TROP2 antibody

Abstract

Provided herein are anti-Trop2 antibodies or their conjugate fragments that bind to Trop2, for example, human Trop2. The anti-Trop2 antibodies of this disclosure are useful for treating proliferative disorders or cells expressing Trop2 or mutant Trop2. Methods of using anti-Trop2 antibodies or their conjugate fragments are also provided herein, as well as bivalent or polyvalent anti-Trop2 antibodies or their conjugate fragments that can form complexes that attract immune effectors or bind to other cells, such as a second antibody, an antigen-binding or fragment thereof of a second antibody; target-binding proteins, cytokines; lectins; or toxins.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 506,268, filed on 5 June 2023, and U.S. Provisional Patent Application No. 63 / 515,375, filed on 25 July 2023, and incorporates all of its contents herein by reference.

[0002] This paper relates to materials and methods for treating cancer, particularly the use of anti-TROP2, and to bispecific antibodies for reducing or eliminating cancer and treating proliferative disorders.

[0003] Integration by referencing materials submitted on compact discs This application includes a sequence listing submitted in ASCII format via EFS-Web, which is incorporated herein by reference in its entirety. 2024 month The aforementioned ASCII copy created on [date] It is named .xml and its size is It's a part-time job. [Background technology]

[0004] Without limiting the scope of this invention, the background is described in relation to the TROP2 antibody.

[0005] Such antibodies have been filed by Cardillo and Goldenberg, respectively, and are taught in U.S. Patent Applications Publications 20180271992 and 20180185351, titled "Treatment of high TROP-2 expressing triple-negative breast cancer (TNBC) with sacituzumab govitecan (IMMU-132) overcomes homologous recombination repair (HRR) rescue mediated by Rad51" and "therapy of small-cell lung cancer (SCLC) with a topoisomerase-i inhibiting antibody-drug conjugate (ADC) targeting Trop-2." These applicants are said to teach the treatment of Trop-2-positive cancer with a combination of an anti-Trop-2 antibody-drug conjugate (ADC) and a Rad51 inhibitor such as Sn-38, where the ADC is sacituzumab govitecan. ADCs may be administered in doses of 4 mg / kg to 16 mg / kg, preferably 4, 6, 8, 9, 10, 12, or 16 mg / kg. When administered according to the specified doses and schedule, the combination of ADCs and Rad51 inhibitors is said to be effective in reducing solid tumor size, reducing or eliminating metastases, and treating cancers resistant to standard therapies such as radiotherapy, chemotherapy, or immunotherapy. Surprisingly, the combination is said to be effective in treating cancers that are refractory to or recurrent from irinotecan or topotecan.

[0006] U.S. Patent Application Publication No. 20130344509 and U.S. Patent No. 10,202,461, filed by Nakamura, et al., are titled “Anti-human Trop-2 antibody having an antitumor activity in vivo” and “Anti-human TROP-2 antibody having an antitumor activity in vivo,” respectively. These inventors claim to teach an antibody (e.g., a humanized antibody) that specifically binds to hTROP-2 and has antitumor activity in vivo, as well as a hybridoma that produces the antibody; and a drug-antibody conjugate; a pharmaceutical composition for diagnosing or treating tumors; a method for detecting tumors; and a kit for detecting or diagnosing tumors.

[0007] Another such antibody has been filed by Guerra and Severio and is taught in U.S. Patent No. 10,501,555, entitled “Disease Therapy By Inducing Immune Response To Trop-2 Expressing Cells.” These inventors are said to teach humanized anti-Trop-2 antibodies, as well as fragments, derivatives, and conjugates thereof, that can recognize specific regions of the Trop-2 molecule and bind with high affinity. These inventors are also said to teach the use of such antibodies and pharmaceutical compositions thereof for the diagnosis and therapy of human lesions such as cancer.

[0008] Another such antibody has been filed by Chang, et al. and is taught in U.S. Patent No. 9,670,286 titled "Disease Therapy By Inducing Immune Response To Trop-2 Expressing Cells". These inventors are said to teach a bispecific antibody having at least one binding site for Trop-2 (EGP-1) and at least one binding site for CD3. The bispecific antibody is said to be used to induce an immune response against Trop-2 expressing tumors such as esophageal cancer, pancreatic cancer, lung cancer, gastric cancer, colorectal cancer, rectal cancer, bladder cancer, breast cancer, ovarian cancer, uterine cancer, kidney cancer, or prostate cancer. The bispecific antibody can be used alone or in combination with one or more therapeutic agents such as antibody-drug conjugates, interferons, and / or checkpoint inhibitory antibodies. The bispecific antibody targets effector T cells, NK cells, monocytes, or neutrophils and is said to have the ability to induce leukocyte-mediated cytotoxicity of Trop-2 + cancer cells. The cytotoxic immune response is enhanced by the co-administration of interferons, checkpoint inhibitory antibodies, and / or ADCs.

[0009] Other patents and applications said to teach anti-TROP2 antibodies include U.S. Patent No. 7,420,040; U.S. Patent No. 7,420,041; U.S. Patent No. 10,501,555; U.S. Patent No. 10,202,461; U.S. Patent No. 9,850,312; U.S. Patent Application Publication No. 2016 / 0053018; U.S. Patent No. 9,399,074; U.S. Patent Application Publication No. 2012 / 0237518; and U.S. Patent No. 9,770,517. [Prior Art Documents] [Patent Documents]

[0010] [Patent Document 1] U.S. Patent Application Publication No. 20180271992 [Patent Document 2] U.S. Patent Application Publication No. 20180185351 [Patent Document 3] U.S. Patent Application Publication No. 20130344509 [Patent Document 4] U.S. Patent No. 10,202,461 [Patent Document 5] U.S. Patent No. 10,501,555 [Patent Document 6] U.S. Patent No. 9,670,286 [Patent Document 7] U.S. Patent No. 7,420,040 [Patent Document 8] U.S. Patent No. 7,420,041 [Patent Document 9] U.S. Patent No. 9,850,312 [Patent Document 10] U.S. Patent Application Publication No. 2016 / 0053018 [Patent Document 11] U.S. Patent No. 9,399,074 [Patent Document 12] U.S. Patent Application Publication No. 2012 / 0237518 [Patent Document 13] U.S. Patent No. 9,770,517 [Overview of the project] [Problems that the invention aims to solve]

[0011] Despite these advances, there is still a need for novel anti-TROP2 antibodies that are free from side effects and for bispecific or multiplespecific anti-TROP2 antibodies, in addition to the known anti-TROP2 antibodies. [Means for solving the problem]

[0012] As illustrated and broadly described herein, aspects of this disclosure relate to an anti-Trop2 antibody or its conjugate fragment, wherein the antibody comprises: a heavy chain variable domain (VH) complementarity-determining region (CDR) 1 containing any one amino acid sequence from SEQ ID NOs: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, or 132; a VH CDR2 containing any one amino acid sequence from SEQ ID NOs: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123, or 133; and a VH CDR2 containing any one amino acid sequence from SEQ ID NOs: 4, 14, 24, 34, 44, 54, 64, 74, 84, 94, 104, 114, 124, or 134. The CDR3 includes a light chain variable domain (VL) CDR1 containing any one amino acid sequence from sequence numbers 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, or 137; a VL CDR2 containing any one amino acid sequence from sequence numbers 8, 18, 28, 38, 48, 58, 68, 78, 88, 98, 108, 118, 128, or 138; and a VL CDR3 containing any one amino acid sequence from sequence numbers 9, 19, 29, 39, 49, 59, 69, 79, 89, 99, 109, 119, 129, or 139. In one embodiment, the antibody comprises VH containing any one amino acid sequence from SEQ ID NOs: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 156, 158, 160, or 162; and VL containing any one amino acid sequence from SEQ ID NOs: 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 164, 166, or 168. In another embodiment, the antibody comprises VH encoded by a nucleic acid sequence containing any one of SEQ ID NOs. 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, 115, 125, 135, 157, 159, 161, or 163, and VL encoded by a nucleic acid sequence containing any one of SEQ ID NOs. 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 165, 167, or 169.In another embodiment, the antibody is a monoclonal antibody, a bispecific antibody, a multivalent antibody, a multiplespecific antibody, a diabody antibody, a chimeric antibody, an scFv antibody, or a fragment thereof. In another embodiment, the antibody is a defucosylated full-length antibody. In another embodiment, the antibody contains one Fc domain from human IgG1, human IgG2, human IgG3, and human IgG4. In another embodiment, the Fc domain is a wild-type Fc domain, a variant Fc domain, or a truncated Fc domain. In another embodiment, the antibody or binding fragment further comprises a second antigen-binding domain that binds to a target other than TROP-2. In another embodiment, the second antigen target is an anti-CD3 antibody having a heavy chain selected from SEQ ID NOs: 174 and 178; the light chain is selected from SEQ ID NOs: 176 and 180. In another embodiment, the antibody is a bispecific antibody containing a heavy chain selected from SEQ ID NOs: 190 and 192, or 194 and 196.

[0013] As illustrated and broadly described herein, aspects of this disclosure relate to methods for treating a disease in a subject requiring such treatment, comprising the step of administering to the subject a therapeutically effective amount of any of the antibodies described above. In another embodiment, the disease is a proliferative disorder. In another embodiment, the proliferative disorder is a cancer selected from cancers expressing Trop2 or a variant thereof. In another embodiment, the subject is a human.

[0014] As illustrated and broadly described herein, aspects of this disclosure relate to bivalent or polyvalent antibodies, wherein the antibody comprises a heavy chain variable domain (VH) complementarity determining region (CDR) 1 containing any one amino acid sequence from SEQ ID NOs: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, or 132; a VH CDR 2 containing any one amino acid sequence from SEQ ID NOs: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123, or 133; and a VH CDR 2 containing any one amino acid sequence from SEQ ID NOs: 4, 14, 24, 34, 44, 54, 64, 74, 84, 94, 104, 114, 124, or 134. An anti-Trop2 antibody or its conjugated fragment comprising CDR3; and a light chain variable domain (VL) CDR1 containing any one amino acid sequence from sequence numbers 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, or 137; VL CDR2 containing any one amino acid sequence from sequence numbers 8, 18, 28, 38, 48, 58, 68, 78, 88, 98, 108, 118, 128, or 138; and VL CDR3 containing any one amino acid sequence from sequence numbers 9, 19, 29, 39, 49, 59, 69, 79, 89, 99, 109, 119, 129, or 139; as well as a second antibody, an antigen-binding agent of the second antibody or its fragment; a target-binding protein, cytokine; lectin; or toxin. In one embodiment, the second antibody targets an immunoeffector cell surface receptor selected from at least one of CTLA-4, PD-1, Lag3, S15, B7H3, B7H4, TCR-alpha, TCR-beta, TIM-3, CD3, 41BB, and OX40. In another embodiment, the antibody is a polyvalent antibody targeting two or more antigens other than Trop2. In yet another embodiment, the antibody is a bispecific antibody containing a heavy chain selected from SEQ ID NOs: 190 and 192, or 194 and 196.In another embodiment, the anti-Trop2 antibody comprises VH containing any one amino acid sequence from SEQ ID NOs: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 156, 158, 160, and 162, and VL containing any one amino acid sequence from SEQ ID NOs: 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 164, 166, or 168. In another embodiment, the anti-Trop2 antibody comprises VH encoded by a nucleic acid sequence containing any one of SEQ ID NOs: 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, 115, 125, 135, 157, 159, 161, or 163, and VL encoded by a nucleic acid sequence containing any one of SEQ ID NOs: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 165, 167, or 169. In another embodiment, the antibody is a monoclonal antibody, a full-length antibody, or an antibody fragment. In another embodiment, the antibody is fused to the Fc domain of any one of human IgG1, human IgG2, human IgG3, and human IgG4. In another embodiment, the target is CD3, and the antibody comprises a heavy chain selected from SEQ ID NOs: 174 and 178; the light chain is selected from SEQ ID NOs: 176 and 180. In another embodiment, the target is CD3, and the antibody comprises a heavy chain encoded by a nucleic acid selected from SEQ ID NOs: 175 and 179; the light chain is encoded by a nucleic acid selected from SEQ ID NOs: 177 and 181. In another embodiment, the Fc domain is a wild-type Fc domain, a variant Fc domain, or a cleaved Fc domain. In another embodiment, the antibody is a defucosylated full-length antibody.

[0015] As illustrated and broadly described herein, aspects of this disclosure relate to methods for treating a disease in a subject requiring such treatment, comprising the step of administering a therapeutically effective amount of the previously described antibody or bivalent antibody to the subject. In another aspect, the disease is a proliferative disorder. In another aspect, the disease is a proliferative disorder selected from cancer cells expressing Trop2 or a variant thereof, selected from breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, certain lung cancers, squamous cell carcinoma of the oral cavity, squamous cell carcinoma of the ovary, squamous cell carcinoma of the pancreas, squamous cell carcinoma of the prostate, squamous cell carcinoma of the stomach, squamous cell carcinoma of the thyroid, squamous cell carcinoma of the bladder, and squamous cell carcinoma of the uterus. In another aspect, the subject is human.

[0016] As illustrated and broadly described herein, aspects of this disclosure include VH encoded by nucleic acid sequences including any one of SEQ ID NOs: 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, 115, 125, 135, 157, 159, 161, 163; and SEQ ID NOs: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 165, 167, 169 The present invention relates to VLs encoded by nucleic acid sequences containing any one of the following; antibodies encoded by the nucleic acid sequences of SEQ ID NOs. 191 and 193, or 195 and 197; or bispecific antibodies encoded by the nucleic acid sequences of SEQ ID NOs. 175 and 177, 179 and 181, 183 and 185, 191 and 193, 195 and 197, or combinations thereof, antibodies, their conjugated fragments, and nucleic acids encoding bivalent or polyvalent nucleic acid sequences.

[0017] As illustrated and broadly described herein, aspects of this disclosure include VH encoded by nucleic acid sequences including any one of SEQ ID NOs: 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, 115, 125, 135, 157, 159, 161, 163; and SEQ ID NOs: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 165, 1 The present invention relates to a vector containing nucleic acids, which may include a VL encoded by a nucleic acid sequence containing any one of 67 or 169; an antibody encoded by the nucleic acid sequences of SEQ ID NOs. 191 and 193, or 195 and 197; or a bispecific antibody encoded by the nucleic acid sequences of SEQ ID NOs. 175 and 177, 179 and 181, 183 and 185, 191 and 193, 195 and 197, or a combination thereof.

[0018] As illustrated and broadly described herein, aspects of this disclosure include VH encoded by nucleic acid sequences including any one of SEQ ID NOs: 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, 115, 125, 135, 157, 159, 161, 163; and SEQ ID NOs: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 165, 167, 16 The present invention relates to a host cell containing a nucleic acid vector comprising a VL encoded by a nucleic acid sequence containing any one of the 9; or an antibody encoded by the nucleic acid sequences of SEQ ID NOs. 187 and 189, or 191 and 193; or a bispecific antibody encoded by the nucleic acid sequences of SEQ ID NOs. 175 and 177, 179 and 181, 183 and 185, 187 and 189, or 191 and 193, or a combination thereof.

[0019] This patent or application file contains at least one drawing made in color. A copy of this patent or patent application publication with the color drawing will be provided by the Secretariat upon request and payment of the necessary fees. This application can be understood by referring to the following description, which includes the attached drawings. [Brief explanation of the drawing]

[0020] [Figure 1A] ~ [Figure 1B] This figure shows a representative sensorgram of the antibody of the present invention. Binding kinetics were measured using a Carterra LSA high-throughput SPR instrument (Carterra) with human Fc capture. None of the tested antibodies bound to human EpCAM, but bound equally to almost all cynomolgus monkey / rhesus monkey TROP2 (cyTROP2). None of the mouse chimeras recognized mouse TROP2 (msTROP2), but almost all chicken chimeras showed affinity for msTROP2 within a range of 10 times that of binding to huTROP2. [Figure 2A] ~ [Figure 2B] Figure 2A shows the MSD binding to TROP2. The dose-dependent binding of anti-TROP2 antibody to immobilized TROP2 was assessed using the MSD assay. Several clones showed lower EC50 values ​​than RS7, indicating greater binding ability. ND: Not determined. Figure 2B shows the dose-dependent binding of anti-TROP2 antibody to immobilized TROP2, assessed using the MSD assay. Several clones showed lower EC50 values ​​than RS7, indicating greater binding ability. ND: Not determined. [Figure 3A] ~ [Figure 3B]Figure 3A shows the epitope binning of the antibodies taught herein. The network plot shown in Figure 3A shows pairwise competition between libraries of 141 antibodies against TROP2 by SPR. Each node represents an individual antibody, and the connecting lines indicate the blockage relationship between two antibodies. Antibodies with similar blockage profiles, indicating a high probability of sharing an epitope, are systematized into five groups. Figure 3B shows various regions of TROP2. Group 1 contains the majority of the evaluated antibodies, including the approved antibody RS7, and is likely to correspond to some of the antibodies that target the immunodominant domain of TROP2. Antibodies in Group 2 form their own clusters, but based on the degree of interaction with antibodies in Group 1, are likely to target the epitope targeted in Group 1 or an epitope near it. Based on peptide mapping data, antibodies in Group 3 are likely to bind at or near the N-terminal domain, and antibodies in Group 4 may bind to a membrane-proximal domain close to the RS7 binding site but different from it. The exact epitopes targeted by group 5 remain unknown. Figure 3B summarizes the vials for each antibody. [Figure 4] This figure shows the peptide mapping of antibodies by bottle group. It is a sequence map showing the locations of eight peptides to which the anti-TROP2 antibody binds, based on SPR data. In this assay, not all of the antibodies tested recognized the linear epitope. The recognition of peptides 1-2 by some antibodies in bottle group 3 suggests an epitope located approximately near the N-terminus of the TROP2 protein. The recognition of peptides 3-6, located in the C-terminal domain close to the presumed RS7 epitope, by some antibodies in groups 1 and 2 that bottled together suggests that those antibodies in those bottles target that site or a nearby region. Finally, some antibodies in group 4 recognized peptides 7-8, and therefore group 4 may broadly target the C-terminal region of the C-terminal domain immediately proximal to the transmembrane domain, which is distant from the RS7 epitope. [Figure 5A] ~ [Figure 5B]This graph shows the results of an ADCC reporter assay to identify the mouse antibodies taught herein. Figure 5A. The dose-response graph shows the ADCC efficacy of anti-human Trop2 antibodies in the human head and neck cancer cell line FaDu. The ADCC efficacy of anti-human Trop2 antibodies and defucosylated anti-human Trop2 antibodies is shown using the NFAT CD16 Jurkat ADCC reporter cell line. The fact that all defucosylated anti-human Trop2 antibodies showed very low EC50 values ​​compared to their fucosylated versions indicates that defucosylated anti-human Trop2 antibodies enhance ADCC efficacy. EC50 values ​​are the average of n=2-3 experiments. Figure 5B. This table shows the ADCC efficacy of anti-human Trop2 antibodies in the FaDu cancer cell line. [Figure 6A] ~ [Figure 6B] This graph shows the results of an ADCC reporter assay to find the chicken antibody taught herein. Figure 6A is a graph showing the ADCC efficacy of the anti-human Trop2 antibody in the FaDu cancer cell line. The dose-response graph shows the ADCC efficacy of the anti-human Trop2 antibody in the FaDu cancer cell line. The ADCC efficacy of the anti-human Trop2 antibody is shown using the NFAT CD16 Jurkat ADCC reporter cell line. Most clones show very similar ADCC efficacy compared to RS7. EC50 values ​​are the average of n=1-3 experiments. ADCC efficacy of the anti-human Trop2 antibody in the FaDu cancer cell line. Figure 6B is a table showing that most clones show very similar ADCC efficacy compared to RS7. EC50 values ​​are the average of n=1-3 experiments. [Figure 7A] ~ [Figure 7B] This figure shows the cell binding results for the antibodies taught herein. The binding efficacy of anti-human Trop2 antibodies against the human head and neck cancer cell line FaDu. In Figure 7A, FACS analysis shows the anti-Trop2 antibody and RS7 bound to FaDu cells endogenously expressing human Trop2. The plotted values ​​are the median fluorescence intensities. In Figure 7B, the EC50 values ​​are the average of n=1-3 experiments. [Figure 8]This graph shows the results of binding of an anti-human Trop2 antibody derived from immunized chickens to ExpiCHO cells overexpressing human Trop2. It also shows a FACS analysis of the anti-Trop2 antibody and RS7, which bind to ExpiCHO cells overexpressing human Trop2 in a dose-dependent manner. The plotted values ​​represent the percentage of antibody binding to the target cells. A histogram comparing the antibody binding levels to human Trop2 for the tested antibodies using the highest dose (66.67 nM) in the binding assay is superimposed. [Figure 9] This graph shows the results of binding of an anti-human Trop2 antibody derived from immunized chickens to ExpiCHO cells overexpressing mouse Trop2. FACS analysis shows that selected anti-Trop2 antibodies, which bind to ExpiCHO cells overexpressing mouse Trop2 in a dose-dependent manner, exhibit cross-reactivity with mouse Trop2. The RS7 control was specific only to human Trop2 and did not show binding to ExpiCHO cells overexpressing mouse Trop2. The plotted values ​​represent the percentage of antibody binding to the target cells. Histograms comparing the antibody binding levels to mouse Trop2 for the tested antibodies using the highest dose (66.67 nM) in the binding assay are superimposed. [Figure 10A] ~ [Figure 10B] This figure shows the killing of peripheral blood mononuclear cells (PBMCs) by the present invention's anti-human Trop2 antibody, which exhibits potent human ovarian cancer (Ovcar3) cell-killing properties. The graph in Figure 10A shows FACS analysis of cell death initiated by the anti-TROP2 molecule and effector cells, human PBMCs, against TROP2-expressing Ovcar3 target cells. Figure 10B contains the EC50 values ​​for each antibody tested. [Figure 11] This is an example of a research design for in vivo efficacy. [Figure 12A] ~ [Figure 12C]This graph shows the results of an in vivo study using FaDu xenografts in nude mice. Randomization and grouping were performed on day 0 based on tumor size. Drug administration was performed on days 0, 3, 7, 10, and 14. Tumor volume (Figure 12A) of the FaDu nude mouse xenograft model. Percentage change in tumor volume (Figure 12B). Mouse body weight (Figure 12C). [Figure 13] This figure shows the humanization of SD-589775. Binding kinetics to huTROP2 were measured using human Fc capture with a Carterra LSA high-throughput SPR instrument (Carterra). Combinations of humanized VH and VL sequences were tested and compared to the parent chimera. Variants with 589-VH_4 or 589-VL_3 did not show measurable binding to huTROP2. 589-VH_5 and 589-VL_4 refer to the parent sequences. [Figure 14] This figure shows ELISA data demonstrating the binding of anti-CD3 antibodies to CD3εδ heterodimers. Antibody-coated plates are then detected by dose-response analysis of CD3εδ-HRP. [Figure 15A] ~ [Figure 15B] This figure shows a dual-specific screening of TROP2 × CD3 TAA-conjugated arms. Cell elimination by co-culture of PBMCs and FaDu tumor cells. Cell lysis % measured after 48 hours. Figure 15A shows mouse-derived TROP2-conjugated antibody (and SD-589775). Figure 15B shows chicken-derived TROP2-conjugated antibody. [Figure 16A] ~ [Figure 16C] This figure characterizes the TROP2×CD3 bispecific cell killing against FaDu. Figure 16A shows cell killing by co-culture of PBMCs and FaDu tumor cells. Cell lysis % measured after 48 hours. Figure 16B shows T cell activation induced by co-culture of PBMCs and FaDu tumor cells. T cell activation measured after 48 hours. Figure 16C shows cytokine production and release induced by co-culture of PBMCs and FaDu tumor cells. Cytokine levels measured after 24 hours. [Figure 17A] ~ [Figure 17C] This figure characterizes the TROP2×CD3 bispecific cell killing against Calu-3. Figure 17A shows cell killing by co-culture of PBMCs and Calu-3 tumor cells. Cell lysis % measured after 48 hours. Figure 17B shows T cell activation induced by co-culture of PBMCs and Calu-3 tumor cells. T cell activation measured after 48 hours. Figure 17C shows cytokine production and release induced by co-culture of PBMCs and Calu-3 tumor cells. Cytokine levels measured after 24 hours. [Figure 18A] ~ [Figure 18C] This figure illustrates the characterization of TROP2×CD3 bispecific cell killing against OvCar-3. Figure 18A shows cell killing by co-culture of PBMCs and OvCar-3 tumor cells. Cell lysis % measured after 48 hours. Figure 18B shows T cell activation induced by co-culture of PBMCs and OvCar-3 tumor cells. T cell activation measured after 48 hours. Figure 18C shows cytokine production and release induced by co-culture of PBMCs and OvCar-3 tumor cells. Cytokine levels measured after 24 hours. [Figure 19] This graph shows the results of an in vivo efficacy study using multiple doses of SD-174078 in FaDu xenografts in humanized NSG MHCI / II dKO mice. Mice were transplanted with 20 × 10⁶ human PBMCs by intravenous injection on day 14. On day 1, 2 × 10⁶ FaDu cells were subcutaneously transplanted. On days 0, 3, and 7, mice were administered either PBS (medium) or SD-174078 (1 mg / kg) by intravenous injection. Tumor volume is shown. Significance was determined using Welch's t-test. [Figure 20]This graph shows the results of an in vivo efficacy study using a single dose of SD-231831. FaDu xenografts in humanized NSG MHCI / II dKO mice. Mice were transplanted with 20 × 10⁶ human PBMCs by intravenous injection on day 14. FaDu cells were subcutaneously transplanted with 2 × 10⁶ cells on day 1. On day 7, mice were given a single dose of either PBS (medium) or SD-231831 (1 mg / kg) by intravenous injection. Tumor volume is shown. Significance was determined using Welch's t-test. [Modes for carrying out the invention]

[0021] While the preparation and use of various embodiments of the present invention will be described in detail below, it should be understood that the present invention provides many applicable inventive concepts that can be exemplified in various specific situations. The specific embodiments described herein are merely examples of specific methods for preparing and using the present invention and do not limit the scope of the invention.

[0022] To facilitate understanding of the present invention, several terms are defined below. Terms as defined herein have meanings that are generally understood by those skilled in the art in the field relating to the present invention. Terms such as "a," "an," and "the" are not intended to refer only to singular entities, but include general classes for which specific examples may be used for illustrative purposes. Terms herein are used to describe specific embodiments of the present invention, but their use is not intended to limit the invention except as outlined in the claims.

[0023] Unless expressly indicated otherwise, in any method described or disclosed herein that involves two or more acts, the order of the acts is not necessarily limited to the order in which the acts of the Method are enumerated; however, it should be understood that this disclosure includes exemplary embodiments in which the order of the acts is very limited.

[0024] TROP2 is a pantumor transmembrane glycoprotein that is overexpressed in many human epithelial cancers, and its expression is positively correlated with tumor growth and metastasis. TROP2 has four N-glycosylation sites, and some tumors exhibit abnormal glycosylation. TROP2 is a novel target in cancer therapy, and the number of ADCs is increasing in clinical and preclinical development (including Troderbi, one of the approved therapies). This invention comprises a novel anti-TROP2 antibody.

[0025] Throughout this specification, the term “antibody” is used in its broadest sense and includes monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, non-human antibodies, chimeric antibodies, monovalent antibodies, antibody fragments, and serial scFv-Fc antibodies.

[0026] The antibody fragments of this disclosure retain antigen-binding specificity. The antibody fragments include an antigen-binding fragment (Fab), a variable fragment (Fv) containing VH and VL sequences, a single-stranded variable fragment (scFv) containing VH and VL sequences linked on a single chain, a single-stranded antibody fragment (scAb), or other antibody variable region fragments that retain antigen-binding specificity.

[0027] Throughout this specification, the term “mesoscale modified molecules (MEM)” includes, as a whole, modified peptides and polypeptides ranging from approximately 1 kDa to approximately 10 kDa. Throughout this specification, the term “MEM-nanoparticles” includes MEM conjugated to nanoparticles (e.g., ferritin nanoparticles).

[0028] In this specification, "subject" may be a mammal. Mammal subjects include humans, non-human primates, rodents (e.g., rats, mice), lagomorphs (e.g., rabbits), and ungulates (e.g., cattle, sheep, pigs, horses, goats, etc.). In some embodiments, the subject is a human. In some embodiments, the subject is a non-human primate, such as a cynomolgus macaque. In some embodiments, the subject is a companion animal (e.g., a cat, a dog).

[0029] All publications, patents, and patent applications described herein are incorporated herein by reference in the same way as each individual publication, patent, or patent application is specifically and individually designated and incorporated herein by reference.

[0030] antibody In this specification, the term “antibody” refers to an intact antibody or its binding fragment that specifically binds to a target antigen. Binding fragments are produced by recombinant DNA technology or by enzymatic or chemical cleavage of an intact antibody. Binding fragments include Fab, Fab', F(ab')2, Fv, and single-strand variable fragment (scFv) antibodies. If an excess of antibody reduces the amount of receptor bound to the counter-receptor by at least about 20%, 40%, 60%, or 80%, and more typically more than about 85% (as measured by an in vitro competitive binding assay), the antibody substantially inhibits the attachment of the receptor to the counter-receptor. The term "antibody" is used in its broadest sense and particularly encompasses monoclonal antibodies (including full-length antibodies or other bivalent, Fc-region-containing antibodies such as bivalent scFv Fc fusion antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments [e.g., Fab, Fab', F(ab')2, Fv, scFv], as long as they exhibit the desired biological activity. Antibodies (Ab) and immunoglobulins (Ig) are glycoproteins with the same structural characteristics. The present invention is fully recombinant, or in other words, includes monoclonal antibodies (and their conjugated fragments) in which a complementarity-determining region (CDR) is genetically inserted into the human antibody backbone, often referred to as veneering antibodies. Therefore, in certain embodiments, monoclonal antibodies are completely synthetic antibodies. In certain embodiments, monoclonal antibodies (and their conjugated fragments) can be produced in eukaryotic cells, including bacterial or plant cells.

[0031] In this specification, the term “antibody fragment” refers to a portion of a full-length antibody, generally known as the antigen-binding or variable region, and includes the Fab, Fab', F(ab')2, Fv, and scFv fragments. Papain digestion of an antibody produces two identical antigen-binding fragments called Fab fragments, each containing one antigen-binding site and the remaining “Fc” fragment, so named due to its ability to readily crystallize. Pepsin treatment produces the F(ab')2 fragment, which has two antigen-binding fragments capable of crosslinking antigens, and another remaining fragment (referred to as pFc'). In this specification, the term “functional fragment” with respect to an antibody refers to the Fv, F(ab), and F(ab')2 fragments.

[0032] In this specification, the "Fv" fragment is a minimal antibody fragment containing a complete antigen recognition and binding site. This region consists of a dimer of one heavy chain and one light chain variable domain that are closely and non-covalently associated (V H -V L (Dimer). In this configuration, the three CDRs of each variable domain interact, V H -V L The antigen-binding site is defined on the surface of the dimer. Overall, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of the Fv containing only three antigen-specific CDRs) has the ability to recognize and bind to the antigen, albeit with lower affinity than the entire binding site.

[0033] The Fab fragment, also represented as F(ab), contains a constant domain of the light chain and a first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment by the addition of several residues containing one or more cysteines derived from the antibody hinge region to the carboxyl terminus of the heavy chain CH1 domain. In this specification, Fab'-SH is the name given to Fab' in which the cysteine ​​residue of the constant domain has a free thiol group. The F(ab') fragment is prepared by cleaving the disulfide bond in the hinge cysteine ​​of the F(ab')2 pepsin digest product. Further chemical coupling of antibody fragments is known to those skilled in the art.

[0034] Natural antibodies and immunoglobulins are typically heterotetrameric glycoproteins with a weight of approximately 150,000 daltons, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by at least one disulfide covalent bond, although the number of disulfide links varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has intrachain disulfide bridges at regularly spaced intervals. Each heavy chain has a variable domain (V) at one end. H Each light chain has a variable domain (V) at one end. L The light chain has a constant domain at one end and the other end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. Certain amino acid residues are thought to form an interface between the light chain and the variable domain of the heavy chain (Clothia et al., J. Mol. Biol. 186, 651-66, 1985); Novotny and Haber, Proc. Natl. Acad. Sci. USA 82 4592-4596 (1985), relevant portions of which are incorporated herein by reference.

[0035] In this specification, “isolated” antibodies are those identified, separated, and / or recovered from components of the environment in which the antibody was produced. Contaminant components of the production environment are materials that would interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In certain embodiments, the antibody will be purified measurably by at least three different methods: 1) more than 50% by weight of the antibody as determined by the Lowry method, such as more than 75% by weight, or more than 85% by weight, or more than 95% by weight, or more than 99% by weight; 2) to a degree sufficient to obtain at least 10 N-terminal or internal amino acid sequences, such as at least 15 residues of sequence, by using a spinning cup sequencer; or 3) homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or preferably silver staining. Since isolated antibodies will be absent from at least one component of the antibody’s natural environment, they will contain the antibody in situ within recombinant cells. However, typically, isolated antibodies will be prepared by at least one purification step.

[0036] In this specification, the terms “antibody mutant” or “antibody variant” refer to an amino acid sequence variant of an antibody in which one or more amino acid residues are modified. Such mutants necessarily have less than 100% sequence identity or similarity to an amino acid sequence having at least 75% amino acid sequence identity or similarity, such as at least 80%, at least 85%, at least 90%, or at least 95%, 96%, 97%, 98%, or 99%, with respect to either the heavy chain or light chain variable domain of the antibody.

[0037] In this specification, the term “variable” in the context of the variable domain of an antibody refers to the fact that certain portions of the variable domain have significantly different sequences between antibodies, and are used for the binding and specificity of each particular antibody to each particular antigen. However, the variability is not uniformly distributed throughout the variable domain of an antibody. It is concentrated in three segments called complementarity-determining regions (CDRs), also known as hypervariable regions, which are present in both the light and heavy chain variable domains. There are at least two techniques for determining CDRs: (1) a method based on interspecies sequence variation [i.e., Kabat et al., Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. 1987)]; and (2) a method based on crystallographic studies of antigen-antibody complexes (Chothia, et al. (1989), Nature 342: 877), or both Chothia and Kabat. The more highly conserved portions of the variable domain are called frameworks (FRs). The variable domains of the natural heavy and light chains each contain four FR regions, primarily in the form of a β-sheet arrangement. These FR regions are connected by three CDRs, which connect the β-sheet structure and, in some cases, form loops that make up that portion. The CDRs within each chain are held together very closely by the FR regions and, together with the CDRs of other chains, contribute to the formation of the antibody's antigen-binding site (see Kabat et al.). The constant domain is not directly involved in the binding of the antibody to its alloantigen, but exhibits various effector functions, such as the antibody's involvement in antibody-dependent cytotoxicity.

[0038] The light chain of an antibody (immunoglobulin) derived from any vertebrate species can be designated as one of two distinct types, called kappa and lambda, based on the amino acid sequence of its constant domain. The amino acid sequence of the heavy chain constant domain can also classify "immunoglobulins" into different classes. There are at least five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG4; IgA-1 and IgA-2. The subunit structures and three-dimensional arrangements of different classes of immunoglobulins are well known.

[0039] In this specification, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within that population are identical except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific and target a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which generally contain different antibodies against different determinants (epitopes), each monoclonal antibody targets a single determinant on an antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by hybridoma culture and are not contaminated with other immunoglobulins. The modifying phrase “monoclonal” indicates a characteristic of antibodies such as those obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring the antibody to be produced by some specific method. For example, the monoclonal antibodies used in the inventions currently disclosed and claimed can be produced by the hybridoma method first described by Kohler and Milstein, Nature 256, 495 (1975), the relevant parts of which are incorporated herein by reference.

[0040] All monoclonal antibodies used in the inventions currently disclosed and claimed are either (1) the result of a planned immunological protocol as described in more detail below; or (2) the result of an immune response that naturally leads to antibody production in the course of disease or cancer.

[0041] The use of the monoclonal antibodies of the currently disclosed and claimed inventions may require the administration of such or similar monoclonal antibodies to subjects such as humans. However, if the monoclonal antibodies are produced in non-human animals such as rodents or chickens, administration of such antibodies to human patients would typically evoke an immune response, which would target the antibody itself. Such a reaction would limit the duration and effectiveness of such therapy. To address such problems, the monoclonal antibodies of the currently disclosed and claimed inventions may be "humanized," that is, the antibody is modified such that its antigenic portion is removed and thus replaced in a manner similar to that of a human antibody, but the antibody's affinity for TROP2 is preserved. This modification may involve only a few amino acids, or it may involve the entire framework region of the antibody, leaving only the complementarity-determining region of the intact antibody. Several methods for humanizing antibodies are known to those skilled in the art and are disclosed in U.S. Patent No. 6,180,370 issued to Queen et al. on 30 January 2001; U.S. Patent No. 6,054,927 issued to Brickell on 25 April 2000; U.S. Patent No. 5,869,619 issued to Studnicka on 9 February 1999; U.S. Patent No. 5,861,155 issued to Lin on 19 January 1999; U.S. Patent No. 5,712,120 issued to Rodriquez et al. on 27 January 1998; and U.S. Patent No. 4,816,567 issued to Cabilly et al. on 28 March 1989, the relevant portions of which are incorporated herein by reference.

[0042] Humanized antibody forms are primarily chimeric immunoglobulins, immunoglobulin chains, or fragments thereof [such as Fab, Fab', F(ab')2, Fv, scFv, or other antigen-binding subsequences of the antibody] composed mainly of human immunoglobulin sequences, and contain minimal sequences derived from non-human immunoglobulins. Humanization can be carried out by substituting non-human (i.e., rodent, chicken) CDRs or CDR sequences with corresponding sequences of human antibodies, according to the methods of Winter and collaborators (Jones et al., 1986; Riechmann et al., 1988; Verhoeyen et al., 1988), see, for example, U.S. Patent No. 5,225,539. In some cases, F of human immunoglobulin may be used. v Framework residues are replaced with corresponding non-human residues from the donor antibody. Humanized antibodies may also contain residues not found in the recipient antibody or in the introduced CDR or framework sequence. Generally, humanized antibodies will contain substantially all, at least one, and generally two variable domains, with all or substantially all of the CDR region corresponding to the variable domains of non-human immunoglobulins, and all or substantially all of the framework region being the common sequence of human immunoglobulins. Humanized antibodies will also optimally contain at least a portion of the immunoglobulin constant region (Fc), generally the constant region of human immunoglobulins.

[0043] The inventions currently disclosed and claimed further include the use of fully human monoclonal antibodies that cross-reactive to Trop2. Fully human antibodies are those in which the entire sequence of both the light and heavy chains, including the CDR, is fundamental to antibody molecules derived from human genes. Such antibodies are referred to herein as “human antibodies” or “fully human antibodies.” Human monoclonal antibodies may be prepared, for example, by trioma technology; human B-cell hybridoma technology [see Kozbor, et al., Hybridoma, 2:7 (1983)] and EBV hybridoma technology for producing human monoclonal antibodies [see Cole, et al., PNAS 82:859 (1985)], or by the techniques taught herein. Human monoclonal antibodies may be used in carrying out the inventions currently disclosed and claimed, and may be produced by using human hybridomas [see Cote, et al., PNAS 80:2026 (1983)] or by transforming human B cells in vitro with Epstein-Barr virus (see Cole et al., 1985), the relevant parts of which are incorporated herein by reference.

[0044] In addition, human antibodies can be produced by introducing human immunoglobulin loci into transgenic animals, such as mice in which the endogenous immunoglobulin genes are partially or completely inactivated. Upon sensitization, the production of human antibodies is observed, which closely resemble those found in humans in every respect, including gene rearrangement, assembly, and antibody repertoire. This method is described, for example, but is not limited to, U.S. Patents 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and Marks et al., J Biol. Chem. 267:16007, (1992); Lonberg et al., Nature, 368:856 (1994); Morrison, 1994; Fishwild et al., Nature Biotechnol. 14:845 (1996); Neuberger, Nat. Biotechnol. 14:826 (1996); and Lonberg and Huszar, Int Rev Immunol. 13:65 (1995), and relevant portions are incorporated herein by reference.

[0045] A method for producing desired antibodies, such as human antibodies, is disclosed in U.S. Patent No. 5,916,771, issued to Hori et al. on June 29, 1999, and is incorporated herein by reference. The method involves introducing an expression vector containing a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses antibodies containing both heavy and light chains.

[0046] In this specification, the term "treatment" refers to both therapeutic measures and preventive or protective measures. Those requiring treatment include those who already have a disability and those for whom disability should be prevented.

[0047] In this specification, the term “disorder” refers to any condition that would benefit from treatment with polypeptides. This includes chronic and acute disorders or diseases, including infectious or pathological conditions that make mammals susceptible to the disorder in question.

[0048] Antibodies or antibody fragments may be produced in a modified sequence or glycosylated state to confer a favorable level of activity in antibody-dependent cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), antibody-dependent neutrophil phagocytosis (ADNP), or antibody-dependent complement deposition (ADCD) function, as measured by bead-based or cell-based assays or in vivo studies in animal models.

[0049] Alternatively, or further, it may be useful to combine amino acid modifications with one or more other amino acid modifications that alter the complement-dependent cytotoxicity (CDC) function of the Fc region of complement component Clq-binding and / or IL-23p19-binding molecules. Particularly interesting binding polypeptides may be those that bind to Clq and exhibit complement-dependent cytotoxicity. Polypeptides that already possess Clq-binding activity and optionally further possess the ability to mediate CDC may be modified so as to enhance one or both of these activities. Amino acid modifications that alter Clq and / or its complement-dependent cytotoxic function are described, for example, in WO / 0042072, which is incorporated herein by reference.

[0050] The Fc region of an antibody can be designed to modify effector function, for example, by modifying Clq binding and / or FcγR binding, thereby changing complement-dependent cytotoxicity (CDC) activity and / or antibody-dependent cell-mediated cytotoxicity (ADCC) activity. These "effector functions" are involved in the activation or attenuation of biological activity (e.g., in a subject). Examples of effector functions include, but are not limited to, Clq binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions may require the Fc region to be combined with a binding domain (e.g., an antibody variable domain) and can be evaluated using various assays (e.g., Fc binding assay, ADCC assay, CDC assay, etc.).

[0051] An antibody can be designed to alter effector function, for example, by binding to an effector molecule of the CD3 complex, thereby changing T cell-mediated cytotoxicity (TDCC) activity. These "effector functions" are involved in the activation or attenuation of biological activity (e.g., in a subject). Examples of effector functions include, but are not limited to: Such effector functions can be evaluated using various assays (e.g., cytokine release assay, T cell proliferation, T H or T C activity, etc.).

[0052] In some embodiments, bispecific or multispecific antibodies are provided, which antibodies include at least one heavy chain variable region of the antibody family of the present invention and may include the heavy and light chain variable regions provided herein. The bispecific antibody includes at least the heavy chain variable regions of antibodies specific for TROP2 and a protein other than TROP2 and may include the heavy and light chain variable regions. Various formats of bispecific antibodies are within the scope of the present invention, including, but not limited to, single-chain polypeptides, double-chain polypeptides, triple-chain polypeptides, quadruple-chain polypeptides, and multiples thereof.

[0053] The Fc region of an antibody may be designed to alter effector function by modifying, for example, Clq binding and / or FcγR binding, thereby altering complement-dependent cytotoxicity (CDC) activity and / or antibody-dependent cell-mediated cytotoxicity (ADCC) activity. These “effector functions” are involved in the activation or attenuation of biological activity (e.g., in a target). Examples of effector functions include, but are not limited to, Clq binding; CDC; Fc receptor binding; ADCC; phagocytosis; and downregulation of cell surface receptors (e.g., B cell receptors; BCRs). Such effector functions may require an Fc region combined with a binding domain (e.g., an antibody variable domain) and can be evaluated using various assays (e.g., Fc binding assays, ADCC assays, CDC assays, etc.).

[0054] The Fc region may be a wild-type Fc region, a mutant Fc region, a monomeric wild-type Fc region, a monomeric mutant Fc region, a dimeric wild-type Fc region or a dimeric mutant Fc region, a second variable weight region and a second Fc region, or a second variable weight region and a second Fc region, and may further include an uncleavable mobile linker and a second variable light region and a second heavy variable region, or a second Fc region and an uncleavable mobile linker and a payload such as a toxin, cytokine or another antibody. Non-limiting examples of Fc variants include mutants such as knob-hole variants that enable directed formation of bispecific or multivalent antibodies.

[0055] For example, a variant Fc region of an antibody can be generated that improves Clq binding and FcγRIII binding (e.g., having both improved ADCC and CDC activity). Alternatively, if a reduction or elimination of effector function is desired, the variant Fc region can be modified to reduce CDC activity and / or ADCC activity. In other embodiments, only one of these activities may be increased, and optionally the other activity may be decreased (e.g., generating an Fc region variant in which ADCC activity is improved but CDC activity is decreased, and vice versa).

[0056] Single-stranded variable fragments (scFvs) are fusions of the variable regions of the heavy and light chains of immunoglobulins, linked together by a short (usually serine, glycine) linker. This chimeric molecule retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of a linker peptide. This modification usually remains unaltered. These molecules were historically created to facilitate phage display, where expressing the antigen-binding domain as a single peptide is very convenient. Alternatively, scFvs can be created directly from subcloned heavy and light chains derived from hybridomas or B cells. Because single-stranded variable fragments lack the constant Fc region found in complete antibody molecules, they lack common binding sites (e.g., protein A / G) used to purify antibodies. These fragments can often be purified / immobilized using protein L, as protein L interacts with the variable region of the kappa light chain.

[0057] Movable linkers are generally composed of helical and turn-promoting amino acid residues such as alanine, serine, and glycine. However, other residues can function similarly. Using phage display, linkers modified for single-chain antibodies (scFv) can be rapidly selected from a protein linker library. A random linker library was constructed, and the genes for the heavy and light chain variable domains were linked by segments encoding 18-amino acid polypeptides of varying compositions. scFv repertoire (approximately 5 × 10⁻¹⁴) 6 Members of different species were presented on the linear phage and subjected to affinity selection by hapten. The selected population of variants exhibited a significant increase in binding activity while retaining considerable sequence diversity. Sequence analysis revealed that conserved proline in the linker two residues after the VH C-terminus, as well as abundant arginine and proline at other positions, were the only common features of the selected chains. In certain embodiments, the antibody fragments were further modified using modifications of the Fc region or mutations in various constant regions, as known to those skilled in the art, to increase their serum half-life.

[0058] In certain embodiments, the antibodies of the present invention are formulated for administration to humans. For example, the antibodies of the present invention may be included in pharmaceutical compositions formulated for administration by intranasal cavity, intrapulmonary cavity, bronchial cavity, vein, oral cavity, intrafat, intraartery, intraarticular cavity, intracranial cavity, intradermal cavity, intrafocal cavity, intramuscular cavity, intrapericardial cavity, intraperitoneal cavity, intrapleural cavity, intrabladder cavity, local, mucosal cavity, parenteral, intraintestinal cavity, subcutaneous cavity, sublingual cavity, topical, transbuccal, transdermal, inhalation, injection, in a cream, in a lipid composition, via a catheter, by gastric lavage, by continuous infusion, by injection, by local delivery, or by local perfusion, and the composition may be serum, drops, gel, ointment, spray, container, or atomizer.

[0059] In this specification, the term “antigen” refers to a molecule containing one or more epitopes (linear, three-dimensional, or both) that stimulate the host’s immune system to elicit a fluid and / or cellular antigen-specific response. This term is used interchangeably with the term “immunogen.” Typically, B cell epitopes will contain at least about 5 amino acids, but may be as small as 3-4 amino acids. T cell epitopes, such as CTL epitopes, will contain at least about 7-9 amino acids, and helper T cell epitopes will contain at least about 12-20 amino acids. Typically, epitopes will contain about 7-15 amino acids, such as 9, 10, 12, or 15 amino acids. This term includes polypeptides that have undergone modifications, such as deletions, additions, and substitutions (generally conserved substitutions in practice), compared to their native sequence, as long as the protein maintains its ability to elicit an immune response as defined herein. These modifications may be planned, such as through site-directed mutagenesis, or accidental, such as due to mutations in the antigen-producing host.

[0060] In this specification, the term “epitope” refers to a specific amino acid sequence or molecule (e.g., carbohydrate, small molecule, lipid, etc.) that, when present in an appropriate form, provides a reactive site for an antibody (e.g., a B cell epitope) or, in the case of a peptide, a reactive site for a T cell receptor (e.g., a T cell epitope).

[0061] A given polypeptide segment containing a B cell epitope can be identified using many epitope mapping techniques known to those skilled in the art (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed., 1996, Humana Press, Totowa, NJ). For example, linear epitopes can be determined, for instance, by simultaneously synthesizing a number of peptides corresponding to segments of a protein molecule on a solid support and reacting the peptides with an antibody while the peptides are still bound to the support. Such techniques are known to those skilled in the art and are described, for example, U.S. Patent No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad Sci. USA 81:3998-4002; Geysen et al. (1986) Molec. Immunol. 23:709-715.

[0062] In this specification, the term “substantially purified” refers to the isolation of a substance (compound, polynucleotide, protein, polypeptide, polypeptide composition) such that it constitutes the majority of the sample present. Generally, substantially purified components in a sample constitute 50%, preferably 80% to 85%, and more preferably 90% to 95% of the sample. Techniques for purifying desired polynucleotides and polypeptides are well known to those skilled in the art and include, for example, ion exchange chromatography, affinity chromatography, and density precipitation.

[0063] In this specification, the term “treatment” means any of the following: (i) prevention of infection or reinfection, such as with conventional vaccines; (ii) reduction or elimination of symptoms; and (iii) substantial or complete elimination of cells containing TROP2 in question. Treatment may be performed prophylactically (before cells become cancerous and / or metastatic) or therapeutically (after cells have become cancerous and / or metastatic).

[0064] Unless otherwise specified, the implementation of this invention will utilize conventional methods of chemistry, biochemistry, molecular biology, immunology, and pharmacology within the scope of the art. Such techniques are described in detail in the literature. For example, Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack Publishing Company, 1990); Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.); and Handbook of Experimental Immunology, Vols. I-IV (DM Weir and CC Blackwell, eds., 1986, Blackwell Scientific Publications); Sambrook, et al. al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989);Short Protocols in Molecular Biology, 4th ed. (Ausubel et al. eds., 1999, John Wiley & Sons);Molecular Biology Techniques: An Intensive Laboratory Course, (Ream et al., eds., 1998, Academic Press);PCR (Introduction to Biotechniques) Series), 2nd ed. (Newton & Graham See also *Fundamental Virology, Second Edition* (Fields & Knipe eds., 1991, Raven Press, New York), and relevant portions are incorporated herein by reference.

[0065] Conservative amino acid substitutions include substitutions of aliphatic or hydrophobic amino acids Ala, Val, Leu, and Ile; substitutions of hydroxyl residues Ser and Thr; substitutions of acidic residues Asp and Glu; substitutions of amide residues Asn and Gln; substitutions of basic residues Lys, Arg, and His; substitutions of aromatic residues Phe, Tyr, and Trp; and substitutions of small amino acids Ala, Ser, Thr, Met, and Gly.

[0066] In some embodiments, the target-binding polypeptide binds to tumor-targeting antigens selected from HER1, HER2, HER3, GD2, carcinoembryonic antigen (CEA), epidermal growth factor receptor activating variant (EGFRVIII), CD133, fibroblast-activating protein alpha (FAP), epidermal cell adhesion molecule (Epcam), glypican 3 (GPC3), EPH receptor A4 (EphA), tyrosine protein kinase Met (cMET), IL-13Ra2, microsomal epoxide hydrolase (mEH), MAGE, mesothelin, MUC16, MUC1, prostate stem cell antigen (PSCA), Wilms tumor-1 (WT-1), and claudin family proteins.

[0067] In some embodiments, the target-binding polypeptide binds to T cell markers selected from CTLA-4, PD-1, Lag3, S15, B7H3, B7H4, TCR-alpha, TCR-beta, and TIM-3, CD3, 41BB, and OX40.

[0068] In some embodiments, the target-binding polypeptide binds to antigen-presenting cell markers selected from PD-L1, CD40, CD24, B7H3, TGF-beta receptor, TNFR family members 1-20, CD80, CD86, FLT3, CD11c, CD8 alpha, 5B6 (CLEC9A), CD1c, CD11b, CD13, CD33, HLA-DR, CD141, CD1a, CD32, CD45, CD80, CD86, CD207, CD2, CD7, CD45RA, CD68, CD123, CD303, and CD304.

[0069] Using an anti-Trop2 antibody or its binding portion (or fragment thereof), cancer cells expressing Trop2 or its variants selected from breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, certain lung cancers, oral squamous cell carcinoma, ovarian squamous cell carcinoma, pancreatic squamous cell carcinoma, prostate squamous cell carcinoma, gastric squamous cell carcinoma, thyroid squamous cell carcinoma, bladder squamous cell carcinoma, and uterine squamous cell carcinoma can be targeted. [Examples]

[0070] Animal immunity Mice were immunized a total of six times over seven weeks using combinations of huTROP2-huFc, huTROP2-His, SK-BR-3 cells, and MCF7 cells. Incomplete Freund's adjuvant was used for protein-based immunization, and the Sigma Adjuvant System was used for cellular immunization. All immunizations were administered by intraperitoneal injection. Spleens and lymph nodes were collected after seven weeks, and cell suspensions were prepared. RNA was isolated from single-cell suspensions using the Direct-zol RNA miniprep kit (Zymo Research, catalog number R2050), and its concentration and purity were determined by measured Abs260, Abs280, and Abs230. Quality was confirmed by microcapillary electrophoresis using a 2100 Bioanalyzer (Agilent Technologies).

[0071] Chickens were immunized a total of five times over 12 weeks using combinations of huTROP2-huFc, msTROP2-His, and SK-Br-3 cells with complete Freund's adjuvant, except for huTROP2-huFc immunization, which used incomplete Freund's adjuvant. All immunizations were performed by subcutaneous injection. Spleens and bone marrow were collected after 12 weeks and cell suspensions were prepared. RNA was isolated from single-cell suspensions using the Direct-zol RNA miniprep kit (Zymo Research, catalog number R2050), and concentration and purity were determined by measured Abs260, Abs280, and Abs230. Quality was confirmed by microcapillary electrophoresis using a 2100 Bioanalyzer (Agilent Technologies).

[0072] Preparation of phage display libraries cDNA was generated from each mouse RNA preparation using the SuperSCript III First-Strand cDNA kit (Invitrogen, catalog no. 18080-51). The VH region was amplified using primer binding in the mouse VH framework 1 region and hinge region, and the VL region was amplified using primer binding in the mouse VL kappa framework 1 region and kappa constant region. Amplification was performed using Expand High Fidelity enzyme (Roche, catalog no. 0473876001). Nested PCR was then performed on each amplified sample to add restriction sites, thereby enabling the cloning of VH and VL sequences for the phage display library.

[0073] cDNA was generated from each chicken RNA preparation using the SuperSCript III First-Strand cDNA kit (Invitrogen, catalog number 18080-51). The VH region was amplified using primer binding in the chicken VH framework 1 and VH framework 4 regions, and the VL region was amplified using primer binding in the chicken VL lambda framework 1 and lambda constant region. Amplification was performed using Expand High Fidelity enzyme (Roche, catalog number 0473876001). Nested PCR was then performed on each amplified sample to add restriction sites, thereby enabling the cloning of VH and VL sequences for phage display libraries.

[0074] The VH and VL sequences were sequentially digested with appropriate restriction enzymes and ligated into digested phagemide vectors for FAB phage display. The ligation products were transformed into ECC TG1 cells (Lucigen, catalog no. 60502-2), and library quality was determined by size and VH / VL insertion percentage. In all cases, the minimum library size was 5 × 10⁶. 7 The number of insertions was 85%, and all libraries exceeded these criteria.

[0075] Phage display screening Selection of mouse library phage displays was performed using both soluble protein antigens and TROP2-expressing cells. In-solution selection was performed using biotinylated huTROP2 with Sera-Mag SPeedBeads Neutravidin-coated magnetic particles (GE Healthcare, catalog no. GE78152104010150) or Sigma Dynabeads MyOne Streptavidin T1 magnetic beads (Invitrogen, catalog no. 65602) for selection with KingFisher Flex. Dissociation rate washing using various concentrations of antigens and, in some cases, non-biotinylated huTROP2 was performed as the final selection. Elution was performed using trypsin (Sigma, catalog no. T1426), and the selection buffer was skim milk in 1×PBS. Cell selection was performed using SK-BR-3, CHO-K1, and 293FF overexpressing huTROP2. For cell selection, 10% FBS in 1×PBS was used as the selection buffer, and trypsin was used for elution.

[0076] After panning, a single clone was isolated and sequenced. The VH sequence was cloned into the huIgG1 expression plasmid, and the VL sequence was cloned into either the huVLkappa or huVLlambda expression plasmid.

[0077] Antibody expression and expression Antibody expression plasmids were transiently introduced into animal cell lines using the ExpiFectamine CHO Transfection Kit (ThermoFisher, catalog number A29129) to obtain transformants producing anti-TROP2 chimeric or humanized antibodies. The host cell lines used were ExpiCHO-S (ThermoFisher, catalog number A29127) or a suspension CHO cell line with α1,6 fucosyltransferase (FUT8) gene knockout (referred to as "WT CHO" and "FUT8 CHO" in further reference). After 6–12 days of growth following DNA introduction, WT CHO or FUT8 CHO cell suspensions were collected by centrifugation at 4000×g for 20 minutes and then filtered using a 0.2 μm disposable PES filter unit (Fisher Scientific, catalog number FB12566504). Anti-TROP2 antibodies were recovered from the filtrate using protein A purification (HiTrap MabSelect SuRe, Cytiva, catalog number GE11-0034-93). Standard glycosylated antibodies were expressed using WT CHO, and defucosylated antibodies with enhanced effector function (indicated by "-afuc") were expressed using FUT8 CHO.

[0078] Cell binding assay FaDu cells (ATCC, catalog number HTB-43) were cultured in EMEM (ATCC, catalog number 30-2003) supplemented with 10% FBS (Sigma, catalog number F5135) and 1× penicillin-streptomycin (Corning, catalog number 30-002-CI). ExpiCHO-S cells (ThermoFisher, catalog number A29127) were cultured in ExpiCHO expression medium (Gibco, catalog number A29100-01).

[0079] For cell binding assays, PBS (Corning, catalog no. 21-040-CV) supplemented with 2% FBS and 2 mM EDTA (Quality Biological, catalog no. 351-027-721) was used as the assay buffer. Cells were counted, resuspended in the assay buffer, and then resuspended in 0.5 or 1 × 10⁶ cells. 5 Individual cells / well were then seeded onto a 96-well plate (VWR, catalog no. 89089-826). The plate was centrifuged, the supernatant was removed, and the plate was then kept on ice for the remaining assay steps. After removing the supernatant, the indicated antibody was diluted in assay buffer and added to the cells on ice at increasing concentrations (0.64 to 10000 ng / mL) for 20 minutes. After incubation, the plate was centrifuged, the supernatant was removed, and the cells were then washed once with assay buffer, followed by centrifugation and washing. After washing, rat anti-human IgG Fc Alexa Fluor 647 (Biolegend, catalog no. 410714) or rabbit anti-goat IgG Alexa Fluor 647 secondary antibody (JIR, catalog no. 305-605-046) was added to the cells on ice for 20 minutes at a 1:200 or 1:800 dilution, respectively, in assay buffer. After incubation, the plate was centrifuged, the supernatant was removed, and the cells were washed once with assay buffer, followed by centrifugation and removal of the wash. After washing, DAPI (Biolegend, catalog no. 422801) was added to the cells in assay buffer at a dilution of 1:5000. Cell binding was analyzed using a Miltenyi MACSQuant 16 flow cytometer. Flow cytometry data was analyzed using FlowJo flow cytometry analysis software. Graphs were created and EC 50 GraphPad Prism 9.3.0 was used to calculate the values.

[0080] Binding kinetics The binding of antibodies to human, mouse, and cynomolgus / rhesus monkey TROP2, as well as human EpCAM, was evaluated by surface plasmon resonance (SPR) using a Carterra LSA (Carterra). Anti-human IgG capture lornes were initially prepared on an HC30M tip (Carterra, catalog no. 4279) by primary amine coupling. Briefly, the tip surface was activated for 10 minutes with a mixture of 133 mM EDC (Thermo Fisher, catalog no. 22980) and 33.3 mM sulfon-NHS (Thermo Fisher, catalog no. 24525) in 100 mM MES pH 5.5 (Carterra, catalog no. 3625), and then goat anti-human IgG (Southern Biotech, catalog no. 2040-01) was coupled at 50 μg / mL in 10 mM sodium acetate buffer (Carterra, catalog no. 3622) pH 4.5 for 15 minutes. Unconjugated areas on the chip surface were blocked with 1M ethanolamine HCl pH 8.5 (Carterra, catalog no. 3626) for 7 minutes. For capture kinetics, the antibody panel was captured for 10 minutes at 1–10 μg / mL in HBSTE buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% Tween20; Carterra, catalog no. 3630) using the prepared anti-human IgG surface and a 96-channel printhead (96PH). Purified recombinant antigens (human TROP2, Acro Biosystems, catalog number TR2-H5223; mouse TROP2, catalog number TR2-M52H6; cynomolgus monkey / rhesus monkey TROP2, catalog number TR2-R52H3; human EpCAM, catalog number EPM-H5223) were subsequently injected onto an antibody panel using a single flow cell (SFC) at five concentrations in a 5-fold dilution series, starting at 500 nM for human TROP2 and 1 μM for the others. Each injection consisted of a 5-minute conjugation phase and a 15-minute dissociation phase. The surface was regenerated between antigens using 0.425% H3PO4 (Carterra, catalog number 3637).The running buffer for antigen infusion was HBSTE supplemented with 0.5 mg / mL BSA (VWR, catalog no. 97061-422). Binding data were double-referenced by subtracting the reference response of the internal spot and the blank response of the buffer only. The resulting sensumograms were globally fitted to a 1:1 Langmuir binding model using Carterra Kinetics software to determine the binding rate constant (k). a ), dissociation rate constant (k d ) and dissociation constant (K D ) was estimated.

[0081] Table 1 shows that binding kinetics were measured using human Fc capture with a Carterra LSA high-throughput SPR instrument (Carterra). Mouse / human chimeric anti-TROP2 chimeric antibodies isolated from immunized mice bound to human TROP2 with low nanomolar / high picomolar affinity. Chicken / human chimeric antibodies also bound to human TROP2 with low nanomolar / high picomolar affinity. Many showed affinity for TROP2 equivalent to or greater than RS7. Some clones also showed non-1:1 dynamic profiles.

[0082] [Table 1] JPEG2026519783000003.jpg133170

[0083] Table 2 shows that binding kinetics were measured using human Fc capture with a Carterra LSA high-throughput SPR instrument (Carterra). None of the tested antibodies bound to human EpCAM, but bound equally to almost all cynomolgus monkey / rhesus monkey TROP2 (cyTROP2). None of the mouse chimeras recognized mouse TROP2 (msTROP2), but almost all chicken chimeras showed affinity for msTROP2 within a range of 10 times that of binding to huTROP2. Table 2. SPR binding to msTROP2, cyTROP2, and EPCAM.

[0084] [Table 2]

[0085] Epitope Binning Antibody binning based on epitope similarity was performed using SPR with Carterra LSA (Carterra). Briefly, antibody-ligand arrays were immobilized on the surface of a sensor chip, and then panels of TROP2 and analyte antibodies were injected separately. A total of 141 antibodies were evaluated using this one-to-many pairwise method. Ligand antibodies may either block analyte binding, suggesting a common or nearby epitope, or they may sandwich the analyte, indicating that both antibodies can bind to the antigen simultaneously and therefore likely target distant epitopes.

[0086] To prepare the ligand array, a mixture of 133 mM EDC (ThermoFisher, catalog no. 22980) and 33.3 mM sulfoNHS (ThermoFisher, catalog no. 24525) in 100 mM MES pH 5.5 (Carterra, catalog no. 3625) was injected across the entire surface of an HC30M tip (Carterra, catalog no. 4279) for 8 minutes using SFC. The array was then coupled using 96PH. Each ligand antibody was printed in 10 mM sodium acetate buffer pH 4.5 (Carterra, catalog no. 3622) at a final concentration of 1–10 μg / mL for 15 minutes. Unconjugated areas on the chip surface were blocked with 1M ethanolamine HCl pH 8.5 (Carterra, catalog no. 3626) for 7 minutes, followed by washing with running buffer (25mM MES + 0.01% Tween20; Carterra, catalog no. 3631). Ligand integrity and regeneration conditions were evaluated by injecting TROP2 antigen (Acro Biosystems, catalog no. TR2-H5223) several times onto the completed surface, followed by injection of different regeneration solutions.

[0087] To prepare the analytes, each antibody was diluted to 1–10 μg / mL in running buffer (HBSTE + 0.5 mg / mL BSA; Carterra, catalog no. 3630; VWR, catalog no. 97061-422) and arranged in a 384 extra-deep well plate for injection by SFC. The TROP2 antigen was prepared at 100 nM in running buffer, and 10 mM glycine pH 2.0 (Carterra, catalog no. 3640) was used for regeneration. Each of the 141 binning cycles consisted of a 1 minute baseline process, a 5 minute antigen injection, a 5 minute analyte injection, two 15-second regeneration solution pulses, and a 1 minute stabilization. Running buffer injection was performed instead of analytes in all 12 cycles. Pairwise binding data were analyzed using Carterra Epitope software.

[0088] Epitope Mapping Antibody binding to a peptide array derived from TROP2 was assessed by SPR using a Carterra LSA (Carterra), which allows for more accurate epitope mapping. Human TROP2 was initially synthesized on a 50 nmol scale as an overlapping set of 15-residue biotinylated peptides containing C-terminal biotin and N-terminal glycinamide, respectively (5 residues overlap, JPT Peptide Technologies). A complete library of 47 peptides was synthesized, each reconstituted in DMSO at 1 mg / mL, and diluted to 2 and 1 μg / mL in HBS (10 mM HEPES pH 7.4, 150 mM NaCl) in 96-well plates. The peptide array was then printed onto the surface of a streptavidin-coated sensor tip (SAHC30M, Carterra, catalog no. 4294) using 96PH. Biotinylated TROP2 (Acro Biosystems, catalog no. TR2-H82E5) was included in the array as a positive control. All arrays were printed in two blocks for 12 minutes each. A panel of 141 antibodies in HBSTE buffer was successively injected onto the arrays one at a time for 5 minutes, and a regeneration solution (0.425% phosphate; Carterra, catalog no. 3637) was pulsed twice for 15 seconds between each antibody. Mapping data was analyzed using Carterra Epitope software.

[0089] TROP2 binding assay The binding of antibody panels to immobilized TROP2 was evaluated using the MSD assay. All incubations were performed at room temperature for 1 hour, and each plate was washed three times with wash buffer (MSD, catalog no. R61AA) between each step. MSD GOLD 96-well Streptavidin QUICKPLEX plates (MSD, catalog no. L55SA) were initially blocked with 150 μL / well of MSD Blocker A (MSD, catalog no. R93BA). The plates were then coated with 0.1 μg / mL biotinylated TROP2 (Acro Biosystems, catalog no. TR2-H82E5) diluted in Diluent 100 (MSD, catalog no. R50AA). Eight dose-response curves were prepared for each antibody using 5-fold serial dilutions starting at 10 μg / mL in Diluent 100. After incubation of antibody samples, the bound antibodies were detected using goat anti-human SULFO-TAG antibody (MSD, catalog number R32AJ). Before reading, 150 μL / well of MSD GOLD Read Buffer A (MSD, catalog number R92TG) was added, and the plates were read using a MESO QuickPlex SQ 120MM instrument. Dose-response curves were analyzed using nonlinear regression and 1 / Y using GraphPad Prism. 2 Fitted under load, EC 50 The result was calculated.

[0090] ADCC Reporter Assay FaDu cells (ATCC, catalog number HTB-43) were cultured in EMEM (ATCC, catalog number 30-2003) supplemented with 10% FBS (Sigma, catalog number F5135) and 1× penicillin-streptomycin (Corning, catalog number 30-002-CI). For the ADCC reporter assay, FaDu cells were counted to evaluate cell number and viability. The cells were centrifuged and measured 5 × 10⁶ cells. 5 The cells were resuspended in growth medium at a concentration of individual cells / mL. 5 × 10⁴ cells per well. 4The cells were seeded into 96-well plates (VWR, catalog number 89131-676) and incubated overnight in a cell culture incubator. Anti-TROP2 or control antibody in Xvivo 15 medium was added to the cells at increasing concentrations (0.32 to 5000 ng / mL) at 37°C and 5% CO2 for 20 minutes. After incubation with the antibody, Jurkat NFAT / CD16 cells were incubated at 1 × 10⁶ levels. 5 Each cell was added to a well and then incubated at 37°C, 5% CO2 for 16–17 hours. After incubation, the assay plate was equilibrated at room temperature for 15 minutes before plate reading. To observe reporter cell activation, 20 μL of medium from each well of the assay plate was added to the corresponding well of a flat-bottomed white 96-well reading plate (VWR, catalog no. 89130-330) along with 50 μL / well of QUANTI-Luc Gold (Invivogen, catalog no. rep-qlcg1) solution. The plate was read using a Promega GloMax plate reader (or Molecular Devices ID5) configured for luminescence reading at an integration time of 0.5 seconds / well. The data were standardized by subtracting the mean baseline luminescence value of the blank well from all other wells and EC was performed using Graphpad Prism 9.3.0. 50 The analysis included the calculation of values.

[0091] ADCC / PBMC assay Ovcar3 cells (ATCC, catalog number HTB-161) were cultured in RPMI (ATCC, catalog number 30-2001) supplemented with 20% FBS (Sigma, catalog number F5135), 1× penicillin-streptomycin (Corning, catalog number 30-002-CI), and 0.01 mg / ml bovine insulin. For the ADCC PBMC assay, Ovcar3 cells were counted to evaluate cell number and viability. Ovcar3 cells were stained with CFSE. Cells were centrifuged and 1 × 10⁶ cells were obtained. 5 Resuspend in growth medium at individual cells / mL, 1 × 10⁶ per well. 4Individual cells were seeded into 96-well plates (VWR, catalog number 89131-676) and incubated overnight in a cell culture incubator. Anti-TROP2 or control antibody from the assay medium was added to the cells at gradually increasing concentrations (0.032 to 500 ng / mL) at 37°C and 5% CO2 for 20 minutes. Subsequently, peripheral blood mononuclear cells (PBMCs) (Stemcell, catalog number 70025.1) 2 × 10⁶ were sampled. 5 The antibody was added to each well of a 96-well plate. The cells and antibody were incubated in a 5% CO2 incubator at 37°C for 18–24 hours. The samples were then stained with LIVE / DEAD® fixable Aqua Dead Cell Stain Kit (Thermo Fisher, catalog number L34957) and analyzed using a flow cytometer. The % of dead target cells were gated with FITC+Live / dead+. EC 50 The values ​​were calculated using Graphpad Prism 9.3.0.

[0092] In vivo efficacy assay Seven-week-old female nude mice (Charles River Laboratories, catalog number 088Nu / Nu) were used in this assay. 2 × 10⁶ FaDu cells were induced in 100 μL of a mixture of PBS and MatriGel (Corning, catalog number 354234) (volume:volume = 1:1). 6 Each individual tumor was inoculated into the upper left side of each nude mouse by sub-Q injection. Tumor growth and mouse body weight were monitored twice a week. For each individual tumor, the longest longitudinal diameter and widest transverse diameter were measured using a Traceable Digital Caliper (VWR, catalog no. 62379-531). Tumor volume (TV) was calculated using the formula TV = [length × (width)] 2 The following calculation was performed using ] / 2. The average tumor volume was 170-175 mm. 3Once the mice reached a certain level, they were randomly divided into groups, each containing 10 mice. Human IgG1 SD-025096 was prepared in a 3 mg / mL storage solution in PBS. For each mouse, the volume of the administered medium control (PBS) or antibody SD-025096 was calculated using the formula: Volume (μL) = Mouse body weight (g) × 10 μL / g. The medium control and antibody drugs were administered intravenously twice a week for a total of 6 times (biw × 6). After the first drug treatment, the percentage change in each tumor was calculated using the formula: TV change % = [(TV - TV day0 ) / TV day0 It was calculated by multiplying by 100.

[0093] Figures 1A and 1B show representative sensorgrams of the antibodies of the present invention. Binding dynamics were measured using human Fc capture with a Carterra LSA high-throughput SPR instrument (Carterra). None of the tested antibodies bound to human EpCAM, but bound equally to almost all cynomolgus monkey / rhesus monkey TROP2 (cyTROP2). None of the mouse chimeras recognized mouse TROP2 (msTROP2), but almost all chicken chimeras showed affinity for msTROP2 within a range of 10 times that of binding to huTROP2.

[0094] Figures 2A and 2B show MSD bound to TROP2. Figure 2A is a graph showing the dose-dependent binding of anti-TROP2 antibody to immobilized TROP2, assessed using the MSD assay. Figure 2B shows the obtained EC 50 The values ​​show that some clones have a lower EC than RS7. 50 A value was shown, indicating greater binding capacity. ND: Not determined.

[0095] Figures 3A and 3B show epitope binning of the antibodies taught herein. Figure 3A. The network plot shown in Figure 3A shows pairwise competition between libraries of 141 antibodies against TROP2 by SPR. Each node represents an individual antibody, and the connecting lines indicate the blockage relationship between two antibodies. Antibodies with similar blockage profiles, indicating a high probability of sharing an epitope, are systematized into five groups. Figure 3B shows various regions of TROP2. Group 1 contains the majority of the evaluated antibodies, including the approved antibody RS7, and is likely to correspond to some of the antibodies that target the immunodominant domain of TROP2. Antibodies in Group 2 form their own clusters, but are likely to target epitopes targeted in Group 1 or nearby epitopes, based on the degree of interaction with antibodies in Group 1. Based on peptide mapping data, antibodies in group 3 are likely to bind at or near the N-terminal domain, while antibodies in group 4 may bind to a membrane-proximal domain close to the RS7 binding site but different from that. The exact epitope targeted by group 5 remains unknown. Figure 3B summarizes the vials for each antibody.

[0096] Figure 4 shows the peptide mapping of antibodies by bin group. The sequence map, based on SPR data, shows the locations of eight peptides to which the anti-TROP2 antibody binds. Not all antibodies tested in this assay recognized the linear epitope. The recognition of peptides 1-2 by some antibodies in bin group 3 suggests an epitope located approximately near the N-terminus of the TROP2 protein. The recognition of peptides 3-6, located in the C-terminal domain close to the presumed RS7 epitope, by some antibodies in groups 1 and 2 suggests that these antibodies target that site or a nearby region. Finally, some antibodies in group 4 recognized peptides 7-8; therefore, group 4 may broadly target the C-terminal region of the C-terminal domain immediately proximal to the transmembrane domain, which is distant from the RS7 epitope.

[0097] Figures 5A and 5B show the results of ADCC reporter assays to identify the mouse antibodies taught herein. The dose-response graph in Figure 5A shows the ADCC efficacy of anti-human Trop2 antibodies in the human head and neck cancer cell line FaDu. The ADCC efficacy of anti-human Trop2 antibodies and defucosylated anti-human Trop2 antibodies is shown using the NFAT CD16 Jurkat ADCC reporter cell line. Figure 5B shows the EC of each antibody tested. 50 The values ​​are shown. All defucosylated anti-human Trop2 antibodies have a very small EC compared to their fucosylated versions. 50 The observed values ​​indicate that defucosylated anti-human Trop2 antibodies enhance their ADCC efficacy. 50 The values ​​are the average of n=2 to 3 experiments.

[0098] Figures 6A and 6B are graphs showing the results of an ADCC reporter assay to find the chicken antibody taught herein. Figure 6A is a graph showing the ADCC efficacy of the anti-human Trop2 antibody in the FaDu cancer cell line. The dose-response graph shows the ADCC efficacy of the anti-human Trop2 antibody in the FaDu cancer cell line. The ADCC efficacy of the anti-human Trop2 antibody is shown using the NFAT CD16 Jurkat ADCC reporter cell line. Most clones show very similar ADCC efficacy compared to RS7. EC 50 The values ​​are the average of n=1-3 experiments. ADCC efficacy of anti-human Trop2 antibody in FaDu cancer cell lines. Figure 6B. Table showing that most clones exhibit very similar ADCC efficacy compared to RS7. EC50 values ​​are the average of n=1-3 experiments.

[0099] Figures 7A and 7B show the cell binding results for the antibodies taught herein. Binding efficacy of anti-human Trop2 antibodies against the human head and neck cancer cell line FaDu. In Figure 7A, FACS analysis shows the anti-Trop2 antibody and RS7 bound to FaDu cells endogenously expressing human Trop2. The plotted values ​​are the median fluorescence intensities. EC in Figure 7B. 50 The values ​​are the average of n=1 to n=3 experiments.

[0100] Figure 8 is a graph showing the results of an anti-human Trop2 antibody derived from immunized chickens binding to ExpiCHO cells overexpressing human Trop2. It is a FACS analysis showing the dose-dependent binding of the anti-Trop2 antibody and RS7 to ExpiCHO cells overexpressing mouse Trop2. The plotted values ​​are the percentage of antibody binding to the target cells. Histograms comparing the antibody binding levels to human Trop2 for the tested antibodies using the highest dose (66.67 nM) in the binding assay are superimposed.

[0101] Figure 9 is a graph showing the results of anti-human Trop2 antibodies derived from immunized chickens binding to ExpiCHO cells overexpressing mouse Trop2. FACS analysis shows that selected anti-Trop2 antibodies, which bind to ExpiCHO cells overexpressing mouse Trop2 in a dose-dependent manner, exhibit cross-reactivity with mouse Trop2. The RS7 control was specific only to human Trop2 and did not show binding to ExpiCHO cells overexpressing mouse Trop2. The plotted values ​​represent the percentage of antibody binding to target cells. Histograms comparing the antibody binding levels to human Trop2 for the tested antibodies using the highest dose (66.67 nM) in the binding assay are superimposed.

[0102] Figures 10A and 10B show the killing of peripheral blood mononuclear cells (PBMCs) by the present invention's anti-human Trop2 antibody, which exhibits potent killing of human ovarian cancer (Ovcar3) cells. The graph in Figure 10A shows FACS analysis of cell death initiated by the anti-TROP2 molecule and effector cells, human PBMCs, against TROP2-expressing Ovcar3 target cells. Figure 10B shows EC against each of the antibodies tested. 50 Contains a value.

[0103] Figure 11 shows an example of a research design for in vivo efficacy.

[0104] Figures 12A–12C are graphs showing the results of an in vivo study using FaDu xenografts in nude mice. Randomization and grouping were performed on day 0 based on tumor size. Drug administration was performed on days 0, 3, 7, 10, and 14. Tumor volume (Figure 12A) of the FaDu nude mouse xenograft model. Percentage change in tumor volume (Figure 12B). Mouse body weight (Figure 12C).

[0105] Figure 13 shows the humanization of SD-589775. Binding kinetics to huTROP2 were measured using human Fc capture with a Carterra LSA high-throughput SPR instrument (Carterra). Combinations of humanized VH and VL sequences were tested and compared to the parent chimera. Variants with 589-VH_4 or 589-VL_3 did not show measurable binding to huTROP2. 589-VH_5 and 589-VL_4 refer to the parent sequences.

[0106] Figure 14 shows ELISA data illustrating the binding of anti-CD3 antibodies to CD3εδ heterodimers. Antibody-coated plates are then detected by dose-response analysis of CD3εδ-HRP.

[0107] Figure 15 shows the TROP2 × CD3 TAA-conjugated arm dual-specific screening. Cell elimination by co-culture of PBMCs and FaDu tumor cells. Cell lysis % measured after 48 hours. Figure 15A shows the mouse-derived TROP2-conjugated antibody (SD-589775). Figure 15B shows the chicken-derived TROP2-conjugated antibody.

[0108] Figures 16A to 16C illustrate the characterization of TROP2×CD3 bispecific cell toxicity against FaDu. Figure 16A shows cell toxicity by co-culture of PBMCs and FaDu tumor cells. Cell lysis % measured after 48 hours. Figure 16B shows T cell activation induced by co-culture of PBMCs and FaDu tumor cells. T cell activation measured after 48 hours. Figure 16C shows cytokine production and release induced by co-culture of PBMCs and FaDu tumor cells. Cytokine levels measured after 24 hours.

[0109] Figures 17A to 17C illustrate the characterization of TROP2 × CD3 bispecific cell toxicity against Calu-3. Figure 17A shows cell toxicity by co-culture of PBMCs and Calu-3 tumor cells. Cell lysis % measured after 48 hours. Figure 17B shows T cell activation induced by co-culture of PBMCs and Calu-3 tumor cells. T cell activation measured after 48 hours. Figure 17C shows cytokine production and release induced by co-culture of PBMCs and Calu-3 tumor cells. Cytokine levels measured after 24 hours.

[0110] Figures 18A to 18C illustrate the characterization of TROP2×CD3 bispecific cell elimination against OvCar-3. Figure 18A shows cell elimination by co-culture of PBMCs and OvCar-3 tumor cells. Cell lysis % measured after 48 hours. Figure 18B shows T cell activation induced by co-culture of PBMCs and OvCar-3 tumor cells. T cell activation measured after 48 hours. Figure 18C shows cytokine production and release induced by co-culture of PBMCs and OvCar-3 tumor cells. Cytokine levels measured after 24 hours.

[0111] In vivo efficacy study design SD-174078, multiple doses.

[0112] Eleven-week-old MHCI / II double knockout female NSG nude mice (Jackson Laboratory, catalog number 025216) were used in this assay. Human PBMCs (STEMCELL Technologies; ID CE0006634) 20×10 6 The cells were resuspended in 100 μL of PBS and transplanted into each mouse by intravenous injection. On day 13 after human PBMC transplantation, the mice were randomly divided into groups so that their average body weights were similar, with each group containing 5-8 mice. At this point, 2×10 FaDu cells in 100 μL of PBS and MatriGel (Corning, catalog number 354234) (volume:volume = 1:1) were transplanted into each mouse. 6Each mouse was inoculated subcutaneously into the upper left side of the tumor. Human bispecific TROP2×CD3 antibody was prepared in a 0.1 mg / ml storage solution in PBS. For each mouse, the volume of administered medium control (PBS) or antibody was calculated using the formula: Volume (μl) = Mouse body weight (g) × 10 μl / g. PBS and SD-174078 were administered intravenously a total of three times (biw × 3) on days 0, 3, and 7. For each individual tumor, the longest longitudinal diameter and widest transverse diameter were measured using a Traceable Digital Calliper (VWR, catalog number 62379-531). Tumor volume (TV) was calculated using the formula: TV = [Length × (Width)] 2 It was then calculated using ] / 2.

[0113] Human bispecific TROP2×CD3 antibodies include: SD-174078_knob:CD3 Gen 1 (SEQ ID NO: 174), SD-174078_hole:813149_VL (SEQ ID NO: 126), SD-231831_knob:CD3 Gen 2 (190), SD-231831_hole:589_VL2 (SEQ ID NO: 166). Those skilled in the art will recognize that the knob-and-hole chains may be interchangeable or modified to other knob-and-hole variants or different Fc variants and / or isotypes.

[0114] Figure 19 is a graph showing the results of an in vivo efficacy study using multiple doses of SD-174078. FaDu xenografts in humanized NSG MHCI / II dKO mice. Mice received intravenous injection of human PBMCs 20×10⁶ on day 14. 6 Individual cells were transplanted. -On day 1, 2 x 10 FaDu cells were transplanted. 6 The tumors were subcutaneously transplanted. On days 0, 3, and 7, the mice were administered either PBS (medium) or SD-174078 (1 mg / kg) by intravenous injection. Tumor volume is shown. Significance was determined using Welch's t-test.

[0115] In vivo efficacy study design - SD-231831 single dose. Eleven-week-old MHCI / II double knockout female NSG nude mice (Jackson Laboratory, catalog number 025216) were used in this assay. Human PBMCs (STEMCELL Technologies; ID CE0006634) 20×10 6 The cells were resuspended in 100 μl of PBS and transplanted into each mouse by intravenous injection. On day 13 after human PBMC transplantation, the mice were randomly divided into groups so that their average body weights were similar, with each group containing 5-8 mice. At this point, 2 × 10⁶ FaDu cells were administered in 100 μl of PBS and MatriGel (Corning, catalog number 354234) (volume:volume = 1:1). 6 Each mouse was inoculated subcutaneously into the upper left side of the tumor. Human bispecific antibody (TROP2×CD3) was prepared in a 0.1 mg / ml storage solution in PBS. For each mouse, the volume of administered medium control (PBS) or antibody was calculated using the formula: Volume (μl) = Mouse body weight (g) × 10 μl / g. PBS and SD-231831 were administered as a single dose by intravenous injection on day 7. For each individual tumor, the longest longitudinal diameter and widest transverse diameter were measured using a Traceable Digital Calliper (VWR, catalog number 62379-531). Tumor volume (TV) was calculated using the formula: TV = [Length × (Width)] 2 It was then calculated using ] / 2.

[0116] Figure 20 is a graph showing the results using a single dose. FaDu xenografts in humanized NSG MHCI / II dKO mice. Mice received human PBMC 20×10⁶ via intravenous injection on day 14. 6 Individual cells were transplanted. -On day 1, 2 x 10 FaDu cells were transplanted. 6 The tumors were subcutaneously transplanted. On day 7, mice were given a single dose of either PBS (medium) or SD-231831 (1 mg / kg) by intravenous injection. Tumor volume is shown. Significance was determined using Welch's t-test.

[0117] A person skilled in the art will recognize that an antibody that shows little or no binding to a target antigen may be described as having low affinity and a high equilibrium dissociation constant (KD) to the target antigen. A person skilled in the art will also recognize that an antibody that shows little or no binding to an assembly of target antigenic epitopes may be described as having low affinity and a high equilibrium dissociation constant (KD) to an assembly of target antigenic epitopes.

[0118] In some embodiments, the Specified provides anti-Trop2 antibodies having binding affinity (KD) to Trop2 in the following ranges: approximately 5 μM to approximately 5 pM, approximately 1 μM to approximately 5 pM, approximately 0.5 μM to approximately 5 pM, approximately 0.1 μM to approximately 5 pM, approximately 50 nM to approximately 5 pM, approximately 10 nM to approximately 5 pM, approximately 5 nM to approximately 5 pM, approximately 1 nM to approximately 5 pM, approximately 0.5 nM to approximately 5 pM, approximately 0.1 nM to approximately 5 pM, approximately 50 pM to approximately 5 pM, and approximately 10 pM to approximately 5 pM.

[0119] In some embodiments, the anti-Trop2 antibody has binding affinity (KD) to Trop2 of approximately 500 nM to 0.1 pM, 100 nM to 0.1 pM, 50 nM to 0.1 pM, 10 nM to 0.1 pM, 5 nM to 0.1 pM, 1 nM to 0.1 pM, 0.5 nM to 0.1 pM, 0.1 nM to 0.1 pM, 50 pM to 0.1 pM, 10 pM to 0.1 pM, 5 pM to 0.1 pM, 1 pM to 0.1 pM, and 0.5 pM to 0.1 pM.

[0120] In some embodiments, the anti-Trop2 antibody is effective at a maximum half-volume effective concentration (EC) of approximately 500 nM to approximately 0.001 nM, approximately 100 nM to approximately 0.001 nM, approximately 50 nM to approximately 0.001 nM, approximately 10 nM to approximately 0.001 nM, approximately 5 nM to approximately 0.001 nM, approximately 1 nM to approximately 0.001 nM, approximately 0.5 nM to approximately 0.001 nM, approximately 0.1 nM to approximately 0.001 nM, approximately 0.05 nM to approximately 0.001 nM, and approximately 0.005 nM to approximately 0.001 nM relative to Trop2. 50 ) has.

[0121] Those skilled in the art will recognize that binding specificity can be determined by a series of competitive binding paradigms, and that a desired antibody exhibits its ability to prevent the binding of a known reference antibody to its target epitope at various concentrations. In some embodiments, the reference antibody is RS7. In some embodiments, the reference RS7 antibody binds to an epitope located at amino acid positions 146–178. Those skilled in the art will also recognize that the reference RS7 antibody can be used as a control antibody in agonist and antagonist assays.

[0122] In some embodiments, the anti-Trop2 antibody is a full-length antibody (an antibody having two heavy chains and two light chains bound to an Fc domain, forming a Y-shape). In some embodiments, the Fc domain (or simply referred to as Fc) is a human Fc domain. In some embodiments, the Fc domain of the anti-Trop2 antibody is derived from human IgG1, human IgG2, human IgG3, or human IgG4.

[0123] Exemplary anti-Trop2 antibody-CDR sequence

[0124] [Table 3] JPEG2026519783000006.jpg251170 JPEG2026519783000007.jpg251170 JPEG2026519783000008.jpg251170 JPEG2026519783000009.jpg251170 JPEG2026519783000010.jpg251170 JPEG2026519783000011.jpg251170 JPEG2026519783000012.jpg251170 JPEG2026519783000013.jpg251170 JPEG2026519783000014.jpg220170

[0125] additional array [Table 4] JPEG2026519783000016.jpg252170 JPEG2026519783000017.jpg249170 JPEG2026519783000018.jpg251170 JPEG2026519783000019.jpg250170 JPEG2026519783000020.jpg116170

[0126] Antibody humanization Humanization of SD-589775 was completed by transplanting both the heavy and light chain CDRs into a closely related human germline sequence, resulting in 589-VH_1 and 589-VL_1. The identified Vernier residues were manually restored to the parent chicken amino acids, resulting in 589-VH_2 and 589-VL_2. Several additional amino acids in VH were restored to the parent chicken sequence to create 589-VH_3. VH and VL were humanized using a publicly available tool (DOI: 10.1080 / 19420862.2021.2020203), resulting in 589-VH_4 and 589-VL_3. The parent chicken sequence was used as 589-VH_5 and 589-VL_4. SD-861408 was generated by combining 589-VH_2 and 589-VL_2. Testing was performed using SPR as described above.

[0127] CD3εδ binding by direct ELISA Antibody binding to recombinant CD3εδ-HRP heterodimer (Acro Biosystems, CDD-HR2W3) was evaluated by direct ELISA. Each antibody was diluted to 5 μg / mL in 50 μL / well of 50 mM carbonate buffer (pH 9.5) and allowed to stand for 1 hour. After the coating step, the plates were blocked at room temperature for 15 minutes using 300 μL / well of SuperBlock® blocking buffer (Thermo Fisher Scientific, #37515). Seven dose-response curves for recombinant CD3εδ-HRP conjugates were generated by 5-fold serial dilutions starting at 10 μg / mL in PBS, and then incubated on the plates for 1 hour. The plates were developed for 5 minutes using 1-Step® TMB ELISA substrate solution (Thermo Fisher Scientific, #34028) and stopped in a 1:1 mixture of stop solution (Thermo Fisher Scientific, #SS04). The absorbance of each well at 450 nm was plotted against the logarithm of the CD3εδ-HRP concentration, and EC50 was calculated by fitting the results using nonlinear regression. Each incubation step was performed at room temperature with gentle stirring for 1 hour, and washing with PBST was performed in between.

[0128] Assembly of a bispecific TROP2 × CD3 antibody Bispecific knob-in-hole antibodies were generated using controlled Fab arm exchange (DOI: 10.1038 / nprot.2014.169). Single-specific parental knob (https: / / doi.org / 10.1038 / nprot.2014.169) (K409R; CD3-bound) and hole (F405L; TROP2 or antigen-bound) antibodies were mixed in PBS in a 1:1 ratio with 75 mM 2-mercaptoethylamine hydrochloride (2-MEA, Millipore Sigma, #M6500) and incubated at 31°C for 5 hours. After incubation, 2-MEA was removed by buffer exchange in PBS using a Zeba® spin desalting column (Thermo Fisher Scientific, #87767), filtered through a 0.2 μm filter, and incubated overnight at 4°C before assembly or activity testing. All bispecific antibodies possessed Fc effector function, which was silenced by the N297A mutation.

[0129] Efficiency of bispecific antibody assembly by cation exchange HPLC The assembly efficiency of bispecific antibodies was evaluated by HPLC (Agilent Infinity 1290) using a ProPac WCX-10 25cm cation exchange column (Thermo Fisher Scientific, #054993). If necessary, the antibody preparation buffer for measurement was replaced with buffer A (20mM NaPi, pH 7.0) using a Zeba® spin desalting column (Thermo Fisher Scientific, #87767) before filtering through a 0.2μm filter. After equilibrating the column with buffer A, the samples were loaded onto 96-well plates and placed in the instrument. 20 μL of each sample was injected into the column under the following gradient conditions: 0–3 min, 100% buffer A; 3–58.5 min, 0–72% buffer B (20 mM NaPi, pH 7.0 + 250 mM NaCl); 58–5–63.5 min, 100% buffer C (20 mM NaPi, pH 7.0 + 750 mM NaCl); 63.5–82 min, 100% buffer A. The resulting peaks were integrated using OpenLab Chemstation (Agilent Technologies). Percentages were calculated by dividing the region corresponding to the bispecific antibody peak by the total peak region.

[0130] T cell-dependent cellular cytotoxicity (TDCC) and T cell activation assay Ovcar3 cells (ATCC, HTB-161) were cultured in RPMI (ATCC, 30-2001) supplemented with 20% FBS (Sigma, F5135), 1× penicillin-streptomycin (Corning, 30-002-CI), and 0.01 mg / ml bovine insulin. FADU cells (ATCC, HTB-43) were cultured in EMEM (ATCC, 30-2003) supplemented with 10% FBS (Sigma, F5135) and 1× penicillin-streptomycin (Corning, 30-002-CI). Calu-3 cells (ATCC, HTB-55) were cultured in EMEM (ATCC, 30-2003) supplemented with 10% FBS (Sigma, F5135) and 1× penicillin-streptomycin (Corning, 30-002-CI).

[0131] In the TDCC PBMC assay, Ovcar3, FADU, or Calu-3 cells were counted to assess cell number and viability. Cells were stained with CFSC. Cells were centrifuged and measured at 1 × 10⁶. 5 Resuspend the cells in growth medium at a concentration of individual cells / ml and place 1 × 10⁶ cells per well on a 96-well plate (VWR, 89131-676). 4 Individual cells were seeded and incubated overnight in a cell culture incubator. Anti-TROP2 or control antibody from the assay medium was added to the cells at gradually increasing concentrations (0.032 to 500 ng / mL) at 37°C and 5% CO2 for 20 minutes. Subsequently, peripheral blood mononuclear cells (PBMCs) (Stemcell, 70025.1) 2 × 10⁶ were analyzed. 5 The cells were added to each well of a 96-well plate. The cells and antibodies were incubated in a 5% CO2 incubator at 37°C for 48 hours. The samples were then stained with the LIVE / DEAD® fixable Aqua Dead Cell Stain Kit (Thermo Fisher, L34957) and an antibody cocktail [CD32 / CD16 blocking antibody, CD3-BV605, CD4-PerCP / Cy5.5, CD8a-APC, CD69-PE, CD25+-APC / cy7 (Biolegend)]. The samples were then analyzed using a flow cytometer (MacsQuant 16, Miltenyi). % of dead target cells were gated with FITC+ Live / dead+. Early activated CD4+ cells were gated as Live / dead- / CD3+ / CD4+ / CD69+. Early activated CD8+ was gated as :Live / dead- / CD3+ / CD8+ / CD69+. Later activated CD4+ was gated as :Live / dead- / CD3+CD4+ / CD25+. Later activated CD8+ was gated as :Live / dead- / CD3+ / CD8+ / CD69+. EC50 values ​​were calculated using Graphpad Prism 9.3.0.

[0132] Measurement of cytokine release Cytokine release was measured using a custom-ordered U-PLEX Biomarker Assay kit (Meso Scale Discovery, K15067M) capable of quantifying IFNγ, IL-2, IL-6, and TNFα, following the kit's instructions. Briefly, biotinylated capture antibodies were conjugated to a specific linker and applied to a plate to capture the antibodies on specific spots. Cell culture supernatant was obtained from the TDCC assay at 24 hours, diluted 1:10 in Diluent 57 before application to the plate, and then a mixture of detection antibodies diluted in Diluent 3 was added to each well. Each incubation step was performed at room temperature for 1 hour with gentle agitation, with washing with 3 × 150 μL of PBST in between. After the final wash, 150 μL / well of MSD GOLD Read Buffer B was added, and the plates were read using a MESO QuickPlex SQ 120MM instrument. Cytokine concentrations were determined using Discovery Workbench software.

[0133] Exemplary anti-Trop2 sequences and the bispecific antibodies of this disclosure are provided herein. These include complementarity-determining region (CDR) sequences and variable weight and light domain sequences (VH, VL) that constitute the anti-Trop2 antigen-binding domain of this disclosure. The discovery of these antibodies is described in detail in the Examples section.

[0134] As will be hereafter referred to, the light chain variable (VL) domain CDR1 region is referred to as CDR-L1; the VL CDR2 region is referred to as CDR-L2; the VL CDR3 region is referred to as CDR-L3; the heavy chain variable (VH) domain CDR1 region is referred to as CDR-H1; the VH CDR2 region is referred to as CDR-H2; and the VH CDR3 region is referred to as CDR-H3. Table 1 provides exemplary CDR combinations of the antibodies of this disclosure.

[0135] scFv-FcTROP2 antibody.

[0136] In some embodiments, the Disclosure provides a series-type scFv antibody having multiple Trop2 binding sites. The series-type scFv-Fc antibody of the Disclosure consists of two or more series-type scFv binding sites on each antibody arm, which may be linked by linkers or by movable linkers. In some embodiments, the series-type scFv antibody has a total of four or more scFv binding sites within one scFv-Fc antibody.

[0137] In some embodiments, scFv1 of each antibody arm comprises a first heavy chain variable domain (VH1) and a first light chain variable domain (VL1); and scFv2 of each antibody arm comprises a first heavy chain variable domain (VH2) and a first light chain variable domain (VL2).

[0138] Therapeutic anti-Trop2 and bispecific antibodies.

[0139] In some embodiments, the anti-Trop2 and bispecific antibodies (TROP2xCD3) provided herein are useful for treating diseases or conditions related to the immune response.

[0140] In some embodiments, anti-Trop2 and the bispecific antibodies provided herein are useful for treating proliferative disorders.

[0141] In vivo administration of the therapeutic anti-Trop2 and bispecific antibodies described herein may be performed intravenously, intramuscularly, subcutaneously, topically, orally, percutaneously, percutaneously, intraperitoneally, intraorbitally, intradurally, intraventricularly, intranasally, transmucosally, implantably, or by inhalation. Intravenous administration may be performed by injection or infusion. In some embodiments, the anti-Trop2 and bispecific antibodies of this disclosure are administered intravenously. In some embodiments, the anti-Trop2 and bispecific antibodies of this disclosure are administered subcutaneously. Administration of the therapeutic anti-Trop2 and bispecific antibodies can be carried out with any suitable excipient, carrier, or other agent to provide appropriate or improved tolerance, transfer, delivery, etc.

[0142] Embodiment Embodiment 1. The antibody is a. Heavy chain variable domain (VH) complementarity-determining region (CDR) 1 containing any one amino acid sequence from sequence numbers 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, or 132; and b. VH CDR2 containing any one of the amino acid sequences among SEQ ID NOs: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123, or 133; and c. VH CDR3 containing any one of the amino acid sequences among SEQ ID NOs: 4, 14, 24, 34, 44, 54, 64, 74, 84, 94, 104, 114, 124, and 134; and d. Light chain variable domain (VL) CDR1 containing any one amino acid sequence from sequence numbers 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, or 137; and e. VL CDR2 containing any one of the amino acid sequences among SEQ ID NOs: 8, 18, 28, 38, 48, 58, 68, 78, 88, 98, 108, 118, 128, or 138; and f. VL CDR3 containing any one of the amino acid sequences among sequence numbers 9, 19, 29, 39, 49, 59, 69, 79, 89, 99, 109, 119, 129, or 139. An anti-Trop2 antibody or its conjugated fragment containing such an antibody.

[0143] Embodiment 2. The antibody is a. VH containing any one of the amino acid sequences of SEQ ID NOs: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 156, 158, 160, or 162, and b. The antibody or conjugated fragment of Embodiment 1, comprising a VL containing any one of the amino acid sequences among SEQ ID NOs: 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 164, 166, or 168.

[0144] Embodiment 3. The antibody is a. VH encoded by a nucleic acid sequence containing any one of sequence numbers 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, 115, 125, 135, 157, 159, 161, or 163, and b. VL encoded by a nucleic acid sequence containing any one of the following sequence numbers: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 165, 167, or 169 The antibody of Embodiment 1, including the antibody of Embodiment 1.

[0145] Embodiment 4. The antibody or conjugated fragment of any of Embodiments 1 to 3, wherein the antibody is a monoclonal antibody, a bispecific antibody, a polyvalent antibody, a multispecific antibody, a diabody antibody, a chimeric antibody, an scFv antibody, or a fragment thereof.

[0146] Embodiment 5. The antibody or conjugated fragment of any of Embodiments 1 to 4, wherein the antibody is a fucosylated full-length antibody.

[0147] Embodiment 6. An antibody or conjugated fragment according to any of Embodiments 1 to 5, wherein the antibody contains one Fc domain from among human IgG1, human IgG2, human IgG3, and human IgG4.

[0148] Embodiment 7. The antibody or conjugated fragment of Embodiment 6, wherein the Fc domain is a wild-type Fc domain, a variant Fc domain, or a cleaved Fc domain.

[0149] Embodiment 8. The antibody or conjugation fragment of Embodiment 1, further comprising a second antigen-binding domain that binds to a target other than TROP-2.

[0150] Embodiment 9. The antibody or conjugate fragment of Embodiment 8, wherein the target is a CD3 antibody having a heavy chain selected from SEQ ID NOs: 174 and 178; and the light chain is selected from SEQ ID NOs: 176 and 180, or from bispecific antibodies including SEQ ID NOs: 174, SEQ ID NOs: 126, SEQ ID NOs: 190 and SEQ ID NOs: 166.

[0151] Embodiment 10. The antibody or conjugate fragment of Embodiment 8, wherein the antibody is a bispecific antibody containing a heavy chain selected from SEQ ID NOs: 190 and 192, or 194 and 196.

[0152] Embodiment 11. A method for treating a disease in a subject requiring it, comprising the step of administering a therapeutically effective amount of any antibody from Embodiments 1 to 8 to the subject.

[0153] Embodiment 12. The method of Embodiment 11, wherein the disease is a proliferative disorder.

[0154] Embodiment 13. The method of Embodiment 12, wherein the proliferative disorder is a cancer selected from cancers expressing Trop2 or its variants.

[0155] Embodiment 14. Any of the methods of Embodiments 11 to 13, wherein the subject is a human.

[0156] Embodiment 15. A bivalent or polyvalent antibody, a. Heavy chain variable domain (VH) complementarity-determining region (CDR) 1 containing any one amino acid sequence from sequence numbers 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, or 132; and b. VH CDR2 containing any one of the amino acid sequences among SEQ ID NOs: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123, or 133; and c. VH CDR3 containing any one of the amino acid sequences among SEQ ID NOs: 4, 14, 24, 34, 44, 54, 64, 74, 84, 94, 104, 114, 124, and 134; and d. Light chain variable domain (VL) CDR1 containing any one amino acid sequence from sequence numbers 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, or 137; and e. VL CDR2 containing any one of the amino acid sequences among SEQ ID NOs: 8, 18, 28, 38, 48, 58, 68, 78, 88, 98, 108, 118, 128, or 138; and f. VL CDR3 containing any one of the amino acid sequences among sequence numbers 9, 19, 29, 39, 49, 59, 69, 79, 89, 99, 109, 119, 129, or 139. Anti-Trop2 antibodies or their conjugated fragments containing such antibodies; and A second antibody, an antigen-binding agent of the second antibody or a fragment thereof; a target-binding protein, cytokine; a lectin; or a toxin. A bivalent or polyvalent antibody containing the above.

[0157] Embodiment 16. The bivalent or polyvalent antibody of Embodiment 15, wherein the second antibody targets an immunoeffector cell surface receptor selected from at least one of CTLA-4, PD-1, Lag3, S15, B7H3, B7H4, TCR-alpha, TCR-beta, TIM-3, CD3, 41BB, and OX40.

[0158] Embodiment 17. The bivalent or polyvalent antibody of Embodiment 16, wherein the antibody is a polyvalent antibody that targets two or three or more antigens other than Trop2.

[0159] Embodiment 18. The bivalent or polyvalent antibody of Embodiment 15, wherein the antibody is a bispecific antibody containing a heavy chain selected from SEQ ID NOs: 190 and 192, 194 and 196, or a bispecific antibody containing SEQ ID NOs: 174, SEQ ID NOs: 126, SEQ ID NOs: 190 and SEQ ID NOs: 166.

[0160] Embodiment 19. The anti-Trop2 antibody is a. VH containing any one of the amino acid sequences of SEQ ID NOs: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 156, 158, 160, or 162, and b. Contains a VL containing any one of the amino acid sequences among SEQ ID NOs: 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 164, 166, or 168. A bivalent or polyvalent antibody according to Embodiment 15.

[0161] Embodiment 20. The anti-Trop2 antibody is a. VH encoded by a nucleic acid sequence containing any one of sequence numbers 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, 115, 125, 135, 157, 159, 161, or 163, and b. VL encoded by a nucleic acid sequence containing any one of the following sequence numbers: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 165, 167, or 169 A bivalent or polyvalent antibody of Embodiment 15, including the above.

[0162] Embodiment 21. A bivalent or polyvalent antibody according to any of Embodiments 15 to 19, wherein the antibody is a monoclonal antibody, a full-length antibody, or an antibody fragment.

[0163] Embodiment 22. A bivalent or polyvalent antibody according to any of Embodiments 15 to 12, wherein the antibody is fused to one of the Fc domains of human IgG1, human IgG2, human IgG3, and human IgG4.

[0164] Embodiment 23. The target is CD3, and the antibody comprises a heavy chain selected from SEQ ID NOs: 174 and 178; and a light chain selected from SEQ ID NOs: 176 and 180, either bivalent or polyvalent, as in any of Embodiments 15 to 21.

[0165] Embodiment 24. A bivalent or polyvalent antibody according to any of Embodiments 15 to 21, wherein the target is CD3, and the antibody comprises a heavy chain encoded by a nucleic acid selected from SEQ ID NOs: 175 and 179; and a light chain encoded by a nucleic acid selected from SEQ ID NOs: 177 and 181.

[0166] Embodiment 25. The divalent or polyvalent form of Embodiment 23, wherein the Fc domain is a wild-type Fc domain, a variant Fc domain, or a cleaved Fc domain.

[0167] Embodiment 26. A divalent or polyvalent antibody according to any of Embodiments 15 to 24, wherein the antibody is a defucosylated full-length antibody.

[0168] Embodiment 27. A method for treating a disease in a subject requiring it, comprising the step of administering a therapeutically effective amount of an antibody of any of Embodiments 1 to 10 or a bivalent antibody of any of Embodiments 15 to 26 to the subject.

[0169] Embodiment 28. The method of Embodiment 27, wherein the disease is a proliferative disorder.

[0170] Embodiment 29. The method of Embodiment 27, wherein the disease is a proliferative disorder selected from cancer cells expressing Trop2 or a variant thereof, selected from breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, certain lung cancers, oral squamous cell carcinoma, ovarian squamous cell carcinoma, pancreatic squamous cell carcinoma, prostate squamous cell carcinoma, gastric squamous cell carcinoma, thyroid squamous cell carcinoma, bladder squamous cell carcinoma, and uterine squamous cell carcinoma.

[0171] Embodiment 30. Any of the methods of Embodiments 27 to 29, wherein the subject is a human.

[0172] Embodiment 31. a. VH encoded by a nucleic acid sequence containing any one of sequence numbers 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, 115, 125, 135, 157, 159, 161, or 163; and b. A VL encoded by a nucleic acid sequence containing any one of sequence numbers 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 165, 167, or 169; or c. Antibodies encoded by the nucleic acid sequences of SEQ ID NOs. 187 and 189, or 191 and 193; or d. A bispecific antibody, or a combination thereof, encoded by the nucleic acid sequences of SEQ ID NOs. 175 and 177, 179 and 181, 183 and 185, 187 and 189, or 191 and 193. Antibodies, their binding fragments, and nucleic acids encoding bivalent or polyvalent nucleic acid sequences.

[0173] Embodiment 32. A vector containing the nucleic acid of Embodiment 31.

[0174] Embodiment 33. A host cell containing the vector of Embodiment 32.

[0175] Any embodiment discussed herein can be realized in connection with any method, kit, reagent, or composition of the present invention, and vice versa. Furthermore, the methods of the present invention can be achieved using the compositions of the present invention.

[0176] It will be understood that the specific embodiments described herein are illustrative and not limit the invention. The main features of the invention can be utilized in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or can verify through routine experimentation, numerous procedures equivalent to the specific procedures described herein. Such equivalent procedures are considered to be within the scope of the invention and are covered by the claims.

[0177] All publications and patent applications cited herein are indicators of the level of skill of those skilled in the art relating to the present invention. All publications and patent applications are incorporated herein by reference as if each individual publication or patent application were specifically and individually designated and incorporated by reference.

[0178] The use of the words “a” or “an,” when used with the term “comprising” in the claims and / or specification, may mean “one,” but also coincide with the meanings of “one or more,” “at least one,” and “one or more than one.” In the claims, the use of the term “or” is used to mean “and / or,” unless it is explicitly indicated that it refers only to the options or that the options are mutually exclusive. However, this disclosure supports the definition of “and / or” as referring only to the options. Throughout this application, the term “about” is used to indicate that a value includes variations of intrinsic error in the variations that exist between the device, the method used to determine the value, or the subject of study.

[0179] As used herein and in the claims, the words “comprising” [and any form of “comprising,” such as “comprise” and “comprises”], “having” [and any form of “having,” such as “have” and “has”], “including” [and any form of “includings” and “include”], or “containing” [and any form of “containings” and “contain”] are all-inclusive or limitless, and do not exclude additional, unlisted elements or method steps. In any embodiment of the compositions and methods provided herein, “comprising” may be replaced with “consisting essentially of” or “consisting of.” In this specification, the phrase “essentially” requires a specified integer or step that does not substantially affect the nature or function of the claimed invention. In this specification, the term “consisting” is used to indicate that only an enumerated integer (e.g., feature, element, characteristic, trait, method / method step or limitation) or a group of integers (e.g., feature, element, characteristic, trait, method / method step or limitation) exists.

[0180] In this specification, the term “or any combination thereof” means any permutation or combination of all the items listed before the term. For example, “A, B, C or any combination thereof” means A, B, C, AB, AC, BC, or ABC, and, where the order is important in a particular context, at least one of BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Following this example, combinations containing repetitions of one or more items or terms, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, are expressly included. A person skilled in the art will understand that, unless otherwise evident from the context, there is generally no limit to the number of items or terms in any combination.

[0181] In this specification, however not limited thereto, approximate words such as “about,” “substantial,” or “effectively” refer to a state that, when modified in this manner, is understood not necessarily absolute or complete, but which a person skilled in the art would consider close enough to justify designating an existing state. The extent to which deviations from this specification may occur will depend on whether, no matter how significant the change, a person skilled in the art can still recognize that the modified feature still possesses the required qualities and capabilities of the unmodified feature. In general, however, in light of the preceding considerations, numerical values ​​in this specification modified by approximate words such as “about” may vary by at least ±1, 2, 3, 4, 5, 6, 7, 10, 12, or 15% from the stated values.

[0182] Furthermore, the headings in this specification are provided to maintain consistency with the proposals of 37 CFR 1.77, and otherwise to provide systematic direction. These headings do not limit or characterize any invention described in any claims that may arise from this disclosure. In particular and as an example, the headings refer to the “Field of Invention,” but such claims should not be limited by the terminology of these headings to describe so-called technical fields. Furthermore, the description of the technology in the “Background Art” section should not be construed as an acknowledgment that the technology is prior art to any invention in this disclosure. In none of these shall the “Summary of the Invention” be considered a characterization of the invention as described in the issued claims. Furthermore, no reference to the singular “Invention” in this disclosure should be used to assert that there is only one novelty in this disclosure. Multiple inventions may be described in accordance with the limitations of multiple claims arising from this disclosure, and such claims shall define the invention and its equivalents protected thereby. In all examples, such claims shall be considered in light of this disclosure on their own basis, but should not be bound by the headings provided herein.

[0183] Each dependent claim may be dependent on both the independent claim and the preceding dependent claim, with respect to each and all claims, insofar as the preceding claim provides an appropriate antecedent for the claim term or element.

[0184] To assist the Patent Office and all readers of any patent issued in this application in interpreting the claims attached hereto, the applicants note that, unless the word “means” or “step” is expressly used in any particular claim, they do not intend to assert in any of the attached claims that paragraph 6 of 35 U.S.SC § 112, paragraph 112(f), or any equivalent, existed as of the filing date of this document.

[0185] All compositions and / or methods disclosed and claimed herein can be carried out and performed without experimentation beyond what is necessary in light of this disclosure. Although the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that modifications can be applied to the compositions and / or methods and the steps or sets of steps of the methods described herein without departing from the concept, spirit and scope of the invention. All such similar substitutions and modifications that are apparent to those skilled in the art are considered to fall within the spirit, scope and concept of the invention as defined by the appended claims.

Claims

1. An anti-Trop2 antibody or its conjugated fragment, The aforementioned antibody a. Heavy chain variable domain (VH) complementarity-determining region (CDR) 1 containing any one amino acid sequence from sequence numbers 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, or 132; and b. VH CDR2 containing any one amino acid sequence from sequence numbers 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123, or 133; and c. VH CDR3 containing any one amino acid sequence from sequence numbers 4, 14, 24, 34, 44, 54, 64, 74, 84, 94, 104, 114, 124, and 134; and d. Light chain variable domain (VL) CDR1 containing any one amino acid sequence from sequence numbers 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, or 137; and e. VL CDR2 containing any one amino acid sequence from sequence numbers 8, 18, 28, 38, 48, 58, 68, 78, 88, 98, 108, 118, 128, or 138; and f. VL CDR3 containing any one of the amino acid sequences among sequence numbers 9, 19, 29, 39, 49, 59, 69, 79, 89, 99, 109, 119, 129, or 139; The antibody or its conjugated fragment, including the antibody.

2. Antibodies, a. VH containing any one amino acid sequence from sequence numbers 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 156, 158, 160, or 162; and b. A VL containing any one of the amino acid sequences of sequence numbers 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 164, 166, or 168; The antibody or conjugated fragment according to claim 1, comprising:

3. a. VH encoded by a nucleic acid sequence containing any one of sequence numbers 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, 115, 125, 135, 157, 159, 161, or 163, and b. VL encoded by a nucleic acid sequence containing any one of sequence numbers 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 165, 167, or 169 The antibody according to claim 1, comprising:

4. The antibody or conjugated fragment according to any one of claims 1 to 3, wherein the antibody is a monoclonal antibody, a bispecific antibody, a polyvalent antibody, a multiple specific antibody, a diabody antibody, a chimeric antibody, an scFv antibody, or a fragment thereof.

5. The antibody or conjugated fragment according to any one of claims 1 to 4, wherein the antibody is a defucosylated full-length antibody.

6. The antibody or conjugated fragment according to any one of claims 1 to 5, wherein the antibody comprises one Fc domain from among human IgG1, human IgG2, human IgG3, and human IgG4.

7. The antibody or conjugated fragment according to claim 6, wherein the Fc domain is wild-type, variant, or cleaved Fc domain.

8. The antibody or binding fragment according to claim 1, further comprising a second antigen-binding domain that binds to a target other than TROP-2.

9. The antibody or conjugated fragment according to claim 8, wherein the target is a CD3 antibody having a heavy chain selected from SEQ ID NO: 174 or 178; and the light chain is selected from SEQ ID NO: 176 or 180, or from a bispecific antibody comprising SEQ ID NO: 174, SEQ ID NO: 126, SEQ ID NO: 190, or SEQ ID NO:

166.

10. The antibody or conjugated fragment according to claim 8, wherein the antibody is a bispecific antibody comprising a heavy chain selected from SEQ ID NOs: 190 and 192, or 194 and 196.

11. A method for treating a disease in a subject requiring such treatment, comprising the step of administering to the subject a therapeutically effective amount of an antibody according to any one of claims 1 to 10.

12. The method according to claim 11, wherein the disease is a proliferative disorder.

13. The method according to claim 12, wherein the proliferative disorder is a cancer selected from cancers expressing Trop2 or a variant thereof.

14. The method according to any one of claims 11 to 13, wherein the subject is a human.

15. A bivalent or polyvalent antibody, a. Heavy chain variable domain (VH) complementarity-determining region (CDR) 1 containing any one amino acid sequence from sequence numbers 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, or 132; and b. VH CDR2 containing any one amino acid sequence from sequence numbers 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123, or 133; and c. VH CDR3 containing any one amino acid sequence from sequence numbers 4, 14, 24, 34, 44, 54, 64, 74, 84, 94, 104, 114, 124, and 134; and d. Light chain variable domain (VL) CDR1 containing any one amino acid sequence from sequence numbers 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, or 137; and e. VL CDR2 containing any one amino acid sequence from sequence numbers 8, 18, 28, 38, 48, 58, 68, 78, 88, 98, 108, 118, 128, or 138; and f. VL CDR3 containing any one of the amino acid sequences among sequence numbers 9, 19, 29, 39, 49, 59, 69, 79, 89, 99, 109, 119, 129, or 139; Anti-Trop2 antibodies or their conjugated fragments containing; and A second antibody, antigen binding of the second antibody or a fragment thereof; target-binding proteins, cytokines; lectins; or toxins; The bivalent or polyvalent antibody, including the aforementioned bivalent or polyvalent antibody.

16. The bivalent or polyvalent antibody according to claim 15, wherein the second antibody targets an immunoeffector cell surface receptor selected from at least one of CTLA-4, PD-1, Lag3, S15, B7H3, B7H4, TCR-alpha, TCR-beta, TIM-3, CD3, 41BB, and OX40.

17. The bivalent or polyvalent antibody according to claim 16, wherein the antibody is a polyvalent antibody that targets two or three or more antigens other than Trop2.

18. The bivalent or polyvalent antibody according to claim 15, wherein the antibody is a bispecific antibody comprising a heavy chain selected from SEQ ID NOs: 190 and 192, 194 and 196, or a bispecific antibody comprising SEQ ID NOs: 174, SEQ ID NOs: 126, SEQ ID NOs: 190 and SEQ ID NOs:

166.

19. Anti-Trop2 antibody, a. VH containing any one amino acid sequence from sequence numbers 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 156, 158, 160, or 162; and b. A VL containing any one of the amino acid sequences of sequence numbers 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 164, 166, or 168; A bivalent or polyvalent antibody according to claim 15, comprising:

20. Anti-Trop2 antibody, a. VH encoded by a nucleic acid sequence containing any one of sequence numbers 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, 115, 125, 135, 157, 159, 161, or 163, and b. VL encoded by a nucleic acid sequence containing any one of sequence numbers 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 165, 167, or 169 A bivalent or polyvalent antibody according to claim 15, comprising:

21. The bivalent or polyvalent antibody according to any one of claims 15 to 19, wherein the antibody is a monoclonal antibody, a full-length antibody, or an antibody fragment.

22. The bivalent or polyvalent antibody according to any one of claims 15 to 12, wherein the antibody is fused to one of the Fc domains of human IgG1, human IgG2, human IgG3, and human IgG4.

23. The bivalent or polyvalent antibody according to any one of claims 15 to 21, wherein the target is CD3, and the antibody comprises a heavy chain selected from SEQ ID NO: 174 or 178; and a light chain selected from SEQ ID NO: 176 or 180.

24. The divalent or polyvalent antibody according to any one of claims 15 to 21, wherein the target is CD3, the antibody is encoded by a nucleic acid selected from SEQ ID NO: 175 or 179, and the light chain is encoded by a nucleic acid selected from SEQ ID NO: 177 or 181.

25. The divalent or polyvalent according to claim 23, wherein the Fc domain is a wild-type Fc domain, a variant Fc domain, or a cleaved Fc domain.

26. The divalent or polyvalent antibody according to any one of claims 15 to 24, wherein the antibody is a defucosylated full-length antibody.

27. A method for treating a disease in a subject requiring such treatment, comprising the step of administering to the subject a therapeutically effective amount of the antibody according to any one of claims 1 to 10 or the bivalent antibody according to any one of claims 15 to 26.

28. The method according to claim 27, wherein the disease is a proliferative disorder.

29. The method according to claim 27, wherein the disease is a proliferative disorder selected from cancer cells expressing Top2 or a variant thereof, selected from breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, certain lung cancers, squamous cell carcinoma of the oral cavity, squamous cell carcinoma of the ovaries, squamous cell carcinoma of the pancreas, squamous cell carcinoma of the prostate, squamous cell carcinoma of the stomach, squamous cell carcinoma of the thyroid, squamous cell carcinoma of the bladder, and squamous cell carcinoma of the uterus.

30. The method according to any one of claims 27 to 29, wherein the subject is a human.

31. a. VH encoded by a nucleic acid sequence containing any one of sequence numbers 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, 115, 125, 135, 157, 159, 161, or 163; and b. A VL encoded by a nucleic acid sequence containing any one of sequence numbers 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 165, 167, or 169; or c. Antibodies encoded by the nucleic acid sequences of SEQ ID NOs: 187 and 189, or 191 and 193; or d. Bispecific antibodies encoded by the nucleic acid sequences of SEQ ID NOs: 175 and 177, 179 and 181, 183 and 185, 187 and 189, or 191 and 193, or combinations thereof; Antibodies containing antibodies, their conjugated fragments, and nucleic acids encoding bivalent or polyvalent nucleic acid sequences.

32. A vector comprising the nucleic acid described in claim 31.

33. A host cell comprising the vector according to claim 32.

Images