein the polypeptide is fused to the C-terminus of one of the two heavy chains of the first full length antibody using the N-terminus of the VH domain, preferably via a peptide linker.

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

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

In another embodiment, the additional residues may be the N-terminal portion of the CH1 domain as described above, e.g., 1-10 residues from the N-terminus of the CH1 domain, e.g., the human IgG1 CH1 domain. For example, the additional residue may be an AST.

In some embodiments, the two antibody heavy chains in part (a) have identical variable domains, optionally identical variable CH1 and/or CH2 domains. Optionally, it may differ only in its CH3 domain, e.g., by the generation of knob-in-hole mutations and other mutations intended to promote proper association of heterodimers.

The second antibody may comprise:

c) a second full length antibody that specifically binds CEA and consists of two antibody heavy chains and two antibody light chains; and

d) a polypeptide comprising or consisting of an antibody light chain variable domain (VL), wherein the light chain variable domain comprises CDRs having SEQ ID NOs 4-6 and/or wherein the light chain variable domain has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO 8;

wherein the polypeptide is fused to the C-terminus of one of the two heavy chains of the second full length antibody using the N-terminus of the VL domain, preferably via a peptide linker, and wherein the second antibody does not comprise a heavy chain domain that associates with the polypeptide of (d) to form a functional binding domain of a radiolabeled compound.

In some embodiments, the two antibody heavy chains in part (c) have variable domains identical to each other, optionally variable CH1 and/or CH2 domains identical. Optionally, it may differ only in its CH3 domain, e.g., by the generation of knob-in-hole mutations and other mutations intended to promote proper association of heterodimers.

In a particular embodiment, the first antibody may have a CEA binding sequence (i.e., a CDR or VH/VL domain) from antibody CH1 A1A.

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

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

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

In another specific embodiment, the first antibody may have a CEA binding sequence (i.e., a CDR or VH/VL domain) from antibody A5B7, including humanized versions thereof.

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

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

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

In another specific embodiment, the first antibody may have a CEA binding sequence (i.e., a CDR or VH/VL domain) from antibody T84.66 (including humanized versions thereof).

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

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

In one particular embodiment, the first antibody may comprise a first heavy chain having SEQ ID NO 86 and a second heavy chain having SEQ ID NO 87 (or variants thereof wherein the C-terminal "AST" is absent or replaced with a different C-terminal extension as disclosed herein) and a light chain having SEQ ID NO 89.

In another particular embodiment, the first antibody may have a CEA binding sequence (i.e., a CDR or VH/VL domain) from antibody 28a9 (including humanized versions thereof).

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

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

In one particular embodiment, the first antibody may comprise a first heavy chain having SEQ ID NO 93 and a second heavy chain having SEQ ID NO 94 (or variants thereof without C-terminal "AST" or with different C-terminal extensions as described herein) and a light chain having SEQ ID NO 96.

In some embodiments, the second antibody may have a CEA binding sequence (i.e., a CDR or VH/VL domain) from antibody CH1 A1A.

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

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

In a particular embodiment, the second antibody may comprise a first heavy chain having SEQ ID NO 30 and a second heavy chain having SEQ ID NO 33 and a light chain having SEQ ID NO 34.

In another specific embodiment, the second antibody may have a CEA binding sequence (i.e., a CDR or VH/VL domain) from A5B7 (including humanized versions thereof).

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

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

In a particular embodiment, the second antibody may comprise a first heavy chain having SEQ ID NO 55 and a second heavy chain having SEQ ID NO 56 and a light chain having SEQ ID NO 58.

In another specific embodiment, the second antibody may have a CEA binding sequence (i.e., a CDR or VH/VL domain) from antibody T84.66 (including humanized versions thereof).

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

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

In a particular embodiment, the second antibody may comprise a first heavy chain having SEQ ID NO 83 and a second heavy chain having SEQ ID NO 84 and a light chain having SEQ ID NO 89.

In another specific embodiment, the second antibody may have a CEA binding sequence (i.e., a CDR or VH/VL domain) from antibody 28a9 (including humanized versions thereof).

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

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

In a particular embodiment, the second antibody may comprise a first heavy chain having SEQ ID NO. 90 and a second heavy chain having SEQ ID NO. 91 and a light chain having SEQ ID NO. 96.

In some embodiments, the first antibody and the second antibody bind the same epitope of CEA. Thus, for example, both the first and second antibodies may have a CEA binding sequence from antibody CH1 A1A; or both the first and second antibodies may have CEA binding sequences from A5B7 (including humanized versions thereof); or both the first and second antibodies may have CEA binding sequences from T84.66 (including humanized versions thereof); or both the first and second antibodies may have CEA binding sequences from 28a9 (including humanized versions thereof); or both the first and second antibodies may have CEA binding sequences from MFE23 (including humanized versions thereof).

Thus, for example:

i) the first antibody may comprise a first heavy chain having SEQ ID NO 28, a second heavy chain having SEQ ID NO 32 (optionally with a C-terminal extension as described herein, e.g., AST), and a light chain having SEQ ID NO 34; and the second antibody may comprise a first heavy chain having SEQ ID NO 30, a second heavy chain having SEQ ID NO 33, and a light chain having SEQ ID NO 34;

ii) the first antibody may comprise a first heavy chain having SEQ ID NO 51, a second heavy chain having SEQ ID NO 52 (optionally with a C-terminal extension as described herein, e.g., AST), and a light chain having SEQ ID NO 54; and the second antibody may comprise a first heavy chain having SEQ ID No. 55, a second heavy chain having SEQ ID No. 56, and a light chain having SEQ ID No. 58;

iii) the first antibody may comprise a first heavy chain having SEQ ID NO 86, a second heavy chain having SEQ ID NO 87 (wherein the C-terminal AST residue is optional and may be absent or replaced by an alternative C-terminal extension), and a light chain having SEQ ID NO 89; and the second antibody may comprise a first heavy chain having SEQ ID NO 83, a second heavy chain having SEQ ID NO 84, and a light chain having SEQ ID NO 89; or

iv) the first antibody may comprise a first heavy chain having SEQ ID NO 93, a second heavy chain having SEQ ID NO 94 (wherein the C-terminal AST residue is optionally selected and may be absent or substituted with an alternative C-terminal extension), and a light chain having SEQ ID NO 96; and the second antibody may comprise a first heavy chain having SEQ ID NO:90, a second heavy chain having SEQ ID NO:91, and a light chain having SEQ ID NO: 96.

In other embodiments, the first and second antibodies bind to different epitopes of CEA as discussed above. Thus, for example, a first antibody may have a CEA binding sequence from antibody CH1A1A and a second antibody may have a CEA binding sequence from A5B 7; or the first antibody may have CEA binding sequences from antibody A5B7 and the second antibody may have CEA binding sequences from CH1 A1A. An example of the use of a pair of two complementary bits (CH1A1A and A5B7) is described in example 6 c.

In yet another particular embodiment, the target antigen may be GPRC5D or FAP and the format may be as shown in figure 25B. Optionally, the first antibody and the second antibody combine to form a functional antigen binding site for a Pb-DOTAM chelate (Pb-DOTAM).

Thus, in one embodiment (wherein the target antigen is GPRC 5D):

i) the first antibody comprises a first heavy chain having SEQ ID NO 104, a second heavy chain having SEQ ID NO 106 (wherein the C-terminal alanine is optionally selected and may be absent or replaced with an alternative C-terminal extension as described herein), and a light chain having SEQ ID NO 107; and

ii) the second antibody comprises a first heavy chain having SEQ ID NO 104, a second heavy chain having SEQ ID NO 105 and a light chain having SEQ ID NO 107.

In another embodiment (wherein the target antigen is FAP):

i) the first antibody comprises a first heavy chain having SEQ ID NO 108, a second heavy chain having SEQ ID NO 110 (wherein the C-terminal alanine is optional and may be absent or replaced with an alternative C-terminal extension as described herein), and a light chain having SEQ ID NO 111; and

ii) the second antibody comprises a first heavy chain having SEQ ID NO 108, a second heavy chain having SEQ ID NO 109 and a light chain having SEQ ID NO 111.

In yet another specific embodiment, the target may be, for example, CEA having a CEA binding sequence from antibody CH1A1A, and the format may be as shown in figure 25C. Optionally, the first antibody and the second antibody combine to form a functional antigen binding site for a Pb-DOTAM chelate (Pb-DOTAM). Thus, in one particular embodiment:

i) the first antibody comprises a first heavy chain having SEQ ID NO 112, a second heavy chain having SEQ ID NO 114, and a light chain having SEQ ID NO 115; and

ii) the second antibody comprises a first heavy chain having SEQ ID NO:112, a second heavy chain having SEQ ID NO:113, and a light chain having SEQ ID NO: 115.

I. Antibody variants

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

Substitution, insertion and deletion variants

In certain embodiments, antibody variants are provided having one or more amino acid substitutions. Alternative mutagenesis sites of interest include HVRs (CDRs) and FRs. Conservative substitutions are shown in table 1 under the heading "preferred substitutions". More substantial changes are provided in table 1 under the heading "exemplary substitutions" and are described further below with reference to amino acid side chain classes. Amino acid substitutions may be introduced into the antibody of interest and the product screened for a desired activity, e.g., retained/improved antigen binding, reduced immunogenicity, or reduced or eliminated ADCC or CDC.

TABLE 1

Amino acids can be grouped according to common side chain properties:

(1) hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilicity: cys, Ser, Thr, Asn, Gln;

(3) acidity: asp and Glu;

(4) alkalinity: his, Lys, Arg;

(5) residues affecting chain orientation: gly, Pro;

(6) aromatic: trp, Tyr, Phe.

Non-conservative substitutions would require the exchange of a member of one of these classes for another.

One type of substitution variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). In general, one or more resulting variants selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, decreased immunogenicity) relative to the parent antibody and/or will substantially retain certain biological properties of the parent antibody. An exemplary alternate variant is an affinity matured antibody that can be conveniently generated, for example, using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more CDR residues are mutated and variant antibodies are presented on phage and screened for a particular biological activity (e.g., binding affinity).

Alterations (e.g., substitutions) can be made in the CDRs, for example, to improve antibody affinity. The alteration may be performed in CDR "hot spots", i.e.residues encoded by codons which undergo high frequency mutations during the process of somatic maturation (see e.g.Chowdhury, Methods mol. biol.207: 179. 196(2008)) and/or antigen-contacting residues, wherein the resulting variant VH or VL is tested for binding affinity. Affinity maturation achieved by constructing secondary libraries and reselecting from the secondary libraries has been described, for example, in Methods in Molecular Biology 178:1-37(O' Brien et al, eds., Human Press, Totowa, NJ, (2001)). In some aspects of affinity maturation, diversity is introduced into variable genes selected for maturation by any of a variety of methods, such as error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis. A secondary library is then generated. The library is then screened to identify any antibody variants with the desired affinity. Another approach for introducing diversity involves a CDR-guided approach, in which several CDR residues (e.g., 4-6 residues at a time) are randomly grouped. Specifically recognizing CDR residues involved in antigen binding can be induced or modeled, for example, using alanine scanning mutagenesis. In particular, CDR-H3 and CDR-L3 are often targeted.

In certain aspects, substitutions, insertions, or deletions may occur within one or more CDRs so long as the alteration does not substantially diminish the ability of the antibody to bind antigen. For example, conservative changes that do not substantially reduce binding affinity (e.g., conservative substitutions as provided herein) may be made in the CDRs. For example, the alteration may be made outside of the residues in the CDRs that contact the antigen. In certain variant VH and VL sequences provided above, each CDR is unaltered or contains no more than one, two, or three amino acid substitutions.

Methods that can be used to identify antibody residues or regions that can be targeted for mutagenesis are referred to as "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science,244: 1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) is recognized and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Additional substitutions may be introduced at amino acid positions that exhibit functional sensitivity to the initial substitution. Alternatively, or in addition, the crystal structure of the antigen-antibody complex may be used to recognize contact points between the antibody and the antigen. The contact residues and adjacent residues may be targeted or eliminated as candidates for substitution. Variants can be screened to determine if they contain the desired property.

Amino acid sequence insertions include amino-terminal and/or carboxy-terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion of the N-or C-terminus of the antibody to an enzyme (e.g., against ADEPT (antibody-directed enzyme prodrug therapy)) or polypeptide that extends the serum half-life of the antibody.

Glycosylation variants

In certain aspects, the antibodies provided herein are altered to increase or decrease the degree of glycosylation of the antibody. The addition of glycosylation sites to an antibody or deletion of glycosylation sites of an antibody can be conveniently achieved by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

Where the antibody comprises an Fc region, the oligosaccharide to which it is attached may be altered. Natural antibodies produced by mammalian cells typically comprise a branched bi-antennary oligosaccharide, generally Asn297 connected to the CH2 domain of the Fc region by an N-bond. See, e.g., Wright et al TIBTECH 15:26-32 (1997). Oligosaccharides may include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "backbone" of the bi-antennary oligosaccharide structure. In some aspects, the oligosaccharides in the antibodies of the invention may be modified in order to produce antibody variants with certain improved properties.

In one aspect, antibody variants are provided having a nonfucosylated oligosaccharide, i.e., an oligosaccharide structure that lacks (directly or indirectly) fucose attached to an Fc region. In particular, the nonfucosylated oligosaccharides (also referred to as "afucosylated" oligosaccharides) are N-linked oligosaccharides lacking the fucose residue attached to the first GlcNAc in the backbone of the biantennary oligosaccharide structure. In one aspect, antibody variants are provided having an increased proportion of nonfucosylated oligosaccharides in the Fc region as compared to the native or parent antibody. For example, the proportion of non-fucosylated oligosaccharides may be at least about 20%, at least about 40%, at least about 60%, at least about 80%, or even about 100% (i.e., no fucosylated oligosaccharides are present). The percentage of non-fucosylated oligosaccharides is the (average) amount of oligosaccharides lacking a fucose residue relative to the sum of all oligosaccharides (e.g. complex, hybrid and high mannose structures) attached to Asn297, as measured by e.g. MALDI-TOF mass spectrometry as described in WO 2006/082515. Asn297 refers to the asparagine residue at about position 297 in the Fc region (EU numbering of Fc region residues); however, due to the small number of sequence variants in antibodies, Asn297 can also be located about ± 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Said antibody having an increased proportion of non-fucosylated oligosaccharides in the Fc region may have improved Fc γ RIIIa receptor binding and/or improved effector functions, in particular improved ADCC functions. See, e.g., US 2003/0157108; US 2004/0093621.

Examples of cell lines capable of producing antibodies with reduced fucosylation include Lec13 CHO cells lacking protein fucosylation (Ripka et al. Arch. biochem. Biophys.249:533-545 (1986); US 2003/0157108; and WO 2004/056312, especially at example 11); and gene knockout cell lines such as alpha-1, 6-fucosyltransferase gene, FUT8, gene knockout CHO cells (see, e.g., Yamane-Ohnuki et al Biotech. Bioeng.87:614-622 (2004); Kanda, Y.et al, Biotechnol. Bioeng.94 (4):680-688 (2006); and WO 2003/085107); or a cell having reduced or eliminated GDP-fucose synthesis or transporter activity (see e.g. US2004259150, US2005031613, US2004132140, US 2004110282).

In another aspect, antibody variants are provided having bisected oligosaccharides, for example, wherein a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. The antibody variants may have reduced fucosylation and/or improved ADCC function as described above. Examples of such antibody variants are described, for example, in the following: umana et al, Nat Biotechnol 17,176-180 (1999); ferrara et al, Biotechn Bioeng 93,851-861 (2006); WO 99/54342; WO 2004/065540; WO 2003/011878.

Antibody variants having at least one galactose residue in an oligosaccharide attached to an Fc region are also provided. The antibody variants may have improved CDC function. Such antibody variants are described, for example, in: WO 1997/30087; WO 1998/58964; and WO 1999/22764.

It may be preferred that the antibody is modified to reduce the degree of glycosylation. In some embodiments, the antibody may be aglycosylated or deglycosylated. The antibody may include a substitution at N297, such as N297D/A.

Fc region variants

In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. A variant Fc region may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

In certain embodiments, the invention contemplates antibody variants having reduced effector function, e.g., reduced or eliminated CDC, ADCC, and/or FcyR binding. In certain aspects, the invention contemplates antibody variants having some, but not all, effector functions that make them desirable candidates for applications in which the in vivo half-life of the antibody is important, but where certain effector functions, such as complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC), are unnecessary or detrimental.

In vitro and/or in vivo cytotoxicity assays may be performed to confirm the reduction/depletion of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays may be performed to ensure that the antibody lacks FcyR binding (and therefore may lack ADCC activity), but retains FcRn binding ability. Primary cell NK cells used to modulate ADCC express Fc γ RIII only, whereas monocytes express Fc γ RI, Fc γ RII and Fc γ RIII. The expression of FcRs on hematopoietic cells is summarized in Table 3 on page 464 of ravatch and Kinet, Annu.Rev.Immunol.9:457-492 (1991). Non-limiting examples of in vitro assays for assessing ADCC activity of molecules of interest are described in the following: U.S. Pat. No. 5,500,362 (see, for example, Hellstrom, I.et al, Proc. nat' l Acad. Sci. USA 83: 7059-; U.S. Pat. No. 5,821,337 (see Brugg)emann, M.et al, J.Exp.Med.166: 1351-. Alternatively, nonradioactive analysis methods may be employed (see, e.g., ACTI for flow cytometry)^TMNonradioactive cytotoxicity assay (cell technology, Mountain View, CA) and CytoToxNon-radioactive cytotoxicity assay (Promega, Madison, WI)). Effector cells useful in the assay include Peripheral Blood Mononuclear Cells (PBMCs) and Natural Killer (NK) cells. Alternatively/additionally, the ADCC activity of a molecule of interest can be assessed in vivo, for example in an animal model such as the animal model disclosed in Clynes et al Proc. Nat' l Acad. Sci. USA 95: 652-. C1q binding assays may also be performed to confirm that the antibody is unable to bind C1q and therefore lacks CDC activity. See, e.g., WO 2006/029879 and WO 2005/100402 for C1q and C3C binding ELISAs. To assess complement activation, CDC assays can be performed (see, e.g., Gazzano-Santoro et al, J.Immunol.methods 202:163 (1996); Cragg, M.S.et al, Blood 101: 1045-. FcRn binding and in vivo clearance/half-life assays may also be performed using methods known in the art (see, e.g., Petkova, s.b.et., Int' l.immunol.18(12): 1759-.

Antibodies with reduced effector function include antibodies with substitutions of one or more of residues 238, 265, 269, 270, 297, 327 and 329 of the Fc region (U.S. Pat. No. 6,737,056), for example P329G. The Fc mutants include Fc mutants having substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including so-called "DANA" Fc mutants having substitutions at residues 265 and 297 to alanine (U.S. Pat. No. 7,332,581).

In certain aspects, an antibody variant comprises an Fc region having one or more amino acid substitutions that reduce FcyR binding, for example substitutions at positions 234 and 235 (EU numbering of residues) of the Fc region. In one aspect, the substitutions are L234A and L235A (LALA). In certain aspects, the antibody variant further comprises D265A and/or P329G in the Fc region derived from the Fc region of human IgG 1. In one aspect, the substitutions are L234A, L235A, and P329G (LALA-PG) in the Fc region derived from the Fc region of human IgG 1. (see, for example, WO 2012/130831). In another aspect, the substitutions are L234A, L235A, and D265A (LALA-DA) in the Fc region derived from the Fc region of human IgG 1.

In other embodiments, it may be possible to use IgG subtypes with reduced effector function, such as IgG4 or IgG 2.

Certain antibody variants with improved or reduced binding to FcR are described. (see, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312; and Shields et al, J.biol. chem.9(2): 6591-.

In some embodiments, alterations are made in the Fc region, resulting in altered (i.e., improved or reduced, preferably reduced) C1q binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat. Nos. 6,194,551, WO 99/51642, and Idusogene et al J.Immunol.164:4178-4184 (2000).

In certain aspects, an antibody variant comprises an Fc region having one or more amino acid substitutions that reduce FcRn binding, e.g., substitutions at positions 253 and/or 310 and/or 435 (EU numbering of residues) of the Fc region. In certain aspects, an antibody variant comprises an Fc region having amino acid substitutions at positions 253, 310, and 435. In one aspect, the substitutions are I253A, H310A, and H435A in the Fc region derived from the Fc region of human IgG 1. See, e.g., grevs, a., et al, j. immunol.194(2015) 5497-5508.

In certain aspects, an antibody variant comprises an Fc region having one or more amino acid substitutions that reduce FcRn binding, e.g., substitutions at positions 310 and/or 433 and/or 436 (EU numbering of residues) of the Fc region. In certain aspects, an antibody variant comprises an Fc region having amino acid substitutions at positions 310, 433, and 436. In one aspect, the substitutions are H310A, H433A, and Y436A in an Fc region derived from a human IgG1 Fc region. (see, for example, WO 2014/177460 Al). For example, in some embodiments, normal FcRn binding may be used.

See also Duncan and Winter, Nature 322:738-40 (1988); U.S. Pat. nos. 5,648,260; U.S. Pat. nos. 5,624,821; and WO 94/29351, which concerns other examples of Fc region variants.

The C-terminus of the heavy chain of a full-length antibody as reported herein may be the complete C-terminus terminated with amino acid residue PGK. The C-terminus of the heavy chain may be the shortened C-terminus, with one or both of the C-terminal amino acid residues removed. The C-terminus of the heavy chain may be a shortened C-terminus terminating in PG. In one of all aspects as reported herein, an antibody comprising a heavy chain comprising a C-terminal CH3 domain comprises a C-terminal glycine residue (G446, EU index numbering of amino acid positions), as specified herein. The term "full-length antibody" or "full-length heavy chain" as used herein still explicitly encompasses the C-terminal glycine residue.

Antibody derivatives

In certain aspects, the antibodies provided herein can be further modified to contain additional non-protein moieties known in the art and readily available. Moieties suitable for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, polydextrose, polyvinyl alcohol, polyvinylpyrrolidone, poly 1, 3-dioxolane, poly 1,3, 6-trioxane, ethylene/maleic anhydride copolymers, polyamino acids (homopolymer or random copolymers) and polydextrose or poly (n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylene polyols (e.g., glycerin), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in production due to its stability in water. The polymer may have any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in therapy under defined conditions, and the like.

J. Recombinant methods and compositions

Antibodies can be produced using recombinant methods and compositions, for example, as described in U.S. Pat. No. 4,816,567. In one embodiment, an isolated nucleic acid or a set of isolated nucleic acids encoding a set of antibodies described herein is provided.

For example, a set of nucleic acids can comprise the following nucleic acids encoding a first antibody:

i) a nucleic acid encoding a first heavy chain of a first antibody, wherein the first heavy chain comprises a heavy chain of a full-length antibody that specifically binds a target antigen, the heavy chain fused via its C-terminus to a polypeptide comprising a VH domain of an antigen-binding site of a radiolabeled compound;

ii) a nucleic acid encoding a second heavy chain of the first antibody, wherein the second heavy chain comprises the heavy chain of the full-length antibody that specifically binds the target antigen and does not comprise the VL domain of the antigen-binding site of the radiolabeled compound (optionally, the second heavy chain consists of the heavy chain of the full-length antibody that specifically binds the target antigen);

iii) a nucleic acid encoding a light chain of the first antibody.

Additionally or alternatively, a set of nucleic acids of the invention may comprise the following nucleic acids encoding a second antibody:

iv) a nucleic acid encoding a first heavy chain of a second antibody, wherein the first heavy chain comprises the heavy chain of a full length antibody that specifically binds to a target antigen, which heavy chain is fused via its C-terminus to a polypeptide comprising the VL domain of the antigen binding site of a radiolabeled compound;

v) a nucleic acid encoding a second heavy chain of a second antibody, wherein the second heavy chain comprises a VH domain of a heavy chain of a full-length antibody that specifically binds to the target antigen and does not comprise the antigen-binding site of the radiolabeled compound (optionally, the second heavy chain consists of a heavy chain of a full-length antibody that specifically binds to the target antigen);

vi) a nucleic acid encoding a light chain of a second antibody.

In some embodiments, certain of these nucleic acids may be identical to each other. For example, the nucleic acid in (iii) may be identical to the nucleic acid in (vi) such that the entire set comprises only 5 different nucleic acid sequences.

The nucleic acid may be contained in one or more nucleic acid molecules or expression vectors.

Thus, in another embodiment, one or more vectors (e.g., expression vectors) comprising the one or more nucleic acids are provided. In one embodiment, each respective heavy and light chain is expressed from a separate plasmid.

In another embodiment, a host cell or a group of host cells comprising the one or more nucleic acids or the one or more vectors is provided. In one embodiment, a first host cell is provided that expresses a first antibody, and a second host cell is provided that expresses a second antibody.

In one such embodiment, the first host cell comprises (e.g., is transformed with): (1) a vector comprising the above nucleic acids (i) - (iii); or (2) a first vector comprising nucleic acid (i), a second vector comprising nucleic acid (ii), and a third vector comprising nucleic acid (iii); or (3) two vectors collectively comprising the above nucleic acids (i) - (iii). The second host cell comprises (e.g., is transformed with): (1) a vector comprising the above nucleic acids (iv) - (vi); or (2) a first vector comprising nucleic acid (iv), a second vector comprising nucleic acid (v), and a third vector comprising nucleic acid (vi); or (3) two vectors collectively comprising the above nucleic acids (iv) - (vi).

In one embodiment, the host cell is a eukaryotic cell, such as a Chinese Hamster Ovary (CHO) cell or a lymphocyte (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of making an antibody of the invention is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody under conditions suitable for expression of the antibody as provided above, and optionally recovering the antibody from the host cell (or host cell culture medium).

For recombinant production of antibodies, nucleic acids encoding the antibodies, e.g., as described above, are isolated and inserted into one or more vectors for further cloning and/or expression in host cells. The nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of an antibody).

Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., US5,648,237, US5,789,199, and US5,840,523. (see also Charlton, Methods in Molecular Biology, Vol.248, Lo, B.K.C. (eds.), Humana Press, Totowa, NJ (2003), pp.245-. After expression, the antibody can be separated from the bacterial cell paste in a soluble fraction and can be further purified.

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

Suitable host cells for the expression of (glycosylated) antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfecting Spodoptera frugiperda (Spodoptera frugiperda) cells.

Plant cell cultures may also be used as hosts. See, e.g., US 5,959,177, US 6,040,498, US 6,420,548, US 7,125,978, and US 6,417,429 (describing the plantibodies technology for antibody production in transgenic plants).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growth in suspension may be suitable. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed with SV40 (COS-7); human embryonic kidney cell lines (such as, for example, 293 or 293T cells as described in Graham, F.L.et al, J.Gen Virol.36(1977) 59-74); baby hamster kidney cells (BHK); mouse sertoli cells (mouse sertoli cells) (such as, for example, TM4 cells as described in Mather, j.p., biol. reprod.23(1980) 243-; monkey kidney cells (CV 1); VERO cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK; buffalo mouse liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumors (MMT 060562); TRI cells (as described, for example, in Mather, J.P.et al., Annals N.Y.Acad.Sci.383(1982) 44-68); MRC 5 cells; and FS4 cell lines other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells (Urlaub, G.et al., Proc.Natl.Acad.Sci.USA 77 (1980)) 4216-20), and myeloma cell lines such as Y0, NS0 and Sp 2/0. for reviews of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki, P.and Wzau, A.M, methodolor, Molecular 255, Molecular Biology, Hutola, Vol.268, Towa J.268 (Towa).

In one aspect, the host cell line is a eukaryotic cell, such as a Chinese Hamster Ovary (CHO) cell or a lymphocyte (e.g., Y0, NS0, Sp20 cell).

K. Analysis of

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

In one aspect, antibodies of the invention are tested for their antigen binding activity, e.g., by known methods such as ELISA, western blotting, and the like.

Affinity of antibody

In certain embodiments, the antibodies provided herein have a dissociation constant (Kd) for a target antigen of less than or equal to 1 μ M, less than or equal to 100nM, less than or equal to 10nM, less than or equal to 1nM, less than or equal to 0.1nM, less than or equal to 0.01nM, or less than or equal to 0.001nM (e.g., 10 nM)^-8M or less, e.g. 10^-8M to 10^{- 13}M, e.g. 10^-9M to 10^-13M) or as otherwise set forth herein.

In certain embodiments, the antigen-binding site of the radiolabeled compound has a dissociation constant (Kd) for the radiolabeled compound of ≦ 1 μ M ≦ 100nM, ≦ 10nM, ≦ 1nM, ≦ 0.1nM, ≦ 0.01nM, or ≦ 0.001nM(e.g., 10)^-8M or less, e.g. 10^-8M to 10^-13M, e.g. 10^-9M to 10^-13M). In some embodiments, the Kd is 1nM or less, 500pM or less, 200pM or less, 100pM or less, 50pM or less, 20pM or less, 10pM or less, 5pM or less or 1pM or less, or as otherwise stated herein. For example, the functional binding site may bind to the radiolabelled compound/metal chelate with a Kd of about 1pM to 1nM, for example about 1 to 10pM, 1 to 100pM, 5 to 50pM, 100pM or 500pM to 1 nM.

In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA). In one embodiment, RIA is performed with Fab versions of the antibody of interest and its antigen. For example, solution binding affinity of Fab to antigen is measured by: (ii) with the lowest concentration in the presence of a titration series of unlabeled antigen¹²⁵I) The Fab is equilibrated with labeled antigen, and the bound antigen is subsequently captured with an anti-Fab antibody coated plate (see, e.g., Chen et al, J.Mol.biol.293:865-881 (1999)). To establish the assay conditions, 50mM sodium carbonate (pH 9.6) containing 5. mu.g/ml capture anti-Fab antibodies (Cappel Labs) was coatedThe well plates (Thermo Scientific) were overnight and then blocked with PBS containing 2% (w/v) bovine serum albumin for two to five hours at room temperature (about 23 ℃). In the nonadsorbent sheet (Nunc No. 269620), 100pM or 26pM [ alpha ], [ beta ] -amylase¹²⁵I]The antigen is mixed with serial dilutions of the Fab of interest (e.g., consistent with the evaluation of anti-VEGF antibody Fab-12 in Presta et al, Cancer Res.57: 4593-. Subsequently, the Fab of interest was incubated overnight; however, incubation may continue for a longer period of time (e.g., about 65 hours) to ensure equilibrium is reached. Thereafter, the mixture is transferred to a capture plate for incubation at room temperature (e.g., for one hour). Subsequently, the solution was removed and replaced with a solution containing 0.1% polysorbate The plate was washed eight times with PBS. When the plate had dried, 150. mu.l/well was addedScintillator (MICROSCINT-20)^TM(ii) a Packard) and in TOPCOUNT^TMPlates were counted for 10 minutes on a gamma counter (Packard). The concentration of each Fab that provides less than or equal to 20% maximal binding is selected for use in competitive binding assays.

According to another embodiment, use is made ofSurface plasmon resonance analysis measures Kd. For example, useOrAnalysis of (BIAcore, Piscataway, NJ) was performed in 10 Response Units (RU) at 25 ℃ using a fixed antigen CM5 chip. In one embodiment, a carboxymethylated polydextrose biosensor chip (CM5, BIACORE) was activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to supplier's instructions. The antigen was diluted to 5 μ g/ml (-0.2 μ M) with 10mM sodium acetate (pH 4.8) before injection at a flow rate of 5 μ l/min to achieve approximately 10 Response Units (RU) of conjugated protein. After injection of the antigen, 1M ethanolamine was injected to block unreacted groups. For kinetic measurements, the sample was tested with 0.05% polysorbate 20 (TWEEN-20) at 25 ℃ at a flow rate of about 25. mu.l/min ^TM) Two-fold serial dilutions (0.78nM to 500nM) of Fab were injected in pbs (pbst) of surfactant. (iii) Using a simple one-to-one Langmuir binding modelEvaluation software version 3.2) to calculate the association rate (k) by simultaneous fitting of the association and dissociation sensor profiles_on) And dissociation rate (k)_off). The equilibrium dissociation constant (Kd) is calculated as the ratio k_off/k_on. See, e.g., Chen et al, J.mol.biol.293:865-881 (1999). If according toThe above surface plasmon resonance analysis shows that the binding rate (on-rate) exceeds 10⁶M^-1s^-1The rate of binding can then be determined by using a fluorescence quenching technique in the presence of a sample such as in a stopped flow equipment type spectrophotometer (Aviv Instruments) or 8000-series SLM-AMINCO^TMIn the case of an antigen of increasing enrichment as measured in the spectrometer of a spectrophotometer (ThermoSpectronic)), the fluorescence emission intensity of PBS (pH 7.2) containing 20nM of anti-antigen antibody (Fab form) was measured at 25 ℃ (excitation 295 nM; emission 340nm, 16nm bandpass) increase or decrease.

In another embodiment, Kd is measured using a SET (solution equilibrium titration) assay. According to this assay, the test antibody is typically administered at a constant concentration and mixed with serial dilutions of the test antigen. After incubation to establish equilibrium, portions of free antibody are captured on the antigen-coated surface and detected with labeled/spiked anti-species antibodies, typically using electrochemiluminescence (e.g., as described in Haene et al, analytical Biochemistry 339(2005) 182-.

For example, in one embodiment, a 384 well streptavidin plate (Nunc, microchat No. 11974998001) was incubated with 25 μ l/well of the antigen-biotin-isomer mixture in PBS-buffer at a concentration of 20ng/ml overnight at 4 ℃. For antibody sample equilibration with free antigen: titrate 0.01nM to 1nM antibody with the relevant antigen in a 1:3, 1:2, or 1:1.7 dilution step starting with a concentration of 2500nM, 500nM, or 100nM antigen. Samples were incubated overnight at 4 ℃ in sealed REMP storage polypropylene microtiter plates (Brooks). After overnight incubation, the streptavidin plates were washed 3 times with 90 μ l PBST/well. 15 μ l of each sample from the equilibration plates were transferred to assay plates and incubated for 15min at RT, followed by 3 90 μ l wash steps with PBST buffer. Detection was performed by adding 25. mu.l of goat anti-human IgG antibody-POD conjugate (Jackson, 109-036-088 in OSEP 1:4000) followed by 6 90. mu.l wash steps with PBST buffer. 25 μ l of TMB substrate (Roche Diagnostics, Inc., Cat. No.: 11835033001) was added to each well. Measurements were made at 370/492nm on a Safire2 reader (Tecan).

In another embodiment, Kd is measured using KinExA (kinetic exclusion) analysis. According to this assay, the antigen is typically titrated into a constant concentration of antibody binding sites, the sample is equilibrated, and then rapidly aspirated through a flow cell where free antibody binding sites are captured on antigen-coated beads while the antigen-saturated antibody complexes are washed away. Subsequently, the bead capture antibody is detected with a labeled anti-species antibody, e.g., a fluorescently labeled anti-species antibody (Bee et al. plos One, 2012; 7(4): e 36261). For example, in one embodiment, the KinExA experiment is performed at Room Temperature (RT) using PBS (pH 7.4) as running buffer. Samples were prepared in running buffer supplemented with 1mg/ml BSA ("sample buffer"). A flow rate of 0.25ml/min was used. A constant amount of antibody with a binding site concentration of 5pM was titrated with antigen by serial dilution (concentration range 0.049pM-100pM) twice starting at 100 pM. An antibody sample without antigen served as 100% signal (i.e., no inhibition). The antigen-antibody complex was incubated at RT for at least 24h to allow equilibration to occur. Subsequently, the equilibrated mixture was aspirated through the antigen-coupled bead column in the KinExA system in a volume of 5ml, permitting capture of unbound antibody by the beads without disturbing the solution equilibrium state. Using a solution containing 250ng/ml Dylight The captured antibody is detected in a sample buffer conjugated with an anti-human Fc fragment-specific secondary antibody. For all equilibrium experiments, each sample was measured in duplicate. KD was obtained from non-linear regression analysis of the data using the "standard analysis" method using a single-site homogeneous binding model contained within KinExA software (version 4.0.11).

Methods and compositions of treatment

The antibody panel as described herein may be used in a method of treatment. In one aspect, a set of antibodies as described herein is provided for use as a medicament. In certain aspects, a panel of antibodies for use in a method of treatment is provided.

As discussed above, in some aspects, the antibody panel of the invention is applicable to any therapy in which delivery of a radionuclide to a target cell in an individual is desired. For example, a set of antibodies as described herein is provided for use in a pre-targeted radioimmunotherapy method, e.g., for cancer treatment.

In certain aspects, the invention provides a set of antibodies for use in a method of pre-targeted radioimmunotherapy in an individual, the method comprising administering to the individual an effective amount of the set of antibodies. The "individual" according to any of the above aspects is preferably a human.

As mentioned above, the treatment may belong to any condition that can be treated by cytotoxic activity targeting diseased cells of the patient. The treatment is preferably of a tumor or cancer. However, the applicability of the present invention is not limited to tumors and cancers. For example, the treatment may also be of a viral infection or of another pathogenic organism, such as a prokaryote. Optionally, T cells can also be targeted to treat T cell driven autoimmune diseases or T cell blood cancers. Thus, conditions to be treated may include viral infections such as HIV, rabies, EBV and kaposi's sarcoma-associated herpes virus, as well as autoimmune diseases such as multiple sclerosis and graft-versus-host disease.

The term "cancer" as used herein includes solid and hematological cancers such as lymphoma, lymphocytic leukemia, lung cancer, non-small cell lung (NSCL) cancer, bronchioloalveolar cell lung cancer, bone cancer, pancreatic cancer including Pancreatic Ductal Adenocarcinoma (PDAC), skin cancer, head and neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, cancer of the anal region, gastric cancer (stomachs/colorectal cancer), colorectal cancer which may be colon cancer and/or rectal cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease (Hodgkin's), cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, renal pelvis cancer, carcinoma of the renal pelvis, carcinoma of the penis, cancer of the non-small cell lung (NSCL lung), cancer of bronchiolar alveolar carcinoma (NSCL), cancer, alveolar carcinoma of the lung cancer, small cell (NSCL cancer, lung cancer, and/colorectal cancer, and/or other cancers, Mesothelioma, hepatocellular carcinoma, biliary cancer, Central Nervous System (CNS) neoplasm, spinal axis tumor, brain stem glioma, glioblastoma multiforme, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, and ewing's sarcoma (Ewings sarcoma), including refractory forms of any of the above cancers, checkpoint inhibitor experienced forms of any of the above cancers, or combinations of one or more of the above cancers.

A method of targeting a radioisotope to a cell, tissue or organ for therapy may comprise:

i) administering to the individual a first antibody and a second antibody (simultaneously or sequentially in either order) as described herein, wherein the antibodies bind to the target antigen and are localized to the surface of a cell expressing the target antigen; and wherein the combination of the first antibody and the second antibody forms a functional binding site for the radiolabeled compound;

and

ii) subsequently administering a radiolabeled compound, wherein the radiolabeled compound binds to a functional binding site of the radiolabeled compound.

The radiolabeled compound is labeled with a radioactive isotope that is cytotoxic to cells. Suitable radioisotopes include alpha and beta emitters as discussed above.

In pre-targeted radioimmunotherapy methods utilizing bispecific antibodies (i.e., non-split "antibodies of the invention), it is common practice to administer a scavenger or blocking agent between administration of the antibody and administration of the radiolabeled compound. The clearing agent binds to the antibody and enhances the rate of its own clearance. It includes anti-idiotypic antibodies. Blocking agents are typically agents that bind to the antigen binding site of a radiolabeled compound, but are not self-radiolabeled. For example, where the radiolabeled compound comprises a chelator loaded with a radioisotope of a particular chemical element (e.g., a metal), the capping reagent may comprise the same chelator loaded with a non-radioisotope of the same element (e.g., a metal), or may comprise a non-loaded chelator or a chelator loaded with a different non-radioactive moiety (e.g., a non-radioisotope of a different element), with the proviso that it remains bound to the antigen-binding site. In some cases, the blocking agent may additionally comprise moieties that increase the size and/or hydrodynamic radius of the molecule. Blocking agents hinder the ability of molecules to access tumors in the circulation without interfering with the ability of the molecules to bind to antibodies. Exemplary moieties include hydrophilic polymers. The moiety may be a polymer or copolymer such as polydextrose, dextrin, PEG, polysialic acid (PSA), hyaluronic acid, hydroxyethyl starch (HES), or poly (2-ethyl 2-oxazoline) (PEOZ). In other embodiments, the moiety may be an unstructured peptide or protein, such as an XTEN polypeptide (unstructured hydrophilic protein polymer), a polymer of high amino acids (HAP), a proline-alanine-serine Polymer (PAS), an elastin-like peptide (ELP), or a gelatin-like protein (GLK). Further exemplary moieties include proteins such as albumin (e.g., bovine serum albumin) or IgG. Suitable molecular weights for the moieties/polymers may range, for example, from at least 50kDa, such as between 50kDa and 2000 kDa. For example, the molecular weight may be 200-800kDa, optionally greater than 300, 350, 400 or 450kDa, and optionally less than 700, 650, 600 or 550kDa, optionally about 500 kDa.

According to certain aspects of the invention, there is no step of administering a clearing or blocking agent to the individual. In certain aspects, there is no step of administering any agent that binds to the first antibody or the second antibody between administration of the antibody and administration of the radiolabeled compound. In certain aspects, there is no step of administering any agent between the administration of the antibody and the radiolabeled compound, except for an optionally selected compound selected from the group consisting of a chemotherapeutic agent, an immunotherapeutic agent, and a radiosensitizer. In some embodiments, no agent is administered between the administration of the antibody and the administration of the radiolabeled compound. In some embodiments, the individual may not be injected or infused with any other agent between administration of the antibody and administration of the radiolabeled compound.

In some embodiments, the method may be a pre-targeted radioimmunotherapy two-step method consisting of or consisting essentially of: i) administering a panel of antibodies (wherein the first and second antibodies may be administered simultaneously or sequentially in either order), and ii) subsequently administering a radiolabeled compound. Treatment may involve multiple cycles of the therapy, i.e., multiple cycles of the two steps. An exemplary treatment cycle duration is 28 days, wherein the antibody panel is administered on cycle day 1, and the radiolabeled compound is optionally administered on cycle days 1, 2, 3, 4, 5, 6, 7 or 8, e.g., on day 7. The number of treatment cycles can vary. In one embodiment, there may be 4, 5 or 6 treatment cycles.

The inventors have unexpectedly determined that it is possible to obtain therapeutically effective uptake of a radiolabeled compound by a tumour using the antibody of the invention, while avoiding excessive accumulation of radioactivity in normal tissues. Indeed, in the examples, the level of radioactive accumulation in non-target tissues was found to be lower than in the three-step PRIT method using bispecific antibodies and a clearing step, while also utilizing a simpler procedure.

In some embodiments, once the first and second antibodies have been administered for a suitable period of time to localize to the target cells, a radiolabeled compound may be administered to the individual. For example, in some embodiments, the radiolabeled compound may be administered to the individual immediately after the first and second antibodies or at least 4 hours, 8 hours, 1 day, or 2 days after the first and second antibodies. Optionally, it may be administered no more than 3 days, 5 days, or 7 days after the first and second antibodies. In a particular embodiment, the radiolabeled compound may be administered to the individual 2 to 7 days after the first and second antibodies.

In some embodiments, the antibodies described herein can be administered as part of a combination therapy. For example, it may be administered in combination with one or more chemotherapeutic agents: the chemotherapeutic agent and antibody may be administered simultaneously or sequentially in either order. Additionally, or alternatively, it may be administered in combination with one or more immunotherapeutic agents: the immunotherapeutic agent and antibody may be administered simultaneously or sequentially in either order.

In some embodiments, additionally and/or alternatively, the antibodies described herein can be administered in combination with a radiosensitizer. The radiosensitizer and the antibody can be administered simultaneously or sequentially in either order.

The antibodies of the invention (and any additional therapeutic agents such as radiolabeled compounds) may be administered by any suitable means including parenteral, intrapulmonary and intranasal, and as required for local treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration may be by any suitable route, for example by injection such as intravenous or subcutaneous injection.

In some embodiments, one or more dosimetry cycles may be used prior to one or more treatment cycles as described above. The dosimetry cycle may comprise the following steps: i) administering a set of antibodies (wherein the first and second antibodies may be administered simultaneously or sequentially in either order), and ii) subsequently administering a gamma-emitter radiolabeled compound suitable for imaging (wherein the radiolabeled compound binds to a functional binding site of the radiolabeled compound). The compound may be the same as that used in subsequent cycles of treatment, except that it is labelled with a gamma emitter rather than an alpha or beta emitter. For example, in one embodiment, the radiolabeled compound used in the dosimetry cycle may be ²⁰³Pb-DOTAM, and the radiolabeled compound used in the treatment cycle may be²¹²Pb-DOTAM. The patient may undergo imaging to determine the uptake of the compound by the tumor and/or to estimate the absorbed dose of the compound. This information can be used to estimate the expected radiation exposure in subsequent treatment steps and to adjust the dose of radiolabeled compound used in the treatment steps to safe levels.

M. pharmaceutical preparation

The first and second antibodies described herein can be formulated in a single pharmaceutical composition or in separate pharmaceutical compositions. Thus, in another aspect, the invention provides a pharmaceutical composition comprising a primary antibody of the invention and a secondary antibody or a first pharmaceutical formulation comprising a primary antibody of the invention and a second pharmaceutical composition comprising a secondary antibody of the invention, e.g., for use in any of the therapeutic or diagnostic methods described herein. In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition further comprises at least one additional therapeutic agent, e.g., as described below.

Pharmaceutical formulations having an antibody as described herein may be prepared in lyophilized formulation or as an aqueous solution by mixing the antibody of the desired purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16 th edition, Osol, a. eds. (1980)).

Pharmaceutically acceptable carriers at the dosages and concentrations employed are generally non-toxic to recipients and include, but are not limited to: buffers such as histidine, phosphate, citrate, acetate and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexa hydroxy quaternary ammonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and/or a non-ionic surfactant, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersants such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), e.g., human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (r: (r) (r)) Halozyme corporation). Certain exemplary shasegps (including rHuPH20) and methods of use are described in U.S. patent publication nos. 2005/0260186 and 2006/0104968. In one aspect, the sHASEGP is combined with one or more of the followingAdditional glycosaminoglycanases combinations such as chondroitinase.

Exemplary lyophilized antibody compositions are described in U.S. Pat. No. 6,267,958. Aqueous antibody compositions include those described in U.S. Pat. No. 6,171,586 and WO 2006/044908, the latter compositions including histidine-acetate buffers.

The formulations herein may also contain more than one active ingredient necessary for the particular indication being treated, preferably active ingredients having complementary activities that do not adversely affect each other. For example, it may be desirable to further provide chemotherapeutic agents, immunotherapeutic agents, and/or radiosensitizers as discussed above. The active ingredients are suitably present in combination in an amount effective to achieve the intended purpose.

The active ingredient may be encapsulated in microcapsules, which are prepared, for example, by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin-microcapsules and poly (methylmethacylate) microcapsules, respectively; encapsulated in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or macroemulsions. The technique is disclosed in Remington's Pharmaceutical Sciences 16 th edition, Osol, A. eds (1980).

Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

The formulations to be used for in vivo administration are generally sterile. Sterility is readily achieved, for example, by filtration through sterile filtration membranes.

Methods and compositions for diagnosis and detection

The antibody panel as described herein may also be used in diagnostic or imaging methods, preferably pre-targeted radioimmunoassay methods or methods comprising pre-targeted radioimmunoassay. Accordingly, the present invention provides diagnostic and imaging methods. It further provides the use of a set of antibodies in an imaging method as described herein, and the use of a set of antibodies as described herein (i.e. a first antibody and a second antibody as described herein) for a diagnostic method performed on an individual, e.g. on a human or animal body.

The imaging method is suitable for imaging the presence and/or distribution of a target antigen in the body. For example, the method may be a method of imaging cells expressing a disease-associated antigen, such as any of the disease conditions discussed above. Optionally, the method is for imaging a tumor or cancer. The method may be used for the purpose of diagnosing an individual suspected of having a proliferative disorder such as cancer or an infectious disease.

In some embodiments, the subject is preferably a human.

A method of targeting a radioisotope to a tissue or organ for imaging or diagnosis may comprise:

i) administering to the individual a first antibody and a second antibody as described herein (either simultaneously or sequentially in either order), wherein the antibodies bind to the target antigen and are localized to the surface of a cell expressing the target antigen, wherein the combination of the first and second antibodies forms a functional binding site for the radiolabeled compound;

and

ii) subsequently administering a radiolabeled compound, wherein the radiolabeled compound binds to a functional binding site of the radiolabeled compound.

Optionally, the method may further comprise:

iii) imaging the tissue or organ in which the radiolabeled compound is located or desired to be located.

Optionally, the method may further comprise one or more steps of developing a diagnosis, delivering a diagnosis to the individual, and/or determining and/or administering an appropriate treatment based on the diagnosis.

In another embodiment, the methods of the invention may comprise imaging a tissue or organ of an individual, wherein the individual has previously been administered:

i) a first antibody and a second antibody (simultaneously or sequentially in either order) as described herein, wherein the antibodies bind to a target antigen and are localized to the surface of a cell expressing the target antigen, and wherein the combination of the first and second antibodies forms a functional binding site for a radiolabeled compound; and

ii) a radiolabeled compound, wherein the radiolabeled compound binds to the antigen binding site of the radiolabeled compound formed by the combination of the first antibody and the second antibody.

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

In conventional pre-targeted radiographic methods, it is common practice to administer a clearing or blocking agent, such as one described above, between administration of the antibody and administration of the radiolabeled compound.

In certain embodiments of the invention, there is no step of administering a scavenger or blocking agent. In certain aspects, there is no step of administering any agent that binds to the first antibody or the second antibody between administration of the antibody and administration of the radiolabeled compound. In certain aspects, there is no step of administering any agent between the administration of the antibody and the radiolabeled compound, except for an optionally selected compound selected from the group consisting of a chemotherapeutic agent, an immunotherapeutic agent, and a radiosensitizer. In some embodiments, no agent is administered between the administration of the antibody and the administration of the radiolabeled compound. In some embodiments, the individual may not be injected or infused with any other agent between administration of the antibody and administration of the radiolabeled compound.

In some embodiments, once the first and second antibodies have been administered for a suitable period of time to localize to the target cells, a radiolabeled compound may be administered to the individual. For example, in some embodiments, the radiolabeled compound may be administered to the individual immediately after the first and second antibodies or at least 4 hours, 8 hours, 1 day, or 2 days after the first and second antibodies. Optionally, it may be administered no more than 3 days, 5 days, or 7 days after the first and second antibodies. In a particular embodiment, the radiolabeled compound may be administered to the individual 2 to 7 days after the first and second antibodies.

In some embodiments, the imaging method can be a pre-targeted radiological imaging method consisting of, or consisting essentially of: i) administering a set of antibodies (wherein the first and second antibodies may be administered simultaneously or sequentially in either order), ii) subsequently administering a radiolabeled compound, and iii) imaging a tissue or organ of interest. The diagnostic method may consist of or consist essentially of: this step, followed by a step of developing a diagnosis, can then be delivered to the patient and can serve as a basis for selecting a treatment regimen and/or administering a treatment regimen.

The target antigen can be any target antigen as discussed herein. In some embodiments, the target antigen may be a tumor-specific antigen as discussed above, and imaging may be a method of imaging one or more tumors. The subject may be known or suspected to have a tumor.

For example, the method may be a method of imaging a tumor in a subject having or suspected of having: lung cancer, non-small cell lung (NSCL) cancer, bronchioloalveolar cell lung cancer, bone cancer, pancreatic cancer including PDAC, skin cancer, cancer of the head and neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, colorectal cancer which may be rectal and/or colon cancer, cancer of the anal region, gastric cancer (stomach cancer), breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, hodgkin's disease, carcinoma of the esophagus, carcinoma of the small intestine, carcinoma of the endocrine system, carcinoma of the thyroid gland, carcinoma of the parathyroid gland, carcinoma of the adrenal gland, sarcoma of soft tissue, carcinoma of the urethra, carcinoma of the penis, carcinoma of the prostate, carcinoma of the bladder, carcinoma of the kidney or ureter, carcinoma of the renal cell, carcinoma of the renal pelvis, mesothelioma, hepatocellular carcinoma, carcinoma of the bile cancer, neoplasms of the Central Nervous System (CNS), neoplasms of the spinal axis, glioma of the brain stem, glioblastoma multiforme, astrocytoma, melanoma, and melanoma, and melanoma Schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, and ewing's sarcoma, including refractory forms of any of the above cancers or checkpoint inhibitors experienced forms of any of these cancers or combinations of one or more of the above cancers.

Sequence III

Example IV

The following are examples of the methods and compositions of the present invention. It is to be understood that various other implementations may be practiced in the context of the general description provided above.

Glossary of abbreviations

ADA anti-drug antibodies

AST alanine, serine, threonine

BsAb bispecific antibody

CA scavenger

CEA carcinoembryonic antigen

DOTAM 1,4,7, 10-tetrakis (carbamoylmethyl) -1,4,7, 10-tetraazacyclododecane

ID injected dose

ELISA enzyme-linked immunosorbent assay

FAP fibroblast activation protein

GPRC5D G protein-coupled receptor family C group 5 member D

IV intravenous

MW molecular weight

PBS phosphate buffered saline

p.i. after injection

PK pharmacokinetics

PRIT pre-targeted radioimmunotherapy

RIT radioimmunotherapy

RT Room temperature

SC subcutaneous tissue

SCID severe combined immunodeficiency disease

Standard deviation of SD

SOPF is free of specific and opportunistic pathogens

TA target antigen

TGI tumor growth inhibition

Regression of TR tumors

Example 1: generation of Rabbit DOTAM binding antibodies

Example 1A: rabbit immunization

The use of 2 enantiomers of Pb-DOTAM-alkyl-PEG as reported in WO 2000/46251, WO 2002/12437, WO 2005/007696, WO 2006/047367, US 2007/0033661 and WO 2008/027986 ₄-1: 1 mixture of KLH fractions (MS2-DOTAM KLH fraction 1 and MS2-DOTAM KLH fraction 2) to immunize New Zealand white rabbits or transgenic rabbits containing human immunoglobulin loci. Each rabbit was immunized by intradermal administration of 500. mu.g of the immunogen mixture emulsified in complete Freund's adjuvant on day 0, and on days 7, 14, and 28And on day 56 by alternate intramuscular and subcutaneous administration of 500 μ g each. Thereafter, the rabbits received 500 μ g subcutaneous immunization once per month, and a small blood sample was collected 7 days after the immunization to determine serum titer. More blood samples (10% of the estimated total blood volume) were taken during the third and ninth months of immunization (5-7 days after immunization) and peripheral monocytes, which served as a source of antigen-specific B cells during the cloning of the B cells, were isolated.

Determination of serum titre (ELISA)

Each of the 2 enantiomeric Pb-DOTAM fractions (PJRD05.133F1 or PJRD05.133F2) was immobilized at 1. mu.g/ml, 100. mu.l/well in PBS on 96-well NUNC Maxisorp plates, followed by: the plate was blocked with 200 μ l/well of 2% Crotein C in PBS; serial dilutions of 100 μ l/well antiserum in PBS containing 0.5% Crotein C were administered in duplicate; detecting with HRP conjugated donkey anti-rabbit IgG antibody (Jackson Immunoresearch/Dianova 711-036-152; 1/16000) and streptavidin-HRP; 100 μ l/well of each was diluted in PBS with 0.5% Crotein C. For all steps, plates were incubated at 37 ℃ for 1 h. Between all steps, plates were washed 3 times with PBS containing 0.05% Tween 20. Signal was visualized by adding 100 μ l/well BM Blue POD soluble substrate (Roche); and stopped by adding 100 μ l/well of 1M HCl. The absorbance was read at 450nm with 690nm as a reference. Titers were defined as the dilution of antisera that gave half-maximal signal.

Example 1B: b cell cloning from rabbits

Isolation of Rabbit Peripheral Blood Mononuclear Cells (PBMC)

Blood samples were collected from the immunized rabbits. Whole blood containing EDTA was diluted two-fold with 1 × PBS (PAA, Pasching, Austria) according to the manufacturer's instructions before density centrifugation using mammalian lymphocytes (Cedarlane Laboratories, Burlington, Ontario, Canada). PBMCs were washed twice with 1 × PBS.

EL-4B5 medium

RP1640 MI (Pan Biotech, Aidenbach, Germany) supplemented with 10% FCS (Hyclone, Logan, UT, USA), 2mM glutamine, 1% penicillin/streptomycin solution (PAA, Pasching, Austria), 2mM sodium pyruvate, 10mM HEPES (PAN Biotech, Aidenbach, Germany) and 0.05mM b-mercaptoethanol (Gibco, Paisley, Scotland) was used.

Coated plate

Sterile cell culture 6-well plates were coated overnight at 4 ℃ with carbonate buffer (0.1M sodium bicarbonate, 34mM disodium bicarbonate, pH 9.55) containing 2. mu.g/ml KLH. Plates were washed three times in sterile PBS prior to use. Sterile streptavidin-coated 6-well plates (Microcoat, Bernarid, Germany) were coated with a 1+1 enantiomeric mixture of biotin-labeled TCMC-Pb-dPEC 3-biotin isomer A (1. mu.g/ml) and B (1. mu.g/ml) in PBS for 3h at room temperature. These 6-well plates were washed three times with sterile PBS prior to the panning step.

Depleting macrophage/monocyte

PBMCs were seeded onto sterile KLH-coated 6-well plates to deplete macrophages and monocytes via non-specific adhesion and to remove cells bound to KLH. Wells were maximally filled with 4ml of medium and up to 6 × 10e6 PBMCs from immunized rabbits and allowed to bind for 1h at 37 ℃ and 5% CO 2. Cells in the supernatant (peripheral blood lymphocytes (PBLs)) were used for the antigen panning step.

Enrichment of B cells on the Pb-containing TCMC enantiomer

A6-well plate coated with TCMC-Pb-dPEC 3-an enantiomeric mixture of biotin isomers A and B was inoculated with up to 6 × 10e6 PBL/4ml of medium and allowed to bind for 1h at 37 ℃ and 5% CO 2. Non-adherent cells were removed by carefully washing the wells 1-3 times with 1 × PBS. The remaining sticky cells were detached by trypsin at 37 ℃ and 5% CO2 for 10 min. Tryptization was stopped with EL-4B5 medium. Cells were kept on ice until immunofluorescent staining.

Immunofluorescent staining and flow cytometry

anti-IgG FITC (AbD Serotec, Dusseldorf, Germany) was used for single cell sorting. For surface staining, cells from depletion and enrichment steps were incubated with anti-IgG FITC antibody in PBS and incubated at 4 ℃ for 45min in the dark. After staining, PBMCs were washed twice with ice-cold PBS. Finally, PBMCs were resuspended in ice-cold PBS and immediately subjected to FACS analysis. Before FACS analysis, propidium iodide (BD Pharmingen, San Diego, Calif., USA) was added at a concentration of 5. mu.g/ml to distinguish dead from live cells.

Single cell sorting was performed using computer equipped Becton Dickinson FACSAria and FACSDiva software (BD Biosciences, USA).

B cell cultures

Rabbit B cell cultures were prepared by the method described in Lightwood et al (J Immunol Methods,2006,316: 133-143). Briefly, single sorted rabbit B cells were incubated in a 96-well plate with 200 μ l/well EL-4B5 medium containing Pansorbin cells (1:100000) (calbiochem (merck), Darmstadt, Deutschland), 5% rabbit thymocyte supernatant (microchoat, Bernried, Germany), and gamma-irradiated murine EL-4B5 thymoma cells (5 × 10e5 cells/well) for 7 days in an incubator at 37 ℃. The supernatant with the B cell culture was removed for screening, and the remaining cells were immediately harvested and frozen at-80 ℃ in 100. mu.l RLT buffer (Qiagen, Hilden, Germany).

Example 1C: expression of Rabbit antibodies

PCR amplification of V-domains

Total RNA was prepared from B cell lysates (resuspended in RLT buffer-Qiagen-Cat No. 79216) using the NucleoSpin 8/96RNA kit (Macherey & Nagel; 740709.4, 740698) according to the manufacturer's protocol. RNA was eluted with 60. mu.l RNase-free water. cDNA was generated by reverse transcriptase reaction using 6. mu.l of RNA using Superscript III first strand synthesis superscalar (Invitrogen 18080-400) and oligo dT primer according to the manufacturer's instructions. All steps were performed on a Hamilton ML Star system. The immunoglobulin heavy and light chain variable regions (VH and VL) were amplified using 4. mu.l cDNA in a 50. mu.l final volume with AccuPrime superslend (Invitrogen 12344-040) using primers rbHC. up and rbHC. do for the heavy chain and primers rbLC. up and rbLC. do for the light chain (Table below). All forward primers are specific for the signal peptide (VH and VL signal peptides, respectively), while the reverse primers are specific for the constant region (VH and VL constant regions, respectively). The PCR conditions for RbVH + RbVL were as follows: hot start at 94 ℃ for 5 min; 35 cycles of 20s at 94 ℃,20 s at 70 ℃, 45s at 68 ℃ and final elongation at 68 ℃ for 7 min.

Primer sequences

Mu.l of 50. mu.l of the PCR solution was loaded on 48E-Gel 2% (Invitrogen G8008-02). Positive PCR reactions were cleaned using the NucleoSpin Extract II kit (Macherey & Nagel; 740609250) according to the manufacturer's protocol and eluted in 50. mu.l of elution buffer. All cleaning steps were performed on a Hamilton ML Starlet system.

Recombinant expression of rabbit monoclonal bivalent antibodies

For recombinant expression of rabbit monoclonal bivalent antibodies, PCR products encoding VH or VL are cloned as cDNA into expression vectors by the bulge cloning method (RS Haun et al, Biotechnicques (1992)13, 515-. The expression vector contains an expression cassette consisting of a 5'CMV promoter including intron a and a 3' BGH polyadenylation sequence. In addition to the expression cassette, the plasmid contains a pUC 18-derived origin of replication and a beta-lactamase gene conferring ampicillin (ampicillin) resistance to plasmid amplification in E.coli. Three variants of the base plasmid were used: one plasmid contained rabbit IgG constant regions designed to accommodate the VH region, while two additional plasmids contained rabbit or human kappa LC constant regions to accommodate the VL region. Overlapping primers were used to amplify the linearized expression plasmid encoding the kappa or gamma constant region and the VL/VH insert by PCR. The purified PCR product was incubated with T4 DNA-polymerase, which generated single-stranded protrusions. The reaction was terminated by dCTP addition. In the next step, the plasmid and insert are combined and incubated with recA, which induces site-specific recombination. The recombinant plasmid was transformed into E.coli. The next day, growing colonies were selected by plasmid preparation, restriction analysis and DNA-sequencing and tested against the correct recombinant plasmid. For antibody expression, isolated HC and LC plasmids were transiently co-transfected into 2ml (96 well plates) FreeStyle HEK293-F cells (Invitrogen R790-07) using 239-Free transfection reagent (Novagen) following the procedure recommended by the reagent supplier. Supernatants were harvested after 1 week and delivered for purification.

Example 1D: selection of Rabbit monoclonal antibodies

SET (solution equilibrium titration) analysis was performed as described below.

SET analysis

Materials:

DOTAM-biotin-isomer mixture:

the mixture concentration of the following components is 20ng/ml

-Pb-Dotam-Bn-Biotin/TCMC-Pb-dPEG 3-Biotin, isomer A

-Pb-Dotam-Bn-Biotin/TCMC-Pb-dPEG 3-Biotin, isomer B

-Pb-Dotam-alkyl-biotin isomer A

-Pb-Dotam-alkyl-biotin isomer B

2.PBS：DPBS、PAN、P04-36500

3.BSA：Roche，10735086001

Tween 20: polysorbate 20(usb, No. 20605, 500ml)

PBST: 10X, Roche, code 11666789001/0.1% Tween 20

OSEP: PBS (10 ×, Roche, No. 11666789001)/0.5% BSA (bovine serum albumin fraction V, no fatty acid, Roche, No. 10735086001)/0.05% Tween 20

Preparing an analysis plate: a384 well streptavidin plate (Nunc, Microcoat No. 11974998001) was incubated overnight at 4 ℃ with 25. mu.l/well of PBS-buffer containing a mixture of DOTAM-biotin-isomers at a concentration of 20 ng/ml.

Equilibration of anti-DOTAM antibody samples with free DOTAM-metal chelates (Pb, Bi, Ca, Cu, Zn, Mg, Fe): the 0.01nM to 1nM antibody is titrated with the relevant DOTAM-metal chelate in a 1:3, 1:2 or 1:1.7 dilution step starting with a concentration of 2500nM, 500nM or 100nM DOTAM-metal chelate. Samples were incubated overnight at 4 ℃ in sealed REMP storage polypropylene microtiter plates (Brooks).

After overnight incubation, the streptavidin plates were washed 3 times with 90 μ l PBST/well. 15 μ l of each sample from the equilibration plates were transferred to assay plates and incubated for 15min at RT, followed by 3 90 μ l wash steps with PBST buffer. Detection was performed by adding 25. mu.l of goat anti-human IgG antibody-POD conjugate (Jackson, 109-036-088 in OSEP 1:4000) followed by 6 90. mu.l wash steps with PBST buffer. 25 μ l of TMB substrate (Roche Diagnostics, Inc., Cat. No.: 11835033001) was added to each well. Measurements were made at 370/492nm on a Safire2 reader (Tecan).

The following table shows the properties of various monoclonal bivalent rabbit antibodies as determined using this assay. PRIT-0128 was chosen as the primary candidate because it has comparable binding to chelated Pb and Bi, reduced binding to other chelated metals, and high affinity (<100 pM).

Binding of monoclonal bivalent rabbit antibodies to chelated metals

WRa: a wild-type rabbit; TgRa: transgenic rabbit

Example 2: humanization

Humanization

Next, the primary candidate PRIT-0128 was subjected to humanization.

For the identification of a suitable human acceptor framework during humanization of DOTAM binder PRIT-0128, a combination of the two approaches was used. In one aspect, the classical approach is performed by searching for acceptor frameworks with high sequence homology to the parent antibody and subsequently grafting the CDR regions onto this acceptor framework. The amino acid differences between the identified framework and the parent antibody are judged for their effect on the structural integrity of the binder, and back mutations are introduced, as appropriate, back to the parent sequence.

On the other hand, in-house developed computer simulation tools were used to predict the orientation of the humanized version of the VH and VL domains towards each other (see WO 2016/062734). This is done for a virtual migration of CDRs across all possible human germline combinations. The results were compared to the VH-VL domain orientation of the parental binders to select framework combinations that are close in geometry to the starting antibody.

In each case, the CDR regions of the following parent antibodies were grafted onto an acceptor framework (numbering according to Kabat):

VH_CDR1：31-35

VH_CDR2：50-65

VH_CDR3：95-102

VL_CDR1：24-34

VL_CDR2：50-56

VL_CDR3：89-97

generating a humanized variant in a format comprising a full length antibody against CEA wherein the C-terminus of one of the heavy chains is fused to the N-terminus of the VH domain of the Dotam-binder and the C-terminus of the other heavy chain is fused to the N-terminus of the VL domain of the Dotam-binder, forming a bispecific antibody having two binding sites for CEA and one functional binding site for Dotam. Thus, the DOTAM binder was fused to the C-terminus of the Fc of tumor-targeting IgG as a VH/VL Fv fusion (without CH1 and Ck, respectively). The parent DOTAM binder PRIT-0128-derived molecule in this bispecific format is called PRIT-0156.

The Herceptin (Herceptin) framework is also included due to suitability for VH/VL prediction and improved framework stability. For all VH humanized variants, human J element hJH2 was used. For all VK humanized variants, human J element hJK4 was used.

HC4 was a transplantation of PRIT-128 onto human germline IGHV3-30-02 with a back mutation of Kabat A49G.

To obtain variable heavy chain HC5, the CDRs were grafted on human germline hVH _2_26 with a49G as a back mutation and the first amino acid deletion to reflect the original rabbit N-terminus.

The variant HC7 grafted onto the herceptin V region (derived from the human germline hVH3_66) was characterized by several modifications in the acceptor framework: deletion of N-terminal E, A49G, A71R and S93A.

For HC10, the CDRs of PRIT-128 were grafted onto the human germline IGHV4_34_ 01.

Here, the N-terminus was modified to start with V2 to reflect the original rabbit antibody starting with Q2. In addition, with respect to Kabat nomenclature, G29F and F31L, as well as V71R and F78V in frame 3, were considered back mutations.

For light chain LC1, the CDRs were grafted on human germline IGKV1_39_01 without any back mutations. The start was selected as I2 to reflect the original rabbit Ab starting with a 2.

Light chain variant LC3 was obtained by grafting CDRs onto human germline hVK1_ 5. D1 was deleted and I2A back-mutation was considered as a new N-terminus. K42Q and a43P were considered as additional back mutations.

Not all possible combinations of humanized matrices are generated, but select defined combinations based on considerations such as VH/VL prediction and risk of sequence for a given combination.

Selecting candidates

The goal of humanization was to obtain a humanized binder that did not lose more than 10-fold in affinity for DOTAM. This target was achieved with several binders with comparable or even more preferred affinity for DOTAM.

As mentioned above, PRIT-0156 is a 2:1 antibody comprising the rabbit DOTAM binder PRIT-0128 in combination with the CEA binder CH1A 1A. PRIT-0178 to PRIT-0204 are humanized variants in the same format with the same CEA binder. PRIT-0205 to PRIT-0221 corresponds to PRIT-0178 to PRIT-0204 humanized variants in the DOTAM binding portion, but changes the CEA binder to T84.66.

Kd determination based on solution equilibration

To screen larger numbers of humanized candidates for their affinity for Pb-DOTAM, Solution Equilibrium Titration (SET) was used. The following table details SET-based affinity assays for selected humanized DOTAM binders to Pb-DOTAM. All antibodies in this table are bispecific antibodies comprising a bivalent binding to CEA and a monovalent binding to Pb-Dotam (2:1 format):

kinexa-based kd determination

For a more detailed analysis of affinity assays and orthogonal methods, Kinexa was used.

Instrumentation and materials

A KinExA3200 instrument with an autosampler from Sapidyne Instruments (Boise, ID) was used. Polymethylmethacrylate (PMMA) beads were purchased from Sapidyne, while PBS (phosphate buffered saline), BSA (bovine serum albumin fraction V) and anti-DOTAM antibodies were prepared internally (Roche). Conjugated affinity purified goat anti-human IgG-Fc fragment cross-adsorbed antibodies were purchased from Bethyl Laboratories (Montgomery, TX). Biotin-labeled Pb-DOTAM antigens (Pb-DOTAM-alkyl-biotin isomers A and B, Pb-DOTAM-Bn-biotin/TCMC-Pb-dPEG 3-biotin isomers A and B) and non-biotin-labeled Pb-DOTAM were obtained from AREVA Med (Bethesda, Md.).

Preparation of antigen-coated beads

The PMMA beads were coated according to the KinExA Handbook protocol (Sapidyne) for labeling molecules with biotin. Briefly, first, 1ml PBS (pH 7.4) containing 10. mu.g biotin-BSA (thermo scientific) was added per vial (200mg) of beads for adsorption coating. After 2h of rotation at room temperature, the supernatant was removed and the beads were washed 5 times with 1ml PBS. Second, 1ml of PBS containing 10mg/ml BSA containing 100 μ g of neutral avidin (NeutrAvidin) biotin-binding protein (Thermo Scientific) was added to the beads and incubated at room temperature for an additional 2h to couple the neutral avidin to the beads and provide additional biotin binding sites for subsequent binding of biotin-labeled protein. Subsequently, the neutral avidin-coated beads were washed 5 times with 1ml PBS. Finally, the beads were coated with PBS containing 200ng/ml of biotinylated Pb-DOTAM-isomer mixture (50 ng of each isomer) and incubated for a further 2h at room temperature. Subsequently, the beads were resuspended in 30ml PBS and used immediately.

KinExA equilibrium analysis

All KinExA experiments were performed at Room Temperature (RT) using PBS (pH 7.4) as running buffer. Samples were prepared in running buffer supplemented with 1mg/ml BSA ("sample buffer"). A flow rate of 0.25ml/min was used. A constant amount of anti-DOTAM antibody with a binding site concentration of 5pM was titrated with the Pb-DOTAM antigen by serial dilution (concentration range 0.049pM to 100pM) starting at twice 100 pM. An antibody sample without antigen served as 100% signal (i.e., no inhibition). The antigen-antibody complex was incubated at RT for at least 24h to allow equilibration to occur. Subsequently, the equilibrated mixture was pumped through a Pb-DOTAM coupled bead column in a KinExA system in a volume of 5ml, permitting capture of unbound antibody by the beads without disturbing the solution equilibrium state. Dylight containing 250ng/ml is usedThe captured antibody is detected in a sample buffer conjugated with an anti-human Fc fragment-specific secondary antibody. For all equilibrium experiments, each sample was measured in duplicate.

KD was obtained from non-linear regression analysis of the data using the "standard analysis" method using a single-site homogeneous binding model contained within KinExA software (version 4.0.11). The software calculates KD and determines a 95% confidence interval by fitting the data points to a theoretical KD curve. The 95% confidence interval (Sapidyne TechNote TN207R0) is given by low KD and high KD.

Thermal stability measurements against humanized PRIT molecules

Method and data analysis

The different variants of the humanized PRIT molecule in final format (in 20mM histidine, 140mM NaCl, pH 6.0) were diluted to 1mg/ml in the same buffer. 30 μ l of each sample was transferred to a 384-well plate filter set (along with the anti-HER 3 antibody as a reference). After centrifugation at 1,000g for 1min, the wells were covered with 10. mu.l of paraffin oil. Plates were centrifuged again (1,000g for 1min) and transferred to a DLS plate reader (Dyna Pro plate reader-II, Wyatt). Starting at 25 ℃, the temperature was increased to 79.9 ℃ at a rate of 0.05 ℃/min. The scattered light was recorded using Dynamics software (version 7.0).

Data were transferred to excel (microsoft), sorted by sample and temperature and melting curves were established using a software plug-in. The temperature at which a clear deviation from the baseline occurs is defined as the "onset of aggregation" and the inflection point of the melting curve is defined as the "melting temperature".

As a result, the

Characteristics of candidate

The following table summarizes the identity of various PRIT molecules and compares their properties. Preferred compounds are PRIT-0213 and PRIT-0214.

Summary of candidates

Comparison of characteristics

Additional affinity value data for PRIT-0213 as determined by Kinexa is provided below. (PRIT-0213 is the same molecule as PRIT-0186, except for another CEA that binds VH/VL).

PRIT-0213

Metal-DOTAM chelate affinity of CEA-DOTAM BsAb

Additional values are shown below:

wide confidence interval indicating measured K_DImprecise(s) of

Assay incomplete optimization for nM affinity

Sequence of

The sequence for this embodiment is provided below. Both PRIT-0213 and PRIT-0214 have a Pb-DOTAM binding site that contains the CDR with SEQ ID NO 116-. The sequences of the heavy and light chain variable domains of the Pb-DOTAM binding site of PRIT-0213 are shown in SEQ ID NO:122-123, and the sequences of the heavy and light chain variable domains of the Pb-DOTAM binding site of PRIT-0214 are shown in SEQ ID NO: 124-125.

PRIT-0214 is comprised of:

i) 126, a first heavy chain having the amino acid sequence of SEQ ID NO;

ii) a second heavy chain having the amino acid sequence of SEQ ID NO: 127; and

iii) two antibody light chains having the amino acid sequence of SEQ ID NO 128.

PRIT-0213 is comprised of:

i) a first heavy chain having the amino acid sequence of SEQ ID NO 129;

ii) a second heavy chain having the amino acid sequence of SEQ ID NO 130; and

iii) two antibody light chains having the amino acid sequence of SEQ ID NO 128.

Example 3: crystallization, data collection and structure determination of Fab P1AA1227 Pb-DOTAM complexes

For complex formation, Fab derived from humanized VH/VL in PRIT-0213, designated P1AA1227, was mixed at 26mg/ml with Pb-DOTAM powder at a molar concentration ratio of 1: 4.2. After incubation for 2 hours at 4 ℃, an initial crystallization experiment was performed in a trickle vapor diffusion setup at 21 ℃ using a JCSG + sieve (Qiagen, Hilden). Crystals appeared on day 5 from 0.2M (NH4)2SO4, 0.1M BIS-TRIS (pH 5.5), 25% w/v PEG 3350. Crystals were harvested directly from the sieve plates without any further optimization steps.

Data collection and structure determination. For data collection, crystals were snap frozen at 100K in a precipitant solution containing 10% ethylene glycol. A PILATUS 6M detector was used at Beam line X10SA of Swiss Light Source (Villigen, Switzerland)And collecting diffraction data at the wavelength of (a). The data were treated with XDS (Kabsch, W.acta Crystal.D66, 133-144(2010)) and scaled with SADABS (BRUKER). The crystals of the compound belong to the group ofAnd a space group C2 of unit cell axes of β 108.36 DEG and diffracts toThe resolution of (2). Structures were determined by molecular replacement with PHASER (McCoy, a.j, gross-Kunstleve, r.w., Adams, p.d., Storoni, l.c., and Read, r.j.j.appl.cryst.40,658-674(2007)) using internal Fab structural coordinates as a search model. Differential electron density was used to place Pb-DOTAM and amino acids were changed by actual spatial optimization according to sequence differences. Constructs were optimized using procedures from the CCP4 suite (colorful computerized Project, Number 4Acta Crystal.D50, 760-763 (1994)) and BUSTER (Bricogne, G., Blanc, E., Brandl, M., Flensburg, C., Keller, P., Paciorek, W., Rovers, P., Sharff, A., Smart, O.S., Vonrein, C., Womack, T.O. (2011) Buster 2.9.5 version Cambridge, United Kingdom: Global Ltd.). Manual reconstitution was performed with COOT (Emsley, p., Lohkamp, b., Scott, w.g., and Cowtan, k.acta Cryst D66, 486-.

Data collection and optimization statistics are summarized below.

All graphic presentations were prepared with PYMOL (The Pymol Molecular Graphics System, version 1.7.4.LLC.)。

Data collection and optimized statistics for Fab P1AA1227-Pb-DOTAM complexes

The values in parentheses are for the highest resolution shell.

Structure of Fab P1AA1227 complexed with Pb-DOTAM

To characterize the details of the interaction of Pb-DOTAM with Fab P1AA1227, we determined thatThe crystal structure of the composite at the resolution of (a). The structure revealed that Fab P1AA1227 was bound to Pb-DOTAM by major contributions of CDR1 and CDR3 of the light chain and by major contributions of CDR2 and CDR3 of the heavy chain.

Binding interface analysis with program PISA revealed the interaction pattern of Fab P1AA1227 with Pb-DOTAM via 3 hydrogen bonds, polar interactions and van der waals contacts. Pb-DOTAM is incorporated in a pocket formed by the heavy and light chains. This bag has the shape of a box open on one side. The side walls and bottom of the pocket promote non-polar interactions, while at the wall edges polar interactions dominate. Side chain hydrogen bonds are formed between CDR3 residues Glu95 and Asp97 of the heavy chain and the DOTAM carbamoyl nitrogen atoms N7 and N8. Another hydrogen bond is established via the backbone carbonyl atom of Arg96 with atom N7 of DOTAM. The complex is further stabilized via a non-polar interaction of the heavy chain CDR2 Phe50 and Tyr58 side chains, the edges of which are oriented facing the azacyclododecane ring. The light chain contributes mainly to the "bottom" of the pocket, with the CDR3 residues Gly91-Tyr96 providing the nonpolar contact with the tetracyclododecane ring. Asp32 focuses the hydrogen bond to the carbamoyl nitrogen atom N6 of DOTAM. (numbering according to Kabat).

Based on analysis with program PISA, the following table shows the heavy chain paratope residues.

Based on analysis with program PISA, the following table shows the paratope residues of the light chain.

Paratope residues in the sequences below are also providedUnderlined：

>P1AA1227_HC

>P1AA1227_LC

Example 4: generation of CEA-division-DOTAM VH/VL antibodies

PRIT (pre-targeted radioimmunotherapy) methods using bispecific antibodies with a binding site for a target antigen and a binding site for a radiolabeled compound typically use a Clearing Agent (CA) between the administered antibody and the radioligand to ensure effective targeting and high tumor to normal tissue uptake dose ratio (see fig. 3). In one example of this approach, the injected BsAb is allowed sufficient time, typically 4-10 days, to penetrate into the tumor, after which the circulating BsAb is neutralized with Pb-DOTAM-polydextrose-500 CA. CA occlusion without penetration into tumor²¹²Pb-DOTAM junctionTo non-targeted BsAb, this will block the pre-targeting site. This pre-targeting regimen allows for the subsequent administration of radiolabeled chelates²¹²Efficient tumor accumulation of Pb-DOTAM.

However, in processes involving scavengers, the use of CA introduces an additional step in the process and is therefore inefficient. Furthermore, it may be important to carefully choose the timing and amount of CA administration, which is a complicating factor.

To address the problems associated with the use of scavengers, the inventors propose a strategy to cleave the DOTAM VL domain and VH domain such that they are found on separate antibodies.

The generation of exemplary split DOTAM VH/VL antibodies is discussed further below.

Generating plasmids for recombinant expression of antibody heavy or light chains

The desired protein was expressed by transient transfection of human embryonic kidney cells (HEK 293). For expression of a desired gene/protein (e.g., a full-length antibody heavy chain, a full-length antibody light chain, or a full-length antibody heavy chain containing an extra domain (e.g., an immunoglobulin heavy chain or light chain variable domain at its C-terminus), a transcriptional unit comprising the following functional elements is used:

an immediate early enhancer from human cytomegalovirus (P-CMV) comprising intron A and a promoter,

human heavy chain immunoglobulin 5 '-untranslated region (5' UTR),

a murine immunoglobulin heavy chain Signal Sequence (SS),

-genes/proteins to be expressed, and

-bovine growth hormone polyadenylation sequence (BGH pA).

In addition to the expression unit/cassette comprising the desired gene to be expressed, the basal/standard mammalian expression plasmid also contains

An origin of replication from the vector pUC18 allowing replication of this plasmid in E.coli, and

A beta-lactamase gene conferring ampicillin resistance in e.

a) Expression plasmids for antibody heavy chains

Antibody heavy chain (VH-CH 1-hinge-CH 2-CH 3-linker-VH or VH-CH 1-hinge-CH 2-CH 3-linker-VL) encoding genes including C-terminal fusion genes comprising a complete and functional antibody heavy chain followed by additional antibody V heavy or light domains are assembled by fusing DNA fragments encoding the respective sequence elements (V heavy or light) each separated by a G4Sx4 linker to the C-terminus of the CH3 domain of a human IgG molecule. Recombinant antibody molecules carrying one VH domain and one VL domain at the C-terminus of the two CH3 domains, respectively, were expressed using node-in-hole technology.

In addition to the antibody heavy chain fragment with the C-terminal VH domain or VL domain expression cassette, the expression plasmid for short expression of the antibody heavy chain with the C-terminal VH domain or VL domain in HEK293 cells also comprises an origin of replication from the vector pUC18 allowing replication of this plasmid in e.coli and a β -lactamase gene conferring ampicillin resistance in e.coli. The transcription unit of the antibody heavy chain fragment with the C-terminal VH domain or VL domain fusion gene comprises the following functional elements:

an immediate early enhancer from human cytomegalovirus (P-CMV) comprising intron A and a promoter,

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

-a murine immunoglobulin heavy chain signal sequence,

antibody heavy chain (VH-CH 1-hinge-CH 2-CH 3-linker-VH or VH-CH 1-hinge-CH 2-CH 3-linker-VL) encoding nucleic acid, and

-bovine growth hormone polyadenylation sequence (BGH pA).

The amino acid sequence of a mature antibody heavy chain fragment with a C-terminal VH domain or VL domain fusion protein is shown below:

PRIT split antibody with DOTAM-VH-P1AD8749

>D1AC4022

>D1AA4507

PRIT split antibody with DOTAM-VL-P1AD8592

>:D1AA4506

>:D1AC4023

b) Expression plasmids for antibody light chains

Antibody light chain encoding genes comprising an intact and functional antibody light chain are assembled by fusing DNA fragments encoding the respective sequence elements.

In addition to the antibody light chain fragment, the expression plasmid used for transient expression of the antibody light chain also contains an origin of replication from the vector pUC18 that allows replication of this plasmid in e.coli and a β -lactamase gene that confers ampicillin resistance in e.coli. The transcriptional unit of the antibody light chain fragment comprises the following functional elements:

an immediate early enhancer from human cytomegalovirus (P-CMV) comprising intron A and a promoter,

human heavy chain immunoglobulin 5 '-untranslated region (5' UTR),

A murine immunoglobulin heavy chain signal sequence,

-nucleic acid encoding the light chain of an antibody (VL-CL), and

-bovine growth hormone polyadenylation sequence (BGH pA).

The amino acid sequence of the mature antibody light chain fragment is identical for P1AD8592 and P1AD 8749.

>D1AA3384

Transient expression of antibody molecules

Antibody molecules were produced in transiently transfected HEK293 cells (human embryonic kidney cell line 293-derived) cultured in F17 medium (Invitrogen). For transfection, a "293-Free" transfection reagent (Novagen) was used. The respective antibody heavy and light chain molecules as described above are expressed from separate expression plasmids. Transfection was performed as specified in the manufacturer's instructions. Three to seven (3-7) days after transfection, immunoglobulin-containing cell culture supernatants were harvested. The supernatant is stored at low temperature (e.g., -80 ℃) until purification.

General information on recombinant expression of human immunoglobulins in, for example, HEK293 cells is provided in Meissner, P.et al, Biotechnol.Bioeng.75(2001) 197-203.

PRIT half antibodies (hemibodies) (split antibodies) were purified by MabSelect Sure (affinity chromatography) followed by Superdex 200 (size exclusion chromatography). For the PRIT split antibody with DOTAM-VL-P1AD8592, 5mg of the PRIT split antibody was produced at a concentration of 1.372mg/mL and a purity of > 96% based on analytical SEC and CE-SDS. For the split PRIT antibody with DOTAM-VH-P1AD8749, a 14mg concentration of 2.03mg/mL and a purity of > 91% was generated based on analytical SEC and CE-SDS.

Antibodies P1AE4956 and P1AE4957 are also generated and their sequences are provided herein. (P1AE4956 has heavy chains with SEQ ID NOS: 51 and 52 and a light chain with SEQ ID NO: 54; P1AE4957 has heavy chains with SEQ ID NOS: 55 and 56 and a light chain with SEQ ID NO: 58). For the PRIT split antibody with DOTAM-VL-P1AE4957, 19mg of the PRIT split antibody was produced at a concentration of 2.6mg/mL and a purity of > 81.6% based on analytical SEC and CE-SDS. For the PRIT split antibody with DOTAM-VH-P1AE4956, 6.9mg of the PRIT split antibody was produced at a concentration of 1.5mg/mL and a purity of > 90% based on analytical SEC and CE-SDS. ESI-MS was used to confirm the identity of the PRIT half-antibody.

Example 5: FACS analysis of split antibody function

To evaluate the function of the split or half-antibody, MKN-45 cells were detached from the culture vessel at 37 ℃ for 10 minutes using Archatase (accutase). Subsequently, the cells were washed twice in PBS and seeded into 96-well v-shaped bottom plates to reach 4 × 10⁶Final density of individual cells/well.

Half antibodies P1AD8749 and P1AD8592 and human ISO control 1:1 were mixed and added to the cells at the concentrations indicated in figure 5. Subsequently, cells were incubated on ice for 1h and washed twice in PBS. The cell pellet was resuspended and 40. mu.l/well of detection reagent, i.e., PBS/5% FCS with < human IgG (H + L) > FITC (10. mu.g/ml) or Pb _ Dotam _ FITC 1:100 ═ 10. mu.g/ml, was added. After incubation on ice for 60min, cells were washed twice in PBS and resuspended in 200 μ Ι PBS/5% FCS to measure FITC fluorescence using FACS canto.

To assess the binding capacity of the half-antibody to CEA on MKN-45 cells, the half-antibody was detected using an antibody, using a human IgG-specific secondary antibody (fig. 5). As expected, no significant human ISO control binding was observed on these cells. When adjusted to the same IgG concentration, both half antibodies and both combinations showed dose-dependent binding to MKN-45 cells with a pronounced hook effect (hook effect) at very high concentrations as expected. This experiment demonstrates that CEA binding plays a role in half antibodies.

To assess the binding ability of the half-antibody to DOTAM, the half-antibody was bound to cells in a 1:1 ratio in the presence of a human ISO control or its respective split antibody partner. After it binds to MKN-45 cells, the cells are washed to remove unbound antibody. Subsequently, Pb-DOTAM-FITC (fluorescently labeled Pb-DOTAM) was added to detect DOTAM binding to competent cell-bound antibody (FIG. 6). As expected, no significant amounts of FITC were observed on these cells when one of the split antibody partners was combined with the human ISO control. The combination of only two half antibodies in a 1:1 ratio showed a dose-dependent FITC signal. This experiment shows that the DOTAM binding site can function when two half antibodies are bound together on one cell.

Example 6: in vivo studies

Example 6 a: materials and methods-summary

All experimental protocols were reviewed and approved by the local authorities (Commite regional D ' Ethique de l ' Exp reiteration animal du Limousin [ CREAL ], laboratory D detailed D ' analytes de recheckes de la Haute-Vienne). Female Severe Combined Immunodeficiency Disease (SCID) mice (Charles River) were maintained under Specific and Opportunistic Pathogen Free (SOPF) conditions with cycles per day/dark (12h/12h) according to ethical guidelines. No action is performed during the first 5 days after arrival to get the animal to a new environment. Animals were controlled daily for clinical symptoms and adverse events were detected.

By Subcutaneous (SC) injectionCEA-expressing tumor cells in a 1:1 mixed cell culture medium of a basement membrane matrix (growth factor-reduced; Cat. No. 354230) to create a solid xenograft. Tumor volume was estimated via 3 manual caliper measurements per week, calculated according to the following formula: volume is 0.5 x length x width². Additional tumor measurements were taken as needed depending on tumor growth rate.

If the mice show signs of distress or pain that are difficult to eliminate due to tumor burden, injection side effects, or other causes, they are euthanized prior to the scheduled endpoint. Indications of pain, distress or discomfort include, but are not limited to, acute weight (BW) loss, rough fur (washfy fur), diarrhea, hunched back posture and lethargy. BW was measured in treated animals 3 times per week with additional measurements made as needed depending on health conditions. All mice were given wet food starting the day after the radiation injection for 7 days or until all individuals had recovered sufficiently from any acute BW loss. The BW loss exceeds 20 percent of the initial BW or the tumor volume reaches 3000mm ³Mice were immediately euthanized. Other factors that are considered for ethical reasons to be euthanized are the tumor status (e.g. necrotic areas, blood/fluid exudation, signs of self-mutilation) and the general appearance of the animal (e.g. fur, posture, action).

To minimize the re-uptake of radioactive urine/feces, administration is ongoing²¹²Place all efficacy study mice in cages with grid floor after Pb-DOTAM4 hours before transferring to a new cage with standard bedding. Subsequently, all cages were replaced 24 hours after injection (p.i.). Mice sacrificed for biodistribution purposes were not subjected to this procedure for 24 hours after the radioactive injection.

As specified by the protocol, at the time of euthanasia, blood was collected from the venous sinus using retro-orbital bleeding on anesthetized mice, followed by termination via cervical dislocation, followed by additional tissue harvesting for radioactivity measurement and/or histological analysis. An unexpected or abnormal condition is noted. The tissues collected for formalin fixation were immediately placed in 10% neutral buffered formalin (4 ℃) and then transferred to phosphate buffered saline (PBS; 4 ℃) after 5 days. Organs and tissues collected for biodistribution purposes were weighed and used 2470WIZARD ²An automatic gamma counter (PerkinElmer) measures radioactivity and then calculates the percent injected dose per gram of tissue (% ID/g), including corrections for decay and background.

Statistical analysis was performed using GraphPad Prism 7(GraphPad Software, inc.) and JMP 12(SAS Institute, inc.). Tumor Growth Inhibition (TGI) curve analysis was performed based on mean tumor volume using the following formula:

where d indicates the study day and 0 indicates the baseline value. The medium is selected as the reference group. Tumor Regression (TR) was calculated according to:

where positive values indicate tumor regression, and values below-1 indicate growth beyond the double baseline value.

Test compounds

The compounds used in the described studies are presented in the table below for bispecific antibody, scavenger and radiolabeled chelate, respectively.

CEA-DOTAM (RO7198427, PRIT-0213) is a fully humanized BsAb targeting the T84.66 epitope of CEA, while DIG-DOTAM (RO7204012) is a non-CEA binding BsAb used as a negative control. P1AD8749, P1AD8592, P1AE4956, and P1AE4957 are CEA-division-DOTAM-VH/VL antibodies targeting the CH1A1A or A5B7 epitope of CEA. All antibody constructs were stored at-80 ℃ until the day of injection, thawed on the day of injection and diluted to their final respective concentrations in standard vehicle buffer (20mM histidine, 140mM NaCl; pH 6.0) or 0.9% NaCl for Intravenous (IV) or Intraperitoneal (IP) administration.

Pb-DOTAM-polydextrose-500 CA (RO7201869) was stored at-20 ℃ until the day of injection, thawed on the day of injection and diluted in PBS for IV or IP administration.

DOTAM chelates for radiolabeling are supplied by Macrocyclics and are maintained at-20 ℃ prior to radiolabeling by Orano Med (Razdes, France).²¹²Pb-DOTAM (RO7205834) was generated from the thorium-generating reagent by elution with DOTAM, and then quenched with Ca after labeling. Will be provided with²¹²The Pb-DOTAM solution was diluted with 0.9% NaCl to obtain the required IV injection²¹²The active concentration of Pb.

Mice in the vehicle control group received multiple substitutions of BsAb, CA and²¹²injection of medium buffer of Pb-DOTAM.

Bispecific antibodies

Scavenging agent

Radiolabelled chelates

Tumor model

The tumor cell lines used and the amounts injected for the inoculation of the mice are described in the table below. BxPC3 is a primary human pancreatic adenocarcinoma cell line that naturally expresses CEA. In RPMI 1640 medium enriched with 10% fetal bovine serum (GE Healthcare Hyclone SH30088.03), GlutaMAX^TMCells were cultured in the supplement HEPES (Gibco, reference 72400-021). By comparison with SCID mice on study day 0Basal membrane matrix (growth factor reduction; catalog No. 354230) cells in 1:1 mixed RPMI medium were injected subcutaneously into the right flank to create a solid xenograft.

Tumor cell strain

European authenticated cell culture Collection (Salisbury, UK)

Example 6 b: scheme 144

The goal of protocol 144 was to provide pretargeting in SCID mice bearing SC BxPC3 tumor after 2-step PRIT using CEA-division-DOTAM-VH/VL BsAb²¹²PK and in vivo profile data for Pb-DOTAM.

By injecting CEA-division-DOTAM-VH and CEA-division-DOTAM-VL (P1AD8749 and P1AD8592) separately or together, 7 days later followed by injection²¹²Pb-DOTAM to perform two-step PRIT. Mice were sacrificed 6 hours after the radioactive injection and blood and organs were harvested for radioactive measurements. The 2-step procedure was compared to 3-step PRIT using standard CEA-DOTAM bispecific antibody, 7 days later followed by Ca-DOTAM-polydextrose-500 CA and 24 hours after CA²¹²Pb-DOTAM。

PK data for CEA-division-DOTAM-VH/VL clearance were collected by repeated blood sampling 1 hour to 7 days after antibody injection and subsequently analyzed by ELISA.

A summary of the study is shown in figure 7. FIG. 7A shows an overview of the 2-step PRIT protocol, which involves blood sampling of CEA-division-DOTAM-VH/VL PK in SCID mice bearing SC BxPC3 tumor. Fig. 7B shows an overview of the 3-step PRIT protocol, which was performed in SCID mice bearing SC BxPC3 tumor (h-hour, d-day).

Design of research

The time course and design of scheme 144 is shown in the table below.

Time course of the plan 144

Study group in protocol 144

By including 5X 10 in each SCID mouse at study day 0⁶Individual cells (passage 26) of RPMI/Matrigel SC were injected into the right flank to establish a solid xenograft. Fourteen days after tumor cell injection at 116mm³The mice were sorted into experimental groups. Injection on day 22 after inoculation²¹²Pb-DOTAM; mean tumor volume 140mm on 21 st scale³。

Blood from mice in the Aa, Ba, and Ca groups was collected via anesthesia retro-orbital bleeding 1h (right eye), 24h (left eye), and 168h (right eye, at termination) after CEA-split-DOTAM-VH/VL injection. Similarly, samples were collected from mice in the Ab, Bb, and Cb groups 4h (right eye), 72h (left eye), and 168h (right eye, at termination) after CEA-split-DOTAM-VH/VL injection.

In the process of injection²¹²At 6 hours after Pb-DOTAM, mice in Aa, Ba, Ca and D groups were sacrificed and necropsied, and the following organs and tissues were harvested to measure radioactivity content: blood, skin, bladder, stomach, small intestine, colon, spleen, pancreas, kidney, liver, lung, heart, cuboid, muscle, brain, tail, ear, and tumor.

Results

Average in all collected tissues 6 hours after injection²¹²Pb accumulation and scavenging are presented in fig. 8. CEA-division-DOTAM-VH or CEA-division-DOTAM-VL pretargeting alone does not cause radiation accumulation in tumors. The two complementary antibodies in combination caused tumor uptake after 2-step PRIT of 65 ± 12% ID/g compared to 87 ± 15% ID/g for the standard 3-step PRIT protocol. Two-way analysis of variance (ANOVA) using the dukel's multiple complexes test showed significant differences in tumor uptake between the two PRIT treatments, as was typical in the bladder (1 ± 2% ID/g and 38 ± 17% ID/g for 2-and 3-step PRIT, respectively); there were no statistically significant other differences in tissue accumulation using this test (p ═ 0.05).

The clearance of IV injected CEA-split-DOTAM-VH/VL constructs as analyzed by enzyme-linked immunosorbent assay (ELISA) is shown in figure 9.

Adverse events and toxicity

There were no adverse events or toxicities associated with this study.

Conclusion

The results of the study demonstrated the proof-of-concept of CA-independent 2-step pretargeting using complementary CEA-division-DOTAM-VH/VL antibodies. Achieved using 2-step PRIT and standard 3-step PRIT²¹²High and specific tumor uptake of Pb-DOTAM with minimal radiation accumulation in normal tissues using complementary CEA-division-DOTAM-VH/VL antibodies.

Example 6 c: scheme 158

The goal of protocol 158 was to evaluate the biparatopic pair (CH1A1A and A5B7) of the CEA-division-DOTAM-VH/VL antibody for scavenger-independent 2-step CEA-PRIT in pre-targeted mice²¹²Pb-DOTAM in combination with subcutaneous BxPC3 tumor. Absorbing and marking the tumorThe tumor uptake of CEA-PRIT was compared in the quasi 3 steps.

Injection of mice bearing subcutaneous BxPC3 tumors

CEA-division-DOTAM-VH/VL antibody, followed by radiolabeling 7 days later²¹²Pb-DOTAM (2-step PRIT), or

CEA-DOTAM BsAb followed by CA after 7 days and finally radiolabelled after 24 hours²¹²Pb-DOTAM (3-step PRIT).

Evaluation 6 hours after radiation injection²¹²In vivo distribution of Pb-DOTAM. A summary of the study is shown in figure 10.

Design of research

The time course and design of scheme 158 is shown in the table below.

Time course of the plan 158

Study group in protocol 158

P1AD8749 dose was adjusted to 154 μ g to compensate for 35% of the pocket/pocket impurities; p1AD8592 dose was adjusted to 167 μ g to compensate for 40% pocket/pocket impurity.

By including 5X 10 in each SCID mouse at study day 0⁶Individual cells (passage 27) of RPMI/Matrigel SC were injected into the right flank to establish a solid xenograft. Fourteen days after tumor cell injection at 177mm ³The mice were sorted into experimental groups. Injection on day 20 after inoculation²¹²Pb-DOTAM; mean tumor volume at 21 st scale 243mm³。

In the process of injection²¹²6 hours after Pb-DOTAM, all groups were addedMice were sacrificed and necropsy was performed, and the following organs and tissues were harvested to measure radioactivity content: blood, skin, bladder, stomach, small intestine, colon, spleen, pancreas, kidney, liver, lung, heart, cuboid, muscle, brain, tail, and tumor.

Results

Average in all collected tissues 6 hours after injection²¹²The Pb distribution is shown in fig. 11. Two-way ANOVA using DuKane's multiple comparison assay showed no significance in normal tissues between the three treatments²¹²Differential Pb uptake, except for the bladder, where the two biparatopic CEA-split-DOTAM-VH/VL pairs produced accumulation below the standard 3-step PRIT. For all three treatments, the kidney uptake was 3-4% ID/g. The double-complementation site combination caused tumor accumulation of about 56% ID/g compared to 67% ID/g for 3-step PRIT; the difference between 2-step and 3-step PRIT is statistically significant (p)<0.0001)。

Adverse events and toxicity

There were no adverse events or toxicities associated with this study.

Conclusion

This study evaluated in mice pre-targeted by biparatopic pairs of CEA-split-DOTAM-VH/VL antibodies for CA-independent 2-step CEA-PRIT compared to standard 3-step PRIT ²¹²Association of Pb-DOTAM with SC BxPC3 tumor. 6 hours after injection for 2-and 3-step PRIT²¹²The Pb distribution is comparable, with high accumulation in tumors and little radioactivity in healthy tissues. This case demonstrates the concept of biparatopic pretargeting of CEA-expressing tumors using 2-step CEA-PRIT with CEA-division-DOTAM-VH/VL antibody.

Example 6 d: scenario 160

The goal of protocol 160 was to compare the therapeutic efficacy after 3 cycles of CA-independent 2-step CEA-PRIT using a complementary CEA-division-DOTAM-VH/VL antibody in SC BxPC3 tumor-bearing mice with that of standard 3-step CEA-PRIT. Also used before injection and²¹²Pb-DOTAM pre-incubated BsAb 1-step CEA-RIT comparisons.

Injection of SC BxPC3 tumor-bearing mice

CEA-DOTAM BsAb followed by CA after 7 days and finally radiolabelled after 24 hours²¹²Pb-DOTAM (3-step PRIT),

CEA-division-DOTAM-VH/VL antibody, followed by radiolabeling 7 days later²¹²Pb-DOTAM (2-step PRIT), or

·²¹²Pb-DOTAM-CEA-DOTAM BsAb (pre-incubated; 1-step RIT).

At 20. mu. Ci²¹²The therapy was administered in 3 repeated cycles of Pb-DOTAM, which also included comparison with non-CEA binding control antibody (DIG-DOTAM) and no treatment (vehicle). Sacrifice of specialized mice for biodistribution purposes to confirm each treatment cycle ²¹²Pb-DOTAM targeting and clearance. Treatment efficacy was assessed in terms of TGI and TR, and mice were carefully monitored for the duration of the study used to assess treatment tolerance. A summary of the study is shown in figure 12.

The time course and design of scheme 160 is shown in the table below.

Time course of the schedule 160

Study group in protocol 160

P1AD 8749: adjusted to 154 μ g to compensate for the dose of 35% sink/sink impurity in the stock solution; p1AD 8592; from 3 to 1 cycle due to acute radiation induced toxicity at the first treatment cycle.

By including 5X 10 in SCID mice on study day 0⁶Individual cells (passage 24) of RPMI/Matrigel SC were injected into the right flank to establish a solid xenograft. Fifteen days after tumor cell injection, 122mm³The mice were sorted into experimental groups. Injection on day 23 after inoculation²¹²Pb-DOTAM; mean tumor volume 155mm on 22 nd scale³。

According to the above table (study group in protocol 160), CEA-DOTAM and DIG-DOTAM antibodies were diluted in vehicle buffer to a final concentration of 100 μ g/200 μ L for IP administration. The CEA-division-DOTAM-VH/VL antibodies were mixed together into a single injection solution for IP administration, containing 100 μ g of each construct per 200 μ L. For P1AD8749, the dose was adjusted to 154 μ g to compensate for 35% of the sink/sink impurities in the stock solution (the molecular side not carrying VH/VL). According to the experimental schedule in FIG. 12, CA-DOTAM-polydextrose-500 CA (25. mu.g/200. mu.L PBS) was IP administered 7 days after BsAb injection, followed by 24 hours later ²¹²Pb-DOTAM (RO 7205834). Mice treated with PRIT (2 and 3 steps) were IV injected with 100. mu.L of Ca-quenched²¹²Pb-DOTAM solution (20. mu. Ci in 100. mu.L of 0.9% NaCl).

Mice treated with 1-step RIT received only one injection: pre-bonding²¹²Pb-DOTAM-CEA-DOTAM (20. mu. Ci/20. mu.g BsAb in 100. mu.L 0.9% NaCl for IV injection). By heating at 37 deg.C²¹²Pb-DOTAM was incubated with CEA-DOTAM BsAb for 10 minutes to prepare a directly labeled antibody.

The following organs and tissues were harvested from mice in groups a-E at euthanasia: serum, liver, spleen, kidney, pancreas and tumor. Prior to euthanasia, live mice were anesthetized to collect retro-orbital blood. The collected blood samples were centrifuged at 10000 rcf over a 5 minute period, and the resulting serum fractions were separated, frozen and stored at-20 ℃. The excised tissue was immediately placed in 10% neutral buffered formalin (4 ℃) and subsequently transferred to 1 x PBS (4 ℃) after 24 hours. Formalin fixed samples were shipped to Roche Pharma Research and Early Development, Roche Innovation Center base for further processing and analysis.

Mice in F, G, J and M groups were injected a first and only time²¹²Pb-DOTAM or²¹²Mice were sacrificed 24 hours after Pb-DOTAM-BsAb and necropsy performed; second injection of mice in groups H and K ²¹²Mice were sacrificed 24 hours after Pb-DOTAM and necropsy was performed; mice in groups I and L were injected a third time²¹²Mice were sacrificed 24 hours after Pb-DOTAM and necropsy was performed. Retroorbital blood collection was used on anesthetized mice at euthanasia to collect blood from the venous sinus prior to termination via cervical dislocation. The following organs and tissues were also harvested for biodistribution purposes: bladder, spleen, kidney, liver, lung, muscle, tail, skin and tumor.

As a result, the

Average in all collected tissues 24 hours after injection²¹²Pb accumulation and clearance is shown in fig. 13 for each therapy and treatment cycle. The negative control caused no uptake in the tumor (0.4% ID/g). Two-way variance analysis (ANOVA) using the dukay multiple comparison assay showed that the distribution at any cycle was not significantly different for 2-step and 3-step PRIT; however, the difference was statistically significant (p) at all cycles compared to the negative control and 1-step RIT<0.05). Tumor uptake was 25-45% ID/g for 3-step PRIT and 25-30% ID/g for 2-step PRIT without any statistically significant difference between any treatment or cycles. For 1-step RIT, tumor uptake at one and only one treatment cycle was 99%. For both PRIT protocols, the uptake in normal tissues was very low, but significantly higher in all organs and tissues after 1-step RIT due to the much longer circulation time of the preincubated antibody compared to the small radiolabeled DOTAM chelate.

Mean tumor progression and individual tumor growth curves are shown in fig. 14 and 15, respectively. Tumors in the untreated vehicle and DIG-DOTAM groups grew steadily, but the doubling rate in the latter was slightly lower after the third treatment. In contrast, tumor size decreased in the PRIT and RIT groups after the first treatment cycle, and tumor control was maintained until about 10 weeks after inoculation, with tumor size beginning to increase at about 10 weeks after inoculation. Both 2-step and 3-step PRIT treatments yielded nearly identical tumor controls. No tumor regressed completely.

On study day 83, i.e., the last day of the total treatment groups could be analyzed based on mean values, the TGI was 91.7% and 88.4% for PRIT with CEA-DOTAM (3 steps) and PRIT with CEA-division-DOTAM-VH/VL (2 steps), respectively, compared to vehicle controls. For the 1-step RIT, the corresponding value was 72.6%, while for the non-specific DIG-DOTAM control, the TGI was-59.7%. On the same day, the TR based on the mean was-1.9 for the 3-step CEA-DOTAM PRIT, -2.9 for the 2-step CEA-division-DOTAM-VH/VL PRIT, -4.7 for the 1-step RIT, -28.8 for the DIG-DOTAM PRIT, and-39.3 for the vehicle control.

Survival analysis was considered statistically irrelevant due to adverse events described below.

Adverse events and toxicity

BW development in all treatment groups is shown in figure 16. Using 20 μ Ci²¹²Multiple cycles of 2-and 3-step PRIT for Pb-DOTAM were well tolerated, but dramatic BW loss occurred in mice receiving a 1-step RIT, after the first RIT cycle (at the time of day)²¹²6-11 days after Pb irradiation) 8/10 mice in group E were euthanized due to a 20% or more BW drop. The remaining 2 RIT mice were not given any additional²¹²Pb-DOTAM-CEA-DOTAM injection, but continuously tracked for tumor growth assessment.

In addition, for ethical reasons, many mice were sacrificed due to weakened tumor status, i.e. tumor patency or leakage. In the DIG-DOTAM group, up to 3000mm is achieved for this reason³9/10 mice were euthanized prior to tumor volume; for the untreated vehicle control, the corresponding number was 5/10. The problem was less pronounced in the PRIT and RIT groups, where for this reason 1/10, 2/10 and 2/10 mice were euthanized in the 3-step PRIT, 2-step PRIT and 1-step RIT groups, respectively. This is reflected in the individual tumor growth curves in fig. 15.

Finally, 1 mouse in group C was euthanized due to the degeneration of the sub-anal wound.

All adverse events are listed in the table below.

Adverse events in protocol 160

Performed on study days 23 (cycle 1), 37 (cycle 2) and 51 (cycle 3)²¹²And (3) irradiating Pb.

Conclusion

In a 3-step protocol (CEA-DOTAM BsAb, CA and²¹²Pb-DOTAM) and 2-step procedure (CEA-split-DOTAM-VH/VL antibodies and²¹²Pb-DOTAM) was not seen as a difference between CEA-PRIT; for both treatments, TGI was quite large and nearly identical, and 3 cycles of 20 μ Ci could be safely administered in both cases. In contrast, 20 μ Ci was pre-bound to CEA-DOTAM prior to injection²¹²Pb-DOTAM (step 1 RIT) was not tolerated by most of the treated mice.

Thus, the study demonstrated the tolerability and therapeutic efficacy of CA-independent 2-step PRIT using the developed CEA-division-DOTAM-VH/VL construct.

Example 7: scheme 175

Protocol 175 is targeted to evaluate the increased amount of pre-targeted antibody injection in subsequent tumors and healthy tissues²¹²The effect of Pb accumulation. Two different doses of CEA-split-DOTAM-VH/VL antibody were compared: standard dose (100 μ g) and 2.5 times higher dose (250 μ g). In addition, the CEA-division-DOTAM-VH construct was modified to extend its VH to avoid the formation of anti-drug antibodies (ADA) for use with previously tested CEA-division-DOTAM-VL constructs. The VH was extended to include the first three amino acids from the antibody CH1 domain: alanine, serine and threonine (AST), and the construct is referred to hereinafter as CEA-cleavage-DOTAM-VH-AST.

The antibody P1AD8592 has been described in example 4 above. P1AF0171 is identical to P1AD8749 except that the fusion HC is extended by residue AST-thus, antibody P1AD0171 consists of light chain D1AA3384(SEQ ID NO:34) as described above, a first heavy chain D1AC4022(SEQ ID NO:28) as described above, and a second heavy chain D1AE3669 as shown below:

d1AE3669(HC node < CEA > CH1A1A Dotam-VH-AST)

Injection of SC BxPC3 tumor-bearing mice

1 × Standard dose of CEA-Split-DOTAM-VH/VL BsAb, followed by radiolabeling 7 days later²¹²Pb-DOTAM, or

2.5 × Standard dose of CEA-Split-DOTAM-VH/VL BsAb, followed by radiolabeling 7 days later²¹²Pb-DOTAM。

Evaluation 24 hours after radiation injection²¹²In vivo distribution of Pb-DOTAM. A summary of the study is shown in figure 17.

Design of research

The timing and design of the protocol 175 is shown below.

Time course of scheme 175

IP injection required due to low compound concentration (200 μ L/construct to 400 μ L total)

Study group in protocol 175

P1AF0171 doses were adjusted to 143 and 357 μ g to compensate-30% of the pocket/pocket impurities.

By including 5X 10 in each SCID mouse on study day 0⁶Individual cells (passage 24) of RPMI/Matrigel SC were injected into the right flank to establish a solid xenograft. After tumor cell injection Eleven days at 310mm³The mice were sorted into experimental groups. Injection on day 29 post inoculation²¹²Pb-DOTAM; mean tumor volume 462mm on 30 th balance³。

In that²¹²All mice were sacrificed 24 hours after Pb-DOTAM injection and necropsy was performed, and the following organs and tissues were harvested to measure radioactivity content: blood, skin, spleen, pancreas, kidney, liver, muscle, tail, and tumor.

Results

Average in all collected tissues 24 hours after injection²¹²The Pb distribution is shown in fig. 18. There was no significance between the two dose levels in tumor or normal tissue²¹²Difference in Pb absorption. For both treatment groups, tumor accumulation was 30% -31% ID/g, and at this time renal uptake was<2% ID/g. One mouse is due to²¹²Pb-DOTAM injection problem with 1% ID/g in the tail, but other collected healthy tissues did not show any appreciable effects²¹²Pb accumulates.

Adverse events and toxicity

There were no adverse events or toxicities associated with this study.

Conclusion

Increasing the dose of the pre-targeted CEA-division-DOTAM-VH/VL antibody by 2.5-fold in this in vivo model does not improve subsequent administration²¹²Tumor accumulation of Pb-DOTAM. However, it also did not increase the accumulation of radioactivity in normal tissues, highlighting the strong specificity achieved using this 2-step pre-targeting protocol. Finally, the results confirm the function of the extended-VH CEA-split-DOTAM-VH-AST construct.

Example 8: scheme 185

Protocol 185 aims at evaluating CEA-cleavage-DOTAM-VH/VL targeting the T84.66 epitope. Provided herein are sequences of P1AF0709 and P1AF 0298. P1AF0709 has a first heavy chain of D1AE4688(SEQ ID NO: 83) and a second heavy chain of D1AA4920(SEQ ID NO: 84). P1AF0298 has a first heavy chain of D1AE4687(SEQ ID NO: 86) and a second heavy chain of D1AE3668(SEQ ID NO: 87). Both had a light chain of D1AA4120(SEQ ID NO: 89).

SC BxPC3 tumor-bearing mice were injected with a standard dose of CEA-split-DOTAM-VH/VL BsAb (100. mu.g/antibody) 6 days later followed by radiolabelling²¹²Pb-DOTAM. Evaluation 6 hours after radiation injection²¹²In vivo distribution of Pb-DOTAM. A summary of the study is shown in figure 19.

Design of research

The timing and design of the scheme 185 is shown below.

Schedule of scenario 185

Day of study	Date	Experimental procedures
			0	2020-03-04	BxPC3 cells were prepared and syringe filled
0	2020-03-04	SC injection of BxPC3 cells
			22	2020-03-26	IV injection of CEA-Split-DOTAM-VH/VL BsAb
27	2020-03-31	Elution is carried out212Pb-DOTAM and fill syringe
			28	2020-04-01	IV injection212Pb-DOTAM
28	2020-04-01	Euthanasia and tissue harvest (6h p.i.) + γ counts

Study group in protocol 185

P1AF0171 dose was adjusted to 143 μ g to compensate-30% of the pocket/pocket impurities.

By including 5X 10 in each SCID mouse at study day 0 ⁶Individual cells (passage 27) of RPMI/Matrigel SC were injected into the right flank to establish a solid xenograft. Twenty-two days after tumor cell injection at 224mm³The mice were sorted into experimental groups. Injection on day 28 after inoculation²¹²Pb-DOTAM when the mean tumor volume reached 385mm³。

In that²¹²All mice were sacrificed 6 hours after Pb-DOTAM injection and necropsy was performed, and the following organs and tissues were harvested to measure radioactivity content: blood, skin, spleen, pancreas, kidney, liver, muscle, tail, and tumor. The collected tumors were divided into two blocks: one piece was measured for radioactivity content and the other piece was placed in a container containingOptimal Cutting Temperature (OCT) in a frozen mold of embedded media and placed on dry ice for rapid solidification. Frozen samples in OCT were maintained at-80 ℃ prior to cryosectioning, immunofluorescent staining, and using Zeiss Axio SA1 modular microscope for analysis.

Results

Average in all collected tissues 6 hours after injection²¹²The Pb distribution is shown in fig. 20. Tumor accumulation was 40% ID/g (CH1A1A) or 44% ID/g (T84.66). The only other appreciable radioactive accumulation is found in the kidney: for both groups, 3% -5% ID/g at 6h p.i..

An example of intratumoral distribution of CEA-split-DOTAM-VH/VL pairs targeting either T84.66 (group a) or CH1A1A (group B) is shown in figure 21. Panels a and C show high and uniform CEA expression in BxPC3 tumors, and panels B and D show that antibody distribution is similar 7 days after injection. However, the samples from group a exhibited overall stronger signals than the tumor samples from group B, providing evidence that T84.66 was a stronger binder than CH1 A1A.

Adverse events and toxicity

There were no adverse events or toxicities associated with this study.

Conclusion

The results demonstrate the function of a CEA-split-DOTAM-VH/VL construct targeting the T84.66 epitope of CEA. The resulting 212Pb accumulation in CEA-expressing tumors is pre-targeted is high and specific, and CEA-division-DOTAM-VH/VL pairs targeting either the CH1A1A or T84.66 epitope are homogenously distributed within CEA-expressing tumors.

Example 9: scheme 189

Protocol 189 was aimed at evaluating the biparatopic CEA-split-DOTAM-VH/VL antibody pair targeting T84.66 VH-AST/CH1A1A VL and T84.66 VL/CH1A1 VH-AST, as compared to a positive control pair targeting CH1A1A VH-AST/VL. This double-complementarity combination precludes the formation of a full Pb-DOTAM binder on soluble CEA that expresses only one of the two epitopes (e.g., T84.66), thereby reducing its potential side effects, such as increased circulating radioactivity and associated radiation-induced toxicity and reduced efficacy in competition with external tumor targets.

SC BxPC3 tumor-bearing mice were injected with standard doses of CEA-split-DOTAM-VH/VL BsAb (100. mu.g/antibody) 7 days later followed by radiolabelling²¹²Pb-DOTAM. Evaluation 6 hours after radiation injection²¹²In vivo distribution of Pb-DOTAM. A summary of the study is shown in figure 22.

Design of research

The timing and design of scheme 189 is shown below.

Time course of ARCOLAB protocol 189

Study group in ARCOLAB protocol 189

P1AF0171 dose was adjusted to 143 μ g to compensate-30% of the pocket/pocket impurities.

By including 5X 10 in each SCID mouse at study day 0⁶Individual cells (passage 31) of RPMI/Matrigel SC were injected into the right flank to establish a solid xenograft. Fourteen days after tumor cell injection at 343mm³The mice were sorted into experimental groups. Injection on day 22 after inoculation²¹²Pb-DOTAM; the mean tumor volume reached 557mm on 21 st scale³。

In that²¹²All mice were sacrificed 6 hours after Pb-DOTAM injection and necropsy was performed, and the following organs and tissues were harvested to measure the radioactivity content: blood, skin, spleen, pancreas, kidney, liver, muscle, tail, and tumor.

Results

Average in all collected tissues 6 hours after injection²¹²The Pb distribution is shown in fig. 23. The tumor accumulation for the biparatopic variant was 71% ID/g and 46% ID/g for T84.66 VH-AST + CH1A1A VL and T84.66 VL + CH1A1A VH-AST, respectively. The positive CH1A1A control caused 37% ID/g. Using a two-way ANOVA display of the DuKai's multiple comparison assay, the method is as follows All three groups differ significantly from each other in terms of tumor uptake (p for T84.66 VH-AST + CH1A1A VL relative to the two other groups<0.0001; p ═ 0.0020 for T84.66 VL + CH1A1A VH-AST versus CH1A1A only). Other organs did not show statistically significant differences between groups, but a slightly higher retention in blood indicates a T84.66 VH-AST + CH1A1A VL combination compared to two other groups: and with<1% ID/g compared to 2% ID/g. Renal absorption was similarly slightly high, but not statistically significantly so: 4.5% ID/g for T84.66 VH-AST + CH1A1A compared to 3% ID/g for the other two.

Adverse events and toxicity

There were no adverse events or toxicities associated with this study. However, BxPC3 tumor growth was significantly faster and accompanied by greater variability in this study compared to the standard growth rate. At necropsy, the conclusion was drawn: large tumors (mostly) filled with liquid, emptied when they were cut in half prior to the radioactivity measurement; this liquid is likely to lead to an acceleration of the growth rate, but does not affect the% IA/g to any great extent, since the tumor is weighed and measured after opening.

Conclusion

Results the tested CEA-split-DOTAM-VH/VL constructs were used to validate the function of biparatopic targeting of the T84.66 and CH1A1A epitopes of CEA, and this combination exhibited unexpectedly high efficacy compared to the positive CH1A1A control. Under the indication of the particular advantage of the T84.66 VH-AST + CH1A1A VL pair, ²¹²The resulting accumulation of Pb in tumors expressing pre-targeted CEA is high and specific.

Example 10

These examples study the recruitment of Pb-DOTA to cells by dividing antibodies as described herein.

P1AF0712 has a first heavy chain with SEQ ID NO 97, a second heavy chain with SEQ ID NO 98 and a light chain with SEQ ID NO 103. P1AF0713 has a first heavy chain with SEQ ID NO 100, a second heavy chain with SEQ ID NO 101 and a light chain with SEQ ID NO 103.

MKN-45 cells were isolated from the culture flask using trypsin and counted using a Casy cell counter. After pelletisation at 4 ℃, 300g of cells were resuspended in FACS buffer (PBS containing 2.5% FCS), adjusted to 2.0E +06 cells/ml, and dispensed into 96-well V-bottom PP plates (25 μ l/well-5.0E +04 cells/well).

FACS staining Using DOTA-FITC

The CEA-specific SPLIT antibody (P1 AF0712 or P1AF0713, respectively) was adjusted to 40. mu.g/mL in FACS buffer to a final concentration of 10. mu.g/mL. The two antibodies were added to the cells in combination or separately, and then added to the buffer, and incubated at 4 ℃ for 1 hour. Subsequently, FITC-labeled Pb-DOTA was added to the cells at an equimolar concentration ratio to the antibody, and incubated at 4 ℃ for 1 hour. Cells were then washed twice in FACS buffer and resuspended in 70 μ Ι/well FACS buffer for measurement using FACS Canto (BD, Pharmingen). It shows (FIG. 24) that neither half of SPLIT produces a fluorescent signal, indicating a lack of Pb-DOTA binding ability. Only the combination of both halves of SPLIT was able to recruit Pb-DOTAM-FITC to the target cells (FIG. 24).

FACS staining Using < huIgG (H + L) A488>

The CEA-specific SPLIT antibody (P1 AF0712 or P1AF0713, respectively) was adjusted to 40. mu.g/mL in FACS buffer to a final concentration of 10. mu.g/mL. The two antibodies were added to the cells, separated, then added to the buffer, or combined, and incubated at 4 ℃ for 1 hour. The cells were then washed twice in FACS buffer. After washing, the cells were resuspended in 50 μ L FACS-buffer containing secondary antibody (< huIgG (H + L) > -Alexa488, c ═ 10 μ g/mL) and incubated at 4 ℃ for 1 hour. Cells were then washed twice in FACS buffer and resuspended in 70 μ Ι/well FACS buffer for measurement using FACS Canto (BD, Pharmingen). EC50 of both SPLIT antibodies were comparable, indicating CEA-specific cell binding of both SPLIT antibodies. Due to the higher amount of antibody in the mixture, lower EC50 was obtained in these cases, as shown in the table below.

EC50 assay for SPLIT antibodies

EC50 for SPLIT antibody was determined using secondary antibody-based detection (< hu >488, upper panel) or Pb-DOTA-FITC (lower panel for DOTA-FITC)

Example 11: biacore binding experiments

This example tested the binding of TA-split-DOTAM-VH and TA-split-DOTAM-VL alone to DOTAM, compared to the reference antibody CEA-DOTAM (RO7198427, PRIT-0213). It was further tested for binding of DOTAM to the TA-split-DOTAM-VH/VL pair compared to the reference antibody.

The correspondence between the codes used in these examples and the protein numbers used elsewhere in the application is shown below. Sequences are also provided. In this example, the reference antibody is encoded as "PRIT _ RS".

For these experiments, the PRIT SPLIT antibody was purified by MabSelect Sure first step (affinity chromatography) and ion exchange chromatography (e.g. POROS XS) as a second step, followed by Superdex 200 (size exclusion chromatography).

The experiment to measure temperature was performed with a Biacore T200 at 25 ℃. All Biacore T200 experiments were performed in HBS-P + (GE Healthcare, Br-1008-27) running buffer pH 7.4. Two experiments were performed on each test antibody/antibody pair using different DOTAM fractions.

1. In a first experiment, the binding of individual TA-division-DOTAM-VH and TA-division-DOTAM-VL antibodies to biotin-labeled DOTAM captured on the chip was assessed relative to the reference antibody.

DOTAM (HBS-P + in 120nM solution) was captured at high density on the CAP chip surface (10. mu.l/min, 60 sec). Subsequently, HBS-P + (10. mu.l/min, 90 seconds) containing 600nM prodrug _ A or prodrug _ B solution was injected on the surface of DOTAM. Dissociation was monitored at a flow rate of 10 μ l/min for 240 seconds. Relative maximal response assays were evaluated using T200 evaluation software.

The results are shown in FIG. 26. None of the individual antibodies showed binding to the captured DOTAM.

2. In a second experiment, individual TA-division-DOTAM-VH and TA-division-DOTAM-VL antibodies were first captured in the chip using immobilized hFab antibodies, and binding of DOTAM-single chain avidin complexes was subsequently assessed (DOTAM + single chain avidin coupling 600nM, 1:1mol, 1h at RT).

HBS-P + (10. mu.l/min, 120 sec) containing 600nM prodrug _ A or prodrug B solution was injected onto the surface of hFab antibody (GE Healthcare, BR-1008-27) CM5 chip. After high density capture of prodrug a or B solution, DOTAM-single chain avidin complex was injected (20 μ l/min, 90 sec). Dissociation was monitored at a flow rate of 20 microliters/min for 180 seconds. For the new cycle, the surface was regenerated at 10 microliters/minute by using glycine 2.1 and 75 seconds regeneration time. Relative maximal response assays were evaluated using T200 evaluation software.

The results are shown in fig. 27. A low percent maximal response (as marked by an x in the figure) is considered a "trace" or non-specific interaction with DOTAM-SA and reflects the need to optimize the assay.

3. In a third experiment, binding of the TA-division-DOTAM-VH/VL pair to DOTAM was assessed compared to the reference antibody. The antibody was first captured in the chip using an immobilized hFab antibody and subsequently the binding of the DOTAM-single chain avidin complex was assessed (DOTAM + single chain avidin coupled 600nM, 1:1mol, 1h at RT).

HBS-P + (10. mu.l/min, 120 sec) containing 300nM prodrug _ A and prodrug B solutions was injected onto the surface of a hFab antibody (GE Healthcare, BR-1008-27) CM5 chip. After high density capture of prodrug a and B solutions, DOTAM-single chain avidin complex was injected (20 μ l/min, 90 sec). Dissociation was monitored at a flow rate of 20 microliters/min for 180 seconds. For the new cycle, the surface was regenerated at 10 microliters/minute by using glycine 2.1 and 75 seconds regeneration time. Relative maximal response assays were evaluated using T200 evaluation software.

The results are shown in FIG. 28. All TA-split-DOTAM-VH/VL pairs showed substantial binding to DOTAM, except the P6_ AB (P1AF0712/P1AF0713) pair, which is a DOTA binder.

Similar results were also obtained for the FAP binders P1AF8286 and P1AF8287, showing that there is substantial DOTAM binding by the TA-split-DOTAM-VH/VL pair, whereas individual members of this pair did not. P1AF8286 consists of the first heavy chain of SEQ ID NO. 108, the second heavy chain of SEQ ID NO. 109 and the light chain of SEQ ID NO. 111, and P1AF8287 consists of the first heavy chain of SEQ ID NO. 108, the second heavy chain of SEQ ID NO. 110 and the light chain of SEQ ID NO. 111. However, the analysis still needs to be optimized.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the description and example should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

SEQUENCE LISTING

<110> Haofmii Roche GmbH (F. Hoffmann-La Roche AG)

<120> antibody binding to cancer cells and targeting radionuclide to the cells

<130> 007681943

<150> EP19186135

<151> 2019-07-12

<160> 180

<170> PatentIn version 3.5

<210> 1

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain CDR1, < Pb-Dotam >

<400> 1

Gly Phe Ser Leu Ser Thr Tyr Ser Met Ser

1 5 10

<210> 2

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain CDR2 < Pb-Dotam >

<400> 2

Phe Ile Gly Ser Arg Gly Asp Thr Tyr Tyr Ala Ser Trp Ala Lys Gly

1 5 10 15

<210> 3

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain CDR3 < Pb-Dotam >

<400> 3

Glu Arg Asp Pro Tyr Gly Gly Gly Ala Tyr Pro Pro His Leu

1 5 10

<210> 4

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> light chain CDR1, < Pb-Dotam >

<400> 4

Gln Ser Ser His Ser Val Tyr Ser Asp Asn Asp Leu Ala

1 5 10

<210> 5

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> light chain CDR2 < Pb-Dotam >

<400> 5

Gln Ala Ser Lys Leu Ala Ser

1 5

<210> 6

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> light chain CDR3 < Pb-Dotam >

<400> 6

Leu Gly Gly Tyr Asp Asp Glu Ser Asp Thr Tyr Gly

1 5 10

<210> 7

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain variable domain < Pb-Dotam > PRIT-0213

<400> 7

Val Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys Pro Thr Glu Thr

1 5 10 15

Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Thr Tyr Ser

20 25 30

Met Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Gly

35 40 45

Phe Ile Gly Ser Arg Gly Asp Thr Tyr Tyr Ala Ser Trp Ala Lys Gly

50 55 60

Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Ser Gln Val Val Leu Thr

65 70 75 80

Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala Arg

85 90 95

Glu Arg Asp Pro Tyr Gly Gly Gly Ala Tyr Pro Pro His Leu Trp Gly

100 105 110

Arg Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 8

<211> 111

<212> PRT

<213> Artificial sequence

<220>

<223> light chain variable domain < Pb-Dotam > PRIT-0213

<400> 8

Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp

1 5 10 15

Arg Val Thr Ile Thr Cys Gln Ser Ser His Ser Val Tyr Ser Asp Asn

20 25 30

Asp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Gln Ala Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

65 70 75 80

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gly Gly Tyr Asp Asp Glu

85 90 95

Ser Asp Thr Tyr Gly Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 9

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain variable domain < Pb-Dotam > PRIT-0214

<400> 9

Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu Thr

1 5 10 15

Leu Ser Leu Thr Cys Ala Val Tyr Gly Phe Ser Leu Ser Thr Tyr Ser

20 25 30

Met Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly

35 40 45

Phe Ile Gly Ser Arg Gly Asp Thr Tyr Tyr Ala Ser Trp Ala Lys Gly

50 55 60

Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Leu Lys

65 70 75 80

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg

85 90 95

Glu Arg Asp Pro Tyr Gly Gly Gly Ala Tyr Pro Pro His Leu Trp Gly

100 105 110

Arg Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 10

<211> 111

<212> PRT

<213> Artificial sequence

<220>

<223> light chain variable domain < Pb-Dotam > PRIT-0214

<400> 10

Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp

1 5 10 15

Arg Val Thr Ile Thr Cys Gln Ser Ser His Ser Val Tyr Ser Asp Asn

20 25 30

Asp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Gln Ala Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

65 70 75 80

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gly Gly Tyr Asp Asp Glu

85 90 95

Ser Asp Thr Tyr Gly Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 11

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain CDR1 < CEA > T84.66

<400> 11

Gly Phe Asn Ile Lys Asp Thr Tyr Met His

1 5 10

<210> 12

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain CDR2 < CEA > T84.66

<400> 12

Arg Ile Asp Pro Ala Asn Gly Asn Ser Lys Tyr Val Pro Lys Phe Gln

1 5 10 15

Gly

<210> 13

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain CDR3 < CEA > T84.66

<400> 13

Phe Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr

1 5 10

<210> 14

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> light chain CDR1 < CEA > T84.66

<400> 14

Arg Ala Gly Glu Ser Val Asp Ile Phe Gly Val Gly Phe Leu His

1 5 10 15

<210> 15

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> light chain CDR2 < CEA > T84.66

<400> 15

Arg Ala Ser Asn Arg Ala Thr

1 5

<210> 16

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> light chain CDR3 < CEA > T84.66

<400> 16

Gln Gln Thr Asn Glu Asp Pro Tyr Thr

1 5

<210> 17

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain variable domain < CEA > T84.66

<400> 17

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Ala Asn Gly Asn Ser Lys Tyr Val Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Pro Phe Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 18

<211> 111

<212> PRT

<213> Artificial sequence

<220>

<223> light chain variable Domain < CEA > T84.66

<400> 18

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Gly Glu Ser Val Asp Ile Phe

20 25 30

Gly Val Gly Phe Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

Arg Leu Leu Ile Tyr Arg Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Thr Asn

85 90 95

Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 19

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain CDR1 < CEA > CH1A1A

<400> 19

Gly Tyr Thr Phe Thr Glu Phe Gly Met Asn

1 5 10

<210> 20

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain CDR2 < CEA > CH1A1A

<400> 20

Trp Ile Asn Thr Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe Lys

1 5 10 15

Gly

<210> 21

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain CDR3 < CEA > CH1A1A

<400> 21

Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr

1 5 10

<210> 22

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> light chain CDR1 < CEA > CH1A1A

<400> 22

Lys Ala Ser Ala Ala Val Gly Thr Tyr Val Ala

1 5 10

<210> 23

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> light chain CDR2 < CEA > CH1A1A

<400> 23

Ser Ala Ser Tyr Arg Lys Arg

1 5

<210> 24

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> light chain CDR3 < CEA > CH1A1A

<400> 24

His Gln Tyr Tyr Thr Tyr Pro Leu Phe Thr

1 5 10

<210> 25

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain variable domain < CEA > CH1A1A

<400> 25

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe

50 55 60

Lys Gly Arg Val Thr Phe Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 26

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> light chain variable domain < CEA > CH1A1A

<400> 26

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Ala Ala Val Gly Thr Tyr

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Lys Arg Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys His Gln Tyr Tyr Thr Tyr Pro Leu

85 90 95

Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 27

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain < CEA > of P1AD8749 without linker and < DOTAM-VH >' plasmid identical to SeqID32, lacking linker and < DOTAM >

<400> 27

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe

50 55 60

Lys Gly Arg Val Thr Phe Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly

450

<210> 28

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> P1AD8749 heavy chain cave < CEA > CH1A1A

<400> 28

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe

50 55 60

Lys Gly Arg Val Thr Phe Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly

450

<210> 29

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain < CEA > of P1AD8592 without linker and < DOTAM-VL >' plasmid identical to SeqID33, lacking linker and < DOTAM >

<400> 29

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe

50 55 60

Lys Gly Arg Val Thr Phe Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly

450

<210> 30

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> P1AD8592 heavy chain segment < CEA > CH1A1A

<400> 30

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe

50 55 60

Lys Gly Arg Val Thr Phe Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly

450

<210> 31

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 31

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

<210> 32

<211> 591

<212> PRT

<213> Artificial sequence

<220>

<223> P1AD8749 heavy chain segment < CEA > CH1A1A < Dotam-VH >

<400> 32

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe

50 55 60

Lys Gly Arg Val Thr Phe Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

450 455 460

Ser Gly Gly Gly Gly Ser Val Thr Leu Lys Glu Ser Gly Pro Val Leu

465 470 475 480

Val Lys Pro Thr Glu Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe

485 490 495

Ser Leu Ser Thr Tyr Ser Met Ser Trp Ile Arg Gln Pro Pro Gly Lys

500 505 510

Ala Leu Glu Trp Leu Gly Phe Ile Gly Ser Arg Gly Asp Thr Tyr Tyr

515 520 525

Ala Ser Trp Ala Lys Gly Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys

530 535 540

Ser Gln Val Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala

545 550 555 560

Thr Tyr Tyr Cys Ala Arg Glu Arg Asp Pro Tyr Gly Gly Gly Ala Tyr

565 570 575

Pro Pro His Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser

580 585 590

<210> 33

<211> 581

<212> PRT

<213> Artificial sequence

<220>

<223> P1AD8592 heavy chain pocket < CEA > CH1A1A < Dotam-VL >

<400> 33

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe

50 55 60

Lys Gly Arg Val Thr Phe Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

450 455 460

Ser Gly Gly Gly Gly Ser Ile Gln Met Thr Gln Ser Pro Ser Ser Leu

465 470 475 480

Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ser Ser His

485 490 495

Ser Val Tyr Ser Asp Asn Asp Leu Ala Trp Tyr Gln Gln Lys Pro Gly

500 505 510

Lys Ala Pro Lys Leu Leu Ile Tyr Gln Ala Ser Lys Leu Ala Ser Gly

515 520 525

Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

530 535 540

Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu

545 550 555 560

Gly Gly Tyr Asp Asp Glu Ser Asp Thr Tyr Gly Phe Gly Gly Gly Thr

565 570 575

Lys Val Glu Ile Lys

580

<210> 34

<211> 215

<212> PRT

<213> Artificial sequence

<220>

<223> P1AD8749 and P1AD8592 light chain < CEA > CH1A1A

<400> 34

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Ala Ala Val Gly Thr Tyr

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Lys Arg Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys His Gln Tyr Tyr Thr Tyr Pro Leu

85 90 95

Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 35

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain CDR1, < C825>

<400> 35

Asp Tyr Gly Val His

1 5

<210> 36

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain CDR2, < C825>

<400> 36

Val Ile Trp Ser Gly Gly Gly Thr Ala Tyr Asn Thr Ala Leu Ile Ser

1 5 10 15

<210> 37

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain CDR3, < C825>

<400> 37

Arg Gly Ser Tyr Pro Tyr Asn Tyr Phe Asp Ala

1 5 10

<210> 38

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> light chain CDR1, < C825>

<400> 38

Gly Ser Ser Thr Gly Ala Val Thr Ala Ser Asn Tyr Ala Asn

1 5 10

<210> 39

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> light chain CDR2, < C825>

<400> 39

Gly His Asn Asn Arg Pro Pro

1 5

<210> 40

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> light chain CDR3, < C825>

<400> 40

Ala Leu Trp Tyr Ser Asp His Trp Val

1 5

<210> 41

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain variable Domain < C825>

<400> 41

His Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asp Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Ser Gly Gly Gly Thr Ala Tyr Asn Thr Ala Leu Ile

50 55 60

Ser Arg Leu Asn Ile Tyr Arg Asp Asn Ser Lys Asn Gln Val Phe Leu

65 70 75 80

Glu Met Asn Ser Leu Gln Ala Glu Asp Thr Ala Met Tyr Tyr Cys Ala

85 90 95

Arg Arg Gly Ser Tyr Pro Tyr Asn Tyr Phe Asp Ala Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 42

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> light chain variable domain, < C825>

<400> 42

Gln Ala Val Val Ile Gln Glu Ser Ala Leu Thr Thr Pro Pro Gly Glu

1 5 10 15

Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Ala Ser

20 25 30

Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Gly

35 40 45

Leu Ile Gly Gly His Asn Asn Arg Pro Pro Gly Val Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Ala Gly Thr

65 70 75 80

Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asp

85 90 95

His Trp Val Ile Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 43

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain CDR1 < CEA > A5B7

<400> 43

Asp Tyr Tyr Met Asn

1 5

<210> 44

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain CDR2 < CEA > A5B7

<400> 44

Phe Ile Gly Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala Ser

1 5 10 15

Val Lys Gly

<210> 45

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain CDR3 < CEA > A5B7

<400> 45

Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr

1 5 10

<210> 46

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> light chain CDR1 < CEA > A5B7

<400> 46

Arg Ala Ser Ser Ser Val Thr Tyr Ile His

1 5 10

<210> 47

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> light chain CDR2 < CEA > A5B7

<400> 47

Ala Thr Ser Asn Leu Ala Ser

1 5

<210> 48

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> light chain CDR3 < CEA > A5B7

<400> 48

Gln His Trp Ser Ser Lys Pro Pro Thr

1 5

<210> 49

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain variable domain < CEA > A5B7

<400> 49

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Phe Ile Gly Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 50

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> light chain variable domain < CEA > A5B7

<400> 50

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Ser Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln His Trp Ser Ser Lys Pro Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 51

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> P1AE4956 heavy chain cave < CEA > A5B7

<400> 51

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Phe Ile Gly Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly

450

<210> 52

<211> 591

<212> PRT

<213> Artificial sequence

<220>

<223> P1AE4956 heavy chain node < CEA > A5B7 < Dotam-VH >

<400> 52

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Phe Ile Gly Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

450 455 460

Ser Gly Gly Gly Gly Ser Val Thr Leu Lys Glu Ser Gly Pro Val Leu

465 470 475 480

Val Lys Pro Thr Glu Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe

485 490 495

Ser Leu Ser Thr Tyr Ser Met Ser Trp Ile Arg Gln Pro Pro Gly Lys

500 505 510

Ala Leu Glu Trp Leu Gly Phe Ile Gly Ser Arg Gly Asp Thr Tyr Tyr

515 520 525

Ala Ser Trp Ala Lys Gly Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys

530 535 540

Ser Gln Val Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala

545 550 555 560

Thr Tyr Tyr Cys Ala Arg Glu Arg Asp Pro Tyr Gly Gly Gly Ala Tyr

565 570 575

Pro Pro His Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser

580 585 590

<210> 53

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain < CEA > of P1AE4956 without linker and DOTAM-VH

<400> 53

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Phe Ile Gly Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro

<210> 54

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<223> P1AE4956 light chain < CEA > A5B7

<400> 54

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Ser Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln His Trp Ser Ser Lys Pro Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 55

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> P1AE4957 heavy chain node < CEA > A5B7

<400> 55

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Phe Ile Gly Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly

450

<210> 56

<211> 581

<212> PRT

<213> Artificial sequence

<220>

<223> P1AE4957 heavy chain pocket < CEA > A5B7 < Dotam-VL >

<400> 56

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Phe Ile Gly Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

450 455 460

Ser Gly Gly Gly Gly Ser Ile Gln Met Thr Gln Ser Pro Ser Ser Leu

465 470 475 480

Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ser Ser His

485 490 495

Ser Val Tyr Ser Asp Asn Asp Leu Ala Trp Tyr Gln Gln Lys Pro Gly

500 505 510

Lys Ala Pro Lys Leu Leu Ile Tyr Gln Ala Ser Lys Leu Ala Ser Gly

515 520 525

Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

530 535 540

Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu

545 550 555 560

Gly Gly Tyr Asp Asp Glu Ser Asp Thr Tyr Gly Phe Gly Gly Gly Thr

565 570 575

Lys Val Glu Ile Lys

580

<210> 57

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain < CEA > of P1AE4957 having no linker and DOTAM-VL

<400> 57

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Phe Ile Gly Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro

<210> 58

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<223> P1AE4957 light chain < CEA > A5B7

<400> 58

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Ser Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln His Trp Ser Ser Lys Pro Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 59

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain CDR1 < CEA > 28A9

<400> 59

Gly Gly Thr Phe Ser Tyr Tyr Ala Ile Ser

1 5 10

<210> 60

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain CDR2 < CEA > 28A9

<400> 60

Gly Ile Leu Pro Ala Phe Gly Ala Ala Asn Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210> 61

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain CDR3 < CEA > 28A9

<400> 61

Leu Pro Pro Leu Pro Gly Ala Gly Leu Asp Tyr

1 5 10

<210> 62

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> light chain CDR1 < CEA > 28A9

<400> 62

Arg Ala Ser Gln Ser Ile Ser Ser Trp Leu Ala

1 5 10

<210> 63

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> light chain CDR2 < CEA > 28A9

<400> 63

Asp Ala Ser Ser Leu Glu Ser

1 5

<210> 64

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> light chain CDR3 < CEA > 28A9

<400> 64

Gln Gln Asn Thr Gln Tyr Pro Met Thr

1 5

<210> 65

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain variable domain < CEA > 28A9

<400> 65

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Tyr Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Leu Pro Ala Phe Gly Ala Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Pro Pro Leu Pro Gly Ala Gly Leu Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 66

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> light chain variable Domain < CEA > 28A9

<400> 66

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asn Thr Gln Tyr Pro Met

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 67

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain CDR1 < GPRC5D >

<400> 67

Gly Phe Thr Phe Ser Lys Tyr Ala Met Ala

1 5 10

<210> 68

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain CDR2 < GPRC5D >

<400> 68

Ser Ile Ser Thr Gly Gly Val Asn Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 69

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain CDR3 < GPRC5D >

<400> 69

His Thr Gly Asp Tyr Phe Asp Tyr

1 5

<210> 70

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> light chain CDR1 < GPRC5D >

<400> 70

Arg Ala Ser Gln Ser Val Ser Ile Ser Gly Ile Asn Leu Met Asn

1 5 10 15

<210> 71

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> light chain CDR2 < GPRC5D >

<400> 71

His Ala Ser Ile Leu Ala Ser

1 5

<210> 72

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> light chain CDR3 < GPRC5D >

<400> 72

Gln Gln Thr Arg Glu Ser Pro Leu Thr

1 5

<210> 73

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain variable Domain < GPRC5D >

<400> 73

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Lys Tyr

20 25 30

Ala Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Thr Gly Gly Val Asn Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr His Thr Gly Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Met

100 105 110

Val Thr Val Ser Ser

115

<210> 74

<211> 111

<212> PRT

<213> Artificial sequence

<220>

<223> light chain variable Domain < GPRC5D >

<400> 74

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ile Ser

20 25 30

Gly Ile Asn Leu Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Gln Pro

35 40 45

Lys Leu Leu Ile Tyr His Ala Ser Ile Leu Ala Ser Gly Ile Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Thr Arg

85 90 95

Glu Ser Pro Leu Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105 110

<210> 75

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain CDR1 < FAP > 4B9

<400> 75

Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser

1 5 10

<210> 76

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain CDR2 < FAP > 4B9

<400> 76

Ala Ile Ile Gly Ser Gly Ala Ser Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 77

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain CDR3 < FAP > 4B9

<400> 77

Gly Trp Phe Gly Gly Phe Asn Tyr

1 5

<210> 78

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> light chain CDR1 < FAP > 4B9

<400> 78

Arg Ala Ser Gln Ser Val Thr Ser Ser Tyr Leu Ala

1 5 10

<210> 79

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> light chain CDR2 < FAP > 4B9

<400> 79

Val Gly Ser Arg Arg Ala Thr

1 5

<210> 80

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> light chain CDR3 < FAP > 4B9

<400> 80

Gln Gln Gly Ile Met Leu Pro Pro Thr

1 5

<210> 81

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain variable domain < FAP > 4B9

<400> 81

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ile Gly Ser Gly Ala Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Gly Trp Phe Gly Gly Phe Asn Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 82

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> light chain variable domain < FAP > 4B9

<400> 82

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Thr Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Asn Val Gly Ser Arg Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Gly Ile Met Leu Pro

85 90 95

Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 83

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> P1AF0709 HC section < CEA > T84.66 (D1AE4688)

<400> 83

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Ala Asn Gly Asn Ser Lys Tyr Val Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Pro Phe Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly

450

<210> 84

<211> 581

<212> PRT

<213> Artificial sequence

<220>

<223> P1AF0709 HC hole < CEA > T84.66 Dotam-VL (D1AA4920)

<400> 84

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Ala Asn Gly Asn Ser Lys Tyr Val Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Pro Phe Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

450 455 460

Ser Gly Gly Gly Gly Ser Ile Gln Met Thr Gln Ser Pro Ser Ser Leu

465 470 475 480

Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ser Ser His

485 490 495

Ser Val Tyr Ser Asp Asn Asp Leu Ala Trp Tyr Gln Gln Lys Pro Gly

500 505 510

Lys Ala Pro Lys Leu Leu Ile Tyr Gln Ala Ser Lys Leu Ala Ser Gly

515 520 525

Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

530 535 540

Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu

545 550 555 560

Gly Gly Tyr Asp Asp Glu Ser Asp Thr Tyr Gly Phe Gly Gly Gly Thr

565 570 575

Lys Val Glu Ile Lys

580

<210> 85

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> P1AF0709 HC pocket < CEA > T84.66 without linker and DOTAM

<400> 85

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Ala Asn Gly Asn Ser Lys Tyr Val Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Pro Phe Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly

450

<210> 86

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> PIAF0298 HC hole < CEA > T84.66 (D1AE4687)

<400> 86

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Ala Asn Gly Asn Ser Lys Tyr Val Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Pro Phe Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly

450

<210> 87

<211> 594

<212> PRT

<213> Artificial sequence

<220>

<223> PIAF0298 HC node < CEA > T84.66 Dotam-VH-AST (D1AE3668)

<400> 87

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Ala Asn Gly Asn Ser Lys Tyr Val Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Pro Phe Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

450 455 460

Ser Gly Gly Gly Gly Ser Val Thr Leu Lys Glu Ser Gly Pro Val Leu

465 470 475 480

Val Lys Pro Thr Glu Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe

485 490 495

Ser Leu Ser Thr Tyr Ser Met Ser Trp Ile Arg Gln Pro Pro Gly Lys

500 505 510

Ala Leu Glu Trp Leu Gly Phe Ile Gly Ser Arg Gly Asp Thr Tyr Tyr

515 520 525

Ala Ser Trp Ala Lys Gly Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys

530 535 540

Ser Gln Val Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala

545 550 555 560

Thr Tyr Tyr Cys Ala Arg Glu Arg Asp Pro Tyr Gly Gly Gly Ala Tyr

565 570 575

Pro Pro His Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala

580 585 590

Ser Thr

<210> 88

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> PIAF0298 HC node < CEA > T84.66 without linker and DOTAM

<400> 88

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Ala Asn Gly Asn Ser Lys Tyr Val Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Pro Phe Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly

450

<210> 89

<211> 218

<212> PRT

<213> Artificial sequence

<220>

<223> P1AF0709 and PIAF0298 light chain (D1AA4120)

<400> 89

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Gly Glu Ser Val Asp Ile Phe

20 25 30

Gly Val Gly Phe Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

Arg Leu Leu Ile Tyr Arg Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Thr Asn

85 90 95

Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 90

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<223> P1AF710 HC node < CEA > 28A9 (D1AE4690)

<400> 90

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Tyr Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Leu Pro Ala Phe Gly Ala Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Pro Pro Leu Pro Gly Ala Gly Leu Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210> 91

<211> 580

<212> PRT

<213> Artificial sequence

<220>

<223> P1AF710 HC pocket < CEA > 28A9 Dotam-VL (D1AC3172)

<400> 91

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Tyr Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Leu Pro Ala Phe Gly Ala Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Pro Pro Leu Pro Gly Ala Gly Leu Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

450 455 460

Gly Gly Gly Gly Ser Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

465 470 475 480

Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ser Ser His Ser

485 490 495

Val Tyr Ser Asp Asn Asp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys

500 505 510

Ala Pro Lys Leu Leu Ile Tyr Gln Ala Ser Lys Leu Ala Ser Gly Val

515 520 525

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

530 535 540

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gly

545 550 555 560

Gly Tyr Asp Asp Glu Ser Asp Thr Tyr Gly Phe Gly Gly Gly Thr Lys

565 570 575

Val Glu Ile Lys

580

<210> 92

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<223> P1AF710 HC pocket < CEA > 28A9 without linker or DOTAM

<400> 92

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Tyr Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Leu Pro Ala Phe Gly Ala Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Pro Pro Leu Pro Gly Ala Gly Leu Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210> 93

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<223> P1AF711 HC pocket < CEA > 28A9 (D1AE4689)

<400> 93

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Tyr Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Leu Pro Ala Phe Gly Ala Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Pro Pro Leu Pro Gly Ala Gly Leu Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210> 94

<211> 593

<212> PRT

<213> Artificial sequence

<220>

<223> P1AF 7111 HC node < CEA > 28A9 Dotam-VH-AST (D1AE3671)

<400> 94

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Tyr Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Leu Pro Ala Phe Gly Ala Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Pro Pro Leu Pro Gly Ala Gly Leu Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

450 455 460

Gly Gly Gly Gly Ser Val Thr Leu Lys Glu Ser Gly Pro Val Leu Val

465 470 475 480

Lys Pro Thr Glu Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser

485 490 495

Leu Ser Thr Tyr Ser Met Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala

500 505 510

Leu Glu Trp Leu Gly Phe Ile Gly Ser Arg Gly Asp Thr Tyr Tyr Ala

515 520 525

Ser Trp Ala Lys Gly Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Ser

530 535 540

Gln Val Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr

545 550 555 560

Tyr Tyr Cys Ala Arg Glu Arg Asp Pro Tyr Gly Gly Gly Ala Tyr Pro

565 570 575

Pro His Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser

580 585 590

Thr

<210> 95

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<223> P1AF711 HC node < CEA > 28A9 has no linker and DOTAM

<400> 95

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Tyr Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Leu Pro Ala Phe Gly Ala Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Pro Pro Leu Pro Gly Ala Gly Leu Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210> 96

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> P1AF710 and P1AF711 light chain (D1AA2299)

<400> 96

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asn Thr Gln Tyr Pro Met

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 97

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> P1AF0712 HC node < CEA > CH1A1A (D1AC4023)

<400> 97

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe

50 55 60

Lys Gly Arg Val Thr Phe Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly

450

<210> 98

<211> 579

<212> PRT

<213> Artificial sequence

<220>

<223> P1AF0712 HC hole < CEA > CH1A1A DOTA-VL (D1AE4684)

<400> 98

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe

50 55 60

Lys Gly Arg Val Thr Phe Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

450 455 460

Ser Gly Gly Gly Gly Ser Gln Ala Val Val Ile Gln Glu Ser Ala Leu

465 470 475 480

Thr Thr Pro Pro Gly Glu Thr Val Thr Leu Thr Cys Gly Ser Ser Thr

485 490 495

Gly Ala Val Thr Ala Ser Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro

500 505 510

Asp His Leu Phe Thr Gly Leu Ile Gly Gly His Asn Asn Arg Pro Pro

515 520 525

Gly Val Pro Ala Arg Phe Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala

530 535 540

Leu Thr Ile Ala Gly Thr Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys

545 550 555 560

Ala Leu Trp Tyr Ser Asp His Trp Val Ile Gly Gly Gly Thr Lys Leu

565 570 575

Thr Val Leu

<210> 99

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> P1AF0712 HC pocket < CEA > without linker or DOTA

<400> 99

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe

50 55 60

Lys Gly Arg Val Thr Phe Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly

450

<210> 100

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> P1AF0713 HC pocket < CEA > CH1A1A (D1AC4022)

<400> 100

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe

50 55 60

Lys Gly Arg Val Thr Phe Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly

450

<210> 101

<211> 592

<212> PRT

<213> Artificial sequence

<220>

<223> P1AF0713 HC section < CEA > CH1A1A DOTA-VH-AST (D1AE3670)

<400> 101

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe

50 55 60

Lys Gly Arg Val Thr Phe Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

450 455 460

Ser Gly Gly Gly Gly Ser His Val Lys Leu Gln Glu Ser Gly Pro Gly

465 470 475 480

Leu Val Gln Pro Ser Gln Ser Leu Ser Leu Thr Cys Thr Val Ser Gly

485 490 495

Phe Ser Leu Thr Asp Tyr Gly Val His Trp Val Arg Gln Ser Pro Gly

500 505 510

Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Ser Gly Gly Gly Thr Ala

515 520 525

Tyr Asn Thr Ala Leu Ile Ser Arg Leu Asn Ile Tyr Arg Asp Asn Ser

530 535 540

Lys Asn Gln Val Phe Leu Glu Met Asn Ser Leu Gln Ala Glu Asp Thr

545 550 555 560

Ala Met Tyr Tyr Cys Ala Arg Arg Gly Ser Tyr Pro Tyr Asn Tyr Phe

565 570 575

Asp Ala Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

580 585 590

<210> 102

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> P1AF0713 HC node < CEA > CH1A1A without linker and DOTA

<400> 102

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe

50 55 60

Lys Gly Arg Val Thr Phe Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly

450

<210> 103

<211> 215

<212> PRT

<213> Artificial sequence

<220>

<223> P1AF0712 and P1AF0713 light chain (D1AA3384)

<400> 103

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Ala Ala Val Gly Thr Tyr

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Lys Arg Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys His Gln Tyr Tyr Thr Tyr Pro Leu

85 90 95

Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 104

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> P1AF8284 and P1AF8285 HC node < GPRC5D > (D1AF6517)

<400> 104

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Lys Tyr

20 25 30

Ala Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Thr Gly Gly Val Asn Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr His Thr Gly Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Met

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 105

<211> 359

<212> PRT

<213> Artificial sequence

<220>

<223> P1AF8284 HC pocket Dotam-VL (D1AG3592)

<400> 105

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

225 230 235 240

Gly Ser Gly Gly Gly Gly Ser Ser Ile Gln Met Thr Gln Ser Pro Ser

245 250 255

Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ser

260 265 270

Ser His Ser Val Tyr Ser Asp Asn Asp Leu Ala Trp Tyr Gln Gln Lys

275 280 285

Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Gln Ala Ser Lys Leu Ala

290 295 300

Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

305 310 315 320

Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr

325 330 335

Cys Leu Gly Gly Tyr Asp Asp Glu Ser Asp Thr Tyr Gly Phe Gly Gly

340 345 350

Gly Thr Lys Val Glu Ile Lys

355

<210> 106

<211> 369

<212> PRT

<213> Artificial sequence

<220>

<223> P1AF 8285H cave Dotam-VHA (D1AG3591)

<400> 106

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

225 230 235 240

Gly Ser Gly Gly Gly Gly Ser Val Thr Leu Lys Glu Ser Gly Pro Val

245 250 255

Leu Val Lys Pro Thr Glu Thr Leu Thr Leu Thr Cys Thr Val Ser Gly

260 265 270

Phe Ser Leu Ser Thr Tyr Ser Met Ser Trp Ile Arg Gln Pro Pro Gly

275 280 285

Lys Ala Leu Glu Trp Leu Gly Phe Ile Gly Ser Arg Gly Asp Thr Tyr

290 295 300

Tyr Ala Ser Trp Ala Lys Gly Arg Leu Thr Ile Ser Lys Asp Thr Ser

305 310 315 320

Lys Ser Gln Val Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr

325 330 335

Ala Thr Tyr Tyr Cys Ala Arg Glu Arg Asp Pro Tyr Gly Gly Gly Ala

340 345 350

Tyr Pro Pro His Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser

355 360 365

Ala

<210> 107

<211> 218

<212> PRT

<213> Artificial sequence

<220>

<223> P1AF8284 and P1AF8285 light chain (D1AF6469)

<400> 107

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ile Ser

20 25 30

Gly Ile Asn Leu Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Gln Pro

35 40 45

Lys Leu Leu Ile Tyr His Ala Ser Ile Leu Ala Ser Gly Ile Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Thr Arg

85 90 95

Glu Ser Pro Leu Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 108

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> P1AF8286 and P1AF8287 HC node < FAP > 4B9 (D1AF6515)

<400> 108

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ile Gly Ser Gly Ala Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Gly Trp Phe Gly Gly Phe Asn Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 109

<211> 359

<212> PRT

<213> Artificial sequence

<220>

<223> P1AF8286 HC pocket Dotam-VL (D1AG3592)

<400> 109

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

225 230 235 240

Gly Ser Gly Gly Gly Gly Ser Ser Ile Gln Met Thr Gln Ser Pro Ser

245 250 255

Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ser

260 265 270

Ser His Ser Val Tyr Ser Asp Asn Asp Leu Ala Trp Tyr Gln Gln Lys

275 280 285

Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Gln Ala Ser Lys Leu Ala

290 295 300

Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

305 310 315 320

Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr

325 330 335

Cys Leu Gly Gly Tyr Asp Asp Glu Ser Asp Thr Tyr Gly Phe Gly Gly

340 345 350

Gly Thr Lys Val Glu Ile Lys

355

<210> 110

<211> 369

<212> PRT

<213> Artificial sequence

<220>

<223> P1AF8287 HC pocket Dotam-VHA (D1AG3591)

<400> 110

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

225 230 235 240

Gly Ser Gly Gly Gly Gly Ser Val Thr Leu Lys Glu Ser Gly Pro Val

245 250 255

Leu Val Lys Pro Thr Glu Thr Leu Thr Leu Thr Cys Thr Val Ser Gly

260 265 270

Phe Ser Leu Ser Thr Tyr Ser Met Ser Trp Ile Arg Gln Pro Pro Gly

275 280 285

Lys Ala Leu Glu Trp Leu Gly Phe Ile Gly Ser Arg Gly Asp Thr Tyr

290 295 300

Tyr Ala Ser Trp Ala Lys Gly Arg Leu Thr Ile Ser Lys Asp Thr Ser

305 310 315 320

Lys Ser Gln Val Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr

325 330 335

Ala Thr Tyr Tyr Cys Ala Arg Glu Arg Asp Pro Tyr Gly Gly Gly Ala

340 345 350

Tyr Pro Pro His Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser

355 360 365

Ala

<210> 111

<211> 215

<212> PRT

<213> Artificial sequence

<220>

<223> P1AF8286 and P1AF8287 light chain (D1AB9974)

<400> 111

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Thr Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Asn Val Gly Ser Arg Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Gly Ile Met Leu Pro

85 90 95

Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 112

<211> 451

<212> PRT

<213> Artificial sequence

<220>

<223> P1AF7782 and P1AF7784 HC node < CEA > CH1A1A (D1AD3419)

<400> 112

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe

50 55 60

Lys Gly Arg Val Thr Phe Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 113

<211> 359

<212> PRT

<213> Artificial sequence

<220>

<223> P1AF7782 HC pocket Dotam-VL (D1AG2237)

<400> 113

Ser Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ser Ser His Ser Val Tyr Ser Asp

20 25 30

Asn Asp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Gln Ala Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe

50 55 60

Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu

65 70 75 80

Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gly Gly Tyr Asp Asp

85 90 95

Glu Ser Asp Thr Tyr Gly Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Ser Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

130 135 140

Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

145 150 155 160

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

165 170 175

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

180 185 190

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

195 200 205

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

210 215 220

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

225 230 235 240

Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

245 250 255

Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

260 265 270

Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp

275 280 285

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

290 295 300

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser

305 310 315 320

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

325 330 335

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

340 345 350

Leu Ser Leu Ser Pro Gly Lys

355

<210> 114

<211> 369

<212> PRT

<213> Artificial sequence

<220>

<223> P1AF7784 HC pocket Dotam-VH (D1AG2236)

<400> 114

Gly Val Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys Pro Thr Glu

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Thr Tyr

20 25 30

Ser Met Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu

35 40 45

Gly Phe Ile Gly Ser Arg Gly Asp Thr Tyr Tyr Ala Ser Trp Ala Lys

50 55 60

Gly Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Ser Gln Val Val Leu

65 70 75 80

Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala

85 90 95

Arg Glu Arg Asp Pro Tyr Gly Gly Gly Ala Tyr Pro Pro His Leu Trp

100 105 110

Gly Arg Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ser Gly Gly Asp Lys

130 135 140

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro

145 150 155 160

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

165 170 175

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

180 185 190

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

195 200 205

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

210 215 220

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

225 230 235 240

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys

245 250 255

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr

260 265 270

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser

275 280 285

Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

290 295 300

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

305 310 315 320

Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys

325 330 335

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

340 345 350

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

355 360 365

Lys

<210> 115

<211> 215

<212> PRT

<213> Artificial sequence

<220>

<223> P1AF7782 and P1AF7784 light chain (D1AD3421)

<400> 115

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Ala Ala Val Gly Thr Tyr

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Lys Arg Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys His Gln Tyr Tyr Thr Tyr Pro Leu

85 90 95

Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 116

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain CDR1 < Pb-Dotam > PRIT-0213

<400> 116

Gly Phe Ser Leu Ser Thr Tyr Ser Met Ser

1 5 10

<210> 117

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain CDR2 < Pb-Dotam > PRIT-0213

<400> 117

Phe Ile Gly Ser Arg Gly Asp Thr Tyr Tyr Ala Ser Trp Ala Lys Gly

1 5 10 15

<210> 118

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain CDR3 < Pb-Dotam > PRIT-0213

<400> 118

Glu Arg Asp Pro Tyr Gly Gly Gly Ala Tyr Pro Pro His Leu

1 5 10

<210> 119

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> light chain CDR1 < Pb-Dotam > PRIT-0213

<400> 119

Gln Ser Ser His Ser Val Tyr Ser Asp Asn Asp Leu Ala

1 5 10

<210> 120

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> light chain CDR2 < Pb-Dotam > PRIT-0213

<400> 120

Gln Ala Ser Lys Leu Ala Ser

1 5

<210> 121

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> light chain CDR3 < Pb-Dotam > PRIT-0213

<400> 121

Leu Gly Gly Tyr Asp Asp Glu Ser Asp Thr Tyr Gly

1 5 10

<210> 122

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> < Pb-Dotam > PRIT-0213 heavy chain variable domain 1

<400> 122

Val Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys Pro Thr Glu Thr

1 5 10 15

Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Thr Tyr Ser

20 25 30

Met Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Gly

35 40 45

Phe Ile Gly Ser Arg Gly Asp Thr Tyr Tyr Ala Ser Trp Ala Lys Gly

50 55 60

Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Ser Gln Val Val Leu Thr

65 70 75 80

Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala Arg

85 90 95

Glu Arg Asp Pro Tyr Gly Gly Gly Ala Tyr Pro Pro His Leu Trp Gly

100 105 110

Arg Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 123

<211> 111

<212> PRT

<213> Artificial sequence

<220>

<223> light chain variable domain < Pb-Dotam > PRIT-0213

<400> 123

Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp

1 5 10 15

Arg Val Thr Ile Thr Cys Gln Ser Ser His Ser Val Tyr Ser Asp Asn

20 25 30

Asp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Gln Ala Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

65 70 75 80

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gly Gly Tyr Asp Asp Glu

85 90 95

Ser Asp Thr Tyr Gly Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 124

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain variable domain < Pb-Dotam > PRIT-0214

<400> 124

Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu Thr

1 5 10 15

Leu Ser Leu Thr Cys Ala Val Tyr Gly Phe Ser Leu Ser Thr Tyr Ser

20 25 30

Met Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly

35 40 45

Phe Ile Gly Ser Arg Gly Asp Thr Tyr Tyr Ala Ser Trp Ala Lys Gly

50 55 60

Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Leu Lys

65 70 75 80

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg

85 90 95

Glu Arg Asp Pro Tyr Gly Gly Gly Ala Tyr Pro Pro His Leu Trp Gly

100 105 110

Arg Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 125

<211> 111

<212> PRT

<213> Artificial sequence

<220>

<223> light chain variable domain < Pb-Dotam > PRIT-0214

<400> 125

Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp

1 5 10 15

Arg Val Thr Ile Thr Cys Gln Ser Ser His Ser Val Tyr Ser Asp Asn

20 25 30

Asp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Gln Ala Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

65 70 75 80

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gly Gly Tyr Asp Asp Glu

85 90 95

Ser Asp Thr Tyr Gly Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 126

<211> 591

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain 1 bispecific, trivalent < CEA/Pb-Dotam > PRIT-0214

VH_84.66

<400> 126

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Ala Asn Gly Asn Ser Lys Tyr Val Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Pro Phe Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

450 455 460

Ser Gly Gly Gly Gly Ser Val Gln Leu Gln Gln Trp Gly Ala Gly Leu

465 470 475 480

Leu Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Phe

485 490 495

Ser Leu Ser Thr Tyr Ser Met Ser Trp Ile Arg Gln Pro Pro Gly Lys

500 505 510

Gly Leu Glu Trp Ile Gly Phe Ile Gly Ser Arg Gly Asp Thr Tyr Tyr

515 520 525

Ala Ser Trp Ala Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys

530 535 540

Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala

545 550 555 560

Val Tyr Tyr Cys Ala Arg Glu Arg Asp Pro Tyr Gly Gly Gly Ala Tyr

565 570 575

Pro Pro His Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser

580 585 590

<210> 127

<211> 581

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain 2 bispecific, trivalent < CEA/Pb-Dotam > PRIT-0214

VL_84.66

<400> 127

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Ala Asn Gly Asn Ser Lys Tyr Val Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Pro Phe Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

450 455 460

Ser Gly Gly Gly Gly Ser Ile Gln Met Thr Gln Ser Pro Ser Ser Leu

465 470 475 480

Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ser Ser His

485 490 495

Ser Val Tyr Ser Asp Asn Asp Leu Ala Trp Tyr Gln Gln Lys Pro Gly

500 505 510

Lys Ala Pro Lys Leu Leu Ile Tyr Gln Ala Ser Lys Leu Ala Ser Gly

515 520 525

Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

530 535 540

Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu

545 550 555 560

Gly Gly Tyr Asp Asp Glu Ser Asp Thr Tyr Gly Phe Gly Gly Gly Thr

565 570 575

Lys Val Glu Ile Lys

580

<210> 128

<211> 218

<212> PRT

<213> Artificial sequence

<220>

<223> light chain < CEA > 84.66

<400> 128

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Gly Glu Ser Val Asp Ile Phe

20 25 30

Gly Val Gly Phe Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

Arg Leu Leu Ile Tyr Arg Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Thr Asn

85 90 95

Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 129

<211> 591

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain 1 bispecific, trivalent < CEA/Pb-Dotam > PRIT-0213

VH _ 84.66- - > segment

<400> 129

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Ala Asn Gly Asn Ser Lys Tyr Val Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Pro Phe Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

450 455 460

Ser Gly Gly Gly Gly Ser Val Thr Leu Lys Glu Ser Gly Pro Val Leu

465 470 475 480

Val Lys Pro Thr Glu Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe

485 490 495

Ser Leu Ser Thr Tyr Ser Met Ser Trp Ile Arg Gln Pro Pro Gly Lys

500 505 510

Ala Leu Glu Trp Leu Gly Phe Ile Gly Ser Arg Gly Asp Thr Tyr Tyr

515 520 525

Ala Ser Trp Ala Lys Gly Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys

530 535 540

Ser Gln Val Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala

545 550 555 560

Thr Tyr Tyr Cys Ala Arg Glu Arg Asp Pro Tyr Gly Gly Gly Ala Tyr

565 570 575

Pro Pro His Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser

580 585 590

<210> 130

<211> 581

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain 2 bispecific, trivalent < CEA/Pb-Dotam > PRIT-0213

VL _ 84.66- - > acupoint

<400> 130

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Ala Asn Gly Asn Ser Lys Tyr Val Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Pro Phe Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

450 455 460

Ser Gly Gly Gly Gly Ser Ile Gln Met Thr Gln Ser Pro Ser Ser Leu

465 470 475 480

Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ser Ser His

485 490 495

Ser Val Tyr Ser Asp Asn Asp Leu Ala Trp Tyr Gln Gln Lys Pro Gly

500 505 510

Lys Ala Pro Lys Leu Leu Ile Tyr Gln Ala Ser Lys Leu Ala Ser Gly

515 520 525

Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

530 535 540

Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu

545 550 555 560

Gly Gly Tyr Asp Asp Glu Ser Asp Thr Tyr Gly Phe Gly Gly Gly Thr

565 570 575

Lys Val Glu Ile Lys

580

<210> 131

<211> 581

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain 1 bispecific, < CEA/Pb-Dotam > rabbit Dotam _84.66

<400> 131

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Ala Asn Gly Asn Ser Lys Tyr Val Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Pro Phe Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

450 455 460

Ser Gly Gly Gly Gly Ser Ala Val Leu Thr Gln Thr Pro Ser Pro Val

465 470 475 480

Ser Pro Ala Val Gly Gly Thr Val Thr Ile Ser Cys Gln Ser Ser His

485 490 495

Ser Val Tyr Ser Asp Asn Asp Leu Ala Trp Tyr Gln Gln Lys Leu Gly

500 505 510

Gln Pro Pro Lys Leu Leu Ile Tyr Gln Ala Ser Lys Leu Ala Ser Gly

515 520 525

Val Ser Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu

530 535 540

Thr Ile Ser Gly Val Gln Ser Asp Asp Ala Ala Thr Tyr Tyr Cys Leu

545 550 555 560

Gly Gly Tyr Asp Asp Glu Ser Asp Thr Tyr Gly Phe Gly Gly Gly Thr

565 570 575

Glu Val Val Val Lys

580

<210> 132

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> HC5

<400> 132

Val Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys Pro Thr Glu Thr

1 5 10 15

Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Thr Tyr Ser

20 25 30

Met Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Gly

35 40 45

Phe Ile Gly Ser Arg Gly Asp Thr Tyr Tyr Ala Ser Trp Ala Lys Gly

50 55 60

Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Ser Gln Val Val Leu Thr

65 70 75 80

Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala Arg

85 90 95

Glu Arg Asp Pro Tyr Gly Gly Gly Ala Tyr Pro Pro His Leu Trp Gly

100 105 110

Arg Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 133

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> LC1

<400> 133

Ser Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ser Ser His Ser Val Tyr Ser Asp

20 25 30

Asn Asp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Gln Ala Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe

50 55 60

Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu

65 70 75 80

Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gly Gly Tyr Asp Asp

85 90 95

Glu Ser Asp Thr Tyr Gly Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 134

<211> 111

<212> PRT

<213> Artificial sequence

<220>

<223> LC3

<400> 134

Ala Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly Asp

1 5 10 15

Arg Val Thr Ile Thr Cys Gln Ser Ser His Ser Val Tyr Ser Asp Asn

20 25 30

Asp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu

35 40 45

Ile Tyr Gln Ala Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln

65 70 75 80

Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Leu Gly Gly Tyr Asp Asp Glu

85 90 95

Ser Asp Thr Tyr Gly Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 135

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> HC7

<400> 135

Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser

1 5 10 15

Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Thr Tyr Ser

20 25 30

Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly

35 40 45

Phe Ile Gly Ser Arg Gly Asp Thr Tyr Tyr Ala Ser Trp Ala Lys Gly

50 55 60

Arg Phe Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln

65 70 75 80

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

85 90 95

Glu Arg Asp Pro Tyr Gly Gly Gly Ala Tyr Pro Pro His Leu Trp Gly

100 105 110

Arg Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 136

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> HC10

<400> 136

Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu Thr

1 5 10 15

Leu Ser Leu Thr Cys Ala Val Tyr Gly Phe Ser Leu Ser Thr Tyr Ser

20 25 30

Met Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly

35 40 45

Phe Ile Gly Ser Arg Gly Asp Thr Tyr Tyr Ala Ser Trp Ala Lys Gly

50 55 60

Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Leu Lys

65 70 75 80

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg

85 90 95

Glu Arg Asp Pro Tyr Gly Gly Gly Ala Tyr Pro Pro His Leu Trp Gly

100 105 110

Arg Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 137

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> T84.66 VH 1

<400> 137

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Ala Asn Gly Asn Ser Lys Tyr Val Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Pro Phe Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 138

<211> 111

<212> PRT

<213> Artificial sequence

<220>

<223> T84.66 VL

<400> 138

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Gly Glu Ser Val Asp Ile Phe

20 25 30

Gly Val Gly Phe Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

Arg Leu Leu Ile Tyr Arg Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Thr Asn

85 90 95

Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 139

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> CH1A1A VH

<400> 139

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe

50 55 60

Lys Gly Arg Val Thr Phe Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 140

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> CH1A1A VL

<400> 140

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Ala Ala Val Gly Thr Tyr

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Lys Arg Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys His Gln Tyr Tyr Thr Tyr Pro Leu

85 90 95

Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 141

<211> 37

<212> DNA

<213> Artificial sequence

<220>

Primer sequence <223> rbHC

<400> 141

aagcttgcca ccatggagac tgggctgcgc tggcttc 37

<210> 142

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> https:// protect-eu. mimecast. com/s/jhrhC2RNxFYMPVS2Ygk4domain = rbhcf. do primer sequence

<400> 142

ccattggtga gggtgcccga g 21

<210> 143

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<223> rbLC.up primer sequence

<400> 143

aagcttgcca ccatggacay gagggccccc actc 34

<210> 144

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> https:// protect-eu. mimecast. com/s/2MppC3lXQckn6XCQnhR8domain = rblc. do primer sequences

<400> 144

cagagtrctg ctgaggttgt aggtac 26

<210> 145

<211> 224

<212> PRT

<213> Artificial sequence

<220>

<223> P1AA1227_HC

<400> 145

Val Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys Pro Thr Glu Thr

1 5 10 15

Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Thr Tyr Ser

20 25 30

Met Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Gly

35 40 45

Phe Ile Gly Ser Arg Gly Asp Thr Tyr Tyr Ala Ser Trp Ala Lys Gly

50 55 60

Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Ser Gln Val Val Leu Thr

65 70 75 80

Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala Arg

85 90 95

Glu Arg Asp Pro Tyr Gly Gly Gly Ala Tyr Pro Pro His Leu Trp Gly

100 105 110

Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

<210> 146

<211> 219

<212> PRT

<213> Artificial sequence

<220>

<223> P1AA1227_LC

<400> 146

Ser Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ser Ser His Ser Val Tyr Ser Asp

20 25 30

Asn Asp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Gln Ala Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe

50 55 60

Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu

65 70 75 80

Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gly Gly Tyr Asp Asp

85 90 95

Glu Ser Asp Thr Tyr Gly Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 147

<211> 594

<212> PRT

<213> Artificial sequence

<220>

<223> D1AE3669 (HC node < CEA > CH1A1A Dotam-VH-AST)

<400> 147

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe

50 55 60

Lys Gly Arg Val Thr Phe Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

450 455 460

Ser Gly Gly Gly Gly Ser Val Thr Leu Lys Glu Ser Gly Pro Val Leu

465 470 475 480

Val Lys Pro Thr Glu Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe

485 490 495

Ser Leu Ser Thr Tyr Ser Met Ser Trp Ile Arg Gln Pro Pro Gly Lys

500 505 510

Ala Leu Glu Trp Leu Gly Phe Ile Gly Ser Arg Gly Asp Thr Tyr Tyr

515 520 525

Ala Ser Trp Ala Lys Gly Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys

530 535 540

Ser Gln Val Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala

545 550 555 560

Thr Tyr Tyr Cys Ala Arg Glu Arg Asp Pro Tyr Gly Gly Gly Ala Tyr

565 570 575

Pro Pro His Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala

580 585 590

Ser Thr

<210> 148

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CDR H1

<400> 148

Gly Phe Ser Leu Thr Asp Tyr Gly Val His

1 5 10

<210> 149

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> human IgG1 CH1 domain

<400> 149

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

1 5 10

<210> 150

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain variable domain band c-terminal extension

<400> 150

Val Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys Pro Thr Glu Thr

1 5 10 15

Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Thr Tyr Ser

20 25 30

Met Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Gly

35 40 45

Phe Ile Gly Ser Arg Gly Asp Thr Tyr Tyr Ala Ser Trp Ala Lys Gly

50 55 60

Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Ser Gln Val Val Leu Thr

65 70 75 80

Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala Arg

85 90 95

Glu Arg Asp Pro Tyr Gly Gly Gly Ala Tyr Pro Pro His Leu Trp Gly

100 105 110

Arg Gly Thr Leu Val Thr Val Ser Ser Ala

115 120

<210> 151

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CDR H1

<400> 151

Gly Phe Thr Phe Thr Asp Tyr Tyr Met Asn

1 5 10

<210> 152

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Flexible Joint

<400> 152

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Ser Gly Gly

20

<210> 153

<211> 592

<212> PRT

<213> Artificial sequence

<220>

<223> variants with c-terminal alanine extension

<400> 153

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe

50 55 60

Lys Gly Arg Val Thr Phe Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

450 455 460

Ser Gly Gly Gly Gly Ser Val Thr Leu Lys Glu Ser Gly Pro Val Leu

465 470 475 480

Val Lys Pro Thr Glu Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe

485 490 495

Ser Leu Ser Thr Tyr Ser Met Ser Trp Ile Arg Gln Pro Pro Gly Lys

500 505 510

Ala Leu Glu Trp Leu Gly Phe Ile Gly Ser Arg Gly Asp Thr Tyr Tyr

515 520 525

Ala Ser Trp Ala Lys Gly Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys

530 535 540

Ser Gln Val Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala

545 550 555 560

Thr Tyr Tyr Cys Ala Arg Glu Arg Asp Pro Tyr Gly Gly Gly Ala Tyr

565 570 575

Pro Pro His Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala

580 585 590

<210> 154

<211> 88

<212> PRT

<213> Artificial sequence

<220>

<223> A5B7 epitope

<400> 154

Pro Lys Pro Phe Ile Thr Ser Asn Asn Ser Asn Pro Val Glu Asp Glu

1 5 10 15

Asp Ala Val Ala Leu Thr Cys Glu Pro Glu Ile Gln Asn Thr Thr Tyr

20 25 30

Leu Trp Trp Val Asn Asn Gln Ser Leu Pro Val Ser Pro Arg Leu Gln

35 40 45

Leu Ser Asn Asp Asn Arg Thr Leu Thr Leu Leu Ser Val Thr Arg Asn

50 55 60

Asp Val Gly Pro Tyr Glu Cys Gly Ile Gln Asn Lys Leu Ser Val Asp

65 70 75 80

His Ser Asp Pro Val Ile Leu Asn

85

<210> 155

<211> 88

<212> PRT

<213> Artificial sequence

<220>

<223> MFE23 epitope

<400> 155

Pro Lys Pro Ser Ile Ser Ser Asn Asn Ser Lys Pro Val Glu Asp Lys

1 5 10 15

Asp Ala Val Ala Phe Thr Cys Glu Pro Glu Thr Gln Asp Ala Thr Tyr

20 25 30

Leu Trp Trp Val Asn Asn Gln Ser Leu Pro Val Ser Pro Arg Leu Gln

35 40 45

Leu Ser Asn Gly Asn Arg Thr Leu Thr Leu Phe Asn Val Thr Arg Asn

50 55 60

Asp Thr Ala Ser Tyr Lys Cys Glu Thr Gln Asn Pro Val Ser Ala Arg

65 70 75 80

Arg Ser Asp Ser Val Ile Leu Asn

85

<210> 156

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain CDR1 < CEA > MFE23

<400> 156

Asp Ser Tyr Met His

1 5

<210> 157

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain CDR2 < CEA > MFE23

<400> 157

Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe Gln

1 5 10 15

Gly

<210> 158

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain CDR2 < CEA > MFE23-H26

<400> 158

Trp Ile Asp Pro Glu Asn Gly Gly Thr Asn Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210> 159

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain CDR3 < CEA > MFE23

<400> 159

Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr

1 5 10

<210> 160

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> light chain CDR1 < CEA > MFE23

<400> 160

Ser Ala Ser Ser Ser Val Ser Tyr Met His

1 5 10

<210> 161

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> light chain CDR1 < CEA > MFE23-L24, L25

<400> 161

Arg Ala Ser Ser Ser Val Ser Tyr Met His

1 5 10

<210> 162

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> light chain CDR1 < CEA > MFE23-L26

<400> 162

Arg Ala Ser Gln Ser Ile Ser Ser Tyr Met Arg Ala Ser Gln Ser Ile

1 5 10 15

Ser Ser Tyr Met

20

<210> 163

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> light chain CDR2 < CEA > MFE23

<400> 163

Ser Thr Ser Asn Leu Ala Ser

1 5

<210> 164

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> light chain CDR2 < CEA > MFE23-L26

<400> 164

Tyr Thr Ser Asn Leu Ala Ser

1 5

<210> 165

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> light chain CDR2 < CEA > MFE23-L29

<400> 165

Ser Thr Ser Ser Leu Gln Ser

1 5

<210> 166

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> light chain CDR3 < CEA > MFE23

<400> 166

Gln Gln Arg Ser Ser Tyr Pro Leu Thr

1 5

<210> 167

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain variable domain < CEA > MFE23

<400> 167

Gln Val Lys Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Ser Gly Thr

1 5 10 15

Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Ser

20 25 30

Tyr Met His Trp Leu Arg Gln Gly Pro Glu Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe

50 55 60

Gln Gly Lys Ala Thr Phe Thr Thr Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Glu Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 168

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> light chain variable domain < CEA > MFE23

<400> 168

Glu Asn Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr

85 90 95

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 169

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> MFE-H24

<400> 169

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Ser

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Glu Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 170

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> MFE-H25

<400> 170

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Lys Asp Ser

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Glu Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 171

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> MFE-H26

<400> 171

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Ser

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asp Pro Glu Asn Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Glu Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 172

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> MFE-H27

<400> 172

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Ser

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 173

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> MFE-H28

<400> 173

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Ser

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Glu Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 174

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> MFE-H29

<400> 174

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Ser

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Thr Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Glu Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 175

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> MFE-L24

<400> 175

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 176

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> MFE-L25

<400> 176

Glu Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 177

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> MFE-L26

<400> 177

Glu Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 178

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> MFE-L27

<400> 178

Glu Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Pro Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 179

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> MFE-L28

<400> 179

Glu Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Pro Tyr Met

20 25 30

His Trp Leu Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 180

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> MFE-L29

<400> 180

Glu Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Pro Tyr Met

20 25 30

His Trp Leu Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ser Thr Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

Claims (103)

1. A set 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 of the antigen-binding site of a radiolabeled compound, but does not comprise a VL domain of the antigen-binding site of the radiolabeled compound; and

ii) a second antibody that binds to the antigen expressed on the surface of the target cell and further comprises a VL domain of the antigen-binding site of the radiolabeled compound but does not comprise a VH domain of the antigen-binding site of the radiolabeled compound,

wherein the VH domain of the first antibody and the VL domain of the second antibody are capable of together forming a functional antigen-binding site for the radiolabeled compound.

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

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

4. The set of antibodies according to claim 3, wherein the first antibody comprises or consists of:

a) fab fragments binding to the antigen, and

b) a polypeptide comprising or consisting of:

i) the antibody heavy chain variable domain (VH) of the antigen binding site of the radiolabeled compound, or

ii) an antibody heavy chain variable domain (VH) and an antibody heavy chain constant domain of the antigen binding site of the radiolabelled compound, wherein the C-terminus of the VH domain is fused to the N-terminus of the constant domain;

wherein the polypeptide is fused via the N-terminus of the VH domain to the C-terminus of the CL or CH1 domain of the Fab fragment;

and wherein the second antibody comprises or consists of:

c) fab fragments binding to the antigen, and

d) a polypeptide comprising or consisting of:

iii) the antibody light chain variable domain (VL) of the antigen binding site of the radiolabeled compound, or

iv) an antibody light chain variable domain (VL) and an antibody light chain constant domain of the antigen binding site of the radiolabeled compound, wherein the C-terminus of the VL domain is fused to the N-terminus of the constant domain;

wherein the polypeptide is fused via the N-terminus of the VL domain to the C-terminus of the CL or CH1 domain of the Fab fragment;

And wherein the antibody heavy chain variable domain (VH) of the polypeptide of (b) and the antibody light chain variable domain (VL) of the polypeptide of (d) are capable together of forming a functional antigen-binding site for the radiolabeled compound.

5. The antibody panel of claim 4, wherein the polypeptide of (b) is fused via a peptide linker through the N-terminus of the VH domain to the C-terminus of the CL or CH1 domain of the Fab fragment of (a); and wherein the polypeptide of (d) is fused via a peptide linker through the N-terminus of the VL domain to the C-terminus of the CL or CH1 domain of the Fab fragment of (C).

6. The antibody panel of claim 3, wherein the first antibody comprises

a) A tandem Fab comprising two Fab fragments, wherein a first Fab fragment and a second Fab fragment each bind to the same epitope of the antigen, and wherein the first Fab fragment and the second Fab fragment are connected via a peptide tether, wherein the first Fab is connected via its C-terminus to the N-terminus of the second Fab; and

b) a polypeptide comprising or consisting of:

i) antibody heavy chain variable domains (VH); or

ii) an antibody heavy chain variable domain (VH) and an antibody constant domain (CH1), wherein the C-terminus of the VH domain is fused to the N-terminus of the CH1 domain;

wherein the polypeptide is fused via the N-terminus of the VH domain to the C-terminus of the CL or CH1 domain of the second Fab fragment;

and the second antibody comprises

c) A tandem Fab comprising two Fab fragments, wherein a first Fab fragment and a second Fab fragment each bind to the same epitope of the antigen, and wherein the first Fab fragment and the second Fab fragment are connected via a peptide tether, wherein the first Fab is connected via its C-terminus to the N-terminus of the second Fab; and

d) A polypeptide comprising or consisting of:

i) an antibody light chain variable domain (VL); or

ii) an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL), wherein the C-terminus of the VL domain is fused to the N-terminus of the constant domain;

wherein the polypeptide is fused via the N-terminus of the VL domain to the C-terminus of the CL or CH1 domain of the second Fab fragment;

and wherein the antibody heavy chain variable domain (VH) of the polypeptide of (b) and the antibody light chain variable domain (VL) of the polypeptide of (d) are capable of together forming a functional antigen binding site for the radiolabeled compound.

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

8. The antibody panel of claim 3, wherein the first antibody comprises

a) A tandem Fab comprising a first fragment and a second fragment, wherein the first fragment is linked through its C-terminus to the N-terminus of the second fragment via a peptide tether, wherein the first fragment binds a first epitope of the antigen and the second fragment binds a second epitope of the antigen, and wherein one of the fragments selected from the first fragment and the second fragment is a Fab and the other is a cross Fab,

b) A polypeptide comprising or consisting of:

i) antibody heavy chain variable domains (VH); or

ii) an antibody heavy chain variable domain (VH) and an antibody heavy chain constant domain (CH1), wherein the C-terminus of the VH domain is fused to the N-terminus of the CH1 domain;

wherein the polypeptide is fused via the N-terminus of the VH domain to the C-terminus of one of the chains of the second fragment; and is provided with

Wherein the second antibody comprises

c) A tandem Fab comprising a first fragment and a second fragment, wherein the first fragment is linked by its C-terminus to the N-terminus of the second fragment, wherein the first fragment binds a first epitope of the antigen and the second fragment binds a second epitope of the antigen, and wherein one of the fragments selected from the first fragment and the second fragment is a Fab and the other is a cross Fab; and

d) a polypeptide comprising or consisting of:

i) an antibody light chain variable domain (VL); or

ii) an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL), wherein the C-terminus of the VL domain is fused to the N-terminus of the light chain constant domain

Wherein the polypeptide is fused via the N-terminus of the VL domain to the C-terminus of one of the strands of the second fragment;

and wherein the antibody heavy chain variable domain (VH) of the polypeptide of (b) and the antibody light chain variable domain (VL) of the polypeptide of (d) are capable of together forming a functional antigen binding site for the radiolabeled compound.

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

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

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

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

13. The antibody panel according to any one of claims 10 to 12, wherein the first antibody comprises or consists of:

i) a complete light chain fragment;

ii) an intact heavy chain;

iii) another Fc chain lacking Fd; and

iv) a VH domain comprising or consisting of the antigen binding site of the radiolabeled compound;

Wherein the light chain of (i) and the heavy chain of (ii) together provide an antigen binding site for the antigen; and wherein the VH domain comprising or consisting of the antigen binding site of the radiolabeled compound is fused via its N-terminus to the C-terminus of (ii) or (iii); and is

Wherein the second antibody comprises or consists of:

v) a complete light chain fragment;

vi) an intact heavy chain;

vii) another Fc chain lacking Fd; and

viii) a VL domain comprising or consisting of the antigen binding site of the radiolabeled compound;

wherein the light chain of (v) and the heavy chain of (vi) together provide an antigen binding site for the antigen; and wherein the VL domain comprising or consisting of the antigen binding site of the radiolabeled compound is fused via its N-terminus to the C-terminus of (vi) or (vii);

wherein the antibody heavy chain variable domain (VH) of the polypeptide of (iv) and the antibody light chain variable domain (VL) of the polypeptide of (viii) are capable of together forming a functional antigen binding site for the radiolabeled compound.

14. The antibody panel according to claim 13, wherein the polypeptide of (iv) is fused via a linker through its N-terminus to the C-terminus of (ii) or (iii); and the polypeptide of (viii) is fused via a linker through its N-terminus to the C-terminus of (vi) or (vii).

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

16. The antibody panel of claim 15, wherein the first antibodies comprise:

i) a complete light chain;

ii) an intact heavy chain;

iii) another Fc chain; and

iv) a VH domain comprising or consisting of the antigen binding site of the radiolabeled compound;

wherein the light chain of (i) and the heavy chain of (ii) together provide an antigen binding site for the target antigen; and wherein the VH domain comprising or consisting of the antigen binding site of the radiolabeled compound is fused via a linker through its C-terminus to the N-terminus of (iii); and is

The second antibody comprises:

v) a complete light chain;

vi) an intact heavy chain;

vii) another Fc chain; and

viii) a VL domain comprising or consisting of the antigen binding site of the radiolabeled compound;

Wherein the light chain of (v) and the heavy chain of (vi) together provide an antigen binding site for the target antigen; and wherein the VL domain comprising or consisting of the antigen binding site of the radiolabeled compound is fused via a linker through its C-terminus to the N-terminus of (vii).

17. The antibody panel according to claim 16, wherein:

i) the first antibody comprises a first heavy chain having SEQ ID NO 112, a second heavy chain having SEQ ID NO 114, and a light chain having SEQ ID NO 115; and

ii) the second antibody comprises a first heavy chain having SEQ ID NO 112, a second heavy chain having SEQ ID NO 113 and a light chain having SEQ ID NO 115.

18. The set of antibodies according to any one of claims 10 to 12, wherein the first antibody comprises

a) A first full-length antibody and consisting of two antibody heavy chains and two antibody light chains, wherein at least one arm of the full-length antibody binds the antigen; and

b) a polypeptide consisting of:

i) antibody heavy chain variable domains (VH); or

ii) an antibody heavy chain variable domain (VH) and an antibody constant domain (CH1),

wherein the polypeptide is fused via the N-terminus of the VH domain to the C-terminus of one of the two heavy chains of the first full-length antibody, an

Wherein the second antibody comprises:

c) A second full-length antibody and consisting of two antibody heavy chains and two antibody light chains, wherein at least one arm of the antibody binds to the antigen; and

d) a polypeptide consisting of:

i) an antibody light chain variable domain (VL); or

ii) an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL),

wherein the polypeptide is fused via the N-terminus of the VL domain to the C-terminus of one of the two heavy chains of the second full-length antibody,

wherein the polypeptide of (b) and the polypeptide of (d) together are capable of forming a functional antigen binding site for the radiolabeled compound.

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

20. The antibody panel of claim 18 or 19, wherein the first antibody and the second antibody are each bivalent to the antigen.

21. The antibody panel of claim 20, wherein both arms of the full length antibody bind to the same epitope of the antigen.

22. The set of antibodies according to claim 18 or 19, wherein for the antigen each of the first and second antibodies is a biparatopic (biparatopic).

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

24. The antibody panel according to any one of the preceding claims, wherein the radiolabeled compound comprises radiolabeled DOTA or a salt or functional variant thereof.

25. The antibody panel of any one of the preceding claims, wherein the radiolabeled compound is DOTA radiolabeled with Lu or Y radioisotopes, or a salt or functional variant thereof.

26. The set of antibodies of claim 24 or 25, wherein the VH domain of the antigen binding site of the radiolabeled compound comprises (a) a CDR-H1 comprising the amino acid sequence of 35; (b) CDR-H2 comprising the amino acid sequence of 36; (c) CDR-H3 comprising the amino acid sequence of 37.

27. The antibody panel of any one of claims 24 to 26, wherein the VH domain of the antigen binding site of the radiolabeled compound comprises the amino acid sequence of SEQ ID No. 41 or a variant thereof comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID No. 41.

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

29. The antibody panel of claim 28, wherein residue AST is added to the C-terminus of the VH domain of the antigen binding site of the radiolabeled compound.

30. The set of antibodies according to any one of claims 24 to 29, wherein the VL domain of the antigen binding site of the radiolabeled compound comprises (d) CDR-L1 comprising the amino acid sequence of 38; (e) CDR-L2 comprising the amino acid sequence of 39; and (f) CDR-L3 comprising the amino acid sequence of 40.

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

32. The antibody panel of any one of claims 1 to 23, wherein the radiolabeled compound comprises Pb-DOTAM.

33. The antibody panel according to claim 32, wherein the functional binding site of Pb-DOTAM binds with a binding affinity Kd value of 100pM, 50pM, 20pM, 10pM, 5pM, 1pM or less, such as 0.9pM or less, 0.8pM or less, 0.7pM or less, 0.6pM or less or 0.5pM or less.

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

35. The antibody panel according to any one of claims 32 to 34, wherein the VH domain of the antigen binding site of the radiolabeled compound comprises:

a) a heavy chain CDR2 comprising amino acid sequence FIGSRGDTYYASWAKG (SEQ ID NO:2) or a variant thereof having up to 1, 2 or 3 substitutions in SEQ ID NO:2, wherein the substitutions do not comprise Phe50, Asp56 and/or Tyr58, and optionally also Gly52 and/or Arg 54;

b) a heavy chain CDR3 comprising amino acid sequence ERDPYGGGAYPPHL (SEQ ID NO:3) or a variant thereof having up to 1, 2 or 3 substitutions in SEQ ID NO:3, wherein the substitutions do not comprise Glu95, Arg96, Asp97, Pro98, and optionally also do not comprise Ala100C, Tyr100D and/or Pro100E, and/or optionally also do not comprise Tyr 99;

and a heavy chain CDR1, which is optionally:

c) Heavy chain CDR1 comprising amino acid sequence GFSLSTYSMS (SEQ ID NO:1) or a variant thereof having up to 1, 2 or 3 substitutions in SEQ ID NO: 1.

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

37. The set of antibodies according to any one of claims 32 to 36, wherein the VH domain of the antigen binding site of the radiolabeled compound comprises an amino acid sequence selected from the group consisting of SEQ ID No. 7 and 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 to SEQ ID No. 7 or SEQ ID No. 9.

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

39. The antibody panel of claim 38, wherein residue AST is added to the C-terminus of the VH domain of the antigen binding site of the radiolabeled compound.

40. The antibody panel of any one of claims 32 to 39, wherein the VL domain of the antigen binding site of the radiolabeled compound comprises

d) A light chain CDR1 comprising amino acid sequence QSSHSVYSDNDLA (SEQ ID NO:4) or a variant thereof having up to 1, 2 or 3 substitutions in SEQ ID NO:4, wherein the substitutions do not include Tyr28 and Asp 32;

e) a light chain CDR3 comprising amino acid sequence LGGYDDESDTYG (SEQ ID NO:6) or a variant thereof having up to 1, 2 or 3 substitutions in SEQ ID NO:6, wherein the substitutions do not include Gly91, Tyr92, Asp93, Thr95c and Tyr96,

and a light chain CDR2, which is optionally

f) Light chain CDR2 comprising the amino acid sequence QASKLA (SEQ ID NO:5) or a variant thereof having at least 1, 2 or 3 substitutions in SEQ ID NO:5, said substitutions optionally not comprising Gln 50.

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

42. The set of antibodies according to any one of claims 32 to 41, wherein the VL domain of the antigen binding site of the radiolabeled compound comprises the amino acid sequence of SEQ ID NO 8 or a variant thereof comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO 8.

43. The antibody panel of any one of the preceding claims, wherein the first antibody and the second antibody bind to the same epitope of the antigen expressed on the surface of a target cell.

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

45. The antibody panel of any one of the preceding claims, wherein the antigen expressed on the surface of a target cell is a tumor associated antigen.

46. The antibody panel of any one of the preceding claims, wherein the antigen expressed on the surface of a 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.

47. The antibody panel of any one of the preceding claims, wherein the antigen expressed on the surface of a target cell is selected from the group consisting of: CEA, GPRC5D, and FAP.

48. The set of antibodies of claim 47, wherein:

i) the first antibody comprises the first heavy chain of SEQ ID NO 104; 106, wherein the C-terminal alanine of SEQ ID NO 106 is optional and may be absent or replaced by an alternative C-terminal extension; and the light chain of SEQ ID NO. 107; and

ii) the second antibody comprises the first heavy chain of SEQ ID NO. 104, the second heavy chain of SEQ ID NO. 105 and the light chain of SEQ ID NO. 107.

49. The antibody panel of claim 47, wherein

i) The first antibody comprises a first heavy chain of SEQ ID NO 108; 110, wherein the C-terminal alanine of SEQ ID NO:110 is optional and may be absent or replaced by an alternative C-terminal extension; and the light chain of SEQ ID NO 111; and

ii) the second antibody comprises a first heavy chain of SEQ ID NO 108, a second heavy chain of SEQ ID NO 109 and a light chain of SEQ ID NO 111.

50. The antibody panel according to any one of claims 1 to 47, wherein the antigen expressed on the surface of target cells is CEA.

51. The antibody panel of claim 50, wherein the first antibody comprises an antigen binding site that binds to CEA, the antigen binding site comprising

A heavy chain variable region comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 19; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO 20; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO 21; and

A light chain variable region comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO. 23; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO. 24.

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

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

54. The antibody panel of claim 50, wherein the first antibody comprises an antigen binding site that binds to CEA comprising: a heavy chain variable region comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 43; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO. 44; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO 45; and a light chain variable region comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 46; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO. 47; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO 48.

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

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

57. The antibody panel of claim 50, wherein the first antibody comprises an antigen binding site that binds to CEA comprising

A heavy chain variable region comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 11; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO 12; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO 13; and

a light chain variable region comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 14; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO. 15; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO 16.

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

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

60. The antibody panel of claim 50, wherein the first antibody comprises an antigen binding site that binds to CEA comprising

A heavy chain variable region comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 59; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO 60; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 61; and

a light chain variable region comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 62; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 63; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO 64.

61. The antibody panel of claim 50 or claim 60, wherein the first antibody comprises the antigen binding site of CEA comprising a VH sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO. 65 or a variant thereof comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO. 65.

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

63. The antibody panel of claim 50, wherein the first antibody comprises an antigen binding site that binds to CEA comprising: a heavy chain variable region comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 156; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO 157 or 158; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO. 159; and a light chain variable region comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:160, 161 or 162; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO 163, 164 or 165; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 166.

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

65. The antibody panel of claim 50, 63, or 64, wherein the first antibody comprises an antigen binding site that binds to CEA comprising:

(a) a VH domain comprising the amino acid sequence of SEQ ID NO 169 and a VL domain comprising the amino acid sequence of SEQ ID NO 179, or

(b) A VH domain comprising the amino acid sequence of SEQ ID NO 173 and a VL domain comprising the amino acid sequence of SEQ ID NO 179, or

(c) A VH domain comprising the amino acid sequence of SEQ ID NO:170 and a VL domain comprising the amino acid sequence of SEQ ID NO:179, or

(d) A VH domain comprising the amino acid sequence of SEQ ID NO. 174 and a VL domain comprising the amino acid sequence of SEQ ID NO. 178, or

(e) A VH domain comprising the amino acid sequence of SEQ ID NO 173 and a VL domain comprising the amino acid sequence of SEQ ID NO 178, or

(f) A VH domain comprising the amino acid sequence of SEQ ID NO. 171 and a VL domain comprising the amino acid sequence of SEQ ID NO. 178, or

(g) A VH domain comprising the amino acid sequence of SEQ ID NO 169 and a VL domain comprising the amino acid sequence of SEQ ID NO 178.

66. The antibody panel according to any one of claims 50 to 65, wherein the second antibody comprises an antigen binding site that binds to CEA comprising

A heavy chain variable region comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 19; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO 20; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO 21; and

a light chain variable region comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO. 23; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO. 24.

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

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

69. The antibody panel according to any one of claims 50 to 65, wherein the second antibody comprises an antigen binding site that binds to CEA comprising: a heavy chain variable region comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 43; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO. 44; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO 45; and a light chain variable region comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 46; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO. 47; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO 48.

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

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

72. The antibody panel according to any one of claims 50 to 65, wherein the second antibody comprises an antigen binding site that binds to CEA comprising

A heavy chain variable region comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 11; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO 12; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO 13; and

a light chain variable region comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 14; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO. 15; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO 16.

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

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

75. The antibody panel according to any one of claims 50 to 65, wherein the second antibody comprises an antigen binding site that binds to CEA comprising

A heavy chain variable region comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 59; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO 60; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 61; and

a light chain variable region comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 62; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO. 63; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 64.

76. The antibody panel according to any one of claims 50 to 65 or 75, wherein the second antibody comprises the antigen binding site of CEA comprising a VH sequence comprising an amino acid sequence selected from the group consisting of SEQ ID No. 65 or a variant thereof comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID No. 65.

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

78. The antibody panel according to claims 50 to 65, wherein the second antibody comprises an antigen binding site that binds to CEA comprising: a heavy chain variable region comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 156; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO 157 or 158; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO. 159; and a light chain variable region comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:160, 161 or 162; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO 163, 164 or 165; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 166.

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

80. The antibody panel according to claim 50 to 65, 78 or 79, wherein the second antibody comprises an antigen binding site that binds to CEA comprising:

(a) a VH domain comprising the amino acid sequence of SEQ ID NO. 169 and a VL domain comprising the amino acid sequence of SEQ ID NO. 179, or

(b) A VH domain comprising the amino acid sequence of SEQ ID NO 173 and a VL domain comprising the amino acid sequence of SEQ ID NO 179, or

(c) A VH domain comprising the amino acid sequence of SEQ ID NO:170 and a VL domain comprising the amino acid sequence of SEQ ID NO:179, or

(d) A VH domain comprising the amino acid sequence of SEQ ID NO. 174 and a VL domain comprising the amino acid sequence of SEQ ID NO. 178, or

(e) A VH domain comprising the amino acid sequence of SEQ ID NO 173 and a VL domain comprising the amino acid sequence of SEQ ID NO 178, or

(f) A VH domain comprising the amino acid sequence of SEQ ID NO. 171 and a VL domain comprising the amino acid sequence of SEQ ID NO. 178, or

(g) A VH domain comprising the amino acid sequence of SEQ ID NO 169 and a VL domain comprising the amino acid sequence of SEQ ID NO 178.

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

82. The set of antibodies according to claim 1, wherein the first antibody comprises a first heavy chain of SEQ ID NO 28, a second heavy chain of SEQ ID NO 32 and a light chain of SEQ ID NO 34.

83. The antibody panel of claim 82, wherein one or more alanine residues are added to the C-terminus of SEQ ID NO. 32, or wherein sequence AST is added to the C-terminus of SEQ ID NO. 32.

84. The set of antibodies of claim 1, wherein the first antibody comprises a first heavy chain of SEQ ID NO 51 and a second heavy chain of SEQ ID NO 52 and a light chain of SEQ ID NO 54.

85. The antibody panel of claim 84, wherein one or more alanine residues are added to the C-terminus of SEQ ID NO. 52, or wherein sequence AST is added to the C-terminus of SEQ ID NO. 32.

86. The set of antibodies according to claim 1, wherein the first antibody comprises a first heavy chain of SEQ ID NO 86; 87, wherein the C-terminal AST residue is optionally selected and may be absent or replaced by a different C-terminal extension; and the light chain of SEQ ID NO. 89.

87. The set of antibodies according to claim 1, wherein the first antibody comprises a first heavy chain of SEQ ID NO 93; 94, wherein the C-terminal AST residue is optionally selected and may be absent or replaced by a different C-terminal extension; and the light chain of SEQ ID NO. 96.

88. The set of antibodies according to any one of claims 1 or 82 to 87, wherein the second antibody comprises a first heavy chain of SEQ ID NO. 30, a second heavy chain of SEQ ID NO. 33 and a light chain of SEQ ID NO. 34.

89. The set of antibodies according to any one of claims 1 or 82 to 87, wherein the second antibody comprises a first heavy chain of SEQ ID NO. 55 and a second heavy chain of SEQ ID NO. 56 and a light chain of SEQ ID NO. 58.

90. The set of antibodies according to any one of claims 1 or 82 to 87, wherein the second antibody comprises a first heavy chain of SEQ ID NO 83 and a second heavy chain of SEQ ID NO 84 and a light chain of SEQ ID NO 89.

91. The set of antibodies according to any one of claims 1 or 82 to 87, wherein the second antibody comprises a first heavy chain of SEQ ID NO. 90 and a second heavy chain of SEQ ID NO. 91 and a light chain of SEQ ID NO. 96.

92. A set of nucleic acids expressing the set of antibodies according to any one of the preceding claims.

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

94. A host cell or a set of host cells comprising an expression vector or set of expression vectors according to claim 93.

95. A method of pre-targeted radioimmunotherapy, the method comprising:

i) Administering to an individual a panel of antibodies according to any one of claims 1 to 91, wherein the first and second antibodies are administered simultaneously or sequentially in either order, wherein the antibodies bind to a target antigen and are localized to the surface of cells expressing the target antigen; and wherein the combination of the first antibody and the second antibody forms a functional binding site for the radiolabeled compound;

and is provided with

ii) subsequently administering a radiolabeled compound, wherein the radiolabeled compound binds to a functional binding site of the radiolabeled compound.

96. The method according to claim 95, wherein the method does not comprise the step of administering a scavenger or a blocking agent.

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

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

99. The antibody panel of any one of claims 1 to 91 for use in a method of pre-targeted radioimmunotherapy according to any one of claims 95 to 98.

100. A method of targeting a radioisotope to a tissue or organ for radiological imaging, the method comprising:

i) Administering to an individual a panel of antibodies according to any one of claims 1 to 91, wherein the first and second antibodies are administered simultaneously or sequentially in either order, wherein the antibodies bind to a target antigen and are localized to the surface of cells expressing the target antigen; and wherein the combination of the first antibody and the second antibody forms a functional binding site for the radiolabeled compound;

and is

ii) subsequently administering a radiolabeled compound, wherein the radiolabeled compound binds to a functional binding site of the radiolabeled compound.

101. The method according to claim 100, wherein the method does not comprise the step of administering a scavenger or a blocking agent.

102. A method according to claim 100 or 101, wherein the method further comprises an imaging step.

103. The method of claim 102, wherein the target antigen is a cancer or tumor-associated antigen and the radiological imaging is used to image a tumor or cancer.