Combination therapy

Abstract

The present invention relates to combination therapy involving the use of pre-targeted radioimmunotherapy (PRIT).

Description

[Technical Field]

[0001] The present invention relates to combination therapies for use in the treatment of cancer. [Background technology]

[0002] Monoclonal antibodies have been developed to target drugs to cancer cells. Attaching toxic agents to antibodies that bind to tumor-associated antigens may provide more specific tumor killing with less damage to surrounding tissue.

[0003] Pretargeted radioimmunotherapy (PRIT) uses antibody constructs with affinity for tumor-associated antigens on the one hand and for radiolabeled compounds on the other. In the first step, the antibody is administered and localized to the tumor. Subsequently, the radiolabeled compound is administered. Because the radiolabeled compound is small, it can be rapidly delivered to the tumor and rapidly cleared, reducing radiation exposure outside the tumor (Goldenberg et al., Theranostics 2012, 2(5), 523-540). Similar procedures can also be used for imaging. Pretargeting can use bispecific antibodies or systems using avidin-biotin, but the latter has the disadvantage that avidin / streptavidin is immunogenic.

[0004] The present invention relates to combination therapies involving the use of PRIT. Summary of the Invention

[0005] The present invention relates to a combination therapy comprising the use of i) a multispecific antibody or a split multispecific antibody having a binding site for a radiolabeled compound and a binding site for a target antigen, ii) a CD40 agonist and iii) an immune checkpoint inhibitor.

[0006] In one aspect, the present invention relates to a pharmaceutical composition for the combined simultaneous or sequential treatment of a proliferative disease, preferably cancer, comprising: A) a composition comprising, as a first component, a multispecific antibody or a split multispecific antibody as an active ingredient, having a binding site for a radiolabeled compound and a binding site for a target antigen; B) a composition comprising, as a second component, a CD40 agonist as an active ingredient; and C) a composition comprising, as a third component, an immune checkpoint inhibitor, preferably a PD-L1 inhibitor as an active ingredient. In some embodiments, the pharmaceutical composition may further comprise a radiolabeled compound.

[0007] In another aspect, the present invention provides a kit comprising a pharmaceutical agent disclosed herein together with instructions for its use.

[0008] In another aspect, the invention relates to a multispecific antibody or a split multispecific antibody having a binding site for a radiolabeled compound and a binding site for a target antigen, for use in a method of treating a proliferative disease, such as cancer, wherein the treatment comprises administering the multispecific antibody or split multispecific antibody, and the treatment further comprises administering i) the radiolabeled compound, ii) a CD40 agonist and iii) an immune checkpoint inhibitor.

[0009] In another aspect, the invention relates to a CD40 agonist for use in a method of treating a proliferative disease, e.g., cancer, wherein the treatment further comprises administering i) a multispecific antibody or a split multispecific antibody having a binding site for a radiolabeled compound and a binding site for a target antigen, ii) the radiolabeled compound, and iii) an immune checkpoint inhibitor.

[0010] In another aspect, the present invention relates to an immune checkpoint inhibitor for use in a method of treating a proliferative disease, e.g., cancer, wherein the treatment further comprises administering i) a multispecific antibody or a split multispecific antibody having a binding site for a radiolabeled compound and a binding site for a target antigen, ii) the radiolabeled compound, and iii) a CD40 agonist.

[0011] In another aspect, the present invention relates to i) a multispecific antibody or a split multispecific antibody having a binding site for a radiolabeled compound and a binding site for a target antigen, ii) a radiolabeled compound, iii) a CD40 agonist and iv) an immune checkpoint inhibitor for use in combination in a method of treating a proliferative disease, e.g., cancer.

[0012] In another aspect, the present invention relates to a method of treating a proliferative disease, e.g., cancer, comprising administering to a patient i) a multispecific antibody or a split multispecific antibody having a binding site for a radiolabeled compound and a binding site for a target antigen, ii) the radiolabeled compound, iii) a CD40 agonist, and iv) an immune checkpoint inhibitor.

[0013] In another aspect, the present invention provides a method of treating a proliferative disease, e.g., cancer, in a subject, comprising: i) a radioimmunotherapy treatment comprising administering to a subject a multispecific antibody or a split multispecific antibody, the multispecific antibody or the split multispecific antibody having a binding site for a radiolabeled compound and a binding site for a target antigen, the radioimmunotherapy treatment further comprising administering to the subject the radiolabeled compound; and ii) immunotherapy treatment comprising administering to a subject a CD40 agonist and an immune checkpoint inhibitor.

[0014] The radiolabeled compound is administered to the patient after the multispecific antibody or split multispecific antibody binds to the target antigen. The radiolabeled compound then binds to the multispecific antibody or split multispecific antibody and is thus localized in the target cells.

[0015] The anti-CD40 antibody and the immune checkpoint inhibitor can be administered simultaneously or sequentially in any order. They can be administered before or after the administration of the multispecific antibody / split multispecific antibody and the radiolabeled compound. Preferably, they are administered after the multispecific antibody / split multispecific antibody and the radiolabeled compound.

[0016] In one embodiment, a cycle of treatment comprises a first step of pre-targeted radioimmunotherapy comprising administering a multispecific antibody or split multispecific antibody followed by administration of a radiolabeled compound, and a second step of immunotherapy comprising administering a CD40 agonist and an immune checkpoint inhibitor, wherein the anti-CD40 antibody and immune checkpoint inhibitor are administered simultaneously or sequentially in either order.

[0017] Treatment may involve one cycle, or may involve multiple cycles, for example, 2, 3, 4, 5 or 6 cycles.

[0018] In some embodiments, not all cycles of treatment are the same. the first cycle comprises a first step of pre-targeted radioimmunotherapy comprising administering a multispecific antibody or split multispecific antibody followed by administering a radiolabeled compound, and a second step of immunotherapy comprising administering a CD40 agonist and an immune checkpoint inhibitor, wherein the anti-CD40 antibody and the immune checkpoint inhibitor are administered simultaneously or sequentially in either order; and One or more subsequent cycles comprise a first step of pre-targeted radioimmunotherapy comprising administering a multispecific antibody or split multispecific antibody followed by administering a radiolabeled compound, and a second step of immunotherapy comprising administering an immune checkpoint inhibitor.

[0019] For example, as described above, there can be 1, 2, 3, 4 or 5 subsequent cycles.

[0020] The multispecific antibody or split multispecific antibody may be a bispecific antibody or a split bispecific antibody.

[0021] In one embodiment, a multispecific antibody (e.g., a bispecific antibody) can be an antibody that contains at least one binding site for a target antigen and at least one binding site for a radiolabeled compound, such as a Pb-DOTAM chelate. Exemplary antibodies are described in WO 2019 / 201959.

[0022] Split multispecific antibodies are composed of two different parts, referred to herein as hemibodies. Each hemibody contains an antigen-binding portion that can bind to a target antigen. The antigen-binding site for the radiolabeled compound is split across the two hemibodies, so that a functional antigen-binding site is formed only when the two hemibodies are associated. Thus, split antibodies are i) a first hemibody that binds to a target antigen (i.e., comprises an antigen-binding portion that is capable of binding to the target antigen) and further comprises a VH domain of an antigen-binding site for a radiolabeled compound, but does not comprise a VL domain of an antigen-binding site for a radiolabeled compound; ii) a second hemibody that binds to the target antigen (i.e., comprises an antigen-binding portion that is capable of binding to the target antigen) and further comprises a VL domain of an antigen-binding site for a radiolabeled compound, but does not comprise a VH domain of an antigen-binding site for a radiolabeled compound; The VH domain of the first hemibody and the VL domain of the second hemibody can together form a functional antigen binding site for a radiolabeled compound.

[0023] Neither the first nor the second hemibody itself contains a functional antigen-binding site for the radiolabeled compound. The first hemibody contains only the VH domain, but not the VL domain, from which the functional binding site for the radiolabeled compound is derived. The second hemibody contains only the VL domain, but not the VH domain.

[0024] The VH and VL domains of the first and second hemibodies associate to form a functional antigen-binding site for the radiolabeled compound, which can occur, for example, when the first and second antibodies are bound to the same individual target cell or adjacent cells.

[0025] The terms "hemibody," "demibody," SPLIT, and "single domain split antibody" may be used interchangeably. Exemplary hemibodies / demibodies are described in co-pending application PCT / EP2020 / 069561.

[0026] When the treatment utilizes a single antibody molecule containing at least one binding site for the target antigen and at least one binding site for the radiolabeled compound (i.e., not a split antibody), the treatment may also include administration of a clearing agent. The clearing agent is administered after the multispecific antibody and before the radiolabeled compound. The clearing agent binds to the antigen-binding site for the radiolabeled compound. The clearing agent blocks the antigen-binding site for the radiolabeled compound, preventing circulating antibodies from binding to the chelated radionuclide. Alternatively or additionally, the clearing agent may increase the clearance rate of the antibody from the body. The "clearing agent" may alternatively be referred to as a "blocking agent," and these terms may be interchangeable in the following description. The clearing agent may be conjugated to a clearing moiety, as further discussed herein.

[0027] When a treatment utilizes a hemibody, the treatment need not include a clearing step. That is, in some embodiments, the method does not include administering a clearing or blocking agent between administration of the first and second hemibody and administration of the radiolabeled compound (i.e., after administration of the hemibodies but before administration of the radiolabeled compound). In another embodiment, no agent, other than an optional radiosensitizer and / or chemotherapeutic agent, is administered between administration of the first and second hemibody and administration of the radiolabeled compound. In another embodiment, no agent is administered between administration of the first and second hemibody and administration of the radiolabeled compound.

[0028] In some embodiments, the combination therapy provides one or more advantages compared to treatment with pre-targeted radioimmunotherapy alone and / or immunotherapy alone. The reference treatment with pre-targeted radioimmunotherapy alone includes administering the same pre-targeted radioimmunotherapy treatment as the combination therapy (i.e., the same compound, dose, administration time, and number of cycles), but without immunotherapy (i.e., without administration of a CD40 agonist or immune checkpoint inhibitor). The reference treatment with immunotherapy alone includes administering the same immunotherapy treatment as the combination therapy (i.e., the same CD40 agonist and immune checkpoint inhibitor compound, dose, administration time, and number of cycles), but without pre-targeted radioimmunotherapy (i.e., without administration of a multispecific / split-multispecific antibody, radiolabeled compound, and, if applicable, ablating agent). In some embodiments, the combination therapy results in a slower tumor growth rate than pre-targeted radioimmunotherapy alone and / or immunotherapy alone. In some embodiments, the combination therapy results in an increased likelihood of patient / subject survival compared to treatment with pre-targeted radioimmunotherapy alone and / or immunotherapy alone. In some embodiments, the combination therapy results in an increased frequency of activated intratumoral CD8 T cells (e.g., as measured by upregulation of 41BB expression), and / or an increased frequency of activated plasmacytoid DCs (pDCs) and classical DCs (cDCs) (e.g., as measured by upregulation of CD86 expression) in the tumor, spleen, and draining lymph nodes (DLNs), and / or an increased frequency of T cells among total immune cells, relative to treatment with pre-targeted radioimmunotherapy and / or immunotherapy alone. In some embodiments, the combination therapy results in an enhanced immune memory response or a reduced likelihood of tumor recurrence relative to treatment with pre-targeted radioimmunotherapy and / or immunotherapy alone.

[0029] In some embodiments, the cancer is resistant to immune checkpoint inhibitors.

[0030] In some embodiments, it may be preferred that the target antigen is CEA.

[0031] In some embodiments, it may be preferred that the radiolabeled compound is Pb-DOTAM. [Brief explanation of the drawings]

[0032] [Figure 1] FIG. 1 shows the schematic structure of a target antigen (TA)-DOTAM bispecific antibody (TA-DOTAM BsAb) and an exemplary TA-split-DOTAM-VH / VL antibody. [Figure 2] Schematic showing assembly of split-VH / VL DOTAM binders on tumor cells. TA-split-DOTAM-VH / VL antibodies do not significantly bind 212Pb-DOTAM unless they bind to a tumor antigen (TA) on the targeted cell where the two domains of the DOTAM binder are assembled. [Figure 3] Schematic of an example of a three-stage TA-PRIT concept including the use of a remover. [Figure 4] Schematic of an example of a two-stage TA-PRIT concept where no remover is used. [Figure 5] Figure 1 shows the binding of split antibodies to MKN45 cells to demonstrate CEA binding capacity. Antibody detection is performed using a human IgG-specific secondary antibody. [Figure 6] Figure 1 shows the binding of split antibodies to MKN45 cells to demonstrate DOTAM binding ability. Antibody detection is performed using Pb-DOTAM-FITC. [Figure 7A] Diagram showing an exemplary protocol of two-stage PRIT with CEA-split-DOTAM-VH / VL performed in SCID mice bearing SC BxPC3 tumors (h=hours, d=days, w=weeks). [Figure 7B] Diagram showing an exemplary protocol of a three-stage PRIT control performed in SCID mice bearing SC BxPC3 tumors (h=hours, d=days, w=weeks). [Figure 8]Biodistribution of pretargeted 212Pb-DOTAM in SCID mice bearing SC BxPC3 tumors 6 hours after injection of 212Pb-DOTAM pretargeted with either CEA-split-DOTAM-VH alone, CEA-split-DOTAM-VL alone, or the two complementary antibodies in combination, or using standard three-step PRIT (%ID / g ± SD, n=4). [Figure 9] FIG. 1 shows the pharmacokinetics of CEA-split-DOTAM-VH / VL after IV injection in SCID mice. [Figure 10] Diagram showing the experimental design of protocol 158 involving CEA-PRIT in two (top) or three (bottom) stages in SCID mice bearing SC BxPC3 tumors. *Dose of CEA split DOTAM BsAb adjusted to compensate for hole / hole impurities in the 2 / 4 construct. [Figure 11] Biodistribution of pretargeted 212Pb-DOTAM in SCID mice bearing SC BxPC3 tumors (6 h p.i.). Distribution of 212Pb in tumor-bearing SCID mice 6 h after injection of 212Pb-DOTAM pretargeted with a biparatopic combination of CEA-DOTAM BsAb or CEA-split-DOTAM antibody. Radioactivity content in organs and tissues is expressed as mean %ID / g ± SD (n=4). [Figure 12] Diagram showing the experimental schedule for protocol 160, which includes one cycle of three-stage CEA-PRIT (top), two-stage CEA-PRIT (middle), or one-stage CEA-RIT in SCID mice bearing SC BxPC3 tumors. Biodistribution (BD) mice were euthanized 24 h after radioactivity injection, while mice in the efficacy group were maintained and carefully monitored until termination criteria were reached. [Figure 13]Figure 1 shows the biodistribution (24 h p.i.) of pretargeted Pb-DOTAM and Pb-DOTAM-CEA-DOTAM in SCID mice bearing SC BxPC3 tumors. Distribution of Pb in tumor-bearing SCID mice 24 h after injection of CEA-DOTAM-pretargeted Pb-DOTAM or preincubated Pb-DOTAM-CEA-DOTAM. Radioactivity content in organs and tissues is expressed as mean %ID / g ± SD (n=3). [Figure 14] Figure showing mean tumor growth with standard error for PRIT-treated groups and controls (groups A-E) in the BxPC3 model (n=10). Curves were truncated at n<5. The dotted vertical line indicates 212Pb-DOTAM administration (20 μCi) to some or all groups according to the study design. [Figure 15] Individual tumor growth curves for PRIT-treated and control groups (Groups A-E) in the BxPC3 model (n=10). The dotted vertical line indicates administration of 212Pb-labeled compound (20 μCi). [Figure 16] Average weight loss in mice treated with CEA-PRIT and CEA-RIT (Groups A-E, n=10) in the BxPC3 model. Curves were truncated at n<5. Dotted vertical lines indicate administration of 212Pb-labeled compound to some or all groups according to the study design. [Figure 17] Diagram showing the experimental design of protocol 175 involving two-stage CEA-PRIT in SCID mice bearing SC BxPC3 tumors, sacrificed and necropsied 24 h after 212Pb-DOTAM injection. The dose of CEA-split-DOTAM-VH-AST was adjusted to compensate for hole / hole impurities. [Figure 18] Distribution of 212Pb in tumor-bearing SCID mice 24 hours after injection of 212Pb-DOTAM pretargeted with CEA-split-DOTAM-VH / VL antibody (Protocol 175). Radioactivity content in organs and tissues is expressed as mean %ID / g ± SD (n=4). [Figure 19]Diagram showing the experimental design of protocol 185 involving two-stage CEA-PRIT in SCID mice bearing SC BxPC3 tumors, sacrificed and necropsied 6 h after 212Pb-DOTAM injection. The dose of CEA-split-DOTAM-VH-AST(CH1A1A) was adjusted to compensate for hole / hole impurities. [Figure 20] Distribution of 212Pb in tumor-bearing SCID mice 6 hours after injection of 212Pb-DOTAM pretargeted with CEA-split-DOTAM-VH / VL antibody (Protocol 185). Radioactivity content in organs and tissues is expressed as mean %ID / g ± SD (n=5). [Figure 21] Figure 1 shows the distribution of CEA-split-DOTAM-VH / VL pairs (VH and VL antibody combinations) in two selected SC BxPC3 tumors 7 days after injection. A and B show sections of tumor from mouse A3 injected with CEA-split-DOTAM-VH / VL targeting T84.66; A shows CEA expression, and B shows the corresponding CEA-split-DOTAM-VH / VL distribution. C and D show sections of tumor from mouse C5 injected with CEA-split-DOTAM-VH / VL targeting CH1A1A; C shows CEA expression, and D shows the corresponding CEA-split-DOTAM-VH / VL distribution. [Figure 22] Diagram showing the experimental design of protocol 189 involving two-stage CEA-PRIT in SCID mice bearing SC BxPC3 tumors, sacrificed and necropsied 6 h after 212Pb-DOTAM injection. The dose of CEA-split-DOTAM-VH-AST(CH1A1A) was adjusted to compensate for hole / hole impurities. [Figure 23] Figure 1 shows the distribution of 212Pb in tumor-bearing SCID mice 6 hours after injection of 212Pb-DOTAM pre-targeted with a biparatopic pair of CEA-split-DOTAM-VH / VL antibodies (T84.66 and CH1A1A) compared to the positive control (CH1A1A only). Radioactivity content in organs and tissues is expressed as mean %ID / g ± SD. [Figure 24]Figure 1 shows the mean fluorescence intensity (MFI) determined by FACS for the SPLIT antibodies. Binding of Pb-DOTA-FITC, as determined by FACS, can only be demonstrated for the co-incubation of both SPLIT antibodies with Pb-DOTA-FITC. A single SPLIT antibody did not produce a significant signal. [Figure 25] 25A-25C show additional exemplary formats of split antibodies. [Figure 26] FIG. 1 shows the results of Example 8, Experiment 1, in which binding of individual TA-split-DOTAM-VH and TA-split-DOTAM-VL antibodies to biotinylated DOTAM captured on a chip was assessed. [Figure 27] FIG. 1 shows the results of Example 8, Experiment 2, which assessed the binding of DOTAM to individual TA-split-DOTAM-VH and TA-split-DOTAM-VL antibodies captured on a chip. [Figure 28] FIG. 1 shows the results of Example 8, Experiment 3, in which the binding of DOTAM to TA-split-DOTAM-VH / VL antibodies (antibody pairs) captured on a chip was evaluated. [Figure 29] Diagram showing study overview of protocol 119, which evaluated CEA-PRIT of orthotopic Panc02-huCEA-luc tumors in B6-huCEA mice (IPANC = intrapancreatic, d = day, h = hour). [Figure 30] Distribution of 212Pb in tumor-bearing B6-huCEA mice 24 h after injection of 212Pb-DOTAM pretargeted with CEA-DOTAM(mu) (cycle 1). Radioactivity content in organs and tissues is expressed as mean %ID / g ± standard deviation (SD, n=3). [Figure 31] Figure 1 shows serum concentrations of anti-CD40 and anti-PD-L1 antibodies, as determined by ELISA, 24 hours after IP administration (200 μg / antibody / mouse) to mice in Groups B and D of Protocol 119. Graphs show individual values ​​as mean ± SD for each treatment cycle. [Figure 32]Figure 1 shows the background-subtracted mean BLI signal for groups A-D in the orthotopic Panc02-huCEA-luc model, expressed as photons (P) / sec / mm ± standard error of the mean (SEM, n=8). Dashed and dotted vertical lines indicate immunotherapy and 212Pb-DOTAM administration (20 μCi) for some or all groups, respectively, according to the study design. [Figure 33] Background-subtracted BLI signals of individual mice in groups A-D in the orthotopic Panc02-huCEA-luc model (n=8), expressed as photons (P) / sec / mm². Dashed and dotted vertical lines indicate immunotherapy and Pb-DOTAM administration (20 µCi) for some or all groups, respectively, according to the study design. The arrow indicates day 88, the last day of imaging; by this time, three mice in group D were still alive and showed no signal. [Figure 34] Kaplan-Meier curves showing survival for groups A–D in the orthotopic Panc02-huCEA-luc model (n=8). Dotted and dashed vertical lines indicate 212Pb-DOTAM (20 μCi) and immunotherapy administration, respectively, for some or all groups according to the study design. [Figure 35] 1 shows the mean change in BW after various treatments, expressed as a percentage of the initial BW±SEM. The dotted and dashed lines indicate 212Pb-DOTAM and immunotherapy administration, respectively, depending on the treatment scheme. [Figure 36] Diagram showing study outline for protocol 136, which evaluated CEA-PRIT of SC Panc02-huCEA-luc tumors in B6-huCEA mice (d = day, h = hour). [Figure 37] Distribution of 212Pb in tumor-bearing B6-huCEA mice 24 hours after injection of 212Pb-DOTAM pre-targeted with CEA-DOTAM(mu) (cycle 1). Radioactivity content in organs and tissues is expressed as mean %ID / g ± SD (n=3). [Figure 38]Figure 1 shows serum concentrations of anti-CD40 and anti-PD-L1 24 hours after IP administration (200 μg / antibody / mouse) to mice in Groups B and D, as determined by ELISA. Graphs show individual values ​​and mean ± SD for each treatment cycle. Asterisks (*) in the right graph indicate skewed mean values ​​due to outliers (not shown; one data point in Cycle 1 and three data points in Cycle 3). [Figure 39] Figure 1 shows mean tumor growth with standard error for groups A-D in the SC Panc02-huCEA-luc model (n=9). Curves were truncated at n<5. Dashed and dotted vertical lines indicate immunotherapy and 212Pb-DOTAM administration (20 μCi) for some or all groups, respectively, according to the study design. [Figure 40] Individual tumor growth curves for groups A to D in the SC Panc02-huCEA-luc model (n = 9). Dashed and dotted vertical lines indicate administration of immunotherapy and 212Pb-DOTAM (20 μCi), respectively. [Figure 41] Figure 1 shows the background-subtracted mean BLI signal for groups A-D in the SC Panc02-huCEA-luc model (n=9), expressed as photons (P) / sec / mm ± SEM. Dashed and dotted vertical lines indicate immunotherapy and 212Pb-DOTAM administration (20 μCi) for some or all groups, respectively, according to the study design. [Figure 42] Figure 1 shows background-subtracted BLI signals of individual mice in groups A-D in the SC Panc02-huCEA-luc model (n=9), expressed as photons (P) / sec / mm. Dashed and dotted vertical lines indicate immunotherapy and Pb-DOTAM administration (20 μCi) to some or all groups, respectively, according to the study design. [Figure 43]Kaplan-Meier curves showing survival for groups A–D in the SC Panc02-huCEA-luc model (n=9) based on the termination criterion of tumor volume ≥3000 mm3. Dashed and dotted vertical lines indicate immunotherapy and 212Pb-DOTAM administration (20 μCi) for some or all groups, respectively, according to the study design. [Figure 44] Figure 1 shows FACS analysis of DLN, spleen, and tumor samples from mice treated with two cycles of immunotherapy, CEA-PRIT, CEA-PRIT + immunotherapy, or no treatment, demonstrating T cell activation. Samples were collected 24 hours after immunotherapy injection, corresponding to 48 hours after 212Pb-DOTAM irradiation. Asterisks indicate significance (one-way ANOVA, p<0.05, n=4). [Figure 45] Figure 1 shows FACS analysis of DLN, spleen, and tumor samples from mice treated with two cycles of immunotherapy, CEA-PRIT, CEA-PRIT + immunotherapy, or no treatment, demonstrating activation of cDCs and pDCs. Markers for the pDC subpopulation: MHCII+CD11cnt CD317+, markers for the CD11b-cDC subpopulation (cross-presenting DCs): MHCII+CD11chigh CD11b-, markers for the CD11b+cDC subpopulation: MHCII+CD11chigh CD11b+. Samples were taken 24 hours after immunotherapy injection, corresponding to 48 hours after 212Pb-DOTAM irradiation. Asterisks indicate significance (one-way ANOVA, p<0.05, n=4). [Figure 46] Figure 1 shows FACS analysis of DLN, spleen, and tumor samples from mice treated with two cycles of immunotherapy, CEA-PRIT, CEA-PRIT + immunotherapy, or no treatment, showing overall T cell frequencies. Samples were collected 24 hours after immunotherapy injection, corresponding to 48 hours after 212Pb-DOTAM irradiation. Asterisks indicate significance (one-way ANOVA, p<0.05, n=4). [Figure 47]Tumor growth curves of rechallenged and naive B6-huCEA mice in the SC Panc02-huCEA-luc model (n=5). Rechallenged mice were initially tumor carriers and became tumor-free after three cycles of CEA-PRIT+CIT (anti-CD40+anti-PD-L1). [Figure 48] Figure 1 shows FACS analysis of blood, spleen, and lymph node (LN) samples from rechallenged and naive mice in the SC Panc02-huCEA-luc model (n=5). Rechallenged mice were initially tumor carriers and became tumor-free after three cycles of CEA-PRIT+CIT (anti-CD40+anti-PD-L1). Asterisks indicate significance (unpaired t-test, p<0.05). dp = double positive, iono = ionomycin. [Figure 49] 1 shows the mean change in BW after various treatments, expressed as a percentage of the initial BW±SEM. The dotted and dashed lines indicate 212Pb-DOTAM and immunotherapy administration, respectively, depending on the treatment scheme. [Figure 50] Diagram showing study outline of protocol 150, evaluating CEA-PRIT of SC MC38-huCEA tumors in B6-huCEA mice (d = day, h = hour). [Figure 51] Distribution of 212Pb in MC38-huCEA tumor-bearing B6-huCEA mice 24 hours after injection of 212Pb-DOTAM pretargeted with CEA-DOTAM(mu) (cycle 1). Radioactivity content in organs and tissues is expressed as mean %ID / g ± SD (n=4). [Figure 52] Figure 1 shows serum concentrations of anti-CD40 and anti-PD-L1 as determined by ELISA 24 hours after IP administration (200 μg / antibody / mouse) to mice in groups B, C, and E. Graphs show individual values ​​as mean ± SD for each treatment cycle. [Figure 53]Figure showing mean tumor growth with standard error for groups A-E in the SC MC38-huCEA model (n=9). Curves were truncated at n<5. Dashed and dotted vertical lines indicate immunotherapy and 212Pb-DOTAM administration (20 μCi) for some or all groups, respectively, according to the study design. Arrows indicate immuno-PD analysis. [Figure 54] Individual tumor growth curves for groups A to E in the SC MC38-huCEA model (n = 9). Dashed and dotted vertical lines indicate administration of immunotherapy and 212Pb-DOTAM (20 μCi), respectively. [Figure 55] Kaplan-Meier curves showing survival for groups A–E in the SC MC38-huCEA model (n=9) based on the termination criterion of tumor volume ≥3000 mm3. Symbols represent censored mice euthanized for reasons other than tumor volume. Dashed and dotted vertical lines indicate immunotherapy and 212Pb-DOTAM administration (20 μCi) for some or all groups, respectively, according to the study design. [Figure 56] Figure showing FACS analysis of lymph node samples from mice treated with two cycles of immunotherapy, CEA-PRIT, CEA-PRIT + immunotherapy, or no treatment. Samples were collected 24 hours after immunotherapy injection, corresponding to 48 hours after 212Pb-DOTAM irradiation. Asterisks indicate significance level (one-way ANOVA with correction for multiple comparisons [Tukey], p<0.05, n=4). MFI = mean fluorescence intensity. [Figure 57] Figure 1 shows tumor growth curves for rechallenged and naive (age-matched) B6-huCEA mice in the SC MC38-huCEA model. Rechallenged mice were initially tumor carriers and became tumor-free after treatment with anti-CD40, anti-CD40 + anti-PD-L1, CEA-PRIT, or CEA-PRIT + anti-CD40 + anti-PD-L1. [Figure 58]Figure 1 shows FACS analysis of re-challenged and naive mice in the SC MC38-huCEA model. Re-challenged mice were initially tumor carriers and became tumor-free after treatment with anti-CD40, anti-CD40 + anti-PD-L1, CEA-PRIT, or CEA-PRIT + anti-CD40 + anti-PD-L1. Asterisks indicate significance (one-way ANOVA with correction for multiple comparisons [Tukey], p<0.05, n=4). dp=double positive. [Figure 59] 1 shows the mean change in BW after various treatments, expressed as a percentage of the initial BW±SEM. The dotted and dashed lines indicate 212Pb-DOTAM and immunotherapy administration, respectively, depending on the treatment scheme. [Figure 60] Diagram showing study outline for protocol 195, which evaluated SPLIT PRIT and / or CIT of SC Panc02-huCEA-Fluc tumors in huCEACAM5 mice (d = day, h = hour). [Figure 61] Distribution of 212Pb in Panc02-huCEA-Fluc tumor-bearing huCEACAM5 mice 24 hours after injection of 212Pb-DOTAM pretargeted by SPLIT CEA-PRIT. Radioactivity content in organs and tissues is expressed as mean %ID / g ± SD (n=3). [Figure 62] Figure 1 shows mean tumor growth with standard error for groups A-D (n=10) in the SC Panc02-huCEA-Fluc model (Protocol 195). Curves were truncated at n<5. Dashed and dotted vertical lines indicate immunotherapy and 212Pb-DOTAM administration (20 μCi) for some or all groups, respectively, according to the study design. [Figure 63] Figure 1 shows individual tumor growth curves for groups A–D in the SC Panc02-huCEA-Fluc model (n = 10) (Protocol 195). Dashed and dotted vertical lines indicate administration of immunotherapy and 212Pb-DOTAM (20 μCi), respectively. [Figure 64]Kaplan-Meier curves showing survival for groups A–D in the SC Panc02-huCEA-Fluc model (protocol 195) based on the termination criterion of tumor volume ≥ 2000 mm (n = 10). Symbols represent censored mice euthanized for reasons other than tumor volume. Dashed and dotted vertical lines indicate immunotherapy and Pb-DOTAM administration (20 μCi) for some or all groups, respectively, according to the study design. [Figure 65] Figure 1 shows mean tumor growth with standard error for rechallenged and naive (age-matched) huCEACAM5 mice in the SC Panc02-huCEA-Fluc model. Rechallenged mice were initially tumor carriers and became tumor-free after treatment with SPLIT CEA-PRIT + anti-CD40 + anti-PD-L1. On day 13 after rechallenge, three mice per group were euthanized for immune-PD analysis (data not shown). [Figure 66] Figure 1 shows tumor growth curves for rechallenged and age-matched huCEACAM5 mice in the SC Panc02-huCEA-Fluc model. Rechallenged mice were initially tumor carriers and became tumor-free after treatment with SPLIT CEA-PRIT + anti-CD40 + anti-PD-L1. On day 13 after rechallenge, three mice per group were euthanized for immune-PD analysis (data not shown). [Figure 67] 1 shows the mean change in BW after various treatments, expressed as a percentage of the initial BW±SEM. The dotted and dashed lines indicate 212Pb-DOTAM and immunotherapy administration, respectively, depending on the treatment scheme. DETAILED DESCRIPTION OF THE INVENTION

[0033] I. Definition For purposes herein, an "acceptor human framework" is a framework that comprises the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence, or it may contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0034] "Affinity" refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y is generally determined by the dissociation constant (K D ) Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative exemplary methods for measuring binding affinity are described below.

[0035] An "affinity matured" antibody refers to an antibody with one or more modifications in one or more complementarity determining regions (CDRs) that result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody that does not possess such modifications.

[0036] The term "binding site for an antigen expressed on the surface of a target cell" or "binding site for a target antigen" refers to a binding site that can bind to the antigen with sufficient affinity so that the antibody is useful as a diagnostic and / or therapeutic agent when targeting that antigen. An antibody comprising a binding site for a target antigen can include any binding moiety that binds to the target antigen with sufficient affinity. In some embodiments, the antigen-binding moiety can be an antibody fragment (e.g., Fv, Fab, cross-Fab, Fab', Fab'-SH, F(ab')2; diabody; linear antibody; single-chain antibody molecule (e.g., scFv or scFab); or single-domain antibody (dAb), e.g., VHH). In other embodiments, it can be a protein-binding scaffold, e.g., DARPin (designed ankyrin repeat protein); affibody; Sso7d; monobody, or anticalin.

[0037] In one embodiment, the extent of binding of the antibody to an unrelated, non-antigenic protein is less than about 10% of the binding of the antibody to the antigen, as measured, for example, by surface plasmon resonance (SPR). In certain embodiments, an antibody that binds to an antigen expressed on the surface of a target cell has a binding affinity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g. 10 -8 M~10 -13 M, e.g. 10 -9 M~10 -13 Dissociation constant (K D The antibody has K D An antibody is said to "specifically bind" to an antigen expressed on the surface of a target cell if its binding affinity is 1 μM or less. In certain embodiments, an antibody binds to an epitope of the antigen that is conserved among the antigens from different species.

[0038] The term "antigen-binding site for a radiolabeled compound" or "functional antigen-binding site for a radiolabeled compound" refers to an antigen-binding site that can bind to a radiolabeled compound with sufficient affinity so that an antibody is useful as a diagnostic and / or therapeutic agent for associating the radiolabeled compound with the antibody. The antigen-binding site for a radiolabeled compound preferably comprises a VH and a VL domain. In one embodiment, the extent of binding of the antigen-binding site to an unrelated, non-antigenic compound is less than about 10% of the binding of the antibody to the radiolabeled compound, as measured, for example, by surface plasmon resonance (SPR). In certain embodiments, the antigen-binding site that binds to a radiolabeled compound has an affinity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g. 10 -8 M~10 -13 M, e.g. 10 -9 M~10- -13 Dissociation constant (K D ) having a K of 100 pM, 50 pM, 20 pM, 10 pM, 5 pM, 1 pM or less, for example, 0.9 pM or less, 0.8 pM or less, 0.7 pM or less, 0.6 pM or less, or 0.5 pM or less. D For example, it may be preferable for a functional binding site to have a K of about 1 pM to 1 nM, e.g., about 1 to 10 pM, 1 to 100 pM, 5 to 50 pM, 100 to 500 pM, or 500 pM to 1 nM. D The antigen-binding site is capable of binding to the radiolabeled compound at a K of 1 μM or less. D A compound is said to "specifically bind" to a radiolabeled compound when it has

[0039] The term "antibody" as used herein is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0040] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, cross-Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv and scFab); single-domain antibodies (dAbs); and multispecific antibodies formed from antibody fragments. For a review of specific antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005). The term "Fab fragment" refers to a protein consisting of the VH and CH1 domains of an immunoglobulin heavy chain and the VL and CL domains of a light chain. "Fab' fragments" differ from Fab fragments by the addition of residues at the carboxy terminus of the CH1 domain, including one or more cysteines from the antibody hinge region. See US Pat. No. 5,869,046 for a description of Fab and F(ab')2 fragments that contain salvage receptor binding epitope residues and have increased in vivo half-lives.

[0041] As used herein, reference to a "Fab fragment" is intended to include cross-Fab fragments or scFabs as well as conventional Fab fragments (i.e., those that contain a light chain comprising the VL and CL domains, and a heavy chain fragment comprising the VH and CH1 domains).

[0042] The term "cross-Fab fragment" or "xFab fragment" or "crossover Fab fragment" refers to a Fab fragment in which either the variable or constant regions of the heavy and light chains have been exchanged. A cross-Fab fragment comprises a polypeptide chain consisting of a light chain variable region (VL) and heavy chain constant region 1 (CH1), and a polypeptide chain consisting of a heavy chain variable region (VH) and light chain constant region (CL). For clarity, in a crossover Fab molecule in which the variable regions of the Fab light chain and the Fab heavy chain have been exchanged, the peptide chain comprising the heavy chain constant region is referred to herein as the "heavy chain" of the crossover Fab molecule. Conversely, in a crossover Fab molecule in which the constant regions of the Fab light chain and the Fab heavy chain have been exchanged, the peptide chain comprising the heavy chain variable region is referred to herein as the "heavy chain" of the crossover Fab molecule.

[0043] As used herein, the term "single chain" refers to a molecule comprising amino acid monomers linearly linked by peptide bonds. A single-chain Fab molecule is a Fab molecule in which the Fab light chain and the Fab heavy chain are connected by a peptide linker to form a single peptide chain. In certain such embodiments, the C-terminus of the Fab light chain is connected to the N-terminus of the Fab heavy chain in the single-chain Fab molecule.

[0044] Asymmetric Fab arms can also be engineered by introducing charged or uncharged amino acid mutations at the domain interface to direct correct Fab pairing. See, e.g., WO 2016 / 172485.

[0045] A "single-chain variable fragment" or "scFv" is a fusion protein of the variable domains of an antibody's heavy chain (VH) and light chain (VL) connected by a peptide linker. In particular, the linker is a short polypeptide of 10 to 25 amino acids, typically rich in glycine for flexibility and serine or threonine for solubility, which can connect the N-terminus of the VH to the C-terminus of the VL, or vice versa. The protein retains the specificity of the original antibody despite the removal of the constant regions and the introduction of the linker. For a review of scFv fragments, see, e.g., Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994). See also WO 93 / 16185 and U.S. Patent Nos. 5,571,894 and 5,587,458.

[0046] A split antibody refers to an antibody in which the antigen-binding site is split between two parts, e.g., two individual antibody molecules. The two parts may be referred to as a "hemibody" or "demibody." When the two parts associate, a functional binding site for the antigen is formed. In the present invention, each hemibody contains an antigen-binding portion for an antigen on the surface of a target cell and either a VH or VL antigen-binding site for a radiolabeled compound. When two hemibodies bind to the same or adjacent target cell, a stable association can be formed between the VH and VL, thus forming a functional binding site for the radiolabeled compound. A "CEA-targeted SPLIT PRIT" refers to a split antibody that targets CEA. The term "SPLIT PRIT" can also be used interchangeably with the term "TA-split-DOTAM-VH / VL." The term "CEA-targeted SPLIT PRIT" can also be used interchangeably with the term "CEA-split-DOTAM-VH / VL."

[0047] The term "clearing agent" refers to an agent that increases the clearance rate of an antibody from the circulation of a subject and / or blocks the binding of an effector molecule, particularly a radiolabeled compound, to a functional binding site for the effector molecule. Generally, the clearing agent binds to the antibody, e.g., specifically binds to the antibody. It can bind to a functional binding site for the effector molecule, e.g., can specifically bind to the functional binding site.

[0048] As used herein, the term "clearing step" or "clearing phase" encompasses the use of agents that increase the rate of clearance of antibodies from the circulation of a subject and / or block effector molecule binding. Some agents can function in both clearing and blocking.

[0049] The term "epitope" refers to a site on an antigen, either proteinaceous or nonproteinaceous, to which an antibody binds. Epitopes may be formed from a contiguous stretch of amino acids (linear epitopes) or may contain noncontiguous amino acids that are spatially adjacent, e.g., due to antigen folding, i.e., tertiary folding of a proteinaceous antigen (conformational epitopes). Linear epitopes are typically still bound by antibodies after exposure of a proteinaceous antigen to a denaturing agent, whereas conformational epitopes are typically disrupted by treatment with a denaturing agent. 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.

[0050] Screening for antibodies that bind to a specific epitope (i.e., antibodies that bind to the same epitope) can be performed using methods routine in the art, such as, but not limited to, alanine scanning, peptide blotting (see Meth. Mol. Biol. 248 (2004) 443-463), peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of antigens (see Prot. Sci. 9 (2000) 487-496), and cross-blocking (see "Antibodies," Harlow and Lane, Cold Spring Harbor Press, Cold Spring Harb., NY).

[0051] Antigen structure-based antibody profiling (ASAP), also known as modification-assisted profiling (MAP), allows antibodies from a large number of monoclonal antibodies that specifically bind to an antigen to be classified into bins based on the binding profile of each antibody to chemically or enzymatically modified antigen surfaces (see, e.g., U.S. Patent Application Publication No. 2004 / 0101920). Antibodies within each bin bind to the same epitope, which may be distinct from or partially overlapping unique epitopes represented by other bins.

[0052] Competitive binding can also be used to easily determine whether an antibody binds to the same epitope as a reference antibody or competes for binding with the reference antibody. For example, an "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competitive assay; conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competitive assay. For example, to determine whether an antibody binds to the same epitope as a reference antibody, the reference antibody is bound to the antigen under saturating conditions. After removing excess reference antibody, the antibody in question is evaluated for its ability to bind to the antigen. If the antibody in question can bind to the antigen after saturating binding of the reference antibody, it can be concluded that the antibody in question binds to a different epitope than the reference antibody. However, if the antibody in question cannot bind to the antigen after saturating binding of the reference antibody, the antibody in question may bind to the same epitope as the epitope bound by the reference antibody. To determine whether the antibodies in question bind to the same epitope or whether binding is simply hindered by steric reasons, routine experiments can be used (e.g., peptide mutations and binding analysis using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art). This assay should be performed in two settings, i.e., when both antibodies are saturating antibodies. If, in both settings, only the first (saturating) antibody can bind to the antigen, it can be concluded that the antibody in question and the reference antibody compete for binding to the antigen.

[0053] In some embodiments, two antibodies are considered to bind to the same or overlapping epitope if a 1-, 5-, 10-, 20-, or 100-fold excess of one antibody inhibits binding of the other by at least 50%, at least 75%, at least 90%, or even 99% or more as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 50 (1990) 1495-1502).

[0054] In some embodiments, two antibodies are considered to bind the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other antibody. Two antibodies are considered to have "overlapping epitopes" if only a subset of amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other antibody.

[0055] 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.

[0056] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In certain embodiments, the antibody is of the IgG1 isotype. In certain embodiments, the antibody is of the IgG1 isotype with P329G, L234A, and L235A mutations to reduce Fc region effector function. In other embodiments, the antibody is of the IgG2 isotype. In certain embodiments, the antibody is of the IgG4 isotype with an S228P mutation in the hinge region to improve the stability of IgG4 antibodies. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The light chain of an antibody can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0057] "Effector function" refers to a biological activity attributable to the Fc region of an antibody, which varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0058] An "effective amount" of an agent, eg, a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic or prophylactic result.

[0059] The term "tandem Fab" refers to an antibody comprising two Fab fragments connected 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.

[0060] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. The term "Fc domain" herein is used to define the C-terminal region of an immunoglobulin, including the constant regions of the two heavy chains, excluding the first constant region. Thus, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM. The term encompasses native-sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Thus, upon expression of a specific nucleic acid molecule encoding a full-length heavy chain, antibodies produced by host cells may contain a full-length heavy chain or a truncated variant of the full-length heavy chain. This may be the case when the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to the EU index). Thus, the C-terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (Lys447) of the Fc region may or may not be present. In one embodiment, a heavy chain comprising an Fc region as specified herein, comprised in an antibody according to the invention, comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to the EU index). In one embodiment, a heavy chain comprising an Fc region as specified herein, comprised in an antibody according to the invention, comprises an additional C-terminal glycine residue (G446, numbering according to the EU index).Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. As used herein, a "subunit" of an Fc domain refers to one of the two polypeptides forming a dimeric Fc domain, i.e., a polypeptide comprising the C-terminal constant region of an immunoglobulin heavy chain that can stably associate with the other of the two polypeptides forming the dimeric Fc domain. For example, a subunit of an IgG Fc domain comprises the IgG CH2 and IgG CH3 constant domains.

[0061] "Framework" or "FR" refers to variable domain residues other than the complementarity-determining regions (CDRs). The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the CDR and FR sequences generally appear in VH (or VL) in the following sequence: FR1-CDR-H1 (CDR-L1)-FR2-CDR-H2 (CDR-L2)-FR3-CDR-H3 (CDR-L3)-FR4.

[0062] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to that of a native antibody or an antibody having a heavy chain including an Fc region as defined herein. As used herein, the term "full-length antibody" refers to an antibody consisting of two "full-length antibody heavy chains" and two "full-length antibody light chains." A "full-length antibody heavy chain" can be a polypeptide consisting, from N- to C-terminus, of an antibody heavy chain variable domain (VH), antibody heavy chain constant domain 1 (CH1), antibody hinge region (HR), antibody heavy chain constant domain 2 (CH2), and antibody heavy chain constant domain 3 (CH3) (abbreviated as VH-CH1-HR-CH2-CH3), and optionally, in the case of an antibody of the subclass IgE, antibody heavy chain constant domain 4 (CH4). Preferably, a "full-length antibody heavy chain" is a polypeptide consisting, from N- to C-terminus, of VH, CH1, HR, CH2, and CH3. The possibility of cross-Mab formation is not intended to be excluded by the reference to "full length"; thus, a heavy chain may have a VH domain exchanged for a VL domain or a CH1 domain exchanged for a CL domain. A "full-length antibody light chain" may be a polypeptide consisting of, from N- to C-terminal, an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL) (abbreviated as VL-CL). Alternatively, in the case of a cross-Mab, the VL domain may be exchanged for a VH domain, or the CL domain may be exchanged for a CH1 domain. The antibody light chain constant domain (CL) may be kappa (kappa) or gamma (lambda). Two full-length antibody chains are linked to each other via interpolypeptide disulfide bonds between the CL and CH1 domains and between the hinge regions of the full-length antibody heavy chains. Typical examples of full-length antibodies are natural antibodies such as IgG (e.g., IgG1 and IgG2), IgM, IgA, IgD, and IgE. A full-length antibody can be derived from a single species, for example, human, or it can be a chimeric or humanized antibody.The full-length antibodies described herein comprise two antigen-binding sites, each formed by a VH and VL pair, which in some embodiments may both specifically bind the same antigen or may bind different antigens. The C-terminus of the heavy or light chain of the full-length antibody refers to the last amino acid at the C-terminus of the heavy or light chain.

[0063] By "fused" is meant that the components are linked by a peptide bond either directly or via one or more peptide linkers.

[0064] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and its progeny without regard to the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0065] A "human antibody" is an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or a human cell, or to an amino acid sequence derived from a non-human source that utilizes the human antibody repertoire, or to a sequence encoding another human antibody. This definition of human antibody specifically excludes humanized antibodies that comprise non-human antigen-binding residues.

[0066] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda, MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup kappa I described in Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III described in Kabat et al., supra.

[0067] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues derived from non-human CDRs and amino acid residues derived from human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, with all or substantially all of the CDRs corresponding to those of a non-human antibody and all or substantially all of the FRs corresponding to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has been humanized.

[0068] As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that is hypervariable in sequence and determines antigen-binding specificity, e.g., the "complementarity-determining region" ("CDR").

[0069] Generally, antibodies comprise six CDRs: three in the VH (CDR-H1, CDR-H2, CDR-H3) and three in the VL (CDR-L1, CDR-L2, CDR-L3). Exemplary CDRs herein include: (a) hypervariable loops located 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, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)), and (c) Antigen contact sites located 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)).

[0070] Unless otherwise specified, CDRs are determined according to Kabat et al., supra. Those skilled in the art will understand that CDR nomenclature can also be determined according to Chothia, supra, McCallum, supra, or any other scientifically accepted nomenclature. Alternatively, the sequence of CDR-H1 described herein can range from Kabat 26 to Kabat 35, for example, for the Pb-DOTAM binding variable domain.

[0071] In one embodiment, the CDR residues include those identified in the sequence listing or elsewhere herein.

[0072] Unless otherwise specified, HVR / CDR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

[0073] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including, but not limited to, cytotoxic agents.

[0074] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, e.g., monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is human.

[0075] The molecules described herein may be "isolated." An "isolated" antibody is one that has been separated from components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, as determined, for example, by electrophoretic (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. Chromatogr. B 848:79-87 (2007).

[0076] The terms "nucleic acid molecule" or "polynucleotide" include 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 the sequence of bases, which represent the primary (linear) structure of the nucleic acid molecule. The sequence of bases is typically presented from 5' to 3'. As used herein, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers containing two or more of these molecules. Nucleic acid molecules can be linear or circular. In addition, the term nucleic acid molecule includes both sense and antisense strands, and both single-stranded and double-stranded forms. Furthermore, 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 with derivatized sugar or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for direct expression of the antibodies of the present invention in vitro and / or in vivo, for example, in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors can be unmodified or modified. For example, mRNA can be chemically modified to increase 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 produce antibodies in vivo (see, e.g., Stadler et al, Nature Medicine 2017, published online 12 June 2017, doi:10.1038 / nm.4356 or European Patent No. 2101823(B1)).

[0077] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule that is contained in a cell that ordinarily contains the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0078] An "isolated nucleic acid encoding an antibody" refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present in one or more locations within a host cell.

[0079] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible variant antibodies (which may contain, for example, naturally occurring mutations or arise during production of the monoclonal antibody preparation and are usually present in small amounts). In contrast to polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies according to the invention can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci; such methods, as well as other exemplary methods for producing monoclonal antibodies, are described herein.

[0080] A "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The naked antibody may be present in a pharmaceutical composition.

[0081] "Native antibodies" refer to naturally occurring immunoglobulin molecules with various structures. For example, native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable domain (VH), also called a variable heavy chain domain or heavy chain variable region, followed by three constant heavy chain domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable domain (VL), also called a variable light chain domain or light chain variable region, followed by a constant light chain (CL) domain.

[0082] The term "package insert" is used to refer to instructions customarily included in commercial packaging of therapeutic products that contain information about the indications, usage, dosage, administration, concomitant therapy, contraindications and / or warnings for use of such therapeutic product.

[0083] "Amino acid sequence identity percentage (%)" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps if necessary to achieve the maximum sequence identity percentage, without considering any conservative substitutions as part of the sequence identity for alignment.Alignment for determining amino acid sequence identity percentage can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or FASTA program package.Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared.Alternatively, the sequence comparison computer program ALIGN-2 can be used to generate identity percentage values. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and its source code, along with user documentation, has been submitted to the U.S. Copyright Office (Washington, DC 20559), where it is registered under U.S. Copyright Registration No. TXU510087, and is described in WO 2001 / 007611.

[0084] Unless otherwise noted, for purposes of this specification, percent amino acid sequence identity values ​​are generated using the ggsearch program of the FASTA package, version 36.3.8c or later, with a BLOSUM50 comparison matrix. The FASTA program package was developed by W.R. Pearson and D.J. Lipman (1988), "Improved Tools for Biological Sequence Analysis," PNAS 85:2444-2448; W.R. Pearson (1996), "Effective protein sequence comparison," Meth. Enzymol. 266:227-258; and Pearson et al. (1997) Genomics 46:24-36, and is publicly available at www.fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml or www.ebi.ac.uk / Tools / sss / fasta. Alternatively, sequences can be compared using the public server accessible at fasta.bioch.virginia.edu / fasta_www2 / index.cgi using the ggsearch(global protein:protein) program and default options (BLOSUM50; open:-10; ext:-2; Ktup=2), ensuring global rather than local alignment. Percent amino acid identity is shown in the output alignment header.

[0085] The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a preparation that is in a form that allows the biological activity of the active ingredients contained in the preparation to be effective, and that does not contain additional ingredients that are unacceptably toxic to the subject to whom the pharmaceutical composition will be administered.

[0086] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition or formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0087] As used herein, unless otherwise specified, the reference to a target antigen refers to any natural target antigen from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). This term encompasses "full-length" unprocessed target antigens, as well as any form of target antigen resulting from intracellular processing. This term also encompasses naturally occurring variants of target antigens, such as splice variants or allelic variants. For example, the target antigen CEA may have the amino acid sequence of human CEA, particularly carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), as shown in UniProt (www.uniprot.org) accession number P06731 (version 119), or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_004354.2.

[0088] As used herein, "treatment" (and grammatical variants thereof, e.g., "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of disease in the individual being treated, and may be performed prophylactically or during the course of clinical pathology. Desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, remission or palliation of disease symptoms, and recovery or improved prognosis. In some embodiments, the antibodies of the invention are used to delay the onset of disease or to slow the progression of disease.

[0089] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains (VH and VL, respectively) of a natural antibody generally have a similar structure, with each domain containing four conserved framework regions (FR) and three complementarity-determining regions (CDR). (See, e.g., Kindt et al., Kuby Immunology, 6th (See, e.g., W.H. Freeman and Co., p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen may be isolated using the VH or VL domain of an antibody that binds to that antigen, and then a library of complementary VL or VH domains, respectively, may be screened. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0090] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0091] As used herein, the term "Pb" or "lead" includes its ions, e.g., Pb(II). References to other metals also include their ions. Thus, those skilled in the art will recognize, e.g., lead, Pb, 212 Pb or 203 It is understood that the term Pb is intended to encompass the ionic forms of the element, particularly Pb(II).

[0092] II. Compositions and Methods A. Radiolabeled compounds According to the present invention, a multispecific antibody or split multispecific antibody comprises a binding site for an effector molecule (in a split multispecific antibody formed from a first hemibody and a second hemibody, the first hemibody comprises the VH domain of the antigen-binding site for the effector molecule, and the second hemibody comprises the VL domain of the antigen-binding site for the effector molecule, and a functional binding site is formed upon association of the two hemibodies).

[0093] Effector molecules according to the present invention are radiolabeled compounds containing a radioisotope, for example, radiolabeled haptens.

[0094] In some embodiments, the effector molecule may comprise a chelated radioisotope.

[0095] In some embodiments, the functional binding site for the effector molecule may bind to a chelate comprising a chelator and a radioisotope, hi other embodiments, the antibody may bind to a moiety conjugated to a chelated radioisotope, such as histamine-succinyl-glycine (HSG), digoxigenin, biotin, or caffeine.

[0096] The chelating agent may be, for example, a multidentate molecule such as an aminopolycarboxylic acid or aminopolythiocarboxylic acid, or a salt or functional variant thereof. The chelating agent may be, for example, bidentate, tridentate, or tetradentate. Examples of suitable metal chelating agents include EDTA (ethylenediaminetetraacetic acid, or a salt form such as CaNa2EDTA), DTPA (diethylenetriaminepentaacetic acid), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), NOTA (2,2',2''-(1,4,7-triazanonane-1,4,7-triyl)triacetic acid), IDA (iminodiacetic acid), MIDA ((methylimino)diacetic acid), TTHA (3,6,9,12-tetrakis(trimethylsilyl)-2-hydroxybenzoates), THIA (thiazolinone ... Tetrakis(carboxymethyl)-3,6,9,12-tetra-azatetradecanedioic acid), TETA (2,2',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) acid, available from Macrocyclics, Inc., Plano, Texas), NTA (nitrilotriacetic acid), EDDHA (ethylenediamine-N,N'-bis(2-hydroxyphenylacetic acid), BAL (2,3-dimercaptopropanol), DMSA (2,3-dimercaptosuccinic acid), DMPS (2,3-dimercapto-1-propanesulfonic acid), D-penicillamine (β-dimethylcysteine), MAG3 (mercaptoacetyltriglycine), Hynic (6-hydrazinopyridine-3-carboxylic acid), p-isothiocyanatobenzyl-desferrioxamine (e.g., zirconium-labeled for imaging), and molecules comprising salts or functional variants / derivatives thereof capable of chelating metals. In some embodiments, it may be preferred that the chelator is DOTA or DOTAM, or a salt or functional variant / derivative thereof capable of chelating metals.Thus, the chelating agent may be or include DOTA or DOTAM to which a radioisotope has been chelated.

[0097] Radiolabeled compounds may comprise or consist of functional variants or derivatives of the above-described chelators together with a radionuclide. Suitable variants / derivatives have a somewhat different structure and retain the ability to function as a chelator (i.e., retain sufficient activity to be used for one or more of the purposes described herein). Functional variants / derivatives may also include the above-described chelators conjugated to one or more additional moieties or substituents, including small molecules, polypeptides, or carbohydrates. This attachment may occur through one constituent carbon, for example, in the backbone portion of the chelator. Suitable substituents may be, for example, hydrocarbon groups such as alkyl, alkenyl, aryl, or alkynyl; hydroxy groups; alcohol groups; halogen atoms; nitro groups; cyano groups; sulfonyl groups; thiol groups; amine groups; oxo groups; carboxy groups; thiocarboxy groups; carbonyl groups; amide groups; ester groups; or heterocycles, including heteroaryl groups. Substituents may include, for example, the following groups "R": 1 ". The small molecule can be, for example, a dye (such as Alexa647 or Alexa488), biotin or a biotin moiety, or a phenyl or benzyl moiety. The polypeptide can be, for example, an oligopeptide, such as an oligopeptide of two or three amino acids. Exemplary carbohydrates include dextrans, linear or branched polymers or copolymers (e.g., polyalkylenes, poly(ethylene-lysine), polymethacrylates, polyamino acids, polysaccharides or oligosaccharides, dendrimers). Derivatives can also include multimers of chelator compounds in which the above compounds are linked via linker moieties. Derivatives can also include functional fragments of the above compounds that retain the ability to chelate metal ions.

[0098] Specific examples of derivatives include benzyl-EDTA and hydroxyethyl-thiouride-benzyl EDTA, DOTA-benzene (e.g., (S-2-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecanetetraacetic acid), DOTA-biotin, and DOTA-TyrLys-DOTA. In some embodiments of the invention, the functional binding site for the radioligand binds to a metal chelate comprising DOTAM and a metal, such as lead (Pb). As noted above, "DOTAM" has the chemical name: 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane, A compound of the formula: JPEG2024503654000001.jpg63170.

[0099] In certain aspects and embodiments, the present invention may also utilize functional variants or derivatives of DOTAM incorporating metal ions. Suitable variants / derivatives of DOTAM have a structure that differs to some extent from the structure of DOTAM and retain the ability to function (i.e., retain sufficient activity to be used for one or more of the purposes described herein). In such aspects and embodiments, DOTAM or a functional variant / derivative of DOTAM may be one of the active variants disclosed in WO 2010 / 099536. Suitable functional variants / derivatives have the following formula: JPEG2024503654000002.jpg69170 (in the formula, R N is H, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-7 Cycloalkyl, C 3-7 Cycloalkyl-C 1-4 Alkyl, C 2-7 Heterocycloalkyl, C 2-7 Heterocycloalkyl-C 1-4 Alkyl, phenyl, phenyl-C 1-4 -Alkyl, C1-7 Heteroaryl, and C 1-7 Heteroaryl-C 1-4 -alkyl, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, and C 2-6 Each alkynyl is selected from 1, 2, 3, or 4 independently selected R w optionally substituted with a group, 3-7 Cycloalkyl, C 3-7 Cycloalkyl-C 1-4 Alkyl, C 2-7 Heterocycloalkyl, C 2-7 Heterocycloalkyl-C 1-4 Alkyl, phenyl, phenyl-C 1-4 -Alkyl, C 1-7 Heteroaryl, and C 1-7 Heteroaryl-C 1-4 -alkyl is selected from 1, 2, 3, or 4 independently selected R x optionally substituted with a group, L 1 independently, C 1-6 Alkylene, C 1-6 Alkenylene, or C 1-6 alkynylene, each of which is independently selected from 1, 2, or 3 R 1 optionally substituted with a group, L 2 is C 2-4 linear alkylene, which is an independently selected R 1 optionally substituted with a C group; 1-4 Alkyl and / or C 1-4 optionally substituted with 1, 2, 3, or 4 groups independently selected from haloalkyl; R 1 is D 1 -D 2 -D 3 , halogen, cyano, nitro, hydroxyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, C 1-6 Alkylthio, C 1-6 Alkylsulfinyl, C 1-6Alkyl sulfonyl, amino, C 1-6 Alkylamino, Di-C 1-6 Alkylamino, C 1-4 Alkylcarbonyl, carboxy, C 1-6 Alkoxycarbonyl, C 1-6 Alkylcarbonylamino, di-C 1-6 Alkylcarbonylamino, C 1-6 Alkoxycarbonylamino, C 1-6 Alkoxycarbonyl-(C 1-6 Alkyl)amino, carbamyl, C 1-6 Alkylcarbamyl, and di-C 1-6 independently selected from alkylcarbamyl; Each D 1 is C 6-10 Aryl-C 1-4 Alkyl, C 1-9 Heteroaryl-C 1-4 Alkyl, C 3-10 Cycloalkyl-C 1-4 Alkyl, C 2-9 Heterocycloalkyl-C 1-4 Alkyl, C 1-8 Alkylene, C 1-8 Alkenylene, and C 1-8 alkynylene, wherein C 1-8 Alkylene, C 1-8 Alkenylene, and C 1-8 Alkynylene can be one, two, three, or four independently selected R 4 optionally substituted with a group, 6-10 Aryl-C 1-4 Alkyl, C 1-9 Heteroaryl-C 1-4 Alkyl, C 3-10 Cycloalkyl-C 1-4 Alkyl, C 2-9 Heterocycloalkyl-C 1-4 Each alkyl is selected from 1, 2, 3, or 4 independently selected R 5 optionally substituted with a group, Each D 2 is independently absent or C 1-20 A straight chain alkylene, 1-20Each of the 1 to 6 non-adjacent methylene groups in the linear alkylene is independently selected from -D 4 -part, but the C 1-20 At least one methylene unit in the linear alkylene is -D 4 - part is not arbitrarily replaced by the C 1-20 Straight chain alkylenes can be substituted with halogen, cyano, nitro, hydroxyl, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, Amino, C 1-4 Alkylamino, Di-C 1-4 Alkylamino, C 1-4 Alkylcarbonyl, carboxy, C 1-4 Alkoxycarbonyl, C 1-4 Alkylcarbonylamino, di-C 1-4 Alkylcarbonylamino, C 1-4 Alkoxycarbonylamino, C 1-4 Alkoxycarbonyl-(C 1-4 Alkyl)amino, carbamyl, C 1-4 Alkylcarbamyl, and di-C 1-4 optionally substituted with one or more groups independently selected from alkylcarbamyl; Each D 3 is H, halogen, cyano, nitro, hydroxyl, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-14 Cycloalkyl, C 3-14 Cycloalkyl-C 1-4 Alkyl, C 2-14 Heterocycloalkyl, C 2-14 Heterocycloalkyl-C 1-4 Alkyl, C 6-14 Aryl, C 6-14 Aryl-C 1-4 Alkyl, C 1-13 Heteroaryl, C 1-13 Heteroaryl-C 1-4 alkyl, wherein C 1-6Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Each alkynyl is selected from 1, 2, 3, or 4 independently selected R 6 optionally substituted with a group, 3-14 Cycloalkyl, C 3-14 Cycloalkyl-C 1-4 Alkyl, C 2-14 Heterocycloalkyl, C 2-14 Heterocycloalkyl-C 1-4 Alkyl, C 6-14 Aryl, C 6-14 Aryl-C 1-4 Alkyl, C 1-13 Heteroaryl, C 1-13 Heteroaryl-C 1-4 Each alkyl is selected from 1, 2, 3, or 4 independently selected R 7 optionally substituted with a group, Each D 4 -O-, -S-, -NR a C(=O)-, -NR a C(=S)-, -NR b C(=O)NR c -, -NR b C(=S)NR c -, -S(=O)-, -S(=O)2-, -S(=O)NR a -, -C(=O)-, -C(=S)-, -C(=O)O-, -OC(=O)NR a -, -OC(=S)NR a -, -NR a -, -NR b S(=O)NR c -, and NR b S(=O)NR O - selected independently from Each R 4 and R 6 is halogen, cyano, nitro, hydroxyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 1-4 Alkylthio, C 1-4 Alkylsulfinyl, C 1-4 Alkyl sulfonyl, amino, C 1-4Alkylamino, Di-C 1-4 Alkylamino, C 1-4 Alkylcarbonyl, carboxy, C 1-4 Alkoxycarbonyl, C 1-4 Alkylcarbonylamino, di-C 1-4 Alkylcarbonylamino, C 1-4 Alkoxycarbonylamino, C 1-4 Alkoxycarbonyl-(C 1-4 Alkyl)amino, carbamyl, C 1-4 Alkylcarbamyl, and di-C 1-4 independently selected from alkylcarbamyl; Each R 5 is halogen, cyano, cyanate, isothiocyanate, nitro, hydroxyl, C 1-4 Alkyl, C 2-4 Alkenyl, C 2-4 Alkynyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 1-4 Alkylthio, C 1-4 Alkylsulfinyl, C 1-4 Alkyl sulfonyl, amino, C 1-4 Alkylamino, Di-C 1-4 Alkylamino, C 1-4 Alkylcarbonyl, carboxy, C 1-4 Alkoxycarbonyl, C 1-4 Alkylcarbonylamino, di-C 1-4 Alkylcarbonylamino, C 1-4 Alkoxycarbonylamino, C 1-4 Alkoxycarbonyl-(C 1-4 Alkyl)amino, carbamyl, C 1-4 Alkylcarbamyl, and di-C 1-4 independently selected from alkylcarbamyl; Each R 7 is halogen, cyano, nitro, hydroxyl, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-7 Cycloalkyl, C 3-7 Cycloalkyl-C 1-4Alkyl, C 2-7 Heterocycloalkyl, C 2-7 Heterocycloalkyl-C 1-4 Alkyl, phenyl, phenyl-C 1-4 Alkyl, C 1-7 Heteroaryl, C 1-7 Heteroaryl-C 1-4 Alkyl, -OR O , -SR O , -S(=O)R P , -S(=O)2R P , -S(=O)NR s R t , -C(=O)R P , -C(=O)OR P , -C(=O)NR s R t , -OC(=O)R P , -OC(=O)NR s R t , -NR s R t , -NR q C(=O)R r , -NR q C(=O)OR r , -NR q C(=O)NR r , -NR q S(=O)2R r , and -NR P S(=O)NR s R t Independently selected from the C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Each alkynyl is optionally substituted with 1, 2, 3, or 4 independently selected R′ groups; 3-7 Cycloalkyl, C 3-7 Cycloalkyl-C 1-4 Alkyl, C 2-7 Heterocycloalkyl, C 2-7 Heterocycloalkyl-C 1-4 Alkyl, phenyl, phenyl-C 1-4 Alkyl, C 1-7 Heteroaryl, C 1-7 Heteroaryl-C 1-4each alkyl is optionally substituted with 1, 2, 3, or 4 independently selected R″ groups; Each R a , R b , and R c is H, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-7 Cycloalkyl, C 3-7 Cycloalkyl-C 1-4 Alkyl, C 2-7 Heterocycloalkyl, C 2-7 Heterocycloalkyl-C 1-4 Alkyl, phenyl, phenyl-C 1-4 Alkyl, C 1-7 Heteroaryl, C 1-7 Heteroaryl-C 1-4 alkyl, wherein C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Each alkynyl is selected from 1, 2, 3, or 4 independently selected R w optionally substituted with a group, 3-7 Cycloalkyl, C 3-7 Cycloalkyl-C 1-4 Alkyl, C 2-7 Heterocycloalkyl, C 2-7 Heterocycloalkyl-C 1-4 Alkyl, phenyl, phenyl-C 1-4 Alkyl, C 1-7 Heteroaryl, C 1-7 Heteroaryl-C 1-4 Each alkyl is selected from 1, 2, 3, or 4 independently selected R x optionally substituted with a group, Each R o , R p , R q , R r , R s and R t is H, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C2-6 Alkynyl, C 3-7 Cycloalkyl, C 3-7 Cycloalkyl-C 1-4 Alkyl, C 2-7 Heterocycloalkyl, C 2-7 Heterocycloalkyl-C 1-4 Alkyl, phenyl, phenyl-C 1-4 Alkyl, C 1-7 Heteroaryl, C 1-7 Heteroaryl-C 1-4 alkyl, wherein C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Each alkynyl is selected from 1, 2, 3, or 4 independently selected R y optionally substituted with a group, 3-7 Cycloalkyl, C 3-7 Cycloalkyl-C 1-4 Alkyl, C 2-7 Heterocycloalkyl, C 2-7 Heterocycloalkyl-C 1-4 Alkyl, phenyl, phenyl-C 1-4 Alkyl, C 1-7 Heteroaryl, C 1-7 Heteroaryl-C 1-4 Each alkyl is selected from 1, 2, 3, or 4 independently selected R z optionally substituted with a group, Each R', R w and R y are hydroxyl, cyano, nitro, C 1-4 Alkoxy, C 1-4 Haloalkoxy, Amino, C 1-4 Alkylamino, and di-C 1-4 alkylamino; Each R'', R x , and R z is hydroxyl, halogen, cyano, nitro, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, Amino, C 1-4Alkylamino, and di-C 1-4 alkylamino; provided that the valence of each atom of the optionally substituted moiety is not exceeded. or a pharmaceutically acceptable salt thereof.

[0100] Suitably, functional variants / derivatives of the above formula have an affinity for the antibodies of the invention that is equal to or greater than that of DOTAM, and have a binding strength for Pb that is equal to or greater than that of DOTAM ("affinity" as measured by the dissociation constant as described above). For example, the dissociation constant of a functional variant / derivative with an antibody of the invention or Pb may be 1.1-fold or less, 1.2-fold or less, 1.3-fold or less, 1.4-fold or less, 1.5-fold or less, or 2-fold or less than the dissociation constant of DOTAM with the same antibody / Pb.

[0101] Each R N is H, C 1-6 Alkyl, or C 1-6 It may be haloalkyl, preferably H, C 1-4 Alkyl, or C 1-4 Most preferably, each R N is H.

[0102] In the case of DOTAM variants, 1, 2, 3 or most preferably each 2 is preferably a C2 alkylene. Advantageously, the C2 alkylene variant of DOTAM can have a particularly high affinity for Pb. 2 The optional substituents of R 1 , C 1-4 Alkyl, or C 1-4 It may be haloalkyl. Preferably, L 2 The optional substituents of C 1-4 Alkyl or C 1-4 It may be haloalkyl.

[0103] Optionally, each L 2 may be an unsubstituted C2 alkylene -CH2CH2-.

[0104] Each L 1 is preferably C 1-4 Alkylene, more preferably C1 alkylene such as -CH2-.

[0105] Functional variants / derivatives of DOTAM have the formula: JPEG2024503654000003.jpg64170, where each Z is independently R as defined above. 1 where p, q, r, and s are 0, 1, or 2, and p+q+r+s is greater than or equal to 1. Preferably, p, q, r, and s are 0 or 1, and / or p+q+r+s is 1. For example, the compound may have p+q+r+s=1, and Z is a p-SCN-benzyl moiety; such compounds are commercially available from Macrocyclics, Inc. (Plano, Texas).

[0106] Radionuclides useful in the present invention may include radioactive isotopes of metals such as lead (Pb), lutetium (Lu), or yttrium (Y).

[0107] Particularly useful radionuclides in therapeutic applications are those that are alpha or beta emitters. For example, they are: 212 Pb, 212 Bi, 213 Bi, 90 Y, 177 Lu, 225 Ac, 211 At, 227 Th, 223 Ra.

[0108] In some embodiments, it may be preferred that DOTAM (or a salt or functional variant thereof) be chelated with Pb or Bi, such as one of the Pb or Bi radioisotopes described above, while in other embodiments, it may be preferred that DOTA (or a salt or functional variant thereof) be chelated with Lu or Y, such as one of the Lu or Y radioisotopes described above.

[0109] In some embodiments, the multivalent antibody or multivalent split antibody may bind to a Pb-DOTAM chelate.

[0110] In some embodiments, the multivalent antibody or multivalent split antibody can specifically bind to a radiolabeled compound. In some embodiments, it can bind to a Pb-DOTAM and / or target with a dissociation constant (K D ) is ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -7 M or less, e.g. 10 -7 ~10 -13 M, 10 -8 M or less, e.g. 10 -8 M~10-13M, e.g., 10 -9 M~10 -13 In some embodiments, it can bind to a radiolabeled compound, e.g., a Pb-DOTAM chelate, with a K of binding affinity of 100 pM, 50 pM, 20 pM, 10 pM, 5 pM, 1 pM or less, e.g., 0.9 pM or less, 0.8 pM or less, 0.7 pM or less, 0.6 pM or less, or 0.5 pM or less. D For example, a functional binding site may have a K of about 1 pM to 1 nM, e.g., about 1 to 10 pM, 1 to 100 pM, 5 to 50 pM, 100 to 500 pM, or 500 pM to 1 nM. D can bind to metal chelates at

[0111] B. Exemplary antigen binding sites for DOTA In one particular embodiment of the invention, the antibody comprises a functional binding site for DOTA (or a functional derivative or variant thereof), or the first hemibody and the second hemibody are associated to bind DOTA (or a functional derivative or variant thereof), for example, DOTA chelated to Lu or Y (e.g., 177 Lu or 90Y). For example, the functional binding site has a K of about 1 pM to 1 nM, e.g., about 1 to 10 pM, 1 to 100 pM, 5 to 50 pM, 100 to 500 pM, or 500 pM to 1 nM. D The radiolabeled compound can be bound to the antibody.

[0112] The C825 is 177 Lu and 90 C825 is a known scFv with high affinity for DOTA-Bn (S-2-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecanetetraacetic acid) conjugated to a radiometal such as Y (see, e.g., Cheal et al. 2018, Theranostics 2018, and WO 2010099536, incorporated herein by reference). The CDR sequences and VL and VH sequences of C825 are provided herein. In one embodiment, the heavy chain variable region forming part of the antigen-binding site for the radiolabeled compound may comprise at least one, two, or all three CDRs selected from (a) CDR-H1 comprising a 35-amino acid sequence, (b) CDR-H2 comprising a 36-amino acid sequence, and (c) CDR-H3 comprising a 37-amino acid sequence. In an alternative embodiment, CDR-H1 may have the sequence GFSLTDYGVH. The light chain variable region that forms part of the binding site for the radiolabeled compound may comprise at least one, two, or all three CDRs selected from (d) CDR-L1 comprising a 38 amino acid sequence, (e) CDR-L2 comprising a 39 amino acid sequence, and (f) CDR-L3 comprising a 40 amino acid sequence.

[0113] In another embodiment, the heavy chain variable domain that forms part of the functional antigen-binding site for a radiolabeled compound (e.g., on the first hemibody in the case of a split antibody) comprises the amino acid sequence of SEQ ID NO: 41, or a variant thereof comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO: 41. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the binding site comprising that sequence retains the ability to bind DOTA complexed to Lu or Y, preferably with the affinity described herein. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 41. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the antibody or first hemibody comprises the VH sequence of SEQ ID NO: 41, including post-translational modifications of that sequence. In certain embodiments, the VH comprises one, two, or three CDRs selected from (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 35 or the sequence GFSLTDYGVH, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 36, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 37.

[0114] Optionally, the light chain variable domain that forms part of the functional antigen-binding site for the radiolabeled compound (e.g., on the second hemibody in the case of a split antibody) 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 contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the binding site comprising that sequence retains the ability to bind DOTA complexed to Lu or Y, preferably with the affinity described herein. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 42. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the antibody or second hemibody comprises the VL sequence of SEQ ID NO: 42, including post-translational modifications of that sequence. In certain embodiments, the VL comprises one, two, or three CDRs selected from (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 38, (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 39, and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 40.

[0115] The embodiments relating to the heavy chain variable region and the light chain variable region are expressly contemplated in combination. Thus, a functional antigen-binding site can be formed from the heavy chain variable region defined above and the light chain variable region defined above. In the case of a split antibody, these can be on the first hemibody and the second hemibody, respectively.

[0116] In any of the above embodiments, the light chain variable region and heavy chain variable region that form the binding site for the DOTA conjugate may be humanized. In one embodiment, the light chain variable region and heavy chain variable region comprise the CDRs of any of the above embodiments and further comprise an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0117] In some embodiments, the heavy chain variable domain may be extended by one or more C-terminal residues, such as one or more C-terminal alanine residues, or one or more residues from the N-terminus of the CH1 domain, as discussed further below.

[0118] C. Exemplary antigen binding sites for DOTAM In another specific embodiment of the invention, the antibody comprises a functional binding site for Pb-DOTAM chelate (Pb-DOTAM), or the first hemibody and second hemibody associate to form a functional antigen-binding site for Pb-DOTAM chelate (Pb-DOTAM).

[0119] Exemplary antigen-binding sites are described in WO 2019 / 201959, which is incorporated by reference in its entirety.

[0120] In certain embodiments, a functional antigen binding site that binds Pb-DOTAM may have one or more of the following properties: ● Specific binding to Pb-DOTAM and Bi-DOTAM. • Selective for Pb-DOTAM (and optionally Bi-DOTAM) compared to other chelated metals such as Cu-DOTAM; ● Binds to Pb-DOTAM with very high affinity, ● Binds to the same epitope on Pb-DOTAM as an antibody described in WO 2019 / 201959, such as PRIT-0213 or PRIT-0214, and / or has the same contact residues as said antibody.

[0121] Radioisotopes of Pb are useful in methods of treatment. Specific radioisotopes of lead that can be used in the present invention include: 212 Examples include Pb.

[0122] Radionuclides that are alpha particle emitters have the potential for less damage to surrounding tissue and more specific tumor cell killing than beta emitters due to a combination of short path lengths and high linear energy deposition. 212 Bi is an alpha particle emitter, but its short half-life precludes its direct use. 212 Pb 212 is the parent radionuclide of Bi, 212 It can act as an in vivo generator of Bi, thereby 212 This effectively overcomes the short half-life of Bi (Yong and Brechbiel, Dalton Trans. 2001 June 21;40(23)6068-6076).

[0123] Generally, radiometals are used in chelated form. In a specific embodiment of the present invention, DOTAM is used as a chelating agent. DOTAM is a stable chelating agent for Pb(II) (Yong and Brechbiel, Dalton Trans. 2001 June 21; 40(23)6068-6076; Chappell et al. Nuclear Medicine and Biology, Vol. 27, pp. 93-100, 2000). Therefore, DOTAM is 212 It is particularly useful in conjunction with the lead isotopes mentioned above, such as Pb.

[0124] In some embodiments, the antibody has a K of binding affinity of 100 pM, 50 pM, 20 pM, 10 pM, 5 pM, 1 pM or less, e.g., 0.9 pM or less, 0.8 pM or less, 0.7 pM or less, 0.6 pM or less, or 0.5 pM or less. D For example, a functional binding site may bind Pb-DOTAM with a K of about 1 pM to 1 nM, e.g., about 1 to 10 pM, 1 to 100 pM, 5 to 50 pM, 100 to 500 pM, or 500 pM to 1 nM. D The radiolabeled compound can be bound to the antibody.

[0125] In certain embodiments, the antibody further binds to Bi chelated by DOTAM. In some embodiments, the antibody has a K of binding affinity of 1 nM, 500 pM, 200 pM, 100 pM, 50 pM, 10 pM or less, e.g., 9 pM, 8 pM, 7 pM, 6 pM, 5 pM or less. D It may be preferable for a functional binding site to bind to Bi-DOTAM (i.e., a chelate comprising DOTAM complexed with bismuth, also referred to herein as a "Bi-DOTAM chelate") with a K value of about 1 pM to 1 nM, e.g., about 1-10 pM, 1-100 pM, 5-50 pM, 100-500 pM, or 500 pM to 1 nM. D can bind to metal chelates at

[0126] In some embodiments, the antibody may bind to Bi-DOTAM and Pb-DOTAM with similar affinity. For example, the ratio of affinities for Bi-DOTAM / Pb-DOTAM, e.g., K D The ratio of the values ​​may preferably be in the range of 0.1 to 10, for example, 1 to 10.

[0127] In one embodiment, the heavy chain variable region that forms part of the antigen-binding site for Pb-DOTAM (e.g., on the first hemibody in the case of a split antibody) 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); and (c) CDR-H3 comprising the amino acid sequence of ERDPYGGGAYPPHL (SEQ ID NO: 3). The light chain variable region that forms part of the binding site for Pb-DOTAM (e.g., on the second hemibody in the case of a split antibody) 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 QASKLAS (SEQ ID NO: 5), and (f) CDR-L3 comprising the amino acid sequence of LGGYDDESDTYG (SEQ ID NO: 6).

[0128] In some embodiments, the antibody may comprise one or more CDR-H1, CDR-H2 and / or CDR-H3, or one or more CDR-L1, CDR-L2 and / or CDR-L3, each having a substitution, e.g., one, two or three substitutions, compared to the amino acid sequence of SEQ ID NOs: 1-6.

[0129] In some embodiments, the antibodies may share the same contact residues as those described herein, e.g., these residues may be invariant. These residues may include: a) in the heavy chain CDR2: Phe50, Asp56 and / or Tyr58, and optionally Gly52 and / or Arg54, b) in the heavy chain CDR3: Glu95, Arg96, Asp97, Pro98, Tyr99, Ala100C and / or Tyr100D, and optionally Pro100E, c) in the light chain CDR1: Tyr28 and / or Asp32, d) in the light chain CDR3: Gly91, Tyr92, Asp93, Thr95c and / or Tyr96, e) in light chain CDR2: optionally Gln50, All numbered according to Kabat.

[0130] For example, in some embodiments, CDR-H2 may comprise the amino acid sequence FIGSRGDTYYASWAKG (SEQ ID NO:2), or a variant thereof having up to one, two, or three substitutions in SEQ ID NO:2, wherein the substitutions do not include Phe50, Asp56, and / or Tyr58, and optionally do not include Gly52 and / or Arg54, all numbered according to Kabat.

[0131] In some embodiments, CDR-H2 may be substituted at one or more positions as shown below. Here, and in the substitution table below, substitutions are based on germline residues (underlined) or with amino acids that theoretically sterically fit and also occur in the crystallization repertoire at that site. In some embodiments, the residues above may be fixed, and other residues may be substituted according to the table below; in other embodiments, substitution of any residue may be made according to the table below. JPEG2024503654000004.jpg124170

[0132] Optionally, CDR-H3 can comprise the amino acid sequence ERDPYGGGAYPPHL (SEQ ID NO: 3), or a variant thereof having up to one, two, or three substitutions in SEQ ID NO: 3, wherein the substitutions do not include Glu95, Arg96, Asp97, Pro98, and optionally do not include Ala100C, Tyr100D, and / or Pro100E, and / or optionally do not include Tyr99. For example, in some embodiments, the substitutions do not include Glu95, Arg96, Asp97, Pro98, Tyr99, Ala100C, and Tyr100D.

[0133] In certain embodiments, CDR-H3 may be substituted at one or more positions as shown below: In some embodiments, the above residues may be fixed and other residues may be substituted according to the table below, and in other embodiments, substitution of any residue may be made according to the table below. JPEG2024503654000005.jpg109170

[0134] Optionally, CDR-L1 may comprise the amino acid sequence QSSHSVYSDNDLA (SEQ ID NO: 4) or a variant thereof having up to one, two, or three substitutions in SEQ ID NO: 4, which substitutions do not include Tyr28 and / or Asp32 (Kabat numbering).

[0135] In certain embodiments, CDR-L1 may be substituted at one or more positions as shown below: Also, in some embodiments, the above residues may be fixed and other residues may be substituted according to the table below, and in other embodiments, substitution of any residue may be made according to the table below. JPEG2024503654000006.jpg101170

[0136] Optionally, CDR-L3 may comprise the amino acid sequence LGGYDDESDTYG (SEQ ID NO: 6) or a variant thereof having up to one, two, or three substitutions in SEQ ID NO: 6, which substitutions do not include Gly91, Tyr92, Asp93, Thr95c, and / or Tyr96 (Kabat).

[0137] In certain embodiments, CDR-L3 may be substituted at the following positions as shown below (many substitutions are possible because most residues are solvent exposed and not in contact with the antigen). Also, in some embodiments, the above residues may be fixed, while other residues may be substituted according to the table below, and in other embodiments, substitution of any residue may be made according to the table below. JPEG2024503654000007.jpg123170

[0138] The antibody may further comprise a CDR-H1 or CDR-L2 optionally having the sequence of SEQ ID NO: 1 or SEQ ID NO: 5, respectively, or a variant thereof having at least one, two or three substitutions therein, optionally conservative substitutions.

[0139] Thus, the heavy chain variable domain that forms part of the antigen binding site for Pb-DOTAM may comprise at least: a) a heavy chain CDR2 comprising the amino acid sequence FIGSRGDTYYASWAKG (SEQ ID NO: 2), or a variant thereof having up to one, two, or three substitutions in SEQ ID NO: 2, wherein these substitutions do not include Phe50, Asp56, and / or Tyr58, and optionally also do not include Gly52 and / or Arg54; b) A heavy chain CDR3 comprising the amino acid sequence ERDPYGGGAYPPHL (SEQ ID NO: 3), or a variant thereof having up to one, two, or three substitutions in SEQ ID NO: 3, wherein these substitutions do not include Glu95, Arg96, Asp97, Pro98, and optionally do not include Ala100C, Tyr100D, and / or Pro100E, and / or optionally do not include Tyr99.

[0140] In some embodiments, the heavy chain variable domain optionally comprises: c) further comprising a heavy chain CDR1 which comprises the amino acid sequence GFSLSTYSMS (SEQ ID NO: 1), or a variant thereof having up to 1, 2 or 3 substitutions in SEQ ID NO: 1.

[0141] 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 existing antibodies that recognize the free VH region. As reported in Holland MC et al. J. Clin Immunol (2013), the free C-terminus appears to be important for the binding of HAVH (human anti-VH domain) autoantibodies to VH domain antibodies, because HAVH autoantibodies do not bind to intact IgG or IgG fragments (fAbs or modified VH molecules) containing the same VH framework sequence, or to VK domain antibodies. Cordy JC et al. Clinical and Experimental Immunology (2015) describe the presence of cryptic epitopes in the C-terminal epitopes of VH dAbs that are not naturally accessible to HAVH antibodies in intact IgG molecules.

[0142] Thus, when an antibody comprises a free VH region (not fused at its C-terminus to any other domain), the sequence may be extended by one or more C-terminal residues. This extension may prevent binding of an antibody that recognizes the free VH region. For example, the extension may be by 1 to 10 residues, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues. In one embodiment, the VH sequence may be extended by one or more C-terminal alanine residues. The VH sequence may also be extended by the N-terminal portion of a CH1 domain, e.g., by 1 to 10 residues from the N-terminus of the CH1 domain, e.g., from the human IgG1 CH1 domain. (The first 10 residues of the human IgG1 CH1 domain are ASTKGPSVFP (SEQ ID NO: 149), and therefore, in one embodiment, 1 to 10 residues may be taken from the N-terminus of this sequence.) For example, in one embodiment, the peptide sequence AST (corresponding to the first three residues of the IgG1 CH1 domain) is added to the C-terminus of the VH region.

[0143] In another embodiment, the light chain variable domain that forms part of the antigen binding site for Pb-DOTAM comprises at least: d) a light chain CDR1 comprising the amino acid sequence QSSHSVYSDNDLA (SEQ ID NO: 4) or a variant thereof having up to one, two, or three substitutions in SEQ ID NO: 4, wherein these substitutions do not include Tyr28 and Asp32; e) A light chain CDR3 comprising the amino acid sequence LGGYDDESDTYG (SEQ ID NO: 6) or a variant thereof having up to one, two, or three substitutions in SEQ ID NO: 6, wherein these substitutions do not include Gly91, Tyr92, Asp93, Thr95c, and Tyr96.

[0144] In some embodiments, the light chain variable domain optionally comprises: f) further comprising a light chain CDR2 comprising the amino acid sequence QASKLAS (SEQ ID NO: 5), or a variant thereof having at least one, two or three substitutions in SEQ ID NO: 5, optionally excluding Gln50.

[0145] In any embodiment of the invention that includes variants of sequences comprising the above CDRs (eg, of the variable domain), the protein may be unchanged in one or more of the CDR residues described above.

[0146] Optionally, the heavy chain variable domain forming part of the functional antigen-binding site for Pb-DOTAM (e.g., on the first hemibody in the case of a split antibody) 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. (The N-terminal amino acids in these reference sequences, shown in parentheses, may be present or absent and, in some embodiments, may be retained in any variant sequence.) In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the binding site comprising that sequence retains the ability to bind Pb-DOTAM, preferably with the affinity described herein. The VH sequence may retain the invariant residues described 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 substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the antibody comprises the VH sequence of SEQ ID NO:7 or SEQ ID NO:9 (with or without the N-terminal residue shown in parentheses), including post-translational modifications of that sequence. In certain embodiments, the VH comprises one, two, or three CDRs selected from (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:1, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:3.

[0147] In some embodiments, as described above, in some variants, SEQ ID NO: 7 or 9 may be extended by one or more additional C-terminal residues, for example, one or more alanine residues, optionally a single alanine residue. Thus, for example, in one particular variant, the sequence of SEQ ID NO: 7 may be extended to become: VTLKESGPVLVKPTETLTLTCTVSGFSLSTYSMSWIRQPPGKALEWLGFIGSRGDTYYASWAKGRLTISKDTSKSQVVLTMTNMMDPVDTATYYCARERDPYGGGAYPPHLWGRGTLVTVSSA

[0148] In other embodiments, the extension may be by the N-terminal portion of the CH1 domain as described above, for example by 1 to 10 residues from the N-terminus of the CH1 domain, for example from the human IgG1 CH1 domain. For example, the extension may be by the peptide sequence AST.

[0149] Optionally, the light chain variable domain forming part of the functional antigen-binding site for Pb-DOTAM (e.g., on the second hemibody in the case of a split 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. (The N-terminal amino acid in this reference sequence, shown in parentheses, may be present or absent and, in some embodiments, may be retained in any variant.) In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an anti-Pb-DOTAM binding site comprising that sequence retains the ability to bind Pb-DOTAM, preferably with the affinity described herein. The VL sequence may retain the invariant residues described above. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 8. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-Pb-DOTAM antibody comprises the VL sequence of SEQ ID NO: 8 (with or without the N-terminal residue shown in parentheses), including post-translational modifications of that sequence. In certain embodiments, the VL comprises one, two, or three CDRs selected from (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

[0150] The embodiments relating to the heavy chain variable region and the light chain variable region are expressly contemplated in combination. Thus, a functional antigen-binding site for Pb-DOTAM can be formed from the heavy chain variable region defined above and the light chain variable region defined above. In the case of a split antibody, these can be on the first hemibody and the second hemibody, respectively.

[0151] Optionally, an antigen-binding site specific for Pb-DOTAM chelate may be formed from a heavy chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 7 or SEQ ID NO: 9 (with or without the N-terminal residue shown in parentheses), or a variant thereof as defined above (including variants with the C-terminal extensions described above), and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 8 (with or without the N-terminal residue shown in parentheses), or a variant thereof as defined above. For example, an antigen-binding site specific for Pb-DOTAM chelate may comprise a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 7 or a variant thereof, and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 8 or a variant thereof, including post-translational modifications of these sequences. In another embodiment, it may comprise a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 9 or a variant thereof (including variants with the C-terminal extensions described above), and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 8 or a variant thereof, including post-translational modifications of these sequences.

[0152] In any of the above embodiments, the light and heavy chain variable regions that form the anti-Pb-DOTAM binding site may be humanized. In one embodiment, the light and heavy chain variable regions comprise the CDRs of any of the above embodiments and further comprise an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework. In another embodiment, the light and / or heavy chain variable regions comprise the CDRs of any of the above embodiments and further comprise framework regions derived from vk139 and / or vh226. For vk139, in some embodiments, there may be no backmutation. For vh226, the germline Ala49 residue may be backmutated to Gly49.

[0153] D. Target antigen The multispecific antibodies (or split multispecific antibodies) used in the combination therapy bind to a target antigen, which is an antigen expressed on the surface of a target cell. This may also be referred to as a "target cell antigen."

[0154] The treatment is preferably the treatment of a tumor or cancer.

[0155] The target antigen can be, for example, a tumor-associated antigen.

[0156] As used herein, the term "tumor-associated antigen" or "tumor-specific antigen" refers to any molecule (e.g., protein, peptide, lipid, carbohydrate, etc.) that is expressed or overexpressed solely or predominantly by tumor cells and / or cancer cells, or other cells in the tumor stroma, such as cancer-associated fibroblasts, such that the antigen is associated with tumor(s) and / or cancer(s). Tumor-associated antigens may also be expressed by normal, non-tumor, or non-cancerous cells. However, in such cases, expression of tumor-associated antigens by normal, non-tumor, or non-cancerous cells is not as robust as expression by tumor or cancer cells. In this regard, tumor or cancer cells may overexpress or express the antigen at significantly higher levels than expression by normal, non-tumor, or non-cancerous cells. Tumor-associated antigens may also be expressed by cells in different states of development or maturation. For example, tumor-associated antigens may also be expressed by cells at embryonic or fetal stages, which are not normally found in adult hosts. Alternatively, tumor-associated antigens may also be expressed by stem or progenitor cells, which are not normally found in the adult host.

[0157] A tumor-associated antigen can be an antigen expressed by any cell of any cancer or tumor, including the cancers and tumors described herein. A tumor-associated antigen can be a tumor-associated antigen of only one type of cancer or tumor, such that the tumor-associated antigen is associated with or characteristic of only one type of cancer or tumor. Alternatively, the tumor-associated antigen can be a tumor-associated antigen of (e.g., characteristic of) two or more types of cancers or tumors. For example, a tumor-associated antigen can be expressed by both breast cancer cells and prostate cancer cells, but not at all by normal cells, non-tumor cells, or non-cancer cells.

[0158] Exemplary tumor-associated antigens to which the antibodies of the invention may bind include, but are not limited to, melanoma-associated chondroitin sulfate proteoglycan (MCSP), mucin 1 (MUCl; tumor-associated epithelial mucin), preferentially expressed antigen of melanoma (PRAME), carcinoembryonic antigen (CEA), prostate-specific membrane antigen (PSMA), PSCA, EpCAM, Trop2 (also known as trophoblast-2, 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 TRP2. In another embodiment, the tumor antigen is cluster of differentiation (CD) 19, CD20, CD21, CD22, CD25, CD30, CD33 (sialic acid-binding Ig-like lectin 3, myeloid cell surface antigen), CD79b, CD123 (interleukin-3 receptor alpha), transferrin receptor, EGF receptor, mesothelin, cadherin, 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), L1 The mesothelin may be selected from the group consisting of CAM (neural cell adhesion molecule L1), endosialin, HER3 (epidermal growth factor receptor family member 3 activated conformation), Alkl / BMP9 complex (anaplastic lymphoma kinase 1 / bone morphogenetic protein 9), TPBG=5T4 (trophoblast glycoprotein), ROR1 (receptor tyrosine kinase-like surface antigen), HER1 (epidermal growth factor receptor activated conformation), and CLL1 (C-type lectin domain family 12, member A). Mesothelin is expressed, for example, in ovarian cancer, mesothelioma, non-small cell lung cancer, lung adenocarcinoma, fallopian tube cancer, head and neck cancer, cervical cancer, and pancreatic cancer. CD22 is expressed, for example, in hairy cell leukemia, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), non-Hodgkin's lymphoma, small lymphocytic lymphoma (SLL), and acute lymphocytic leukemia (ALL).CD25 is expressed in leukemia and lymphoma, including hairy cell leukemia and Hodgkin's lymphoma.Lewis Y antigen is expressed in bladder cancer, breast cancer, ovarian cancer, colorectal cancer, esophageal cancer, gastric cancer, lung cancer and pancreatic cancer.CD33 is expressed in acute myeloid leukemia (AML), chronic myelomonocytic leukemia (CML) and myeloproliferative disorders.

[0159] Exemplary antibodies that specifically bind to tumor-associated antigens include, but are not limited to, antibodies against the transferrin receptor (e.g., HB21 and its variants), antibodies against CD22 (e.g., RFB4 and its variants), antibodies against CD25 (e.g., anti-Tac and its variants), antibodies against mesothelin (e.g., SS1, MORAb-009, SS, HN1, HN2, MN, MB, and variants thereof), and antibodies against the Lewis Y antigen (e.g., B3 and its variants). In this regard, the targeting moiety (cell-binding agent) can be an antibody selected from the group consisting of B3, RFB4, SS, SS1, MN, MB, HN1, HN2, HB21, and MORAb-009, and antigen-binding portions thereof. Further exemplary targeting moieties suitable for use in the chimeric molecules of the present invention are described, for example, in U.S. Pat. Nos. 5,242,824 (anti-transferrin receptor); 5,846,535 (anti-CD25); 5,889,157 (anti-Lewis Y); 5,981,726 (anti-Lewis Y); 5,990,296 (anti-Lewis Y); 7,081,518 Nos. 7,355,012 (anti-mesothelin), 7,368,110 (anti-mesothelin), 7,470,775 (anti-CD30), 7,521,054 (anti-CD25), and 7,541,034 (anti-CD22), U.S. Patent Application Publication No. 2007 / 0189962 (anti-CD22), Frankel et al., Clin. Cancer Res., 6:326-334 (2000), and Kreitman et al., AAPS Journal, 8(3):E532-E551 (2006), each of which is incorporated herein by reference.

[0160] Additional antibodies have been produced to target specific tumor-associated antigens: Cripto, CD30, CD19, CD33, glycoprotein NMB, CanAg, Her2 (ErbB2 / Neu), CD56 (NCAM), CD22 (Siglec2), CD33 (Siglec3), CD79, CD138, PSCA, PSMA (prostate-specific membrane antigen), BCMA, CD20, CD70, E-selectin, EphB2, melanotransferrin, Mucl6, and TMEFF2. Any of these, or antigen-binding fragments thereof, may be useful in the present invention, i.e., may be incorporated into the antibodies described herein.

[0161] In some embodiments of the present invention, it may be preferred that the tumor-associated antigen is carcinoembryonic antigen (CEA).

[0162] CEA is advantageous in the context of the present invention because it is internalized relatively slowly, and therefore a high percentage of antibody remains available on the cell surface for binding to the radionuclide after initial treatment. Other low-internalization targets / tumor-associated antigens may also be preferred. Other examples of tumor-associated antigens include CD20 or HER2. In yet further embodiments, the target may be EGP-1 (epithelial glycoprotein-1, also known as trophoblast-2), colon-specific antigen-p (CSAp), or pancreatic mucin MUC1. See, for example, Goldenberg et al. 2012 (Theranostics 2(5)), which is incorporated herein by reference. This reference also describes antibodies such as Mu-9, which binds to CSAp (see also Sharkey et al Cancer Res. 2003;63:354-63), hPAM4, which binds to MUC1 (see also Gold et al Cancer Res. 2008:68:4819-26), valtuzumab, which binds to CD20 (see also Sharkey et al Cancer Res. 2008;68:5282-90), and hRS7, which binds to EGP-1 (see also Cubas et al Biochim Biophys Acta 2009;1796:309-14). Any of these, or antigen-binding portions thereof, may be useful in the present invention, i.e., may be incorporated into the antibodies described herein. One example of an antibody that has been raised against CEA is T84.66 (set forth in NCBI Accession Nos. CAA36980 (heavy chain) and CAA36979 (light chain), or set forth in SEQ ID NOs: 317 and 318 of WO 2016 / 075278), as well as humanized and chimeric versions thereof, such as T84.66-LCHA, described in WO 2016 / 075278(A1) and / or WO 2017 / 055389. Another example is CH1A1a, an anti-CEA antibody described in WO 2012 / 117002 and WO 2014 / 131712, and CEA hMN-14 (see also U.S. Pat. Nos. 6,676,924 and 5,874,540).Another anti-CEA antibody is A5B7, described in MJ Banfield et al., Proteins 1997, 29(2), 161-171. Humanized antibodies derived from the murine antibody A5B7 are disclosed in WO 92 / 01059 and WO 2007 / 071422. See also co-pending application PCT / EP2020 / 067582. An example of a humanized version of A5B7 is A5H1EL1(G54A). Further exemplary antibodies against CEA are MFE23 and its humanized versions, described in U.S. Patent No. 7,626,011 and / or co-pending application PCT / EP2020 / 067582. Yet another example of an antibody against CEA is 28A9. Any of these or antigen-binding fragments thereof may be useful in forming a CEA-binding portion in the present invention.

[0163] In some embodiments, antibodies of the invention can specifically bind to a target antigen (e.g., any of the target antigens discussed herein). In some embodiments, they have an affinity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -7 M or less, e.g. 10 -7 ~10 -13 M, 10 -8 M or less, e.g. 10 -8 M~10 -13 M, e.g. 10 -9 M~10 -13 Dissociation constant (K D ) can be bonded.

[0164] E. Exemplary antigen-binding sites for CEA In certain embodiments of the invention that may be combined with the above-described embodiments, such as exemplary binding sites for radiolabeled compounds and DOTA / DOTAM, the target antigen bound by the multispecific antibody or by the first and / or second hemibody of a split multispecific antibody may be CEA (carcinoembryonic antigen). Antibodies that have been produced against CEA include T84.66 and its humanized and chimeric versions, such as T84.66-LCHA, CH1A1a, as described in WO 2016 / 075278(A1) and / or WO 2017 / 055389, the anti-CEA antibodies described in WO 2012 / 117002 and WO 2014 / 131712, and CEA hMN-14 or labetuzimab (as described, for example, in U.S. Pat. Nos. 6,676,924 and 5,874,540). Another exemplary antibody against CEA is A5B7 (e.g., as described in MJ Banfield et al., Proteins 1997, 29(2), 161-171), or a humanized antibody derived from murine A5B7 described in WO 92 / 01059 and WO 2007 / 071422. See also co-pending application PCT / EP2020 / 067582. An example of a humanized version of A5B7 is A5H1EL1(G54A). Further exemplary antibodies against CEA are MFE23 and humanized versions thereof, described in U.S. Pat. No. 7,626,011 and / or co-pending application PCT / EP2020 / 067582. Yet another example of an anti-CEA antibody is 28A9. Any of these, or antigen-binding fragments thereof, can be used to form a CEA-binding portion in the present invention.

[0165] Optionally, the antigen-binding portion that binds CEA has a K for monovalent binding of 1 nM or less, 500 pM or less, 200 pM or less, or 100 pM or less. D Can be combined by value.

[0166] In some embodiments, the first hemibody and / or the second hemibody of the multispecific antibody or split multispecificity may bind to the CH1A1a epitope, the A5B7 epitope, the MFE23 epitope, the T84.66 epitope, or the 28A9 epitope of CEA.

[0167] In some embodiments, the first and / or second hemibody of a multispecific antibody or split multispecific antibody binds to a CEA epitope that is not present on soluble CEA (sCEA). Soluble CEA is the portion of the CEA molecule that is cleaved by GPI phospholipase and released into the blood. An example of an epitope that is not found on soluble CEA is the CH1A1A epitope. Optionally, in the case of a split antibody, one of the first and / or second hemibody binds to an epitope that is not present on soluble CEA, and the other binds to an epitope that is present on soluble CEA.

[0168] The epitope for CH1A1a and its parent murine antibody PR1A3 are described in International Publication No. 2012 / 117002 (A1) and Durbin H. et al., Proc. Natl. Scad. Sci. USA, 91:4313-4317, 1994. Antibodies that bind to the CH1A1a epitope bind to a conformational epitope within the B3 domain and GPI anchor of the CEA molecule. In one embodiment, the antibody binds to the same epitope as the CH1A1a antibody having VH of SEQ ID NO: 25 and VL of SEQ ID NO: 26 herein. The A5B7 epitope is described in co-pending application PCT / EP2020 / 067582. Antibodies that bind to the A5B7 epitope bind to the A2 domain of CEA, i.e., the domain containing the amino acid sequence of SEQ ID NO: 141:

[0169] PKPFITSNNSNPVEDEDAVALTCEPEIQNTTYLWWVNNQSLPVSPRLQLSNDNRTLTLLSVTRNDVGP YECGIQNKLSVDHSDPVILN (SEQ ID NO: 141).

[0170] In one aspect, the antibody binds to the same epitope as the A5B7 antibody having a VH of SEQ ID NO: 49 and a VL of SEQ ID NO: 50 herein.

[0171] In one embodiment, the antibody binds to the same epitope as T84.66 described in WO 2016 / 075278. The antibody may bind to the same epitope as the antibody having VH of SEQ ID NO: 17 and VL of SEQ ID NO: 18 herein.

[0172] The MFE23 epitope is described in co-pending application PCT / EP2020 / 067582. Antibodies that bind to the MFE23 epitope bind to the A1 domain of CEA, i.e., the domain comprising the amino acid sequence of SEQ ID NO: 142: PKPSISSNNSKPVEDKDAVAFTCEPETQDATYLWWVNNQSLPVSPRLQLSNGNRTLTLFNVTRNDTAS YKCETQNPVSARRSDSVILN (SEQ ID NO: 142).

[0173] In one aspect, the antibody may bind to the same epitope as an antibody having a VH domain of SEQ ID NO:127 and a VL domain of SEQ ID NO:128 herein.

[0174] In some embodiments of split multispecific antibodies, the first and second hemibodies bind to the same epitope on CEA, for example, the first and second hemibodies may both bind to the CH1A1a, A5B7, MFE23, T84.66, or 28A9 epitope.

[0175] In some embodiments, both the first and second hemibodies may have CEA-binding sequences (i.e., CDRs and / or VH / VL domains) from CH1A1A, or both the first and second hemibodies may have CEA-binding sequences from A5B7 or a humanized version thereof, or both the first and second hemibodies may have CEA-binding sequences from T84.66 or a humanized version thereof, or both the first and second hemibodies may have CEA-binding sequences from MFE23 or a humanized version thereof, or both the first and second hemibodies may have CEA-binding sequences from 28A9 or a humanized version thereof. Exemplary sequences are disclosed herein.

[0176] In other embodiments, the first and second hemibody bind to different epitopes of CEA. Thus, for example, i) one hemibody may bind to the CH1A1A epitope and the other may bind to the A5B7, T84.66, MFE23, or 28A9 epitope; ii) one hemibody may bind to the A5B7 epitope and the other may bind to the CH1A1A, T84.66, MFE23, or 28A9 epitope; or iii) one hemibody may bind to the MFE23 epitope and the other may bind to the CH1A1A epitope. iv) one hemibody can bind the T84.66 epitope and the other can bind the CH1A1A, A5B7, MFE23, or 28A9 epitope; or v) one hemibody can bind the 28A9 epitope and the other can bind the CH1A1a, A5B7, MFE23, or T84.66 epitope.

[0177] In some embodiments, i) one hemibody may have a CEA-binding sequence (i.e., CDRs or VH / VL domains) from CH1A1A and the other may have a CEA-binding sequence from A5B7 or a humanized version thereof, T84.66 or a humanized version thereof, MFE23 or a humanized version thereof, or 28A9 or a humanized version thereof; ii) one hemibody may have a CEA-binding sequence from A5B7 or a humanized version thereof and the other may have a CEA-binding sequence from CH1A1A, T84.66 or a humanized version thereof, MFE23 or a humanized version thereof, or 28A9 or a humanized version thereof; iii) one hemibody may have a CEA-binding sequence from MFE23 or a humanized version thereof and the other , CH1A1A, A5B7 or a humanized version thereof, T84.66 or a humanized version thereof, or 28A9 or a humanized version thereof; iv) one hemibody may have a CEA-binding sequence derived from T84.66 or a humanized version thereof, and the other may have a CEA-binding sequence derived from CH1A1A, A5B7 or a humanized version thereof, MFE23 or a humanized version thereof, or 28A9 or a humanized version thereof; v) one hemibody may have a CEA-binding sequence derived from 28A9 or a humanized version thereof, and the other may have a CEA-binding sequence derived from CH1A1A, A5B7 or a humanized version thereof, T84.66 or a humanized version thereof, or MFE23 or a humanized version thereof.

[0178] In one specific embodiment, one hemibody can bind to the CH1A1A epitope and the other can bind to the A5B7 epitope. The first hemibody can have the CEA-binding sequence from antibody CH1A1A and the second hemibody can have the CEA-binding sequence from A5B7 (including humanized versions thereof), or the first hemibody can have the CEA-binding sequence from antibody A5B7 (including humanized versions thereof) and the second hemibody can have the CEA-binding sequence from CH1A1A.

[0179] In another specific embodiment, one hemibody can bind to the CH1A1A epitope and the other can bind to the T84.66 epitope. The first hemibody can have a CEA-binding sequence from antibody CH1A1A, and the second hemibody can have a CEA-binding sequence from T84.66 (including humanized versions thereof). Alternatively, the first hemibody can have a CEA-binding sequence from antibody T84.66 (including humanized versions thereof), and the second hemibody can have a CEA-binding sequence from CH1A1A. In some embodiments, the first hemibody can bind to the T84.66 epitope and / or have an antigen-binding site as described in (i) below, and the second hemibody can bind to the CH1A1A epitope and / or have an antigen-binding site as described in (ii) below.

[0180] Exemplary CEA-binding sequences i)-v) are disclosed below, which provide examples of CEA-binding sequences from i) T84.66, ii) CH1A1A, iii) A5B7, iv) 28A9, and v) MFE23 (or humanized versions thereof).

[0181] i). In one embodiment, the antigen-binding site that binds to CEA may comprise at least one, two, three, four, five, or six CDRs selected from (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 11, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 12, (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 13, (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 14, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 15, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 16.

[0182] 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.

[0183] 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.

[0184] 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.

[0185] In another aspect, the antigen-binding site that binds to CEA comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 11, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 12, (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 13, (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 14, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 15, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 16.

[0186] In any of the above embodiments, the multispecific antibody may be humanized. In one embodiment, the anti-CEA antigen-binding site comprises the CDRs of any of the above embodiments and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0187] In another embodiment, the antigen-binding site that binds to CEA comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 17. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the antigen-binding site comprising that sequence retains the ability to bind to CEA, preferably with the affinity described above. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 17. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the antigen-binding site that binds to CEA comprises the VH sequence of SEQ ID NO: 17, including post-translational modifications of that sequence. In certain embodiments, the VH comprises one, two, or three CDRs selected from (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 11, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 12, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 13.

[0188] In another embodiment, the antigen-binding site that binds to CEA comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 18. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the antigen-binding site comprising that sequence retains the ability to bind to CEA, preferably with the affinity described above. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 18. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the antigen-binding site against CEA comprises the VL sequence of SEQ ID NO: 18, including post-translational modifications of that sequence. In certain embodiments, the VL comprises one, two, or three CDRs selected from (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 14, (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 15, and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 16.

[0189] In another embodiment, the antigen-binding site that binds to CEA comprises a VH of any of the embodiments provided above and a VL of any of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 17 and SEQ ID NO: 18, respectively, including post-translational modifications of those sequences.

[0190] ii) In further specific embodiments, the antigen-binding site that binds to CEA may comprise at least one, two, three, four, five, or six CDRs selected from (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 19, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 20, (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 21, (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 24.

[0191] 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.

[0192] Optionally, the antigen-binding site that binds to CEA comprises at least one, at least two, or all three VL CDR sequences selected from (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22, (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 24.

[0193] 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.

[0194] In another embodiment, the antigen-binding site that binds to CEA comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 19, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 20, (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 21, (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 24.

[0195] In any of the above embodiments, the multispecific antibody may be humanized. In one embodiment, the anti-CEA antigen-binding site comprises the CDRs of any of the above embodiments and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0196] In another embodiment, the antigen-binding site that binds to CEA comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 25. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the antigen-binding site comprising that sequence retains the ability to bind to CEA, preferably with the affinity described above. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 25. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the antigen-binding site that binds to CEA comprises the VH sequence of SEQ ID NO: 25, including post-translational modifications of that sequence. In certain embodiments, the VH comprises one, two, or three CDRs selected from (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 19, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 20, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 21.

[0197] In another embodiment, the antigen-binding site that binds to CEA comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 26. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the antigen-binding site comprising that sequence retains the ability to bind to CEA, preferably with the affinity described above. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 26. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVR (i.e., in the FR). Optionally, the antigen-binding site against CEA comprises the VL sequence of SEQ ID NO: 26, including post-translational modifications of that sequence. In certain embodiments, the VL comprises one, two, or three CDRs selected from (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22, (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23, and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 24.

[0198] In another embodiment, the antigen-binding site that binds to CEA comprises a VH of any of the embodiments provided above and a VL of any of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 25 and SEQ ID NO: 26, respectively, including post-translational modifications of those sequences.

[0199] iii) In further specific embodiments, 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: 43, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 44, (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 45, (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 46, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 47, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 48. In some embodiments, CDR-H1 may have the sequence GFTFTDYYMN (SEQ ID NO: 151).

[0200] 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, CDR-H1 may have the sequence GFTFTDYYMN (SEQ ID NO: 151).

[0201] 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.

[0202] 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, CDR-H1 may have the sequence GFTFTDYYMN (SEQ ID NO: 151).

[0203] In another aspect, the antigen-binding site that binds to CEA comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 43, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 44, (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 45, (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 46, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 47, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 48. In some embodiments, CDR-H1 may have the sequence GFTFTDYYMN (SEQ ID NO: 151).

[0204] In any of the above embodiments, the multispecific antibody may be humanized. In one embodiment, the anti-CEA antigen-binding site comprises the CDRs of any of the above embodiments and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0205] In another embodiment, an antigen-binding site that binds to CEA comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 49. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the antigen-binding site comprising that sequence retains the ability to bind to CEA, preferably with the affinity described above. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 49. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the antigen-binding site that binds to CEA comprises the VH sequence of SEQ ID NO: 49, including post-translational modifications of that sequence. In certain embodiments, the VH comprises one, two, or three CDRs selected from (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 43 or the sequence GFTFTDYYMN (SEQ ID NO: 151), (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 44, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 45.

[0206] In another embodiment, the antigen-binding site that binds to CEA comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 50. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the antigen-binding site comprising that sequence retains the ability to bind to CEA, preferably with the affinity described above. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 50. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the antigen-binding site against CEA comprises the VL sequence of SEQ ID NO: 50, including post-translational modifications of that sequence. In certain embodiments, the VL comprises one, two, or three CDRs selected from (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 46, (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 47, and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 48.

[0207] In another embodiment, the antigen-binding site that binds to CEA comprises a VH of any of the embodiments provided above and a VL of any of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 49 and SEQ ID NO: 50, respectively, including post-translational modifications of those sequences.

[0208] iv) In still further specific embodiments, 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.

[0209] 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.

[0210] 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.

[0211] 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.

[0212] In another aspect, the antigen-binding site that binds to CEA comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 59, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 60, (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 61, (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 62, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 63, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 64.

[0213] In any of the above embodiments, the multispecific antibody may be humanized. In one embodiment, the anti-CEA antigen-binding site comprises the CDRs of any of the above embodiments and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0214] In another embodiment, the antigen-binding site that binds to CEA comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 65. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the antigen-binding site comprising that sequence retains the ability to bind to CEA, preferably with the affinity described above. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 65. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the antigen-binding site that binds to CEA comprises the VH sequence of SEQ ID NO: 65, including post-translational modifications of that sequence. In certain embodiments, the VH comprises one, two, or three CDRs selected from (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 59, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 60, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 61.

[0215] In another embodiment, the antigen-binding site that binds to CEA comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 66. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the antigen-binding site comprising that sequence retains the ability to bind to CEA, preferably with the affinity described above. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 66. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the antigen-binding site against CEA comprises the VL sequence of SEQ ID NO: 66, including post-translational modifications of that sequence. In certain embodiments, the VL comprises one, two, or three CDRs selected from (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 62, (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 63, and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 64.

[0216] In another embodiment, the antigen-binding site that binds to CEA comprises a VH of any of the embodiments provided above and a VL of any of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 65 and SEQ ID NO: 66, respectively, including post-translational modifications of those sequences.

[0217] v). In still further specific embodiments, 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: 116, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 117 or 118, (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 119, (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 120, 121, or 122, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 123, 124, or 125, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 126.

[0218] Optionally, the antigen binding site that binds to CEA is VH CDR sequences: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 116, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 117 or 118, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 119, and / or The VL CDR sequences may include (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 120, 121 or 122, (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 123, 124 or 125, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 126.

[0219] In one embodiment, the antigen-binding site against CEA comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 127, or (more preferably) an amino acid sequence selected from SEQ ID NOs: 129, 130, 131, 132, 133 or 134, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 128, or (more preferably) an amino acid sequence selected from SEQ ID NOs: 135, 136, 137, 138, 139 or 140.

[0220] In any of the above embodiments, the multispecific antibody may be humanized. In one embodiment, the anti-CEA antigen-binding site comprises the CDRs of any of the above embodiments and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0221] In a particular embodiment, the antigen binding domain capable of binding to CEA is (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 129 and a VL domain comprising the amino acid sequence of SEQ ID NO: 139, or (b) a VH domain comprising the amino acid sequence of SEQ ID NO: 133 and a VL domain comprising the amino acid sequence of SEQ ID NO: 139; or (c) a VH domain comprising the amino acid sequence of SEQ ID NO: 130 and a VL domain comprising the amino acid sequence of SEQ ID NO: 139; or (d) a VH domain comprising the amino acid sequence of SEQ ID NO: 134 and a VL domain comprising the amino acid sequence of SEQ ID NO: 138; or (e) a VH domain comprising the amino acid sequence of SEQ ID NO: 133 and a VL domain comprising the amino acid sequence of SEQ ID NO: 138; or (f) a VH domain comprising the amino acid sequence of SEQ ID NO: 131 and a VL domain comprising the amino acid sequence of SEQ ID NO: 138; or (g) A VH domain comprising the amino acid sequence of SEQ ID NO: 129 and a VL domain comprising the amino acid sequence of SEQ ID NO: 138.

[0222] 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 described in a) through g) above. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the antigen-binding site comprising that sequence retains the ability to bind to CEA, preferably with the affinity described above. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs).

[0223] 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 set forth in a) through g) above. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the antigen-binding site comprising that sequence retains the ability to bind to CEA, preferably with the affinity described above. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs).

[0224] In another embodiment, an antigen binding site that binds to CEA comprises a VH of any of the embodiments provided above and a VL of any of the embodiments provided above.

[0225] F. Exemplary Multispecific Antibodies The multispecific antibodies used in the present invention can be in various formats. Exemplary formats are described in International Publication No. 2019 / 201959, which is incorporated herein by reference, and any of the formats described therein can be applied. Specific exemplary antibodies are also described in International Publication No. 2019 / 201959, and any of these specific antibodies can also be selected for use in the present invention.

[0226] In some embodiments, the multispecific antibody may contain an Fc domain. The presence of an Fc region has advantages in the context of radioimmunotherapy and radioimaging, for example, by extending the circulating half-life of the protein and / or resulting in higher tumor uptake than can be observed with smaller fragments. The Fc domain may be engineered to reduce or eliminate Fc effector function.

[0227] One exemplary format comprises a full-length antibody (e.g., an IgG) comprising a first hemibody and a second antibody heavy chain and a first hemibody and a second antibody light chain, where the first heavy chain and the first light chain assemble to form an antigen binding site for a radiolabeled compound, and the second heavy chain and the second light chain assemble to form an antigen binding site for a target antigen.

[0228] Correct assembly of the heavy heterodimer chains can be aided, for example, by the use of knob-into-hole mutations and / or other modifications as discussed further below.

[0229] The correct assembly of a light chain and its respective heavy chain can be aided by using cross-mab technology. In this approach, either a first heavy chain and a first light chain, or a second heavy chain and a second light chain, can assemble to form a cross-Fab fragment (while the other assembles to form a conventional Fab). Thus, in one embodiment, the first heavy chain can contain a VL domain instead of a VH domain (e.g., VL-CH1-hinge-CH2-CH3), the first light chain can contain a VH domain swapped with a VL domain (e.g., VH-CL), or the first heavy chain can contain a CL domain instead of an HC1 domain (e.g., VH-CL-hinge-CH2-CH3), and the first light chain can contain a CH1 domain instead of a CL domain (e.g., VL-CH1). In this embodiment, the second heavy chain and the second light chain have conventional domain structures (e.g., VH-CH1-hinge-CH2-CH3 and VL-CL, respectively). In alternative embodiments, the second heavy chain may contain a VL domain instead of a VH domain (e.g., VL-CH1-hinge-CH2-CH3), the second light chain may contain a VH domain exchanged with a VL domain (e.g., VH-CL), or the second heavy chain may contain a CL domain instead of an HC1 domain (e.g., VH-CL-hinge-CH2-CH3), and the second light chain may contain a CH1 domain instead of a CL domain (e.g., VL-CH1). In this embodiment, the first heavy chain and the first light chain have conventional domain structures.

[0230] In some embodiments, the correct assembly of a light chain with its respective heavy chain may additionally or alternatively be aided by the use of charge modifications, as discussed further below.

[0231] In some embodiments of the above format, the format can be bivalent. In another possible embodiment, an additional antigen-binding moiety can be fused, for example, to the first hemibody and / or the second heavy chain to increase the valency of one or both antigens. For example, an additional antigen-binding moiety for a target antigen can be fused to the N-terminus of one or both heavy chain molecules. In some embodiments, the antibody can be multivalent, for example, bivalent, for tumor-associated antigens and monovalent for radiolabeled compounds.

[0232] The additional antigen-binding portion may be, for example, an scFab, comprising an antigen-binding site for a first antigen (e.g., a tumor-associated antigen). In another embodiment, the additional antigen-binding portion is a Fab or cross-Fab. For example, the N-terminus or C-terminus of one of the heavy chains may be linked via a polypeptide linker to a first polypeptide consisting of a VH domain and a CH1 domain, and the first polypeptide is associated with a second polypeptide consisting of a VL domain and a CL domain to form a Fab. In another embodiment, the N-terminus or C-terminus of one of the heavy chains may be linked via a polypeptide linker to a first polypeptide consisting of a VL domain and a CH1 domain, and the first polypeptide is associated with a second polypeptide consisting of a VH domain and a CL domain. In another embodiment, the N-terminus or C-terminus of one of the heavy chains may be linked via a polypeptide linker to a first polypeptide consisting of a VH domain and a CL domain, and the first polypeptide is associated with a second polypeptide consisting of a VL domain and a CH1 domain.

[0233] In this format, it may be preferable that binding arms of the same antigen specificity are formed by association with the same light chain. Thus, the antigen-binding moiety / arm for the target antigen can be a cross-Fab, and the antigen-binding moiety(s) / arm(s) for the radiolabeled compound can be a conventional Fab. Alternatively, the antigen-binding moiety / arm for the target antigen can be a conventional Fab, and the antigen-binding moiety(s) / arm(s) for the radiolabeled compound can be a cross-Fab.

[0234] The format may also incorporate charge modifications, as discussed further below.

[0235] Another exemplary format includes a full-length antibody, such as IgG, containing an antigen-binding site for the target antigen (which may be, for example, bivalent for the target antigen), linked to an antigen-binding moiety for a radiolabeled compound.

[0236] For example, the antigen-binding portion for a radiolabeled compound can be an scFab containing an antigen-binding site for a radiolabeled compound (e.g., Pb-DOTAM chelate). In some embodiments, the scFab can be fused to the C-terminus of one of the two heavy chains of a full-length antibody, e.g., the C-terminus of its CH3 domain. Correct assembly of the heterodimeric heavy chains can be aided, for example, by the use of knob-into-hole mutations and / or other modifications as discussed further below.

[0237] Another exemplary format comprises a full-length antibody comprising an antigen-binding site for a target antigen (e.g., which may be bivalent for the target antigen), wherein the N- or C-terminus of one of the heavy chains is linked via a polypeptide linker to a first polypeptide, and the first polypeptide associates with a second polypeptide to form a Fab or cross-Fab comprising a binding site for a radiolabeled compound. For example, this format can be i) a first polypeptide consisting of a VH domain and a CH1 domain associated with a second polypeptide consisting of a VL domain and a CL domain; or ii) a first polypeptide consisting of a VL domain and a CH1 domain associated with a second polypeptide consisting of a VH domain and a CL domain; or iii) a first polypeptide consisting of a VH domain and a CL domain associated with a second polypeptide consisting of a VL domain and a CH1 domain, The first polypeptide and the second polypeptide may be comprised such that together they form an antigen binding site for a radiolabeled compound.

[0238] Correct assembly of the heavy chain heterodimers can be aided by the use of knob-into-hole mutations and / or other modifications, as discussed further below, including charge modifications. For example, the Fab domain of a full-length antibody can contain charge modifications.

[0239] In another exemplary format, the antibody is a) a full-length antibody that specifically binds to a target antigen and is composed of two antibody heavy chains and two antibody light chains; b) i) an antibody heavy chain variable domain (VH), or ii) an antibody heavy chain variable domain (VH) and an antibody heavy chain constant domain (CH1), or iii) a polypeptide consisting of an antibody heavy chain variable domain (VH) and an antibody light chain constant domain (CL), a polypeptide in which the polypeptide is fused by the N-terminus of the VH domain to the C-terminus of one of the two heavy chains of the full-length antibody via a peptide linker; c) i) an antibody light chain variable domain (VL), or ii) an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL), or iii) a polypeptide consisting of an antibody light chain variable domain (VL) and an antibody heavy chain constant domain (CH1), a polypeptide in which the polypeptide is fused by the N-terminus of the VL domain to the C-terminus of the other of the two heavy chains of the full-length antibody via a peptide linker; The antibody heavy chain variable domain (VL) of peptide (b) and the antibody light chain variable domain of peptide (c) together form an antigen-binding site for the radiolabeled compound.

[0240] In this format, if the first polypeptide is as shown in b(i), the second polypeptide is as shown in c(i), if the first polypeptide is as shown in b(ii), the second polypeptide is as shown in c(ii), and if the first polypeptide is as shown in b(iii), the second polypeptide is as shown in c(iii). Charge-modifying substitutions may also be used, for example, in the Fab of a full-length antibody.

[0241] Optionally, the structure may be stabilized, whereby the antibody heavy chain variable region (VH) of the polypeptide of (b) and the antibody light chain variable domain (VL) of the polypeptide of (c) are linked and stabilized via an interchain disulfide bridge by introducing a disulfide bond between the following positions (numbering always according to the Kabat EU index): i) from position 44 in the heavy chain variable domain to position 100 in the light chain variable domain; ii) from position 105 of the heavy chain variable domain to position 43 of the light chain variable domain, or iii) from position 101 in the heavy chain variable domain to position 100 in the light chain variable domain.

[0242] Examples of the above formats in which the antibody (b) consists of a VH domain and the antibody (c) consists of a VL domain are PRIT213 (also called PRIT-0213) and PRIT214 (also called PRIT-0214) described in WO 2019 / 201959. Thus, in certain embodiments, multispecific antibodies for use in the present invention comprise: i) a first heavy chain having the amino acid sequence of SEQ ID NO: 22; ii) a second heavy chain having the amino acid sequence of SEQ ID NO: 23, and iii) may comprise two antibody light chains having the amino acid sequence of SEQ ID NO: 21; The sequence numbering is that of WO 2019 / 201959.

[0243] In another embodiment, the multispecific antibody for use in the present invention comprises: i) a first heavy chain having the amino acid sequence of SEQ ID NO: 19; ii) a second heavy chain having the amino acid sequence of SEQ ID NO: 20, and iii) may comprise two antibody light chains having the amino acid sequence of SEQ ID NO: 21; The sequence numbering is that of WO 2019 / 201959.

[0244] G. Exemplary Formats of Split Multispecific Antibodies In other embodiments, the antibody for use in the combination therapy may be a split multispecific antibody. A split multispecific antibody comprises: i) a first hemibody that binds to an antigen expressed on the surface of a target cell and further comprises a VH domain of an antigen-binding site for a radiolabeled compound, but does not comprise a VL domain of an antigen-binding site for a radiolabeled compound; ii) a second hemibody that binds to an antigen expressed on the surface of a target cell and further comprises a VL domain of an antigen-binding site for a radiolabeled compound, but does not comprise a VH domain of an antigen-binding site for a radiolabeled compound; The VH domain of the first hemibody and the VL domain of the second hemibody can together form a functional antigen binding site for a radiolabeled compound.

[0245] The first hemibody and the second hemibody can bind to the same target antigen at the same or different epitopes.

[0246] In some embodiments, the first and second hemibodies can each comprise an Fc domain, which can be engineered to reduce or eliminate Fc effector function.

[0247] In some embodiments, as described above, when the VH domain of the antigen-binding site for a radiolabeled compound is free at its C-terminus (e.g., not fused to another domain via its C-terminus), it can be extended by one or more residues to avoid binding of HAVH autoantibodies. For example, the extension can be by 1 to 10 residues, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues. In one embodiment, it can be extended by one or more alanine residues, optionally one alanine residue. The VH sequence can also be extended by the N-terminal portion of the CH1 domain, e.g., by 1 to 10 residues from the N-terminus of the CH1 domain, e.g., from the human IgG1 CH1 domain. (Because the first 10 residues of the human IgG1 CH1 domain are ASTKGPSVFP, in one embodiment, 1 to 10 residues can be removed from the N-terminus of this sequence.) For example, in one embodiment, the peptide sequence AST (corresponding to the first three residues of the IgG1 CH1 domain) is added to the C-terminus of the VH region. In some embodiments, the first hemibody and / or the second hemibody can each be multivalent, e.g., bivalent, with respect to the target antigen (e.g., tumor-associated antigen), which has the advantage of increasing avidity.

[0248] In some embodiments, it may be preferable that when the first and second hemibodies associate, they form a monovalent antibody complex for the radiolabeled compound. Thus, the first hemibody may contain only one VH domain of the antigen-binding site for the radiolabeled compound, and the second hemibody may contain only one VL domain of the antigen-binding site for the radiolabeled compound, so that together they form only one complete functional binding site for the radiolabeled compound.

[0249] Each hemibody can comprise: i) at least one antigen-binding moiety (e.g., an antibody fragment) capable of binding to a target antigen; ii) either a VL domain or a VH domain of an antigen-binding site for a 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 the target antigen. The antigen-binding moiety (e.g., an antibody fragment) can be fused a) to either the VL domain or the VH domain of the antigen-binding site for a radiolabeled compound, or b) if the antibody comprises an Fc region, to an Fc region fused to either the VL domain or the VH domain of the antigen-binding site for a radiolabeled compound. In some embodiments, the C-terminus of the Fc region is fused to the N-terminus of the VL domain or the VH domain.

[0250] Fusion may be direct or indirect. In some embodiments, fusion may be via a linker. For example, an Fc region may be fused to an antibody fragment via a hinge region or another suitable linker. Similarly, the VL or VH domain of the antigen-binding site for a radiolabeled compound may be connected to the rest of the antibody structure via a linker. In one particular embodiment, the first hemibody comprises: a) an scFv fragment, wherein the scFv fragment binds to a target antigen; b) i) an antibody heavy chain variable domain (VH), or ii) a polypeptide comprising or consisting of 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, The polypeptide may comprise or consist of a polypeptide fused by the N-terminus of the VH domain, preferably via a peptide linker, to the C-terminus of the scFv fragment.

[0251] The second hemibody is c) a second scFv that binds to the target antigen; and d) i) an antibody light chain variable domain (VL), or ii) a polypeptide comprising or consisting of 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, The polypeptide may comprise or consist of a polypeptide fused by the N-terminus of the VL domain, preferably via a peptide linker, to the C-terminus of the scFv fragment.

[0252] The antibody heavy chain variable domain (VH) of the first hemibody and the antibody light chain variable domain (VL) of the second hemibody together form a functional antigen binding site for a radiolabeled compound upon association of the two hemibodies.

[0253] Optionally, the polypeptide of portion b(i) may further comprise one or more residues, optionally one or more alanine residues, optionally a single alanine residue, at the C-terminus of the VH domain. Optionally, the additional residues may be the N-terminal portion of the CH1 domain described above, e.g., 1 to 10 residues from the N-terminus of the CH1 domain, e.g., from the human IgG1 CH1 domain. For example, the additional residues may be AST.

[0254] The target antigen-recognizing variable domains of the heavy and light chains of scFv can be connected by a peptide tether. Such a peptide tether can contain 1 to 25 amino acids, preferably 12 to 20 amino acids, and preferably 12 to 16 or 15 to 20 amino acids. The tether can contain one or more (G3S) and / or (G4S) motifs, particularly one, two, three, four, five, or six (G3S) and / or (G4S) motifs, preferably three or four (G3S) and / or (G4S) motifs, and more preferably three or four (G4S) motifs.

[0255] Optionally, the first hemibody can consist essentially of components (a) and (b) above, and the second hemibody can consist essentially of components (c) and (d) above. In either case, the first hemibody does not contain an antibody light chain variable domain (VL) capable of associating with component (b) of the first hemibody to form a functional antigen-binding site for a radiolabeled compound, and the second hemibody does not contain an antibody heavy chain variable domain (VH) capable of associating with component (d) of the second hemibody to form a functional antigen-binding site for a radiolabeled compound.

[0256] In another specific embodiment, the first hemibody is a) a Fab fragment that binds to a target antigen; b) i) an antibody heavy chain variable domain (VH) of the antigen-binding site for a radiolabeled compound, or ii) a polypeptide comprising or consisting of an antibody heavy chain variable domain (VH) and an antibody heavy chain constant domain of an antigen-binding site for a radiolabeled compound, wherein the C-terminus of the VH domain is fused to the N-terminus of the constant domain, The polypeptide may comprise or consist of a polypeptide fused by the N-terminus of the VH domain, preferably via a peptide linker, to the C-terminus of the CL or CH1 domain of the Fab fragment.

[0257] The second hemibody is c) a Fab fragment that binds to the target antigen; d) iii) an antibody light chain variable domain (VL) of the antigen-binding site for a radiolabeled compound; or iv) a polypeptide comprising or consisting of an antibody light chain variable domain (VL) and an antibody light chain constant domain of an antigen-binding site for a radiolabeled compound, wherein the C-terminus of the VL domain is fused to the N-terminus of the constant domain, The polypeptide may comprise or consist of a polypeptide fused by the N-terminus of the VL domain, preferably via a peptide linker, to the C-terminus of the CL or CH1 domain of the Fab fragment.

[0258] The antibody heavy chain variable domain (VH) of the polypeptide of (b) and the antibody light chain variable domain (VL) of the polypeptide of (d) together (i.e., upon association of the two hemibodies) form a functional antigen-binding site for a radiolabeled compound.

[0259] Optionally, the polypeptide of portion b(i) may further comprise one or more residues, optionally one or more alanine residues, optionally a single alanine residue, at the C-terminus of said VH domain. Optionally, the additional residues may be the N-terminal portion of said CH1 domain, e.g., 1 to 10 residues from the N-terminus of the CH1 domain, e.g., from the human IgG1 CH1 domain. For example, the additional residues may be AST.

[0260] Optionally, the first hemibody can consist essentially of components (a) and (b) above, and the second hemibody can consist essentially of components (c) and (d) above. In either case, the first hemibody does not contain an antibody light chain variable domain (VL) capable of associating with component (b) of the first hemibody to form a functional antigen-binding site for a radiolabeled compound, and the second hemibody does not contain an antibody heavy chain variable domain (VH) capable of associating with component (d) of the second hemibody to form a functional antigen-binding site for a radiolabeled compound.

[0261] The chains of the Fab fragments fused to the polypeptides can be selected independently for the first and second hemibodies. Thus, in one embodiment, the polypeptide (b) is fused to the C-terminus of the CH1 domain of the Fab fragment of the first hemibody, and the polypeptide (d) is fused to the C-terminus of the CH1 domain of the Fab fragment of the second hemibody. In another embodiment, the polypeptide (b) is fused to the C-terminus of the CL domain of the Fab fragment of the first hemibody, and the polypeptide (d) is fused to the C-terminus of the CL domain of the Fab fragment of the second hemibody. In another embodiment, the polypeptide (b) is fused to the C-terminus of the CH1 domain of the Fab fragment of the first hemibody, and the polypeptide (d) is fused to the C-terminus of the CL domain of the Fab fragment of the second hemibody. In a further embodiment, the polypeptide of (b) is fused to the C-terminus of the CL domain of the Fab fragment of the first hemibody, and the polypeptide of (d) is fused to the C-terminus of the CH1 domain of the Fab fragment of the second hemibody.

[0262] As described above, in some embodiments, the first hemibody and / or the second hemibody can each be multivalent, e.g., bivalent, with respect to the target antigen (e.g., a tumor-associated antigen). This has the advantage of increasing avidity. The hemibodies can be multivalent, e.g., bivalent, and each can be monospecific for a particular epitope (which may be the same epitope for the first hemibody and the second hemibody, or may be different epitopes for the first hemibody and the second hemibody). Thus, in some embodiments, the first hemibody can comprise: i) two or more antigen-binding moieties (e.g., antibody fragments) capable of binding to the same epitope of the target antigen; ii) either a VL domain or a VH domain (but not both) of the antigen-binding site for the radiolabeled compound; and iii) optionally an Fc region. The second hemibody may comprise: i) two or more antigen-binding moieties (e.g., antibody fragments) capable of binding to the same epitope of the target antigen; ii) either a VL domain or a VH domain (but not both) of an antigen-binding site for a radiolabeled compound; and iii) optionally an Fc region. As noted above, the epitopes may be the same for the first and second hemibody or may be different for the first and second hemibody.

[0263] For example, each of the first and second hemibodies can comprise a tandem Fab, i.e., two Fab fragments connected via a peptide linker (Fab-linker-Fab), in which the first Fab is connected via its C-terminus to the N-terminus of the second Fab.

[0264] In one embodiment, the first hemibody is a) a tandem Fab comprising two Fab fragments, wherein a first Fab fragment and a second Fab fragment bind to the same target antigen ("target antigen A"), the epitope bound by the first Fab fragment is the same as the epitope bound by the second Fab fragment, and the first Fab fragment and the second Fab fragment are connected via a peptide linker, and the first Fab is connected via its C-terminus to the N-terminus of the second Fab; b) i) an antibody heavy chain variable domain (VH), or ii) a polypeptide comprising or consisting of an antibody heavy chain variable domain (VH) and an antibody constant domain (CH1), wherein the C-terminus of the VH domain is fused to the N-terminus of the CH1 domain, a polypeptide fused by the N-terminus of the VH domain, preferably via a peptide linker, to the C-terminus of the CL or CH1 domain of a second Fab fragment, The second hemibody is c) a tandem Fab comprising two Fab fragments, wherein a first Fab fragment and a second Fab fragment bind to target antigen A, the epitope bound by the first Fab fragment is the same as the epitope bound by the second Fab fragment, the first Fab fragment and the second Fab fragment are connected via a peptide linker, and the first Fab is connected via its C-terminus to the N-terminus of the second Fab; d) i) an antibody light chain variable domain (VL), or ii) a polypeptide comprising or consisting of an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL), wherein the C-terminus of the VH domain is fused to the N-terminus of the constant domain, a polypeptide in which the polypeptide is fused by the N-terminus of the VL domain, preferably via a peptide linker, to the C-terminus of the CL or CH1 domain of a second Fab fragment.

[0265] The antibody heavy chain variable domain (VH) of portion b (in the first hemibody) and the antibody light chain variable domain (VL) of portion (d) (in the second hemibody) together, i.e., upon association of the two hemibodies, form a functional antigen-binding site for a radiolabeled compound.

[0266] Optionally, the polypeptide of portion b(i) may further comprise one or more residues, optionally one or more alanine residues, optionally a single alanine residue, at the C-terminus of the VH domain. Optionally, the additional residues may be the N-terminal portion of the CH1 domain described above, e.g., 1 to 10 residues from the N-terminus of the CH1 domain, e.g., from the human IgG1 CH1 domain. For example, the additional residues may be AST.

[0267] The chain of the Fab tandem fused to the polypeptide (i.e., whether the polypeptide is fused to the CL domain or CH1 domain of the second Fab fragment) can be selected independently for the first hemibody and the second hemibody.

[0268] As described above, the first Fab fragment of the Fab tandem is connected to the N-terminus of the second Fab fragment. In one embodiment, the C-terminus of the heavy chain fragment of the first Fab fragment is connected to the N-terminus of the heavy or light chain fragment of the second Fab fragment. In another embodiment, the C-terminus of the light chain fragment of the first Fab fragment is connected to the N-terminus of the heavy or light chain fragment of the second Fab fragment. Thus, in some embodiments, the Fab tandem of the first hemibody and / or second hemibody may comprise three chains as follows: 1) a light chain fragment of a first Fab fragment ((VLCL)1), a heavy chain fragment of a first Fab fragment connected via a peptide linker to a heavy chain fragment of a second Fab fragment ((VHCH1)1-linker-(VHCH1)2), and a light chain fragment of a second Fab fragment ((VLCL)2); or 2) a light chain fragment of a first Fab fragment ((VLCL)1), a heavy chain fragment of a first Fab fragment connected via a peptide linker to a light chain fragment of a second Fab fragment ((VHCH1)1-linker-(VLCL)2), and a heavy chain fragment of a second Fab fragment ((VH-CH1)2); or 3) a heavy chain fragment of a first Fab fragment (VHCH1), a light chain fragment of a first Fab fragment connected via a peptide linker to a light chain fragment of a second Fab fragment ((VLCL)1-linker-(VLCL)2), and a heavy chain fragment of a second Fab fragment; or 4) a heavy chain fragment of a first Fab fragment (VHCH1), a light chain fragment of a first Fab fragment connected via a peptide linker to a heavy chain fragment of a second Fab fragment ((VLCL)1-linker-(VHCH1)2), and a light chain fragment of a second Fab fragment ((VLCL)2).

[0269] In another embodiment, the first hemibody and / or the second hemibody can each bind to two or more, optionally two different, epitopes of the target antigen. Thus, one or both of the hemibodies can be biparatopic with respect to the target antigen. In some embodiments, the first hemibody and the second hemibody can each comprise: i) an antigen-binding portion (e.g., an antibody fragment) capable of binding to a first epitope of the target antigen; ii) an antigen-binding portion (e.g., an antibody fragment) capable of binding to a second epitope of the target antigen; iii) either a VL domain or a VH domain (but not both) of an antigen-binding site for a radiolabeled compound; and iv) optionally an Fc region.

[0270] In such embodiments, the correct assembly of a light chain with its respective heavy chain can be aided by using cross-mab technology. For example, in one embodiment, each hemibody can comprise a tandem Fab, including one Fab and one cross-Fab, where one fragment selected from the Fab and the cross-Fab is specific for a first epitope and the other is specific for a second epitope.

[0271] In one particular example, the first hemibody comprises: a) a tandem Fab comprising a first fragment and a second fragment, wherein the first fragment is connected by its C-terminus to the N-terminus of the second fragment via a peptide linker, the first fragment binds to a first epitope of a target antigen, and the second fragment binds to a second epitope of the target antigen, 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) i) an antibody heavy chain variable domain (VH), or ii) a polypeptide comprising or consisting of an antibody heavy chain variable domain (VH) and an antibody heavy chain constant domain (CH1), wherein the C-terminus of the VH domain is fused to the N-terminus of the CH1 domain, a polypeptide in which the polypeptide is fused by the N-terminus of the VH domain, preferably via a peptide linker, to the C-terminus of one of the chains of a second fragment.

[0272] The second hemibody is c) a tandem Fab comprising a first fragment and a second fragment, wherein the first fragment is connected by its C-terminus to the N-terminus of the second fragment, the first fragment binds to a first epitope of a target antigen, and the second fragment binds to a second epitope of the target antigen, and one of the fragments selected from the first fragment and the second fragment is a Fab and the other is a cross-Fab; d) i) an antibody light chain variable domain (VL), or ii) a polypeptide comprising or consisting of an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL), wherein the C-terminus of the VL domain is fused to the N-terminus of the light chain constant domain, a polypeptide in which the polypeptide is fused by the N-terminus of the VL domain, preferably via a peptide linker, to the C-terminus of one of the chains of a second fragment.

[0273] The antibody heavy chain variable domain (VH) of the first hemibody and the antibody light chain variable domain (VL) of the second hemibody together form a functional antigen binding site for a radiolabeled compound.

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

[0275] Either the first or second fragment can be a cross-Fab, so long as the tandem Fab contains one conventional Fab and one cross-Fab.

[0276] In any of the above tandem Fab embodiments (including those comprising cross-Fabs), optionally, the first hemibody can consist essentially of or consist of components (a) and (b), and the second hemibody can consist essentially of or consist of components (c) and (d). In either case, the first hemibody does not contain an antibody light chain variable domain (VL) capable of associating with component (b) of the first hemibody to form a functional antigen-binding site for a radiolabeled compound, and the second hemibody does not contain an antibody heavy chain variable domain (VH) capable of associating with component (d) of the second hemibody to form a functional antigen-binding site for a radiolabeled compound.

[0277] As noted above, in some embodiments, the first and second hemibodies may each comprise an Fc domain that is optionally engineered to reduce or eliminate effector function.

[0278] In one embodiment, each of the first hemibody and the second hemibody may comprise: i) an Fc domain; ii) at least one antigen-binding moiety (e.g., an antibody fragment, e.g., an scFv, Fv, Fab, or cross-Fab fragment) capable of binding to a target antigen; and iii) either a VL domain or a VH domain (but not both) of an antigen-binding site for a radiolabeled compound.

[0279] Optionally, hemibodies comprising an Fc domain can be monovalent with respect to binding to a target antigen. In other embodiments, they can be multivalent, e.g., bivalent. The first and second hemibodies can each be multivalent and monospecific for the same epitope of the target antigen. In yet other embodiments, the first and second hemibodies can each have binding sites for different epitopes of the target antigen, e.g., they can be biparatopic.

[0280] The antibody fragment may be an scFv. Thus, in one embodiment, the first hemibody comprises: a) an scFv fragment, wherein the scFv fragment binds to a target antigen; b) an Fc domain; c) i) an antibody heavy chain variable domain (VH), or ii) a polypeptide comprising or consisting of 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; The scFv in (a) is fused to the N-terminus of the Fc domain, and the polypeptide in c) is fused to the C-terminus of the Fc domain via the N-terminus of the VH domain, preferably via a peptide linker.

[0281] Optionally, the polypeptide of portion c(i) may further comprise one or more residues, optionally one or more alanine residues, optionally a single alanine residue, at the C-terminus of the VH domain. Optionally, the additional residues may be from the N-terminal portion of the CH1 domain as described above, e.g., 1 to 10 residues from the N-terminus of the CH1 domain, e.g., from the human IgG1 CH1 domain. For example, the additional residues may be AST.

[0282] The second hemibody is d) a second scFv that binds to the target antigen; and e) an Fc domain; f) i) an antibody light chain variable domain (VL), or ii) a polypeptide comprising or consisting of 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; The scFv of (d) is fused to the N-terminus of the Fc domain, and the polypeptide of (f) is fused to the C-terminus of the Fc domain via the N-terminus of the VH domain, preferably via a peptide linker.

[0283] In another embodiment, the first hemibody and the second hemibody can each be a one-arm IgG comprising a Fab against a target antigen (e.g., a single Fab against a target antigen) and an Fc domain. i) an intact light chain fragment; and ii) an intact heavy chain; and iii) an additional Fc chain lacking Fd; iv) a polypeptide comprising or consisting of a VH domain of an antigen-binding site for a radiolabeled compound; The light chain of (i) and the heavy chain of (ii) together provide an antigen-binding site for the target antigen, and a polypeptide comprising or consisting of a VH domain of an antigen-binding site for a radiolabeled compound is fused by its N-terminus to the C-terminus of either (ii) or (iii), preferably via a linker.

[0284] The second hemibody is v) an intact light chain fragment; and vi) an intact heavy chain; vii) an additional Fc chain lacking Fd; viii) a polypeptide comprising or consisting of a VL domain of an antigen-binding site for a radiolabeled compound, The light chain of (v) and the heavy chain of (vi) together provide an antigen-binding site for the target antigen, and a polypeptide comprising or consisting of a VL domain of an antigen-binding site for a radiolabeled compound is fused by its N-terminus to the C-terminus of either (vi) or (vii), preferably via a linker.

[0285] The polypeptide comprising or consisting of a VH domain of the antigen-binding site for a radiolabeled compound is i) an antibody heavy chain variable domain (VH), wherein the polypeptide may further 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) It may be a polypeptide comprising or consisting of 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.

[0286] The polypeptide comprising or consisting of the VL domain of the antigen-binding site for the radiolabeled compound is i) an antibody heavy chain variable domain (VL), or ii) It may be a polypeptide comprising or consisting of an antibody heavy chain variable domain (VL) and an antibody light chain constant domain, wherein the C-terminus of the VL domain is fused to the N-terminus of the constant domain.

[0287] If the first and second hemibody are heterodimers, e.g., one-arm IgG, their assembly can be assisted by the use of knob-into-hole technology, as described further below.

[0288] In another embodiment, each hemibody may comprise the above-described tandem Fab (e.g., comprising two Fab fragments, wherein a first Fab fragment and a second Fab fragment both bind to the same epitope on target antigen A, or comprising a Fab and a cross-Fab, one of which binds to a first epitope on target antigen A and the other of which binds to a second epitope on target antigen A), wherein the Fab tandem is fused to the N-terminus (e.g., via its C-terminus) of an Fc domain, and a peptide comprising or consisting of a VH or VL domain of an antigen-binding site for a radiolabeled compound is fused to the C-terminus (e.g., via its N-terminus) of the Fc domain.

[0289] Therefore, the first hemibody a) i) a tandem Fab comprising two Fab fragments, wherein a first Fab fragment and a second Fab fragment bind to a target antigen A, the epitope bound by the first Fab fragment is the same as the epitope bound by the second Fab fragment, and the first Fab fragment and the second Fab fragment are connected via a peptide tether, and the first Fab is connected via its C-terminus to the N-terminus of the second Fab; and ii) a tandem Fab comprising a first fragment and a second fragment, wherein the first fragment is connected by its C-terminus to the N-terminus of the second fragment via a peptide tether, the first fragment binds to a first epitope of target antigen A, and the second fragment binds to a second epitope of target antigen A, wherein one of the fragments selected from the first fragment and the second fragment is a Fab and the other is a cross-Fab; b) an Fc domain; c) i) an antibody heavy chain variable domain (VH), or ii) a polypeptide comprising or consisting of 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 tandem Fab is fused to the N-terminus of one of the chains of the Fc domain, and the polypeptide of c) is fused to the C-terminus of one of the chains of the Fc domain by the N-terminus of the VH domain, preferably via a peptide linker.

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

[0291] The second hemibody is d) i) a tandem Fab comprising two Fab fragments, wherein a first Fab fragment and a second Fab fragment bind to a target antigen A, the epitope bound by the first Fab fragment is the same as the epitope bound by the second Fab fragment, and the first Fab fragment and the second Fab fragment are connected via a peptide tether, and the first Fab is connected via its C-terminus to the N-terminus of the second Fab; and ii) a tandem Fab comprising a first fragment and a second fragment, wherein the first fragment is connected by its C-terminus to the N-terminus of the second fragment via a peptide tether, the first fragment binds to a first epitope of target antigen A, and the second fragment binds to a second epitope of target antigen A, wherein one of the fragments selected from the first fragment and the second fragment is a Fab and the other is a cross-Fab; e) an Fc domain; f) i) an antibody heavy chain variable domain (VL), or ii) a polypeptide comprising or consisting of an antibody heavy chain variable domain (VL) and an antibody light chain constant domain, wherein the C-terminus of the VL domain is fused to the N-terminus of the light chain constant domain; The tandem Fab of (d) is fused to the N-terminus of one chain of the Fc domain, and the polypeptide of (f) is fused to the C-terminus of one chain of the Fc domain by the N-terminus of the VL domain, preferably via a peptide linker.

[0292] The VH domain of the first hemibody and the VL domain of the second hemibody together, i.e., upon association of the two antibodies, form the antigen binding site for the radiolabeled compound.

[0293] If the first hemibody comprises a tandem Fab according to (a)(i), the second hemibody will generally comprise a tandem Fab according to d(i), and if the first hemibody comprises a tandem Fab according to (a)(ii), the second hemibody will generally comprise a tandem Fab according to d(ii).

[0294] The tandem Fab may generally be as described above. For example, the tandem Fab may be composed of any of the sets of chains described above. Generally, the heavy chain fragment of the second Fab (which may be a cross-Fab) may be linked to an Fc domain.

[0295] In a further embodiment, each of the first hemibody and the second hemibody may comprise: a) an Fc domain comprising a first subunit and a second subunit; b) at least one antigen-binding portion capable of binding to a target antigen (e.g., an antibody fragment such as an scFv, Fv, Fab, or cross-Fab fragment comprising an antigen-binding site for the target antigen); and c) a polypeptide comprising either the VL domain or VH domain of the antigen-binding site of a radiolabeled compound, wherein the C-terminus of the antigen-binding portion (e.g., antibody fragment) of (b) is fused to the N-terminus of the first Fc domain subunit, and the C-terminus of the polypeptide of (c) is fused to the N-terminus of the second Fc domain subunit. Fusion of the antibody fragment of (b) is preferably via the hinge region. Fusion of the polypeptide of (c) can be via a linker disposed between the C-terminus of the polypeptide and the N-terminus of the Fc region and / or via part or all of the upper hinge region (e.g., Asp221 and residues C-terminal thereto according to the EU numbering index). In one embodiment, the antibody fragment of (b) can be a Fab fragment. In one embodiment, in the first hemibody, the polypeptide of (c) consists of the VH domain of the antigen-binding site for the radiolabeled compound, and in the second hemibody, the polypeptide of (c) consists of the VL domain of the antigen-binding site for the radiolabeled compound.

[0296] Thus, in one embodiment, the first hemibody is i) an intact light chain; ii) an intact heavy chain; and iii) an additional Fc chain; iv) a polypeptide comprising or consisting of a VH domain of an antigen-binding site for a radiolabeled compound; The light chain of (i) and the heavy chain of (ii) together provide an antigen-binding site for the target antigen, and a polypeptide comprising or consisting of a VH domain of an antigen-binding site for a radiolabeled compound is fused by its C-terminus to the N-terminus of (iii), preferably via a linker.

[0297] The second hemibody is v) an intact light chain; vi) an intact heavy chain; vii) an additional Fc chain; viii) a polypeptide comprising or consisting of a VL domain of an antigen-binding site for a radiolabeled compound, The light chain of (v) and the heavy chain of (vi) together provide an antigen-binding site for the target antigen, and a polypeptide comprising or consisting of a VL domain of an antigen-binding site for a radiolabeled compound is fused by its C-terminus to the N-terminus of (vii), preferably via a linker.

[0298] The linker can include any flexible linker known to one of skill in the art or described herein, such as the linker GGGGSGGGGSGGGGSGGSGG (SEQ ID NO: 152). The linker may further include a portion of the entire upper hinge region, for example, extending from Asp221 to the beginning of the Fc chain (e.g., at Cys226).

[0299] In still further embodiments, the first hemibody and / or second hemibody each comprise a full-length antibody having an antigen-binding site for a target antigen and further comprise either a VL domain or a VH domain of an antigen-binding site for a radiolabeled compound.

[0300] In one particular embodiment, the first hemibody comprises: 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 a target antigen; b) i) a further antibody heavy chain variable domain (VH), or ii) a polypeptide comprising or consisting of 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, The polypeptide may comprise a polypeptide fused by the N-terminus of the VH domain to the C-terminus of one of the two heavy chains of the first full-length antibody, preferably via a peptide linker.

[0301] The second hemibody is c) a second full-length antibody consisting of two antibody heavy chains and two antibody light chains, wherein at least one arm of the full-length antibody binds to a target antigen; and d) i) a further antibody light chain variable domain (VL), or ii) a polypeptide comprising or consisting of a further antibody light chain variable domain (VL) and a further antibody light chain constant domain (CL), wherein the C-terminus of the VL domain is fused to the N-terminus of the CL domain, The polypeptide may comprise a polypeptide fused by the N-terminus of the VL domain to the C-terminus of one of the two heavy chains of the second full-length antibody, preferably via a peptide linker.

[0302] The antibody heavy chain variable domain (VH) of the first hemibody and the antibody light chain variable domain (VL) of the second hemibody together, i.e., upon association of the two antibodies, form a functional antigen-binding site for the radiolabeled compound.

[0303] Optionally, the polypeptide of portion b(i) may further comprise one or more residues, optionally one or more alanine residues, optionally a single alanine residue, at the C-terminus of the VH domain. Optionally, the additional residues may be the N-terminal portion of the CH1 domain described above, e.g., 1 to 10 residues from the N-terminus of the CH1 domain, e.g., from the human IgG1 CH1 domain. For example, the additional residues may be AST.

[0304] Optionally, the first hemibody can consist essentially of or consist of components (a) and (b) above, and the second hemibody can consist essentially of or consist of components (c) and (d) above. In either case, the first hemibody does not contain an antibody light chain variable domain (VL) that can associate with component (b) of the first hemibody to form a functional antigen-binding site for a radiolabeled compound, and the second hemibody does not contain an antibody heavy chain variable domain (VH) that can associate with component (b) of the second hemibody to form a functional antigen-binding site for a radiolabeled compound.

[0305] It may be preferable for both arms of a full-length antibody to have binding specificity for the same target antigen. If an antibody is bivalent for a target antigen, both arms of the full-length antibody may bind to the same epitope on the same target antigen.

[0306] In another embodiment, the antibody may be biparatopic to the target antigen; for example, one arm of the full-length antibody may bind to a first epitope of the target antigen, and one arm may bind to a second epitope of the target antigen. In such an embodiment, one arm of the antibody may comprise a Fab and one arm may comprise a cross-Fab, which aids in the correct assembly of the light chain and its respective heavy chain. Thus, in one embodiment, the first heavy chain of the full-length antibody may comprise a VL domain instead of a VH domain (e.g., VL-CH1-hinge-CH2-CH3), the first light chain may comprise a VH domain swapped with a VL domain (e.g., VH-CL), or the first heavy chain may comprise a CL domain instead of an HC1 domain (e.g., VH-CL-hinge-CH2-CH3), and the first light chain may comprise a CH1 domain instead of a CL domain (e.g., VL-CH1). In this embodiment, the second heavy chain and the second light chain have conventional domain structures (e.g., VH-CH1-hinge-CH2-CH3 and VL-CL, respectively). In an alternative embodiment, the second heavy chain of the full-length antibody may comprise a VL domain instead of a VH domain (e.g., VL-CH1-hinge-CH2-CH3), the second light chain may comprise a VH domain swapped with a VL domain (e.g., VH-CL), or the second heavy chain may comprise a CL domain instead of an HC1 domain (e.g., VH-CL-hinge-CH2-CH3), and the second light chain may comprise a CH1 domain instead of a CL domain (e.g., VL-CH1). In this embodiment, the first heavy chain and the first light chain have conventional domain structures.

[0307] Yet another possible format for the hemibody is i) The first hemibody is a) an antigen-binding moiety (e.g., an antibody fragment, e.g., a Fab fragment) capable of binding to an antigen expressed on the surface of a target cell; b) a polypeptide comprising or consisting of an antibody heavy chain variable domain (VH) of an antigen-binding site for a radiolabeled compound; c) an Fc domain comprising two subunits; the polypeptide of (b) is fused by its N-terminus to the C-terminus of the antigen-binding portion of (a) (e.g., to the C-terminus of one of the chains of the Fab fragment of (a)) and by its C-terminus to the N-terminus of one of the subunits of the Fc domain of (c); The first hemibody does not contain a VL domain of the antigen binding site for the radiolabeled compound; and ii) The second hemibody is d) an antigen-binding moiety (e.g., an antibody fragment, e.g., a Fab) capable of binding to an antigen expressed on the surface of a target cell; e) a polypeptide comprising or consisting of an antibody light chain variable domain (VL) of an antigen-binding site for a radiolabeled compound; f) an Fc domain comprising two subunits; The polypeptide of (e) is fused by its N-terminus to the C-terminus of the antigen-binding portion of (d) (e.g., to the C-terminus of one of the chains of the Fab fragment of (d)) and by its C-terminus to the N-terminus of one of the subunits of the Fc domain of (f); the second hemibody does not contain the VH domain of the antigen binding site for the radiolabeled compound; The VH domain of the first hemibody and the VL domain of the second hemibody can together form a functional antigen binding site for a radiolabeled compound.

[0308] The fusion can be direct or indirect, for example, via a peptide linker.

[0309] In some embodiments, the first hemibody and / or the second hemibody further comprises another antigen-binding moiety (e.g., an additional antibody fragment), e.g., another Fab fragment, that binds to the target antigen. Thus, in some embodiments, the first hemibody and / or the second hemibody (generally both) each comprise two antigen-binding moieties capable of binding to the target antigen. The two antigen-binding moieties of a hemibody preferably bind to the same target antigen, either at the same or different epitopes. Optionally, the first antibody and the second antibody each comprise no more than two antigen-binding moieties capable of binding to the target antigen. In other embodiments, they may comprise three or more antigen-binding moieties capable of binding to the target antigen.

[0310] In one embodiment, this additional antibody binding moiety (e.g., an antibody fragment, e.g., a Fab fragment) is fused by its C-terminus (e.g., of one of its chains, e.g., the heavy chain) to the N-terminus of the other subunit of the Fc domain. Thus, in one embodiment, the first hemibody and / or second hemibody may be a two-arm hemibody, with each arm carrying a binding moiety for a target antigen.

[0311] Thus, in one embodiment, the split antibody comprises: i) a first hemibody, a) a first antigen-binding portion (e.g., a Fab fragment), where the antigen-binding portion (e.g., a Fab fragment) binds to an antigen expressed on the surface of a target cell; and b) a polypeptide comprising or consisting of an antibody heavy chain variable domain (VH) of an antigen-binding site for a radiolabeled compound; c) an Fc domain comprising a first subunit and a second subunit; the polypeptide of (b) is fused by its N-terminus to the C-terminus of the antigen-binding portion of (a) (e.g., to the C-terminus of one of the chains of the Fab fragment of (a)) and by its C-terminus to the N-terminus of the first subunit of the Fc domain of (c); and further comprising a second antigen-binding portion (e.g., a second Fab fragment) that binds to an antigen expressed on the surface of a target cell, wherein the second antigen-binding portion (e.g., Fab) is fused by its C-terminus (e.g., by the C-terminus of one of its chains) to the N-terminus of the second subunit of the Fc domain of (c); a first hemibody that does not contain a VL domain of an antigen binding site for a radiolabeled compound; ii) a second hemibody, d) a first antigen-binding portion (e.g., a Fab fragment), wherein the antigen-binding portion (e.g., a Fab fragment) binds to an antigen expressed on the surface of a target cell; and e) a polypeptide comprising or consisting of an antibody light chain variable domain (VL) of an antigen-binding site for a radiolabeled compound; f) an Fc domain comprising a first subunit and a second subunit; The polypeptide of (e) is fused by its N-terminus to the C-terminus of the antigen-binding portion of (d) (e.g., to the C-terminus of one of the chains of the Fab fragment of (d)) and by its C-terminus to the N-terminus of the first subunit of the Fc domain of (f); and further comprising a second antigen-binding portion (e.g., a second Fab fragment) that binds to an antigen expressed on the surface of a target cell, wherein the second antigen-binding portion (e.g., Fab) is fused by its C-terminus (e.g., by the C-terminus of one of its chains) to the N-terminus of the second subunit of the Fc domain of (f); a second hemibody that does not contain a VH domain of an antigen-binding site for a radiolabeled compound; The VH domain of the first hemibody and the VL domain of the second hemibody can together form a functional antigen binding site for a radiolabeled compound.

[0312] In other embodiments, which may be preferred in some instances, the first hemibody and / or the second hemibody each have a single antigen-binding moiety capable of specifically binding to a target antigen. Thus, the first hemibody and / or the second hemibody can be monospecific and monovalent with respect to the target antigen. Preferably, the first hemibody and the second hemibody bind to the same target antigen, either at the same or different epitopes.

[0313] In one embodiment, the first hemibody and / or the second hemibody is a one-armed antibody. In such an embodiment, the Fc subunit of the first hemibody that is not fused to the polypeptide of (b) is also not fused to any other antigen-binding domain / moiety, and / or the Fc subunit of the second hemibody that is not fused to the polypeptide of (e) is also not fused to any other antigen-binding domain / moiety. Thus, the Fc domain can include a subunit that lacks an Fd. In some embodiments, one of the polypeptides that make up a hemibody can consist of or consist essentially of an Fc subunit.

[0314] Thus, in some embodiments, the first hemibody comprises the following polypeptide: i) a polypeptide comprising, from N-terminus to C-terminus, a Fab heavy chain (e.g., VH-CH1), an optional linker, a VH domain of an antigen binding site for a radiolabeled compound, an optional linker, and an Fc subunit (e.g., CH2-CH3); ii) a Fab light chain polypeptide (e.g., VL-CL), and iii) an Fc subunit polypeptide (e.g., CH2-CH3), The Fab heavy chain of (i) and the Fab light chain of (ii) form a Fab fragment that is capable of binding to a target antigen.

[0315] The second hemibody comprises the following polypeptide: iv) a polypeptide comprising, from N-terminus to C-terminus, a Fab heavy chain (e.g., VH-CH1), an optional linker, a VL domain of an antigen binding site for a radiolabeled compound, an optional linker, and an Fc subunit (e.g., CH2-CH3); v) a Fab light chain polypeptide (e.g., VL-CL), and vi) an Fc subunit polypeptide (e.g., CH2-CH3), The Fab heavy chain of (iv) and the Fab light chain of (v) form a Fab fragment capable of binding to a target antigen.

[0316] In some embodiments of these one-arm hemibodies, the Fab heavy chains of (i) and (iv) can have the same sequence as each other, and the Fab light chain polypeptides of (ii) and (v) can have the same sequence as each other.

[0317] In some embodiments of any of the above formats in which Fabs with different specificities are present, the correct assembly of a light chain with its respective heavy chain can be aided by the use of charge modifications, as discussed further below.

[0318] The correct assembly of the heavy heterodimer chains can be assisted by knob-into-hole techniques, as discussed further below.

[0319] H. Exemplary Split Multispecific Split Antibodies Aspects and embodiments relating to target binding (e.g., CEA binding) and aspects and embodiments relating to DOTA binding may be combined in some embodiments. In one embodiment, a multispecific antibody may comprise a binding site for CEA having any of the above sequences and a binding site for a DOTA chelate having any of the above sequences. In another embodiment, the first hemibody and the second hemibody each comprise a binding site for CEA, e.g., comprising any of the above sequences, and associate to form a binding site for a DOTA chelate having any of the above sequences. It is also expressly contemplated that aspects and embodiments relating to CEA binding and / or DOTA binding may be combined with the preferred antibody formats described above; i.e., in any of the preferred formats, the moiety that binds to the target antigen may be a CEA binder comprising the above CDR or variable region sequences, and / or the moiety that binds to the radionuclide-labeled compound may be a DOTA binder having the above CDR and / or variable region sequences.

[0320] In one particular embodiment of a split antibody, the first hemibody comprises: a) a first full-length antibody that specifically binds to CEA and consists of two antibody heavy chains and two antibody light chains; b) a polypeptide comprising or consisting of an antibody heavy chain variable domain (VH), wherein the heavy chain variable domain comprises the heavy chain CDRs of SEQ ID NOs: 35-37 (or CDR-H1 has the sequence GFSLTDYGVH) and / or 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; The polypeptide is fused by the N-terminus of the VH domain to the C-terminus of one of the two heavy chains of the first full-length antibody, preferably via a peptide linker.

[0321] The first hemibody does not include a light chain domain that associates with the polypeptide of (b) to form a functional binding domain for the radiolabeled compound.

[0322] It may be preferred that the polypeptide of (b) further comprises one or more residues, for example, 1 to 10 residues, at the C-terminus of the VH domain. Optionally, these may be one or more alanine residues, optionally a single alanine residue. In another embodiment, the further residues may be 1 to 10 residues from the N-terminal portion of the CH1 domain described above, for example, from the N-terminus of the CH1 domain, for example, from the human IgG1 CH1 domain. For example, the further residues may be AST.

[0323] In some embodiments, the two antibody heavy chains of portion (a) have identical variable domains, optionally identical variable domains, CH1 domains and / or CH2 domains, and may optionally differ only in their CH3 domains, for example, by making knob-into-hole mutations and other mutations intended to promote correct association of the heterodimer.

[0324] The second hemibody is c) a second full-length antibody that specifically binds to CEA and consists of two antibody heavy chains and two antibody light chains; d) a polypeptide comprising or consisting of an antibody light chain variable domain (VL), wherein the light chain variable domain comprises the CDRs of SEQ ID NOs: 38-40 and / or 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; The polypeptide is fused by the N-terminus of the VL domain to the C-terminus of one of the two heavy chains of the second full-length antibody, preferably via a peptide linker, and the second hemibody does not include a heavy chain domain that associates with the polypeptide of (d) to form a functional binding domain for a radiolabeled compound.

[0325] In some embodiments, the two antibody heavy chains of portion (c) have identical variable domains, optionally identical variable domains, CH1 domains and / or CH2 domains, and may optionally differ only in their CH3 domains, for example, by making knob-into-hole mutations and other mutations intended to promote correct association of the heterodimer.

[0326] The CEA binding site / sequence can be any of the CEA binding sites / sequences described above.

[0327] In one specific embodiment, the first hemibody can have a CEA-binding sequence (ie, CDRs or VH / VL domains) from antibody CH1A1A.

[0328] For example, the two light chains in (a) may comprise the CDRs of SEQ ID NOs: 22-24 and / or may comprise a light chain variable domain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 26. In some embodiments, they may have at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 103. In some embodiments, it may be preferred that the two light chains in (a) are identical to each other.

[0329] The two antibody heavy chains of portion (a) may comprise the CDRs of SEQ ID NOs: 19-21 and / or the two antibody heavy chains of portion (a) comprise variable domains having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO: 25. In one embodiment, one heavy chain of portion (a) has the sequence of SEQ ID NO: 100 and the other has the sequence of SEQ ID NO: 102.

[0330] In one particular embodiment, the first hemibody may comprise a first heavy chain of SEQ ID NO: 100, a second heavy chain of SEQ ID NO: 101 (the C-terminal AST is optional and may be absent or replaced with another C-terminal extension described herein), and a light chain of SEQ ID NO: 103.

[0331] The second hemibody can also have a CEA-binding sequence (ie, CDRs or VH / VL domains) from antibody CH1A1A.

[0332] For example, the two light chains of (c) may comprise the CDRs of SEQ ID NOs: 22-24 and / or may comprise a light chain variable domain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 26. In some embodiments, they may have at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 103. In some embodiments, it may be preferred that the two light chains of (c) are identical to each other. In some embodiments, it may be preferred that the two light chains of (c) have the same sequence as the light chain of (a) of the first hemibody, e.g., all of the light chains of portions (a) and (c) have the same sequence.

[0333] In some embodiments, the two antibody heavy chains of portion (c) comprise the CDRs of SEQ ID NOs: 19-21 and / or the two antibody heavy chains of portion (c) comprise variable domains having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO: 25. In one embodiment, one heavy chain of portion (c) has the sequence of SEQ ID NO: 97 and the other has the sequence of SEQ ID NO: 99.

[0334] In one particular embodiment, the second hemibody may comprise a first heavy chain of SEQ ID NO:97, a second heavy chain of SEQ ID NO:98, and a light chain of SEQ ID NO:103.

[0335] Similarly, aspects and embodiments relating to target binding (e.g., CEA binding) and aspects and embodiments relating to Pb-DOTAM binding may be combined in some embodiments. In one embodiment, a multispecific antibody may comprise a binding site for CEA having any of the above sequences and a binding site for a Pb-DOTAM chelate having any of the above sequences. In another embodiment, the first hemibody and the second hemibody may each comprise a binding site for CEA, e.g., comprising any of the above sequences, and associate to form a binding site for a Pb-DOTAM chelate having any of the above sequences. It is also expressly contemplated that aspects and embodiments relating to CEA binding and / or Pb-DOTAM binding may be combined with the preferred antibody formats described above; i.e., in any of the preferred formats, the moiety that binds to the target antigen may be a CEA binder comprising the above CDR or variable region sequences, and / or the moiety that binds to the radionuclide-labeled compound may be a Pb-DOTAM binder having the above CDR and / or variable region sequences.

[0336] In one particular embodiment of a split antibody, the first hemibody comprises: a) a first full-length antibody that specifically binds to CEA and consists of two antibody heavy chains and two antibody light chains; b) a polypeptide comprising or consisting of an antibody heavy chain variable domain (VH), wherein the heavy chain variable domain comprises the heavy chain CDRs of SEQ ID NOs: 1-3 and / or the heavy chain variable domain has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO: 7; The polypeptide is fused by the N-terminus of the VH domain to the C-terminus of one of the two heavy chains of the first full-length antibody, preferably via a peptide linker.

[0337] The first hemibody does not include a light chain domain that associates with the polypeptide of (b) to form a functional binding domain for the radiolabeled compound.

[0338] It may be preferred that the polypeptide of (b) further comprises one or more residues, optionally one or more alanine residues, optionally a single alanine residue, at the C-terminus of the VH domain. For example, the polypeptide of (b) may comprise SEQ ID NO: 7 with a C-terminal alanine extension, e.g., the sequence VTLKESGPVLVKPTETLTLTCTVSGFSLSTYSMSWIRQPPGKALEWLGFIGSRGDTYYASWAKGRLTISKDTSKSQVVLTMTNMDPVDTATYYCARERDPYGGGAYPPHLWGRGTLVTVSSA.

[0339] In another embodiment, the additional residues may be 1 to 10 residues from the N-terminal portion of the CH1 domain described above, e.g., from the N-terminus of the CH1 domain, e.g., from the human IgG1 CH1 domain. For example, the additional residues may be AST.

[0340] In some embodiments, the two antibody heavy chains of portion (a) have identical variable domains, optionally identical variable domains, CH1 domains and / or CH2 domains, and may optionally differ only in their CH3 domains, for example, by making knob-into-hole mutations and other mutations intended to promote correct association of the heterodimer.

[0341] The second hemibody is c) a second full-length antibody that specifically binds to CEA and consists of two antibody heavy chains and two antibody light chains; d) a polypeptide comprising or consisting of an antibody light chain variable domain (VL), wherein the light chain variable domain comprises the CDRs of SEQ ID NOs: 4-6 and / or the light chain variable domain has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO: 8; The polypeptide is fused by the N-terminus of the VL domain to the C-terminus of one of the two heavy chains of the second full-length antibody, preferably via a peptide linker, and the second hemibody does not include a heavy chain domain that associates with the polypeptide of (d) to form a functional binding domain for a radiolabeled compound.

[0342] In some embodiments, the two antibody heavy chains of portion (c) have identical variable domains, optionally identical variable domains, CH1 domains and / or CH2 domains, and may optionally differ only in their CH3 domains, for example, by making knob-into-hole mutations and other mutations intended to promote correct association of the heterodimer.

[0343] In certain embodiments, the first hemibody may have a CEA-binding sequence (ie, CDRs or VH / VL domains) from antibody CH1A1A.

[0344] For example, the two light chains in (a) may comprise the CDRs of SEQ ID NOs: 22-24 and / or may comprise a light chain variable domain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 26. In some embodiments, they may have at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 34. In some embodiments, it may be preferred that the two light chains in (a) are identical to each other.

[0345] The two antibody heavy chains of portion (a) may comprise the CDRs of SEQ ID NOs: 19-21 and / or the two antibody heavy chains of portion (a) comprise variable domains having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO: 25. In one embodiment, one heavy chain of portion (a) has the sequence of SEQ ID NO: 27 and the other has the sequence of SEQ ID NO: 28.

[0346] In one particular embodiment, the first hemibody may comprise a first heavy chain of SEQ ID NO: 28, a second heavy chain of SEQ ID NO: 32 (or a variant thereof comprising an additional C-terminal alanine or other C-terminal extension described herein, such as an extension with AST), and a light chain of SEQ ID NO: 34. A variant of SEQ ID NO: 32 with a C-terminal alanine extension is shown below: QVQLVQSGAEVKKPGASVKVSCKASGYTFTEFGMNWVRQAPGQGLEWMGWINTKTGEATYVEEFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARWDFAYYVEAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGGGGGSGGGGSGGGGSGGGGSVTLKESGPVLVKPTETLTLTCTVSGFSLSTYSMSWIRQPPGKALEWLGFIGSRGDTYYASWAKGRLTISKDTSKSQVVLTMTNMDPVDTATYYCARERDPYGGGAYPPHLWGRGTLVTVSSA

[0347] In another specific embodiment, the first hemibody may have CEA binding sequences (ie, CDRs or VH / VL domains) from antibody A5B7 (including humanized versions thereof).

[0348] For example, the two light chains in (a) may comprise the CDRs of SEQ ID NOs: 46-48 and / or may comprise a light chain variable domain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 50. In some embodiments, they may have at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 54. In some embodiments, it may be preferred that the two light chains in (a) are identical to each other.

[0349] In some embodiments, the two antibody heavy chains of portion (a) may comprise the CDRs of SEQ ID NOs: 43-45 and / or the two antibody heavy chains of portion (a) comprise variable domains having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO: 49. In one embodiment, one heavy chain of portion (a) has the sequence of SEQ ID NO: 51 and the other has the sequence of SEQ ID NO: 53.

[0350] In one particular embodiment, the first hemibody may comprise a first heavy chain of SEQ ID NO: 51, a second heavy chain of SEQ ID NO: 52 (or a variant thereof having a C-terminal alanine extension or other C-terminal extension described herein, e.g., an extension with AST), and a light chain of SEQ ID NO: 54.

[0351] In another specific embodiment, the first hemibody may have CEA-binding sequences (ie, CDRs or VH / VL domains) from antibody T84.66 (including humanized versions thereof).

[0352] For example, the two light chains in (a) may comprise the CDRs of SEQ ID NOs: 14-16 and / or may comprise a light chain variable domain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 18. In some embodiments, they may have at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 89. In some embodiments, it may be preferred that the two light chains in (a) are identical to each other.

[0353] In some embodiments, the two antibody heavy chains of portion (a) may comprise the CDRs of SEQ ID NOs: 11-13 and / or the two antibody heavy chains of portion (a) comprise variable domains having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO: 17. In one embodiment, one heavy chain of portion (a) has the sequence of SEQ ID NO: 86 and the other has the sequence of SEQ ID NO: 88.

[0354] In one particular embodiment, the first hemibody may comprise a first heavy chain of SEQ ID NO: 86, a second heavy chain of SEQ ID NO: 87 (or a variant thereof in which the C-terminal "AST" is absent or replaced with a different C-terminal extension as disclosed herein), and a light chain of SEQ ID NO: 89.

[0355] In another specific embodiment, the first hemibody may have CEA-binding sequences (ie, CDRs or VH / VL domains) from antibody 28A9 (including humanized versions thereof).

[0356] For example, the two light chains in (a) may comprise the CDRs of SEQ ID NOs: 62-64 and / or may comprise a light chain variable domain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 66. In some embodiments, they may have at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 96. In some embodiments, it may be preferred that the two light chains in (a) are identical to each other.

[0357] In some embodiments, the two antibody heavy chains of portion (a) may comprise the CDRs of SEQ ID NOs: 59-61 and / or the two antibody heavy chains of portion (a) comprise variable domains having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO: 65. In one embodiment, one heavy chain of portion (a) has the sequence of SEQ ID NO: 93 and the other has the sequence of SEQ ID NO: 95.

[0358] In one particular embodiment, the first hemibody may comprise a first heavy chain of SEQ ID NO: 93, a second heavy chain of SEQ ID NO: 94 (or a variant thereof without the C-terminal "AST" or with a different C-terminal extension as described herein), and a light chain of SEQ ID NO: 96.

[0359] In some embodiments, the second hemibody can have a CEA-binding sequence (ie, CDRs or VH / VL domains) from antibody CH1A1A.

[0360] For example, the two light chains of (c) may comprise the CDRs of SEQ ID NOs: 22-24 and / or may comprise a light chain variable domain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 26. In some embodiments, they may have at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 34. In some embodiments, it may be preferred that the two light chains of (c) are identical to each other. In some embodiments, it may be preferred that the two light chains of (c) have the same sequence as the light chain of (a) of the first hemibody, e.g., all of the light chains of portions (a) and (c) have the same sequence.

[0361] In some embodiments, the two antibody heavy chains of portion (c) comprise the CDRs of SEQ ID NOs: 19-21 and / or the two antibody heavy chains of portion (c) comprise variable domains having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO: 25. In one embodiment, one heavy chain of portion (c) has the sequence of SEQ ID NO: 29 and the other has the sequence of SEQ ID NO: 30.

[0362] In one particular embodiment, the second hemibody may comprise a first heavy chain of SEQ ID NO:30, a second heavy chain of SEQ ID NO:33, and a light chain of SEQ ID NO:34.

[0363] In another specific embodiment, the second hemibody may have a CEA-binding sequence (ie, CDRs or VH / VL domains) from A5B7 (including humanized versions thereof).

[0364] For example, the two light chains of (c) may comprise the CDRs of SEQ ID NOs: 46-48 and / or may comprise a light chain variable domain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 50. In some embodiments, they may have at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 58. In some embodiments, it may be preferred that the two light chains of (c) are identical to each other. In some embodiments, it may be preferred that the two light chains of (c) have the same sequence as the light chain of (a) of the first hemibody, e.g., all of the light chains of portions (a) and (c) have the same sequence.

[0365] In some embodiments, the two antibody heavy chains of portion (c) comprise the CDRs of SEQ ID NOs: 43-45 and / or the two antibody heavy chains of portion (c) comprise variable domains having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO: 49. In one embodiment, one heavy chain of portion (c) has the sequence of SEQ ID NO: 55 and the other has the sequence of SEQ ID NO: 57.

[0366] In one particular embodiment, the second hemibody may comprise a first heavy chain of SEQ ID NO:55, a second heavy chain of SEQ ID NO:56, and a light chain of SEQ ID NO:58.

[0367] In another specific embodiment, the second hemibody may have CEA-binding sequences (ie, CDRs or VH / VL domains) from antibody T84.66 (including humanized versions thereof).

[0368] For example, the two light chains of (c) may comprise the CDRs of SEQ ID NOs: 14-16 and / or may comprise a light chain variable domain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 18. In some embodiments, they may have at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 89. In some embodiments, it may be preferred that the two light chains of (c) are identical to each other.

[0369] In some embodiments, the two antibody heavy chains of portion (c) may comprise the CDRs of SEQ ID NOs: 11-13 and / or the two antibody heavy chains of portion (c) comprise variable domains having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO: 17. In one embodiment, one heavy chain of portion (c) has the sequence of SEQ ID NO: 83 and the other has the sequence of SEQ ID NO: 85.

[0370] In one particular embodiment, the second hemibody may comprise a first heavy chain of SEQ ID NO:83, a second heavy chain of SEQ ID NO:84, and a light chain of SEQ ID NO:89.

[0371] In another specific embodiment, the second hemibody can have CEA-binding sequences (ie, CDRs or VH / VL domains) from antibody 28A9 (including humanized versions thereof).

[0372] For example, the two light chains of (c) may comprise the CDRs of SEQ ID NOs: 62-64 and / or may comprise a light chain variable domain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 66. In some embodiments, they may have at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 96. In some embodiments, it may be preferred that the two light chains of (c) are identical to each other.

[0373] In some embodiments, the two antibody heavy chains of portion (c) may comprise the CDRs of SEQ ID NOs: 59-61, and / or the two antibody heavy chains of portion (a) comprise variable domains having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 65. In one embodiment, one heavy chain of portion (c) has the sequence of SEQ ID NO: 90, and the other has the sequence of SEQ ID NO: 92.

[0374] In one particular embodiment, the second hemibody may comprise a first heavy chain of SEQ ID NO:90, a second heavy chain of SEQ ID NO:91, and a light chain of SEQ ID NO:96.

[0375] In some embodiments, the first hemibody and the second hemibody bind to the same epitope of CEA. Thus, for example, the first hemibody and the second hemibody may both have a CEA-binding sequence derived from antibody CH1A1A, or the first hemibody and the second hemibody may both have a CEA-binding sequence derived from A5B7 (including humanized versions thereof), or the first hemibody and the second hemibody may both have a CEA-binding sequence derived from T84.66 (including humanized versions thereof), or the first hemibody and the second hemibody may both have a CEA-binding sequence derived from 28A9 (including humanized versions thereof), or the first hemibody and the second hemibody may both have a CEA-binding sequence derived from MFE23 (including humanized versions thereof).

[0376] So, for example, i) a first hemibody may comprise a first heavy chain of SEQ ID NO: 28, a second heavy chain of SEQ ID NO: 32 (optionally with a C-terminal extension described herein, e.g., AST), and a light chain of SEQ ID NO: 34; and a second hemibody may comprise a first heavy chain of SEQ ID NO: 30, a second heavy chain of SEQ ID NO: 33, and a light chain of SEQ ID NO: 34; ii) a first hemibody may comprise a first heavy chain of SEQ ID NO: 51, a second heavy chain of SEQ ID NO: 52 (optionally with a C-terminal extension described herein, e.g., AST), and a light chain of SEQ ID NO: 54; and a second hemibody may comprise a first heavy chain of SEQ ID NO: 55, a second heavy chain of SEQ ID NO: 56, and a light chain of SEQ ID NO: 58; iii) the first hemibody may comprise a first heavy chain of SEQ ID NO: 86, a second heavy chain of SEQ ID NO: 87 (the C-terminal AST residue is optional and may be absent or replaced by an alternative C-terminal extension), and a light chain of SEQ ID NO: 89, and the second hemibody antibody may comprise a first heavy chain of SEQ ID NO: 83, a second heavy chain of SEQ ID NO: 84, and a light chain of SEQ ID NO: 89; or iv) The first hemibody may comprise a first heavy chain of SEQ ID NO:93, a second heavy chain of SEQ ID NO:94 (the C-terminal AST residue is optional and may be absent or replaced by an alternative C-terminal extension), and a light chain of SEQ ID NO:96; the second hemibody may comprise a first heavy chain of SEQ ID NO:90, a second heavy chain of SEQ ID NO:91, and a light chain of SEQ ID NO:96.

[0377] In other embodiments, the first and second hemibodies bind to different epitopes of CEA, as described above. Thus, for example, the first hemibody may have a CEA-binding sequence derived from antibody CH1A1A, and the second hemibody may have a CEA-binding sequence derived from A5B7, or the first hemibody may have a CEA-binding sequence derived from antibody A5B7, and the second hemibody may have a CEA-binding sequence derived from CH1A1A. An example of the use of a biparatopic (CH1A1A and A5B7) pair is described in Example 6c.

[0378] In yet a further specific embodiment, the target may be CEA, e.g., with a CEA-binding sequence from antibody CH1A1A, and the format may be as shown in Figure 25C. Optionally, the first hemibody and second hemibody associate to form a functional antigen-binding site for Pb-DOTAM chelate (Pb-DOTAM).

[0379] I. Polypeptide Linker In multispecific antibodies or split multispecific antibodies used in combination therapy, components or domains (e.g., Fc domains, antibody binding portions, VH, VL) can be indirectly fused to other components or domains via peptide linkers.

[0380] The linker (e.g., the linker between the Fab fragment and the VH / VL for the radiolabeled compound, and / or the linker between the VH / VL for the radiolabeled compound and the Fc domain) can be a peptide of at least 5 or at least 10 amino acids, preferably 5 to 100, e.g., 5 to 70, 5 to 60, or 5 to 50, or 10 to 100, 10 to 70, 10 to 60, or 10 to 50 amino acids. In some embodiments, the linker may be preferably 15 to 30 amino acids in length, e.g., 15 to 25, e.g., 16, 17, 18, 19, 20, 21, 22, 23, or 24 amino acids in length. The linker can be rigid or flexible. In some embodiments, it is a flexible linker comprising or consisting of Thr, Ser, Gly, and / or Ala residues. For example, it can comprise or consist of Gly and Ser residues. In some embodiments, it may have a repeat motif such as (Gly-Gly-Gly-Gly-Ser)n, where n is, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Suitable non-immunogenic peptide linkers include, for example, (G4S)n, (SG4)n, (G4S)n, or G4(SG4)n peptide linkers, where "n" is generally a number from 1 to 10, typically 2 to 4. In another embodiment, the peptide linker is (GxS)n or (GxS)nGm, where G=glycine, S=serine, and (x=3, n=3, 4, 5, or 6, and m=0, 1, 2, or 3) or (x=4, n=2, 3, 4, or 5, and m=0, 1, 2, or 3), for example, x=4 and n=2 or 3, for example, x=4, n=2. In some embodiments, the linker can be or include the sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 31). In another embodiment, the linker can be or include GGGGSGGGGSGGGGSGGSGG or GGGGSGGGGSGGGGSGGSGGGS or GGGGSGGGGSGGGGGSGGSGGG. Another exemplary peptide linker is EPKSC(D)-(GS)2.Furthermore, when fused to the N-terminus of an Fc domain subunit, the antigen-binding portion may be fused via an immunoglobulin hinge region or a portion thereof, with or without an additional peptide linker.

[0381] The present inventors have determined that in a peptide linker consisting of y amino acids, Ser at position y (i.e., Ser as the last / C-terminal amino acid of the linker) can induce glycosylation of the y+2 amino acid (i.e., the amino acid located two residues C-terminally from the last amino acid of the linker), depending on the nature of this y+2 amino acid. Thus, it may be preferable for the last serine residue of the linker to be located at position y-2 or y-3 (i.e., the last serine residue of the linker is located 2 or 3 amino acids N-terminally from the last amino acid of the linker). In some embodiments, the linker may consist of y consecutive amino acid residues selected from the group consisting of Gly and Ser, e.g., y=5 or more, e.g., y=5 to 100, 5 to 70, 5 to 60, 5 to 50, or 10 to 100, 10 to 70, 10 to 60, or 10 to 50, e.g., 15 to 31 or 15 to 30, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, with the final serine at position y-2 or y-3. (Thus, a serine may be present at position y-2 and a glycine at positions y-1 and y, or a serine may be present at position y-3 and a glycine at positions y-2, y-1, and y.) In some embodiments, y=20 or 21 may be preferred. In some embodiments, it may be preferred that the linker is (GxS)n(GGSGG) or (GxS)n(GGSGGG), where G=glycine, S=serine, x=4 and n=1 to 20, or 2 to 20, or 1 to 10, or 2 to 10, e.g., n=2, 3, 4, 5, 6, 7, 8, or 9, e.g., n=2 to 5, or 2 to 4. For example, the linker can be GGGGSGGGGSGGGGSGGSGG or GGGGSGGGGSGGGGSGGSGGG.

[0382] J. CD40 agonist The combination therapy of the present invention includes a CD40 agonist.

[0383] The human CD40 antigen is a 50 kDa cell surface glycoprotein belonging to the tumor necrosis factor receptor (TNF-R) family (Stamenkovic et al., EMBO J. 8:1403-10 (1989)). It is also known as "tumor necrosis factor receptor superfamily member 5." Alternative names include B cell surface antigen 40, Bp50, CD40L receptor, CDw40, CDW40, MGC9013, p50, or TNFRSF5. It is registered, for example, under UniProt entry number P25942. In one embodiment, the human CD40 antigen has the sequence shown below (see Table 1). [Table 1]

[0384] CD40 is expressed by antigen-presenting cells (APCs), and binding of its natural ligand on T cells activates APCs, including dendritic cells and B cells.

[0385] As used herein, a "CD40 agonist" includes any moiety that agonizes the CD40 / CD40L interaction. Typically, these moieties will be agonist CD40 antibodies or agonist CD40L polypeptides. An "agonist" binds to a receptor on a cell and initiates a similar or identical reaction or activity to that initiated by the receptor's natural ligand. In one embodiment, a "CD40 agonist" induces any or all of the following responses, including, but not limited to, B cell proliferation and / or differentiation; upregulation of cell-cell adhesion via molecules such as ICAM-1, E-selectin, VC AM; secretion of pro-inflammatory cytokines such as IL-1, IL-6, IL-8, IL-12, TNF; signaling through the CD40 receptor by pathways such as TRAFs (e.g., TRAF2 and / or TRAF3), MAP kinases, e.g., NIK (NF-kB-inducing kinase), I-kappa B kinase (IKK / .beta.), transcription factors NF-kB, Ras and the MEK / ERK pathway, the PI3K AKT pathway, the p38 MAPK pathway, and the like; transduction of anti-apoptotic signals by molecules such as XIAP, mcl-1, bcl-x, and the like; generation of B and / or T cell memory; B cell antibody production; B cell isotype switching, upregulation of MHC class II and CD80 / 86 cell surface expression, and the like.

[0386] Exemplary agonists include the CD40 ligand CD40L (including functional variants thereof), or a nucleic acid expressing CD40L or a functional variant thereof, such as recombinant human CD40L, or an adenoviral vector expressing CD40L.

[0387] In other embodiments, the agonist may be an anti-CD40 antibody, such as a monoclonal antibody. For example, the antibody may be a human or humanized antibody or a chimeric antibody. The antibody may be an IgG, such as an IgG1, IgG2, IgG3, or IgG4.

[0388] In some embodiments, the antibody has a cytotoxicity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g. 10 -8 M~10 -13 M, e.g. 10 -9 M~10 -13 Dissociation constant (K D In another embodiment, the antibody can bind to CD40 at 4×10 -10 K below M D It can bind to human CD40 at 1000 kJ / s.

[0389] Exemplary anti-CD40 agonist antibodies are known in the art, and any of these or functional variants thereof may be used in embodiments of the present invention.

[0390] CP-870,893 (Pfizer) (also known as cericlerumab) is a fully human CD40 agonist IgG2 mAb that exhibits immune- and non-immune-mediated effects on tumor cell death (Vonderheide RH, Flaherty KT, Khalil M, Stumacher MS, Bajor DL, Hutnick NA, et al. Clinical activity and immune modulation in cancer patients treated with CP-870,893, a novel CD40 agonist monoclonal antibody. J Clin Oncol. 2007;25:876-83).

[0391] Dacetuzumab (Seattle Genetics) is a humanized mAb IgG1 against CD40 (Khubchandani S, Czuczman MS, Hernandez-Ilizaliturri FJ. Dacetuzumab, a humanized mAb against CD40 for the treatment of hematological malignancies. Curr Opin Investig Drugs. 2009;10:579-87.).

[0392] Chi Lob 7 / 4(University of Southampton) is a specific IgG1 (Johnson PW, Steven NM, Chowdhury F, Dobbyn J, Hall E, Ashton-Key M, et al monoclonal antibody(MAb),Chi Lob 7 / 4.J Clin Oncol.2010;28:2507).

[0393] APX005M contains active IgG1(Bjorck P,Filbert E,Zhang Y,Yang X,Trifan O.The CD40 agonistic monoclonal antibody APX005M has potent immune stimulatory capabilities.J Immunother Cancer.2015;3:P198.doi:10.1186 / 2051-1426-3-S2-P198.).

[0394] ADC-1013 is a specific IgG1 (Mangsbo SM,Broos S,Fletcher E,Veitonmaki N,Furebring C,Dahlen E,Norlen P,Lindstedt M,Totterman TH,Ellmark P.The human agonistic CD40 antibody ADC-1013 eradicates Clin Cancer Res.2015;21:1115-1126.doi:10.1158 / 1078-0432.CCR-14-0913.) bladder tumors and generates T-cell-dependent tumor immunity.

[0395] CDX-1140 is a fully human IgG2 (Vitale LA, Thomas LJ, He LZ, O'Neill T, Widger J, Crocker A, Sundarapandiyan K, Storey JR, Forsberg EM, Weidlick J, et al. Development of CDX-1140, an agonist CD40 antibody for cancer immunotherapy. Cancer Immunol Immunother. 2019;68:233-245. doi:10.1007 / s00262-018-2267-0.).

[0396] K. Immune Checkpoint Inhibitors: The combination therapy of the present invention includes an immune checkpoint inhibitor.

[0397] Exemplary immune checkpoint inhibitors include inhibitors of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-1 5049, CHK1, CHK2, A2aR, B-7, or combinations thereof.

[0398] In some embodiments, the checkpoint inhibitor can be an inhibitor of PD1, PDL1, or CTLA4.

[0399] The human PD-L1 (or PDL1) antigen is also known as "programmed cell death 1 ligand 1" or the CD274 molecule. Alternative designations include B7-H, B7H1, B7-H1, B7 homolog 1, MGC142294, MGC142296, PDCD1L1, PDCD1LG1, PDCD1 ligand 1, PDL1, PD-L1, and programmed death ligand 1. In one embodiment, the human PD-L1 antigen has the sequence shown below (Table 2), as found, for example, in UniProt entry number Q9NZQ7: [Table 2]

[0400] In some embodiments, the inhibitor can be, for example, a small molecule or peptide that can bind to PD-1 or PD-L1 and block the association of PD1 with PD-L1.

[0401] In some embodiments, the inhibitor is an antibody against a checkpoint inhibitor, such as an anti-PD1, anti-PDL1, or anti-CTLA4 antibody. The antibody can be a monoclonal antibody. In some embodiments, the antibody can be a human or humanized antibody or a chimeric antibody. The antibody can be an IgG, such as an IgG1, IgG2, IgG3, or IgG4.

[0402] In some embodiments, the antibody has a cytotoxicity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g. 10 -8 M~10 -13 M, e.g. 10 -9 M~10 -13 Dissociation constant (K D ) and may bind to checkpoint inhibitors, such as PD1, PDL1, or CTLA4. The antibody may bind to human PD1, PDL1, or CTLA4.

[0403] Exemplary antibodies are known in the art. Any of these or functional variants thereof may be used in embodiments of the present invention. Examples include: nivolumab (anti-PD-1 mAb, BMS-936558 / ONO-4538, Bristol-Myers Squibb, formerly MDX-1 106); pembrolizumab (anti-PD1 mAb, MK-3475, lambrolizumab, Keytruda®, Merck), cemiplimab (anti-PD-1, Regeneron), atezolizumab (anti-PD-L1 mAb, Tecentriq®, MPDL3280A / RG7446, Roche / Genentech); avelumab (Bavencio), a fully human IgG1 anti-PD-L1 antibody developed by Merck Serono and Pfizer; Durvalumab (Imfinzi), a fully human IgG1 anti-PD-L1 antibody developed by AstraZeneca.

[0404] Exemplary PD-1 inhibitors may also be selected from JTX-4014 by Jounce Therapeutics; spartalizumab (PDR001); camrelizumab (SHR1210); sintilimab (IBI308); tislelizumab (BGB-A317); toripalimab (JS 001); dostarimab (TSR-042, WBP-285); INCMGA00012 (MGA012), a humanized IgG4 monoclonal antibody developed by Incyte and MacroGenics; AMP-224 by AstraZeneca / MedImmune and GlaxoSmithKline; AMP-514 (MEDI0680) by AstraZeneca.

[0405] Exemplary PD-L1 inhibitors may also be selected from KN035; CK-301 by Checkpoint Therapeutics; AUNP12; CA-170; or BMS-986189.

[0406] CTLA-4 (cytotoxic T lymphocyte-associated protein 4), also known as CD152, is another inhibitory member of the CD28 family of receptors and is expressed on T cells.

[0407] Antibodies that bind to and inhibit CTLA-4 are known in the art. In one example, the antibody is ipilimumab (trade name Yervoy®, Bristol-Myers Squibb), a human IgG antibody.

[0408] In another example, the anti-CTLA-4 antibody is tremelimumab (formerly ticilimumab, CP-675,206), a human IgG2 antibody.

[0409] L. Remover As mentioned above, in some embodiments of the present invention, a scavenger may be used.

[0410] Exemplary agents bind to antibodies and increase their rate of clearance from the body, including anti-idiotypic antibodies.

[0411] Other exemplary agents are those that bind to the antigen binding site of the radiolabeled compound, but are not themselves radiolabeled.For example, if the radiolabeled compound comprises a chelating agent that carries a radioisotope of a specific chemical element (e.g., metal), the agent can comprise the same chelating agent that carries a non-radioactive isotope of the same element (e.g., metal), or can comprise an unloaded chelating agent or a chelating agent that carries a different non-radioactive moiety (e.g., a non-radioactive isotope of a different element), as long as it can still be bound by the antigen binding site.

[0412] In some cases, the removing / blocking agent may further comprise a moiety that increases the size and / or hydrodynamic radius of the molecule. These prevent the molecule's ability to access the tumor without interfering with its ability to bind to circulating antibodies. Exemplary moieties include hydrophilic polymers. The moiety may be, for example, a polymer or copolymer of dextran, dextrin, PEG, polysialic acid (PSA), hyaluronic acid, hydroxyethyl starch (HES), or poly(2-ethyl-2-oxazoline) (PEOZ). In other embodiments, the moiety may be an unstructured peptide or protein, such as an XTEN polypeptide (an unstructured hydrophilic protein polymer), a homoamino acid polymer (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 moieties / polymers can be, for example, at least 50 kDa, for example, in the range of 50 kDa to 2000 kDa. For example, the molecular weight can be 200 to 800 kDa, optionally greater than 300, 350, 400, or 450 kDa, and optionally less than 700, 650, 600, or 550 kDa, optionally about 500 kDa.

[0413] Exemplary clearing agents are described in International Publication No. 2019 / 202399, which is incorporated herein by reference. This publication describes dextran-based clearing agents containing dextran or its derivatives, such as aminodextran, conjugated to M-DOTAM (M-DOTAM is DOTAM or a functional variant thereof incorporating a metal ion), and the complex is recognized by the antigen binding site for Pb-DOTAM. The metal present in the clearing agent may be a stable (non-radioactive) isotope of lead, or a stable or essentially stable isotope of another metal ion, as long as the metal ion-DOTAM complex is recognized with high affinity by the antibody.

[0414] "Stable isotope" means an isotope that does not undergo radioactive decay. "Inherently stable isotope" means an isotope that undergoes radioactive decay with a very long half-life, making it safe for use. Preferably, the metal ion is selected from ions of Pb, Ca, and Bi. For example, the scavenger may be a stable isotope of Pb complexed with DOTAM or a functional variant thereof, Ca complexed with DOTAM or a functional variant thereof, or Bi complexed with DOTAM or a functional variant thereof. 209 Bi(1.9×10 19 Pb may be naturally occurring lead, which is a stable (non-radioactive) isotope. 204 Pb, 206 Pb, 207 Pb and 208 It is a mixture of Pb.

[0415] M. Therapeutic Methods and Compositions In certain aspects, the present invention provides a combination therapy as described herein for the treatment of a proliferative disease or disorder, e.g., a tumor or cancer, in an individual. An "individual" or "subject" according to any of the above aspects is preferably a mammal, more preferably a human.

[0416] The term "cancer" as used herein includes both 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, cancer of the head and neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, cancer of the anal region, stomach cancer, gastric cancer, and the like. cancer), colorectal cancer, which may be colon 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, 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, carcinoma of the renal pelvis, mesothelioma, hepatocellular carcinoma, biliary tract cancer, neoplasms of the central nervous system (CNS), spinal axis tumors, brain stem glioma, glioblastoma multiforme, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, and Ewing's sarcoma (including refractory versions of any of the above cancers, versions of any of the above cancers that have experienced a checkpoint inhibitor, or a combination of one or more of the above cancers). In one embodiment, such "cancer" is a solid tumor selected from breast cancer, lung cancer, colon cancer, ovarian cancer, melanoma cancer, bladder cancer, renal cancer, kidney cancer, liver cancer, head and neck cancer, colorectal cancer, pancreatic cancer, gastric carcinoma cancer, esophageal cancer, mesothelioma, or prostate cancer. In another embodiment, such "cancer" is a hematological tumor, such as, for example, leukemia (AML, CLL, etc.), lymphoma, or myeloma. In yet another embodiment, the "cancer" is breast cancer, lung cancer, colon cancer, colorectal cancer, pancreatic cancer, gastric cancer, or prostate cancer.

[0417] In some embodiments, the cancer may be resistant to immune checkpoint inhibitors as monotherapy. Examples may include human melanoma, renal cell carcinoma (RCC), NSCLC, gastrointestinal cancer, breast cancer, pancreatic cancer, prostate cancer, sarcoma, and colorectal cancer, such as pancreatic ductal adenocarcinoma.

[0418] A method of treating a proliferative disorder or cancer can include administering to a patient i) a multispecific antibody or a split multispecific antibody having a binding site for a radiolabeled compound and a binding site for a target antigen, ii) a radiolabeled compound, iii) a CD40 agonist, and iv) an immune checkpoint inhibitor.

[0419] The radiolabeled compound is administered to the patient after the multispecific antibody or split multispecific antibody binds to the target antigen. The radiolabeled compound then binds to the multispecific antibody or split multispecific antibody and is thus localized in the target cells.

[0420] The anti-CD40 antibody and the immune checkpoint inhibitor can be administered simultaneously or sequentially in any order. They can be administered before or after the administration of the multispecific antibody / split multispecific antibody and the radiolabeled compound. Preferably, they are administered after the multispecific antibody / split multispecific antibody and the radiolabeled compound.

[0421] In one embodiment, the treatment comprises a treatment cycle comprising a first step of pre-targeted radioimmunotherapy comprising administering a multispecific antibody or split multispecific antibody followed by administration of a radiolabeled compound, and a second step of immunotherapy comprising administering a CD40 agonist and an immune checkpoint inhibitor, wherein the anti-CD40 antibody and the immune checkpoint inhibitor are administered simultaneously or sequentially in either order.

[0422] In some embodiments, the second step (immunotherapy) may include repeated administration of one or both of an anti-CD40 antibody and an immune checkpoint inhibitor. For example, the second step may include administration of both an anti-CD40 antibody and an immune checkpoint inhibitor (simultaneously or sequentially in any order), followed by one or more administrations of the immune checkpoint inhibitor alone. The repeated administration may be performed at suitable intervals, as can be determined by those skilled in the art.

[0423] Treatment may involve one cycle, or preferred embodiments may involve multiple cycles, for example, 2, 3, 4, 5 or 6 cycles.

[0424] In some embodiments, not all cycles of treatment are the same. the first treatment cycle comprises a first step of pre-targeted radioimmunotherapy comprising administering a multispecific antibody or split multispecific antibody followed by administering a radiolabeled compound, and a second step of immunotherapy comprising administering a CD40 agonist and an immune checkpoint inhibitor, wherein the anti-CD40 antibody and the immune checkpoint inhibitor are administered simultaneously or sequentially in either order; and One or more subsequent cycles comprise a first step of pre-targeted radioimmunotherapy comprising administering a multispecific antibody or split multispecific antibody followed by administering a radiolabeled compound, and a second step of immunotherapy comprising administering an immune checkpoint inhibitor.

[0425] For example, there may be 1, 2, 3, 4, or 5 subsequent cycles. The radiolabeled compound is labeled with a radioisotope that is cytotoxic to cells. Suitable radioisotopes include alpha and beta emitters, as described above.

[0426] In some embodiments, the radiolabeled compound can be administered to a subject after allowing a suitable period for the multispecific antibody or split multispecific antibody to localize to target cells. For example, in some embodiments, the radiolabeled compound can be administered to a subject immediately after the multispecific antibody or split multispecific antibody, or at least 4 hours, 8 hours, 1 day, or 2 days after the multispecific antibody or split multispecific antibody. Optionally, it can be administered up to 3 days, 5 days, or 7 days after the multispecific antibody or split multispecific antibody. In one particular embodiment, the radiolabeled compound can be administered to a subject 2 to 7 days after the multispecific antibody or split multispecific antibody.

[0427] In some embodiments, the immunotherapy may be administered after the radiolabeled compound.

[0428] An exemplary treatment cycle duration is 14 days, with the multispecific antibody or split multispecific antibody being administered on day 1 of the cycle, the radiolabeled compound being administered for the next 7 days of the cycle, e.g., no later than day 8 in this example, and the immunotherapy (e.g., comprising a CD40 agonist and an immune checkpoint inhibitor, where the CD40 agonist and immune checkpoint inhibitor are administered simultaneously or sequentially in either order, or comprising an immune checkpoint inhibitor without anti-CD40) being administered at least one day after administration of the radiolabeled compound, e.g., on day 9. In one embodiment, the CD40 agonist is administered only once in the first treatment cycle. Treatment schedules involving combination therapies comprising an anti-CD40 antibody and an anti-PD-L1 antibody are disclosed, for example, in WO 2016 / 023875.

[0429] In methods of pre-targeted radioimmunotherapy using bispecific antibodies (i.e., not "split" antibodies according to the present invention), it is common practice to administer a clearing or blocking agent between administration of the antibody and the radiolabeled compound.

[0430] In some aspects of the invention, the clearing agent is administered after the multispecific antibody and before the radiolabeled compound.

[0431] In some embodiments, the clearing agent can be administered several hours or days after treatment with the multispecific antibody. In some embodiments, it may be preferable to administer the clearing agent at least 2, 4, 6, 8, 10, 12, 16, 18, 22, or 24 hours, or at least 1, 2, 3, 4, 5, 6, or 7 days after the multispecific antibody. In some embodiments, it may be preferable to administer the clearing agent within 14 days, e.g., within 10, 9, 8, 7, 6, 5, 4, 3, or 2 days, after the antibody.

[0432] Optionally, the clearing agent is administered within a period of 4 to 10 days, 4 to 7 days, 2 to 7 days, or 2 to 4 days after the multispecific antibody.

[0433] In some embodiments, the radionuclide is administered a few minutes, a few hours, or a few days after the clearing agent. In some embodiments, it may be preferable to administer the radionuclide at least 30 minutes after the administration of the clearing agent, and optionally within 48 hours, 24 hours, 8 hours, or 4 hours after the administration of the clearing agent. In some embodiments, the radionuclide can be administered the day after the administration of the clearing agent. Thus, for example, if the radiolabeled compound is administered on the 8th day of the cycle, the clearing agent can be administered on the 7th day.

[0434] In other aspects of the invention, such as those involving split multispecific antibodies, there is no step of administering a clearing or blocking agent to the subject. In certain aspects, there is no step of administering any agent that binds to the first hemibody or the second hemibody, or the split antibody formed from the first hemibody and the second hemibody, between the administration of the split antibody and the administration of the radiolabeled compound. In certain aspects, there is no step of administering any agent between the administration of the split antibody and the administration of the radiolabeled compound, except optionally for compounds selected from chemotherapeutic agents and radiosensitizers. In some embodiments, no agent is administered between the administration of the antibody and the administration of the radiolabeled compound. In some embodiments, there may be no injection or infusion of any other agent into the subject between the administration of the antibody and the administration of the radiolabeled compound.

[0435] In some embodiments, the antibodies described herein may additionally or alternatively be administered in combination with a radiosensitizer. The radiosensitizer and antibody may be administered simultaneously or sequentially in either order.

[0436] The multispecific or split multispecific antibody, radiolabeled compound, anti-CD40 antibody, and immune checkpoint inhibitor may be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for localized treatment, intralesional administration. Parenteral infusion or injection includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In some embodiments, administration may be by intravenous or subcutaneous injection. In some embodiments, the multispecific or split multispecific antibody, and / or anti-CD40 antibody and / or immune checkpoint inhibitor may be administered by IV infusion. In some embodiments, the radiolabeled compound may be administered by IV injection, and the anti-CD40 antibody and / or immune checkpoint inhibitor may be administered subcutaneously (sc).

[0437] In some embodiments, one or more dosimetry cycles may be used prior to one or more treatment cycles described above. A dosimetry cycle may comprise i) administering a multispecific antibody or split multispecific antibody, followed by ii) administering a compound suitable for imaging radiolabeled with a gamma emitter, which radiolabeled compound binds to a functional binding site for the radiolabeled compound. The compound may be the same as the compound used in the subsequent treatment cycle, except that it is labeled with a gamma emitter rather than an alpha or beta emitter. For example, in one embodiment, the radiolabeled compound used in the dosimetry cycle is 203 The radiolabeled compound used in the treatment cycle can be Pb-DOTAM. 212 The compound may be Pb-DOTAM. The patient may undergo imaging to determine the uptake of the compound into the tumor and / or to estimate the absorbed dose of the compound. This information can be used to estimate the expected radiation exposure during subsequent treatment steps and to adjust the dose of the radiolabeled compound used in the treatment steps to a safe level.

[0438] N. Pharmaceutical Preparations Pharmaceutical formulations of the antibodies described herein can be prepared in the form of a lyophilized formulation or aqueous solution by mixing such antibodies having the desired degree of purity with one or more pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations used, 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 (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, 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; serum albumin, gelatin, or immunoglobulins. proteins such as globulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG), poloxamer (e.g., poloxamer 188), and polysorbates (e.g., PS20, PS80, high-grade PS80, i.e., PS80 containing more than 98% oleic acid). Pharmaceutical compositions for some of the cancer immunotherapy components used according to the present invention are disclosed, for example, in WO 2003 / 040170 (for anti-CD40 antibodies) and WO 2010 / 77634 (for PD-L1 antibodies) or are available as commercial pharmaceuticals.

[0439] Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersing agents, such as soluble neutral-active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Halozyme, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one embodiment, a sHASEGP is combined with one or more additional glycosaminoglycanases (e.g., chondroitinases).

[0440] Exemplary lyophilized antibody compositions are described in U.S. Patent No. 6,267,958. Aqueous antibody compositions include those described in U.S. Patent No. 6,171,586 and WO 2006 / 044908, the latter compositions comprising a histidine-acetate buffer.

[0441] When the antibody is a split antibody, the first hemibody and the second hemibody can be formulated in a single pharmaceutical composition or in separate pharmaceutical compositions.

[0442] The formulations herein may also contain two or more active ingredients as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to additionally provide the aforementioned chemotherapeutic agents and / or radiosensitizers. Such active ingredients are suitably present in combination in amounts effective for the intended purpose.

[0443] The active ingredient can also be incorporated into colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or into macroemulsions, for example, by coacervation techniques or by microcapsules prepared by interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0444] Sustained-release preparations may also be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

[0445] Formulations to be used for in vivo administration are generally sterile, which may be readily accomplished, for example, by filtration through sterile filtration membranes.

[0446] O. Antibody Variants In certain embodiments, amino acid sequence variants of any of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions 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, as long as the final construct possesses the desired characteristics (e.g., antigen binding).

[0447] Substitution, insertion, and deletion mutants In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include HVRs (CDRs) and FRs. Conservative substitutions are shown in Table 3 under the heading of "Preferred Substitutions." More substantial changes are shown in Table 3 under the heading of "Exemplary Substitutions" and are further described below with respect to amino acid side chain classes. Amino acid substitutions may be introduced into the antibody of interest, and the products screened for a desired activity, such as retained / improved antigen binding, reduced immunogenicity, or reduced or eliminated ADCC or CDC. [Table 3]

[0448] Amino acids can be classified according to general side chain properties. (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0449] Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0450] Certain substitutional variants involve substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further testing will have modified (e.g., improved) certain biological properties (e.g., increased affinity, reduced immunogenicity) compared to the parent antibody and / or will have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity-matured antibody, which can be conveniently generated using, for example, phage-display-based affinity maturation techniques as described herein. Briefly, one or more CDR residues are mutated, and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0451] For example, modifications (e.g., substitutions) may be made in CDRs to improve antibody affinity. Such modifications may be made in CDR "hot spots," i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or residues that contact antigen, and the resulting mutant VH or VL are tested for binding affinity. Affinity maturation by construction of and reselection from secondary libraries is described, for example, in Hoogenboom et al. Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. This library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves a CDR-directed approach, in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 are often targeted in particular.

[0452] In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, as long as such modifications do not substantially reduce the antibody's ability to bind to the antigen. For example, conservative modifications (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in the CDRs. Such modifications may, for example, be outside the antigen-contacting residues in the CDRs. In the specific variant VH and VL sequences provided above, each CDR is either unmodified or contains no more than one, two, or three amino acid substitutions.

[0453] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) is identified and substituted with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction between the antibody and antigen is affected. Further substitutions may be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitution. Alternatively, or additionally, a crystal structure of an antigen-antibody complex may be used to identify contact points between the antibody and antigen. Such contact and adjacent residues may be targeted as candidates for substitution or eliminated. Mutants may be screened to determine whether they contain desired properties.

[0454] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT (antibody-directed enzyme prodrug therapy)) or a polypeptide which increases the serum half-life of the antibody.

[0455] Glycosylation variants In certain embodiments, the antibodies provided herein are modified to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0456] If an antibody contains an Fc region, the oligosaccharides attached thereto can be modified. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides that are generally attached to Asn297 in the CH2 domain of the Fc region via an N-linkage. See, for example, Wright et al., TIBTECH 15:26-32 (1997). Oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the antibodies of the present invention can be performed to create antibody variants with specific improved properties.

[0457] In one embodiment, antibody variants are provided that have nonfucosylated oligosaccharides, i.e., oligosaccharide structures lacking fucose attached (directly or indirectly) to the Fc region. Such nonfucosylated oligosaccharides (also referred to as "afucosylated" oligosaccharides) are particularly N-linked oligosaccharides that lack a fucose residue attached to the first GlcNAc in the stem of the biantennary oligosaccharide structure. In one embodiment, antibody variants are provided that have an increased proportion of nonfucosylated oligosaccharides in the Fc region compared to the native or parent antibody. For example, the proportion of nonfucosylated oligosaccharides can be at least about 20%, at least about 40%, at least about 60%, at least about 80%, or even about 100% (i.e., no fucosylated oligosaccharides are present). The percentage of nonfucosylated oligosaccharides is the (average) amount of oligosaccharides lacking a fucose residue relative to the sum of all oligosaccharides (e.g., complex, hybrid, and high-mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, for example, as described in WO 2006 / 082515. Asn297 refers to an asparagine residue located at approximately position 297 (Fc region residues according to EU numbering) within the Fc region; however, due to slight sequence variations in antibodies, Asn297 may also be located ±3 amino acids upstream or downstream from position 297, i.e., between positions 294 and 300. Such antibodies with an increased percentage of nonfucosylated oligosaccharides in the Fc region may have improved FcγRIIIa receptor binding and / or improved effector function, particularly improved ADCC function. See, for example, U.S. Patent Application Publication Nos. 2003 / 0157108 and 2004 / 0093621.

[0458] Examples of cell lines capable of producing antibodies with reduced fucosylation include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986), U.S. Patent Application Publication No. 2003 / 0157108, and WO 2004 / 056312, especially Example 11), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene FUT8 knockout CHO cells (e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614-622 (2004), Kanda, Y. et al. al., Biotechnol. Bioeng., 94(4):680-688 (2006), and WO 2003 / 085107), or cells with reduced or eliminated activity of GDP-fucose synthesis or transporter proteins (see, e.g., U.S. Patent Application Publication Nos. 2004259150, 2005031613, 2004132140, and 2004110282).

[0459] In a further embodiment, antibody variants are provided with bisected oligosaccharides, e.g., biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function, as described above. Examples of such antibody variants are described, for example, in Umana et al., Nat Biotechnol 17, 176-180 (1999), Ferrara et al., Biotechn Bioeng 93, 851-861 (2006), WO 99 / 54342, WO 2004 / 065540, and WO 2003 / 011878.

[0460] Also provided are antibody variants that have at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants can have improved CDC function. Examples of such antibody variants are described in, for example, WO 1997 / 30087, WO 1998 / 58964, and WO 1999 / 22764.

[0461] It may be preferable to modify the antibody to reduce the degree of glycosylation. In some embodiments, the antibody may be aglycosylated or deglycosylated. The antibody may include a substitution at N297, for example, N297D / A.

[0462] Fc region variants In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0463] In certain embodiments, the present invention contemplates antibody variants that have reduced effector function, e.g., reduced or eliminated CDC, ADCC, and / or FcγR binding. In certain aspects, the present invention contemplates antibody variants that possess some, but not all, effector functions, making them desirable candidates for applications in which antibody half-life in vivo is important, but where certain effector functions (e.g., complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC)) are unnecessary or deleterious.

[0464] To confirm reduced / depleted CDC and / or ADCC activity, in vitro and / or in vivo cytotoxicity assays can be performed. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985), 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be used (e.g., the ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, CA) and the CytoTox96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest can be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay may also be performed to confirm that the antibody is unable to bind C1q and therefore lacks CDC activity.See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). Determination of FcRn binding and in vivo clearance / half-life can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006); WO 2013 / 120929 A1).

[0465] Antibodies with reduced effector function include those with substitutions at one or more of residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region (U.S. Patent No. 6,737,056), e.g., P329G. Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0466] In certain embodiments, the antibody variant comprises an Fc region having one or more amino acid substitutions that reduce FcγR binding, e.g., substitutions at positions 234 and 235 (EU numbering) of the Fc region. In one embodiment, the substitutions are L234A and L235A (LALA). In certain embodiments, the antibody variant further comprises D265A and / or P329G in an Fc region derived from a human IgG1 Fc region. In one embodiment, the substitutions are L234A, L235A, and P329G (LALA-PG) in an Fc region derived from a human IgG1 Fc region. (See, e.g., WO 2012 / 130831). In another embodiment, the substitutions are L234A, L235A, and D265A (LALA-DA) in an Fc region derived from a human IgG1 Fc region. Alternative substitutions include L234F and / or L235E, optionally in combination with D265A and / or P329G and / or P331S.

[0467] In other embodiments, it may be possible to use IgG subtypes with reduced effector function, such as IgG4 or IgG2. Certain antibody variants have been described with improved or diminished binding to FcRs (see, e.g., U.S. Pat. No. 6,737,056, WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0468] In some embodiments, modifications are made within the Fc region that result in altered (i.e., either improved or decreased, preferably decreased) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0469] In certain embodiments, the antibody variant comprises an Fc region having one or more amino acid substitutions that reduce FcRn binding, e.g., substitutions at positions 253, 310, and / or 435 (EU numbering) of the Fc region. In certain embodiments, the antibody variant comprises an Fc region having amino acid substitutions at positions 253, 310, and 435. In one embodiment, the substitutions are I253A, H310A, and H435A in the Fc region derived from a human IgG1 Fc region. See, e.g., Grevys, A., et al., J. Immunol. 194 (2015) 5497-5508.

[0470] In certain aspects, the antibody mutant comprises an Fc region having one or more amino acid substitutions that reduce FcRn binding, for example, substitutions at positions 310, 433, and / or 436 (EU numbering) of the Fc region. In certain aspects, the antibody mutant 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 the Fc region derived from a human IgG1 Fc region. (See, e.g., WO 2014 / 177460 A1.) For example, in some embodiments, normal FcRn binding may be used.

[0471] See also Duncan & Winter, Nature 322:738-40 (1988), U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821, and WO 94 / 29351 for other examples of Fc region variants.

[0472] The C-terminus of the heavy chain of a full-length antibody reported herein can be a complete C-terminus ending at amino acid residue PGK. The C-terminus of the heavy chain can be a shortened C-terminus in which one or two of the C-terminal amino acid residues are removed. The C-terminus of the heavy chain can be a shortened C-terminus ending at PG. In one ...

Claims

1. 1. A medicament for treating a proliferative disorder in a subject, comprising a multispecific antibody or a split multispecific antibody, said multispecific antibody or split multispecific antibody having a binding site for a radiolabeled compound and a binding site for a target antigen, wherein i) said radiolabeled compound, ii) a CD40 agonist, and iii) an immune checkpoint inhibitor are further administered to said subject.

2. A pharmaceutical for treating a proliferative disorder in a subject comprising a CD40 agonist, wherein i) a multispecific antibody or a split multispecific antibody having a binding site for a radiolabeled compound and a binding site for a target antigen, ii) the radiolabeled compound, and iii) an immune checkpoint inhibitor are further administered to the subject.

3. A pharmaceutical for treating a proliferative disorder in a subject comprising an immune checkpoint inhibitor, wherein i) a multispecific antibody or a split multispecific antibody having a binding site for a radiolabeled compound and a binding site for a target antigen, ii) the radiolabeled compound, and iii) a CD40 agonist are further administered to the subject.

4. A pharmaceutical for treating a proliferative disorder in a subject, comprising a multispecific antibody or a split multispecific antibody having a binding site for a radiolabeled compound and a binding site for a target antigen, a radiolabeled compound, a CD40 agonist and an immune checkpoint inhibitor.

5. The medicament according to any one of claims 1 to 4, wherein the treatment cycle comprises a first step of pre-targeted radioimmunotherapy comprising administering said multispecific antibody or split multispecific antibody followed by administering said radiolabeled compound, and a second step of immunotherapy comprising administering a CD40 agonist and an immune checkpoint inhibitor, said CD40 agonist and said immune checkpoint inhibitor being administered simultaneously or sequentially in any order.

6. 6. The medicament of claim 5, wherein one or more additional treatment cycles are included, each additional cycle comprising a first step of pre-targeted radioimmunotherapy comprising administering said multispecific antibody or split multispecific antibody followed by administering said radiolabeled compound, and a second step of immunotherapy comprising administering an immune checkpoint inhibitor.

7. The method of claim 6, wherein 2, 3, 4 or 5 additional cycles are included.

8. The pharmaceutical described in any one of claims 1 to 4, wherein the radioactive label is an alpha emitter.

9. The radiolabeled compound is ２１２ Pb, ２１２ Bi or ２１３ The pharmaceutical according to any one of claims 1 to 4, which is DOTAM chelated with Bi.

10. The pharmaceutical composition according to any one of claims 1 to 4, wherein the target antigen is CEA.

11. The pharmaceutical according to any one of claims 1 to 4, wherein the multispecific antibody comprises an Fc domain, and optionally the Fc domain has been modified to reduce or eliminate effector function.

12. The split multispecific antibody, i) a first hemibody that binds to a target antigen and further comprises a VH domain of a functional antigen binding site for a radiolabeled compound, but does not comprise a VL domain of said functional antigen binding site for said radiolabeled compound; ii) a second hemibody that binds to the target antigen and further comprises a VL domain of a functional antigen binding site for said radiolabeled compound, but does not comprise a VH domain of said functional antigen binding site for said radiolabeled compound; Including, The medicament of any one of claims 1 to 4, wherein the VH domain of the first hemibody and the VL domain of the second hemibody are capable together of forming a functional antigen binding site for the radiolabeled compound.

13. The pharmaceutical of claim 12, wherein the first hemibody and the second hemibody each comprise an Fc domain, and optionally the Fc domain has been modified to reduce or eliminate effector function.

14. The pharmaceutical according to any one of claims 1 to 4, wherein the CD40 agonist is an agonistic anti-CD40 antibody.

15. The pharmaceutical according to any one of claims 1 to 4, wherein the immune checkpoint inhibitor is selected from an inhibitor of PD1, PDL1 or CTLA4, optionally the immune checkpoint inhibitor is an antibody selected from an antibody against PD1, an antibody against PDL1 and an antibody against CTLA4, and further optionally the immune checkpoint inhibitor is an antibody against PDL1.

16. The pharmaceutical composition according to any one of claims 1 to 4, wherein the proliferative disorder is cancer.

17. The pharmaceutical composition according to any one of claims 1 to 4, wherein the subject is a human.

18. The pharmaceutical composition comprises: i) results in a slower rate of tumor growth than treatment with pre-targeted radioimmunotherapy and / or immunotherapy alone; and / or ii) results in an increased likelihood of survival for a subject relative to treatment with pre-targeted radioimmunotherapy and / or immunotherapy alone; and / or iii) results in an increased frequency of activated intratumoral CD8 T cells and / or an increased frequency of activated plasmacytoid DCs (pDCs) and classical DCs (cDCs) in the tumor, spleen and draining lymph nodes (DLNs) compared to treatment with prior targeted radioimmunotherapy and / or immunotherapy alone; and / or iv) results in an enhanced immune memory response relative to prior targeted radioimmunotherapy and / or immunotherapy treatment alone; The pharmaceutical composition according to any one of claims 1 to 4.

19. A kit for the treatment of a proliferative disease, comprising: A) as a first component, a composition comprising as an active ingredient a multispecific antibody or a split multispecific antibody having a binding site for a radiolabeled compound and a binding site for a target antigen; B) as a second component, a composition comprising as an active ingredient a CD40 agonist; and C) as a third component, a composition comprising as an active ingredient an immune checkpoint inhibitor, preferably a PD-L1 inhibitor, wherein said components are administered in combination, simultaneously or sequentially.

20. 20. The kit of claim 19, wherein the proliferative disease is cancer.

21. The kit according to claim 19 or 20, further comprising, as a fourth component, a composition comprising the radiolabeled compound as an active ingredient.