Gpa33 antibody compositions and uses thereof

Abstract

The present disclosure relates generally to immunoglobulin-related compositions (e.g., antibodies or antigen-binding fragments thereof) that can bind to and neutralize the activity of GPA33 protein. Antibodies of the present technology are useful in methods for detecting and treating GPA33-positive cancers in a subject in need thereof.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit and priority of U.S. Provisional Application No. 63 / 583,662, filed September 19, 2023, which is incorporated herein by reference in its entirety for any and all purposes. Technical Field

[0003] This invention generally relates to the preparation of immunoglobulin-associated compositions (e.g., antibodies or antigen-binding fragments thereof) that specifically bind to the GPA33 protein, and the use of such immunoglobulin-associated compositions. Specifically, this invention relates to the preparation of antibodies that neutralize GPA33 and their use in the detection and treatment of GPA33-related cancers. Background Technology

[0004] The following description of the technical background of the present invention is provided only to help understand the technology of the present invention, and does not acknowledge any prior art that describes or constitutes the technology of the present invention.

[0005] T-cell acute lymphoblastic leukemia (T-ALL) accounts for 12-15% of childhood T-ALL and 20-25% of adult T-ALL. The 5-year overall survival rate for relapsed patients is less than 25%. CAR T-cell therapy has been tested against T-ALL targets. For example, CD1a CAR T and CD5 CAR T have been shown to preserve normal T progenitor cells and at least some mature T cells. However, CAR T-cell therapy is associated with adverse side effects such as cytokine storms.

[0006] Arming T cells with bispecific antibodies (T-BsAbs) in vitro significantly reduced cytokine release. However, no T-BsAbs have yet been clinically approved for the treatment of T-ALL. Therefore, there is an urgent need for T-BsAbs that effectively target T-ALL cells and induce cannibalism. Summary of the Invention

[0007] On one hand, this disclosure provides an antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising a heavy chain immunoglobulin variable domain (V). H ) and light chain immunoglobulin variable domain (V L ), wherein (a) V H V containing SEQ ID NO: 2 or SEQ ID NO: 7 H -CDR1 sequence, V of SEQ ID NO: 3 or SEQ ID NO: 8 H -CDR2 sequence and V of SEQ ID NO: 4 or SEQ ID NO: 9 H-CDR3 sequence; and (b) the V L V contains groups consisting of the following items L -CDR1 sequence, V L -CDR2 sequence and V L -CDR3 sequences: SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14; and SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19. Alternatively or additionally, in some embodiments, (a) the V H Contains an amino acid sequence comprising either SEQ ID NO: 1 or SEQ ID NO: 6; and / or (b) the V L Contains the amino acid sequence of either SEQ ID NO: 11 or SEQ ID NO: 16. In some embodiments, (a) the V H Contains the amino acid sequence of SEQ ID NO: 1, and the V L The amino acid sequence comprising SEQ ID NO:16; or (b) the V H Contains the amino acid sequence of SEQ ID NO: 6, and the V L It contains the amino acid sequence of SEQ ID NO: 11.

[0008] The antibody may further include an isotype Fc domain selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, and IgE. In some embodiments, the antibody comprises an IgG1 constant region containing one or more amino acid substitutions selected from the group consisting of N297A, K322A, L234A, and L235A. Additionally or alternatively, in some embodiments, the antibody comprises an IgG4 constant region including an S228P mutation. In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab, F(ab')2, Fab', and scF. v or F v In some embodiments, the antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, a multispecific antibody, or a bispecific antibody.

[0009] On the other hand, this disclosure provides an antibody comprising a heavy chain (HC) amino acid sequence and a light chain (LC) amino acid sequence selected from the group consisting of: SEQ ID NO: 35 and SEQ ID NO: 36; SEQ ID NO: 35 and SEQ ID NO: 37; SEQ ID NO: 35 and SEQ ID NO: 38; SEQ ID NO: 39 and SEQ ID NO: 40; SEQ ID NO: 39 and SEQ ID NO: 41; SEQ ID NO: 39 and SEQ ID NO: 42; SEQ ID NO: 47 and SEQ ID NO: 48; SEQ ID NO: 47 and SEQ ID NO: 49; SEQ ID NO: 50 and SEQ ID NO: 51; and SEQ ID NO: 50 and SEQ ID NO: 52.

[0010] In any of the foregoing embodiments, the antibody or antigen-binding fragment binds to an epitope of the GPA33 protein comprising at least five to eight consecutive amino acid residues of SEQ ID NO: 53. In some embodiments, the epitope is a conformational epitope.

[0011] On one hand, this disclosure provides a multispecific antigen-binding fragment comprising a first polypeptide chain, wherein: the first polypeptide chain comprises, in the direction from the N-terminus to the C-terminus: a heavy chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope; a flexible peptide linker comprising the amino acid sequence (GGGGS)6; a light chain variable domain of the first immunoglobulin; a flexible peptide linker comprising the amino acid sequence (GGGGS)4; a heavy chain variable domain of a second immunoglobulin capable of specifically binding to a second epitope; a flexible peptide linker comprising the amino acid sequence (GGGGS)6; a light chain variable domain of the second immunoglobulin; a flexible peptide linker sequence comprising the amino acid sequence TPLDTTHT; and a self-assembly and disassembly (SADA) polypeptide; wherein the heavy chain variable domain of the first immunoglobulin or the heavy chain variable domain of the second immunoglobulin is selected from any one of the following: SEQ ID NO: 1 and SEQ ID NO: 6, and / or the light chain variable domain of the first immunoglobulin or the light chain variable domain of the second immunoglobulin is selected from any one of the following: SEQ ID NO: 11 and SEQ ID NO: 6. ID NO: 16. On the other hand, this disclosure provides a multispecific antigen-binding fragment comprising a first polypeptide chain, wherein: the first polypeptide chain comprises, in the direction from the N-terminus to the C-terminus: a light chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope; a flexible peptide linker comprising the amino acid sequence (GGGGS)6; a heavy chain variable domain of the first immunoglobulin; a flexible peptide linker comprising the amino acid sequence (GGGGS)4; a heavy chain variable domain of a second immunoglobulin capable of specifically binding to a second epitope; a flexible peptide linker comprising the amino acid sequence (GGGGS)6; a light chain variable domain of the second immunoglobulin; a flexible peptide linker sequence comprising the amino acid sequence TPLDTTHT; and a self-assembly and disassembly (SADA) polypeptide; wherein the heavy chain variable domain of the first immunoglobulin or the heavy chain variable domain of the second immunoglobulin is selected from any one of the following: SEQ ID NO: 1 and SEQ ID NO: 6, and / or the light chain variable domain of the first immunoglobulin or the light chain variable domain of the second immunoglobulin is selected from any one of the following: SEQ ID NO: 11 and SEQ ID NO: 6. NO: 16. The SADA polypeptide may comprise a tetramerizing, pentamerizing, or hexamerizing domain. In some embodiments, the SADA polypeptide comprises a tetramerizing domain of any of the following: p53, p63, p73, hnRNPC, SNA-23, StefinB, KCNQ4, or CBFA2T1. Additionally or alternatively, in some embodiments, the antigen-binding fragment comprises an amino acid sequence selected from the following: SEQ ID NO: 43 to 46.

[0012] On one hand, this disclosure provides a multispecific antibody comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain are covalently bonded to each other, the second polypeptide chain and the third polypeptide chain are covalently bonded to each other, and the third polypeptide chain and the fourth polypeptide chain are covalently bonded to each other, and wherein: each of the first polypeptide chain and the fourth polypeptide chain comprises, in the direction from the N-terminus to the C-terminus,: a light chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope; a light chain constant domain of the first immunoglobulin; a flexible peptide linker comprising the amino acid sequence (GGGGS)3; and a second immunoglobulin linked to a complementary heavy chain variable domain of the second immunoglobulin. A light chain variable domain, or a heavy chain variable domain of the second immunoglobulin linked to a complementary light chain variable domain of the second immunoglobulin, wherein the light chain variable domain and the heavy chain variable domain of the second immunoglobulin are specifically capable of binding to a second epitope and are linked together via a flexible peptide linker comprising the amino acid sequence (GGGGS)6 to form a single-chain variable fragment; and each of the second polypeptide chain and the third polypeptide chain comprises, in the direction from the N-terminus to the C-terminus: a heavy chain variable domain of the first immunoglobulin capable of specifically binding to the first epitope; and a heavy chain constant domain of the first immunoglobulin; and wherein the heavy chain variable domain of the first immunoglobulin is selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 6, and / or the light chain variable domain of the first immunoglobulin is selected from the group consisting of SEQ ID NO: 11 and SEQ ID NO: 16.

[0013] In any and all embodiments of the multispecific antibody or multispecific antigen-binding fragment disclosed herein, the antibody or antigen-binding fragment binds to the following: CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, CD32, CD64, TCR γ / δ, NKp46, KIR, PD-1, PD-L1, LAG3, CD28, B7H3, STEAP1, HER2, EGFR, CEA, CECAM5, transferrin receptor, FAP, NKG2D-ligand, TRAIL, FasL, cathepsin G, granzyme, carboxypeptidase, β-lactamase, DOTA (metal) complex, benzyl-DOTA (metal) complex, Proteus spp.-DOTA (metal) complex, NOGADA-Proteus spp.-DOTA (metal) complex, astr-DFO (metal) complex, DFO (metal) complex, or small molecule DOTA hapten. Additionally or alternatively, in some embodiments, the multispecific antibody or multispecific antigen-binding fragment binds to T cells, B cells, myeloid cells, plasma cells, or mast cells.

[0014] On the other hand, this disclosure provides a heterodimeric multispecific antibody comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain are covalently bonded to each other, the second polypeptide chain and the third polypeptide chain are covalently bonded to each other, and the third polypeptide chain and the fourth polypeptide chain, wherein: (a) the first polypeptide chain comprises, in the direction from the N-terminus to the C-terminus,: (i) a light chain variable domain (VL-1) of a first immunoglobulin capable of specifically binding to a first epitope; (ii) a light chain constant domain (CL-1) of the first immunoglobulin; (iii) a flexible peptide linker comprising the amino acid sequence (GGGGS)3; and ( iv) a light chain variable domain (VL-2) of the second immunoglobulin linked to a complementary heavy chain variable domain (VH-2) of the second immunoglobulin, or a heavy chain variable domain (VH-2) of the second immunoglobulin linked to a complementary light chain variable domain (VL-2) of the second immunoglobulin, wherein VL-2 and VH-2 are capable of specifically binding to the second epitope and are linked together via a flexible peptide linker comprising an amino acid sequence (GGGGS)6 to form a single-chain variable fragment; (b) the second polypeptide comprises, in the direction from the N-terminus to the C-terminus: (i) a heavy chain variable domain (VH-1) of the first immunoglobulin capable of specifically binding to the first epitope; (ii) the... The first immunoglobulin comprises: (i) a first CH1 domain (CH1-1); and (iii) a first heterodimerizing domain of the first immunoglobulin, wherein the first heterodimerizing domain cannot form a stable homodimer with another first heterodimerizing domain; (c) the third polypeptide comprises, in the N-terminal to C-terminal direction: (i) a heavy chain variable domain (VH-3) of the third immunoglobulin capable of specifically binding to a third epitope; (ii) a second CH1 domain (CH1-3) of the third immunoglobulin; and (iii) a second heterodimerizing domain of the third immunoglobulin, wherein the second heterodimerizing domain comprises a domain different from that of the first immunoglobulin. The amino acid sequence or nucleic acid sequence of the first heterodimerizing domain, wherein the second heterodimerizing domain cannot form a stable homodimer with another second heterodimerizing domain, and wherein the second heterodimerizing domain of the third immunoglobulin is configured to form a heterodimer with the first heterodimerizing domain of the first immunoglobulin; (d) the fourth polypeptide comprises, in the direction from the N-terminus to the C-terminus: (i) a light chain variable domain (VL-3) of the third immunoglobulin capable of specifically binding to the third epitope; (ii) a light chain constant domain (CL-3) of the third immunoglobulin; and (iii) a flexible peptide linker comprising the amino acid sequence (GGGGS)3.And (iv) the light chain variable domain (VL-4) of the fourth immunoglobulin linked to the complementary heavy chain variable domain (VH-4) of the fourth immunoglobulin, or the heavy chain variable domain (VH-4) of the fourth immunoglobulin linked to the complementary light chain variable domain (VL-4) of the fourth immunoglobulin, wherein VL-4 and VH-4 are capable of specifically binding to the fourth epitope and are linked together via a flexible peptide linker comprising an amino acid sequence (GGGGS)6 to form a single-chain variable fragment; wherein VL-1 and / or VL-3 contain V selected from any of the following: L Amino acid sequences: SEQ ID NO: 11 and SEQ ID NO: 16, wherein VH-1 and / or VH-3 contain V selected from any of the following. H Amino acid sequences: SEQ ID NO: 1 and SEQ ID NO: 6. In some embodiments, the multispecific antibody binds to GPA33 and at least one of the following: CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, CD32, CD64, TCR γ / δ, NKp46, KIR, PD-1, PD-L1, LAG3, CD28, B7H3, STEAP1, HER2, EGFR, CEA, CECAM5, transferrin receptor, FAP, NKG2D-ligand, TRAIL, FasL, cathepsin G, granzyme, carboxypeptidase, β-lactamase, DOTA (metal) complex, benzyl-DOTA (metal) complex, Proteus spp.-DOTA (metal) complex, NOGADA-Proteus spp.-DOTA (metal) complex, astr-DFO (metal) complex, DFO (metal) complex, or small molecule DOTA hapten. Additionally or alternatively, in some embodiments, the multispecific antibody binds to T cells, B cells, myeloid cells, plasma cells, or mast cells.

[0015] In any and all embodiments of the antibody described herein, the antibody lacks α-1,6-fucose modification.

[0016] On one hand, this disclosure provides a recombinant nucleic acid sequence that encodes any antibody of the antibodies described herein. In some embodiments, the recombinant nucleic acid sequence is selected from the group consisting of SEQ ID NO: 5, 10, 15, and 20.

[0017] On the other hand, this disclosure provides a host cell or vector containing any recombinant nucleic acid sequence disclosed herein.

[0018] On one hand, this disclosure provides a composition comprising any and all embodiments of an antibody or antigen-binding fragment of the present invention and a pharmaceutically acceptable carrier, wherein the antibody or antigen-binding fragment is optionally conjugated with a pharmaceutical agent selected from the group consisting of: isotopes, dyes, chromophores, contrast agents, drugs, toxins, cytokines, enzymes, enzyme inhibitors, hormones, hormone antagonists, growth factors, radionuclides, metals, liposomes, nanoparticles, RNA, DNA, or any combination thereof.

[0019] On the other hand, this disclosure provides a method for treating GPA33-related cancer in a subject of need, the method comprising administering to the subject an effective amount of either an antibody or an antigen-binding fragment disclosed herein, wherein the antibody or antigen-binding fragment specifically binds to GPA33 and neutralizes its activity. In some embodiments, the GPA33-related cancer is T-cell acute lymphoblastic leukemia (T-ALL), colorectal cancer, pseudomyxoma peritonei, appendiceal cancer, pancreatic cancer, or gastric cancer. The GPA33-related cancer may be colorectal cancer with an MSI genotype or an MSS genotype. Additionally or alternatively, in some embodiments, the colorectal cancer is associated with a KRAS G12D mutation or a p53 mutation.

[0020] Alternatively or concurrently, in some embodiments of the method, the antibody or antigen-binding fragment is administered to the subject alone, sequentially, or simultaneously with another therapeutic agent. Examples of other therapeutic agents include one or more of the following: alkylating agents, platinum-based agents, taxanes, vinca extract, anti-estrogens, aromatase inhibitors, ovarian inhibitors, VEGF / VEGFR inhibitors, EGF / EGFR inhibitors, PARP inhibitors, cell-inhibiting alkaloids, cytotoxic antibiotics, antimetabolites, endocrine / hormone agents, and bisphosphonate therapeutic agents.

[0021] On the other hand, this disclosure provides a method for detecting tumors in a subject in vivo, the method comprising: (a) administering an effective amount of an antibody of the present invention to the subject, wherein the antibody is configured to target a tumor expressing GPA33 and is labeled with a radioactive isotope; and (b) detecting the presence of a tumor in the subject by detecting a level of radioactivity emitted by the antibody that is above a reference value. In some embodiments, the subject is diagnosed with or suspected of having cancer. The level of radioactivity emitted by the antibody can be detected using positron emission tomography (PET) or single-photon emission computed tomography (SPECT).

[0022] Alternatively or additionally, in some embodiments, the method further includes administering an effective amount of an immunoconjugate to the subject, the immunoconjugate comprising an antibody of the present invention conjugated to a radionuclide. In some embodiments, the radionuclide is an alpha particle emission isotope, a beta particle emission isotope, an Auger emitter, or any combination thereof. Examples of beta particle emission isotopes include... 86 Y、 90 Y、 89 Sr、 165 Dy、 186 Re、 188 Re、 177 Lu and 67 Cu. In some embodiments of the method, nonspecific FcR-dependent binding in normal tissue is eliminated or reduced (e.g., via an N297A mutation in the Fc region, which leads to glycosylation).

[0023] This document also discloses kits for detecting and / or treating GPA33-related cancers, the kits comprising at least one immunoglobulin-associated composition of the present invention (e.g., any antibody or antigen-binding fragment described herein) or a functional variant thereof (e.g., a substituted variant) and instructions for use. In some embodiments, the immunoglobulin-associated composition is conjugated to one or more detectable markers. In one embodiment, the one or more detectable markers include radiolabels, fluorescent markers, or chromogenic markers.

[0024] Alternatively or additionally, in some embodiments, the kit further includes a secondary antibody that specifically binds to the anti-GPA33 immunoglobulin-associated composition described herein. In some embodiments, the secondary antibody is conjugated to at least one detectable label selected from the group consisting of: a radiolabel, a fluorescent label, or a chromogenic label.

[0025] On one hand, this disclosure provides a method for detecting solid tumors in a subject of need, the method comprising: (a) administering to the subject an effective amount of a complex comprising a radiolabeled DOTA hapten and a multispecific antibody of the present invention that binds to the radiolabeled DOTA hapten and a GPA33 antigen, wherein the complex is configured to target a solid tumor expressing the GPA33 antigen recognized by the multispecific antibody of the complex; and (b) detecting the presence of a solid tumor in the subject by detecting a radioactivity level above a reference value emitted by the complex.

[0026] On the other hand, this disclosure provides a method for selecting a subject for pre-targeted radioimmunotherapy, the method comprising: (a) administering an effective amount of a complex to the subject, the complex comprising a radiolabeled DOTA hapten and a multispecific antibody of the present invention binding to the radiolabeled DOTA hapten and GPA33 antigen, wherein the complex is configured to target a solid tumor expressing the GPA33 antigen recognized by the multispecific antibody of the complex; (b) detecting the level of radioactivity emitted by the complex; and (c) selecting the subject for pre-targeted radioimmunotherapy when the level of radioactivity emitted by the complex is higher than a reference value.

[0027] On one hand, this disclosure provides a method for increasing the tumor sensitivity to radiotherapy in a subject diagnosed with GPA33-positive cancer, the method comprising administering to the subject an effective amount of a complex comprising a radiolabeled DOTA hapten and a multispecific antibody of the present invention that recognizes and binds to the radiolabeled DOTA hapten and a GPA33 antigen target, wherein the complex is configured to target a tumor expressing the GPA33 antigen target recognized by the multispecific antibody of the complex.

[0028] On the other hand, this disclosure provides a method for treating cancer in a subject in need, the method comprising administering to the subject an effective amount of a complex comprising a radiolabeled DOTA hapten and a multispecific antibody of the present invention that recognizes and binds to the radiolabeled DOTA hapten and a GPA33 antigen target, wherein the complex is configured to target a tumor expressing the GPA33 antigen target recognized by the multispecific antibody of the complex.

[0029] In any of the above embodiments of the methods disclosed herein, the complex is administered intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intra-sacrally, intraorally, intraperitoneally, via the trachea, subcutaneously, intravenously, orally, or intranasally. In some embodiments of the methods disclosed herein, the subject is a human. Additionally or alternatively, in any of the above embodiments of the methods disclosed herein, the radiolabeled DOTA hapten comprises 213 Bi、 211 At、 225 Ac、 152 Dy、 212 Bi、 223 Ra、 219 Rn、 215 Po、 211 Bi、 221 Fr、 217 At、255 Fm、 86 Y、 90 Y、 89 Sr、 165 Dy、 186 Re、 188 Re、 177 Lu、 67 Cu、 111 In、 67 Ga、 51 Cr 58 Co、 99m Tc, 103m Rh、 195m Pt, 119 Sb, 161 Ho、 189m Os、 192 Ir、 201 Tl、 203 Pb, 68 Ga、 227 Th or 64 Cu, and optionally includes alpha particle emission isotopes, beta particle emission isotopes or Auger emitters.

[0030] On one hand, this disclosure provides a method for increasing the tumor sensitivity to radiotherapy in a subject diagnosed with GPA33-positive cancer, the method comprising: (a) administering to the subject an effective amount of an anti-DOTA multispecific antibody of the present invention, wherein the anti-DOTA multispecific antibody is configured to target a tumor expressing a GPA33 antigen; and (b) administering to the subject an effective amount of a radiolabeled DOTA hapten, wherein the radiolabeled DOTA hapten is configured to bind to the anti-DOTA multispecific antibody. On the other hand, this disclosure provides a method for treating cancer in a subject of need, the method comprising: (a) administering to the subject an effective amount of an anti-DOTA multispecific antibody of the present invention, wherein the anti-DOTA multispecific antibody is configured to target a tumor expressing a GPA33 antigen; and (b) administering to the subject an effective amount of a radiolabeled DOTA hapten, wherein the radiolabeled DOTA hapten is configured to bind to the anti-DOTA multispecific antibody. In some embodiments, the method of the present invention further comprises administering to the subject an effective amount of a scavenger prior to administering the radiolabeled DOTA hapten.

[0031] Alternatively or alternatively, in any of the above embodiments of the methods disclosed herein, the radiolabeled DOTA hapten comprises 213 Bi、 211 At、 225Ac、 152 Dy、 212 Bi、 223 Ra、 219 Rn、 215 Po、 211 Bi、 221 Fr、 217 At、 255 Fm、 86 Y、 90 Y、 89 Sr、 165 Dy、 186 Re、 188 Re、 177 Lu、 67 Cu、 111 In、 67 Ga、 51 Cr 58 Co、 99m Tc, 103m Rh、 195m Pt, 119 Sb, 161 Ho、 189m Os、 192 Ir、 201 Tl、 203 Pb, 68 Ga、 227 Th or 64 Cu, and optionally includes an alpha particle emission isotope, a beta particle emission isotope, or an Auger emitter. In any of the above embodiments of the methods disclosed herein, the subject is a human.

[0032] This document also discloses ex vivo armed T cells coated with or compounded with an effective amount of any and all embodiments of the multispecific antibody disclosed herein, wherein the multispecific antibody binds to at least CD3 and GPA33 antigens. In some embodiments, the multispecific antibody comprises a CD3-binding domain. Alternatively or additionally, in some embodiments, the multispecific antibody is an immunoglobulin comprising two heavy chains and two light chains, wherein each of the light chains is fused to a single-chain variable fragment (scFv), and wherein at least one scFv of the multispecific antibody comprises the CD3-binding domain. On the other hand, this disclosure provides a method for treating a subject with GPA33-related cancer, the method comprising administering an effective amount of the ex vivo armed T cells described herein to the subject. Attached Figure Description

[0033] Figure 1A summary of the properties of the different antibody clones described herein is shown. The humanized GPA33 (BC123) obtained via Eureka is referred to as GPA33 (v1), the parent GPA33 H1L4 is referred to as GPA33 (v2), the affinity-matured GPA33 H1L4 of this invention is referred to as GPA33 (v3), the parent GPA33 H3L3 is referred to as GPA33 (v4), and the affinity-matured GPA33 H3L3 of this invention is referred to as GPA33 (v5).

[0034] Figure 2 The density / number of GPA33 molecules expressed on the surface of each cell in T-cell acute lymphoblastic leukemia (T-ALL) (MOLT16, PF-382, CEM.NKR and 8402) (highlighted in gray) and colorectal cancer (CRC) (LS174T and SW1222) cell lines is shown.

[0035] Figure 3A The KD values ​​of the affinity-mature H3L3 and H1L4 anti-GPA33 antibody sequences of the present invention are shown compared with the parental H3L3 and H1L4 clones and the alternative anti-A33 BC123 antibody clone.

[0036] Figure 3B This demonstrates the beneficial effects of the affinity-matured H3L3 anti-GPA33 antibody sequence of the present invention on antitumor activity and survival relative to the H3L3 parent clone in the PF-382 leukemia xenograft model.

[0037] Figure 3C This study demonstrates the beneficial effects of the affinity-matured H3L3 anti-GPA33 antibody sequence of the present invention on antitumor activity and survival relative to the H3L3 parent clone in a CEM.NKR leukemia xenograft model.

[0038] Figure 3D This study demonstrates the beneficial effects of the affinity-matured H1L4 anti-GPA33 antibody sequence of the present invention on antitumor activity and survival relative to the H1L4 parent clone in the PF-382 leukemia xenograft model.

[0039] Figure 3E The invention demonstrates T-cell-dependent cell activity of mature H3L3 and H1L4 anti-GPA33 antibody sequences with higher affinity compared to other T-ALL targets in CEM.NKR leukemia cells.

[0040] Figure 3FThe invention demonstrates T-cell-dependent cell activity of mature H3L3 and H1L4 anti-GPA33 antibody sequences with higher affinity compared to other T-ALL targets in PF-382 leukemia cells.

[0041] Figure 4A The design of a self-assembled and self-disassembled bispecific antibody (GPA33 × DOTA SADA) is shown. Figure 4B The design of the IgG-[L]-scFv bispecific antibody (GPA33 × DOTA IgG-[L]-scFv) is shown. Figure 4C The design of a GPA33 × CD3 SADA bispecific antibody targeting tumor antigens GPA33 and CD3 on T cells is shown. Figure 4D The design of a GPA33 × CD3 IgG-[L]-scFv bispecific antibody targeting tumor antigens GPA33 and CD3 on T cells is shown.

[0042] Figure 5 The amino acid and nucleic acid sequences of the humanized heavy chains of the anti-GPA33 antibodies of the present invention, designated G3A-H1 and G3A-H3, are shown. The amino acid and nucleic acid sequences of G3A-H1 are represented by SEQ ID NO: 1 and SEQ ID NO: 5, respectively. The amino acid and nucleic acid sequences of G3A-H3 are represented by SEQ ID NO: 6 and SEQ ID NO: 10, respectively. The CDR1, CDR2, and CDR3 regions of G3A-H1 (SEQ ID NO: 2 to 4) and G3A-H3 (SEQ ID NO: 7 to 9) are indicated by underlined text.

[0043] Figure 6A The amino acid and nucleic acid sequences of the humanized light chains of the anti-GPA33 antibodies of the present invention, designated G3A-L3 (AM2 LC N53D H91Y) and G3A-L4 (AM2 LC N53D H91Y), are shown. The amino acid and nucleic acid sequences of G3A-L3 (AM2 LCN53D H91Y) are represented by SEQ ID NO: 11 and SEQ ID NO: 15, respectively. The amino acid and nucleic acid sequences of G3A-L4 (AM2 LC N53D H91Y) are represented by SEQ ID NO: 16 and SEQ ID NO: 20, respectively. The CDR1, CDR2, and CDR3 regions of G3A-L3 (AM2 LC N53D H91Y) (SEQ ID NO: 12 to 14) and G3A-L4 (AM2 LC N53D H91Y) (SEQ ID NO: 17 to 19) are indicated by underlined text.

[0044] Figure 6B The amino acid and nucleic acid sequences of the humanized light chains of the parental clones resistant to GPA33 H3L3 and H1L4 are shown. The amino acid and nucleic acid sequences of G3A-L4 are represented by SEQ ID NO: 21 and SEQ ID NO: 25, respectively. The amino acid and nucleic acid sequences of G3A-L3 are represented by SEQ ID NO: 26 and SEQ ID NO: 30, respectively. The CDR1, CDR2, and CDR3 regions of G3A-L4 (SEQ ID NO: 22 to 24) and G3A-L3 (SEQ ID NO: 27 to 29) are indicated by underlined text.

[0045] Figure 7 The heavy and light chain amino acid sequences of the GPA33 × CD3 IgG-[L]-scFv bispecific antibody containing the H1L4 parental sequence are shown, denoted as SEQ ID NO: 31 and SEQ ID NO: 32, respectively. V H and V L The amino acid sequences are shown in italics, and the G4S peptide linker sequence is shown in bold.

[0046] Figure 8 The heavy and light chain amino acid sequences of the GPA33 × CD3 IgG-[L]-scFv bispecific antibody containing the H3L3 parental sequence are shown, denoted as SEQ ID NO: 33 and SEQ ID NO: 34, respectively. V H and V L The amino acid sequences are shown in italics, and the G4S peptide linker sequence is shown in bold.

[0047] Figure 9 Exemplary heavy and light chain amino acid sequences, represented as SEQ ID NO: 35 and SEQ ID NO: 36 to 38, respectively, of the GPA33 × CD3 IgG-[L]-scFv bispecific antibody comprising the affinity-mature H3L3 sequence of the present invention. H and V L The amino acid sequences are shown in italics, and the G4S peptide linker sequence is shown in bold.

[0048] Figure 10 Exemplary heavy and light chain amino acid sequences, represented as SEQ ID NO: 39 and SEQ ID NO: 40 to 42, respectively, of the GPA33 × CD3 IgG-[L]-scFv bispecific antibody comprising the affinity-mature H1L4 sequence of the present invention. H and V LThe amino acid sequences are shown in italics, and the G4S peptide linker sequence is shown in bold.

[0049] Figure 11 Exemplary amino acid sequences of GPA33 × DOTA SADA bispecific antibodies are shown, comprising the humanized C825 (huC825) antigen-binding domain of the present invention and an affinity-matured H1L4 sequence (denoted as SEQ ID NO: 43) or an affinity-matured H3L3 sequence (denoted as SEQ ID NO: 44). H and V L Amino acid sequences are indicated in italics. Linker and spacer amino acid sequences are indicated in bold. P53 SADA peptide and His-tagged amino acid sequences are indicated with dashed underlines.

[0050] Figure 12 An exemplary amino acid sequence of a GPA33 × DOTA SADA bispecific antibody is shown, comprising the mouse C825 (C825) antigen-binding domain and an affinity-matured H1L4 sequence (denoted as SEQ ID NO: 45) or an affinity-matured H3L3 sequence (denoted as SEQ ID NO: 46) of the present invention. H and V L Amino acid sequences are indicated in italics. Linker and spacer amino acid sequences are indicated in bold. P53 SADA peptide and His-tagged amino acid sequences are indicated with dashed underlines.

[0051] Figure 13 Exemplary heavy and light chain amino acid sequences, represented as SEQ ID NO: 47 and SEQ ID NO: 48 to 49, respectively, of the GPA33 × DOTA IgG-[L]-scFv bispecific antibody comprising the affinity-mature H3L3 sequence of the present invention. H and V L The amino acid sequences are shown in italics, and the G4S peptide linker sequence is shown in bold.

[0052] Figure 14 Exemplary heavy and light chain amino acid sequences, represented as SEQ ID NO: 50 and SEQ ID NO: 51 to 52, respectively, of the GPA33 × DOTA IgG-[L]-scFv bispecific antibody comprising the affinity-mature H1L4 sequence of the present invention. H and V L The amino acid sequences are shown in italics, and the G4S peptide linker sequence is shown in bold.

[0053] Figure 15The biodistribution of GPA33 × DOTA SADA bispecific antibody (TC386), which contains the affinity-mature H1L4 sequence, is shown compared to that of GPA33 × DOTA SADA bispecific antibody (TC170), which contains the parental H1L4 clone sequence. Detailed Implementation

[0054] It should be understood that certain aspects, methods, embodiments, variations and features of the methods of the present invention are described below at different levels of detail in order to provide a substantial understanding of the technology of the present invention.

[0055] This disclosure generally provides immunoglobulin-associated compositions (e.g., antibodies or antigen-binding fragments thereof) that can specifically bind to and neutralize the biological activity of GPA33 peptides. The immunoglobulin-associated compositions of the present invention are useful in methods for detecting or treating GPA33-related cancers in subjects of need. Therefore, various aspects of the methods of the present invention relate to the preparation, characterization, and manipulation of anti-GPA33 antibodies. The immunoglobulin-associated compositions of the present invention can be used alone or in combination with other therapeutic agents for the treatment of cancer. In some embodiments, the immunoglobulin-associated compositions are humanized antibodies, chimeric antibodies, or bispecific antibodies.

[0056] In practicing the methods of this invention, many conventional techniques from molecular biology, protein biochemistry, cell biology, immunology, microbiology, and recombinant DNA were used. See, for example, Sambrook and Russell, eds. (2001) *Molecular Cloning: A Laboratory Manual*, 3rd edition; Ausubel et al., eds., series (2007) *Current Protocols in Molecular Biology*; *Methods in Enzymology* series (AcademicPress, Inc., NY).MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane (eds.) (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th Edition; Gait (ed.) (1984) Oligonucleotide Synthesis; US Patent No. 4,683,195; Hames and Higgins (eds.) (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins (eds.) (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos (eds., 1987) Gene Transfer Vectors for Mammalian Cells (ColdSpring Harbor Laboratory); Makrides (ed., 2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker (ed., 1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. (eds., 1996) Weir's Handbook of Experimental Immunology. Methods for detecting and measuring peptide gene expression product levels (i.e., gene translation levels) are well known in the art and include peptide detection methods such as antibody detection and quantitative techniques. (See also Strachan and Read, Human Molecular Genetics, 2nd Edition.)(John Wiley and Sons, Inc., NY, 1999)). .

[0057] CAR T-cell therapy has been tested against T-ALL targets. However, CAR T-cell therapy is accompanied by adverse side effects, such as cytokine storms. "Arming" T cells in vitro with bispecific antibodies (T-BsAbs) significantly reduces cytokine release. However, no T-BsAbs have yet been clinically approved for the treatment of T-ALL.

[0058] Therefore, we created T-BsAbs using proven T-ALL targets: GPA33, CD1a, CD5, CD7, CD99, and the T-cell receptor β chain (TRBC1) to compare their in vitro and in vivo antileukemic effects. See also Figure 1 .like Figure 2 As shown, the density of GPA33 expression on T-ALL cell lines was significantly lower (ranging from less than 1,000 to approximately 21,000 molecules per cell) compared to CRC cell lines. Surprisingly, the maximum killing effect achieved with GPA33 in T-ALL cell lines was high compared to other known T-ALL targets, despite its low density on T-ALL. Figures 3E to 3F Furthermore, the mature GPA33 T-BsAb of this invention exhibits superior antitumor activity in treated animals compared to its corresponding parental T-BsAb clone. Without being bound by theory, it is believed that the high affinity of the GPA33 T-BsAb of this invention contributes to its superior efficacy in eradicating T-ALL in xenograft models.

[0059] definition

[0060] Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this art pertains. As used in this specification and the appended claims, unless otherwise expressly indicated, the singular forms “a / an” and “the” include plural referents. For example, reference to “cell” includes a combination of two or more types of cells, etc. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry, and nucleic acid chemistry and hybridization described below are those well-known and commonly used in the art.

[0061] As used herein, the term “about” when referring to a number means a number that is generally included in the range of 1%, 5% or 10% in either direction of the number, unless otherwise stated or otherwise obvious from the context (unless such number is less than 0% or more than 100% of the possible value).

[0062] As used herein, “administering” a drug or medicine to a subject involves any route of introducing or delivering a compound to the subject to exert its intended function. Administration may be performed via any suitable route, including but not limited to oral, intranasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), rectal, intrathecal, intratumoral, or local administration. Administration includes self-administration and administration by another person.

[0063] "Adjuvant" refers to one or more substances that stimulate the immune system. In this context, adjuvants are used to enhance the immune response to one or more vaccine antigens or antibodies. Adjuvants may be administered to subjects before, in combination with, or after vaccination. Examples of chemical compounds used as adjuvants include aluminum compounds, oils, block polymers, immunostimulatory complexes, vitamins and minerals (e.g., vitamin E, vitamin A, selenium, and vitamin B12), Quil A (saponins), bacterial and fungal cell wall components (e.g., lipopolysaccharides, lipoproteins, and glycoproteins), hormones, cytokines, and co-stimulatory factors.

[0064] As used herein, the term "antibody" refers collectively to immunoglobulins or immunoglobulin-like molecules, including, but not limited to, IgA, IgD, IgE, IgG, and IgM, combinations thereof, and similar molecules produced in any vertebrate, such as mammals like humans, goats, rabbits, and mice, as well as non-mammalian species, such as shark immunoglobulins. As used herein, "antibody" (including complete immunoglobulins) and "antigen-binding fragment" specifically bind to the molecule of interest (or a highly similar group of molecules of interest) to substantially exclude binding to other molecules (e.g., the binding constant for the molecule of interest is at least 10 greater than the binding constant for other molecules in the biological sample). 3 M -1 At least 10 4 M -1 Or at least 10 5 M -1(Antibodies and antibody fragments). The term "antibody" also includes genetically engineered forms such as chimeric antibodies (e.g., humanized mouse antibodies) and heteroconjugated antibodies (such as bispecific antibodies). See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology, 3rd edition, WHFreeman & Co., New York, 1997.

[0065] More specifically, an antibody is a polypeptide ligand comprising at least one variable region of a light chain immunoglobulin or a variable region of a heavy chain immunoglobulin that specifically recognizes and binds to an epitope of an antigen. Antibodies consist of a heavy chain and a light chain, each of which has a variable region, referred to as a variable weight (VJ). H ) zone and variable light (V L ) area. V H District and V L Together, these regions are responsible for binding to antigens recognized by antibodies. Typically, immunoglobulins have heavy (H) chains and light (L) chains linked by disulfide bonds. There are two types of light chains, λ (lambda) and κ (kappa). There are five major heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each heavy and light chain contains constant and variable regions (also referred to as “domains”). In summary, the variable regions of the heavy and light chains specifically bind to antigens. The variable regions of both the light and heavy chains contain “framework” regions interrupted by three hypervariable regions also known as “complementarity-determining regions” or “CDRs”. The extent of the framework regions and CDRs has been defined (see Kabat et al., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991, which is incorporated herein by reference). The Kabat database is now maintained online. The sequences of the framework regions of different light or heavy chains are relatively conserved within species. The framework region of an antibody, namely the combined framework region comprising the light and heavy chains, primarily adopts a β-sheet conformation, and the CDR forms loops that connect the β-sheet structure and, in some cases, form part of the β-sheet structure. Therefore, the framework region serves to form a scaffold that positions the CDR in the correct orientation through non-covalent interchain interactions.

[0066] CDRs are primarily responsible for binding to epitopes of the antigen. The CDRs on each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are usually further identified by the chain to which the specific CDR resides. Therefore, V H CDR3 is located in the variable domain of the heavy chain of the antibody that discovered it, while V L CDR1 is derived from the variable domain of the light chain of the antibody that discovered it. Antibodies binding to the GPA33 protein will possess a specific V... H District and V L Antibodies have specific CDR sequences and therefore specific CDR sequences. Antibodies with different specificities (i.e., different binding sites against different antigens) have different CDRs. Although it is the CDR that varies from antibody to antibody, only a limited number of amino acid positions within the CDR are directly involved in antigen binding. These positions within the CDR are called specificity-determining residues (SDRs). As used herein, “immunoglobulin-associated composition” refers to antibodies (including monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, multispecific antibodies, bispecific antibodies, etc.) and antibody fragments. Antibodies or their antigen-binding fragments bind specifically to antigens.

[0067] As used herein, the term "antibody-associated polypeptide" means an antigen-binding antibody fragment including a single-chain antibody, which may include a variable region, alone or in combination with all or part of the following polypeptide elements: a hinge region of the antibody molecule, and CH1, CH2, and CH3 domains. This technique also includes any combination of the variable region and the hinge region, and the CH1, CH2, and CH3 domains. Antibody-associated molecules that can be used in the methods of this invention include, for example, but not limited to, Fab, Fab', and F(ab')2, Fd, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (sdFv), and those containing V L or V H Fragments of a structural domain. Examples include: (i) Fab fragments, which are formed by V L V H C L (ii) a monovalent segment consisting of a CH1 domain; (iii) a divalent segment consisting of two Fab segments connected by a disulfide bridge at the hinge region; and (iv) an Fd segment consisting of a V... H (iv) The Fv fragment, which consists of the V of a single arm of the antibody, and the CH1 domain; L and V H The domain is composed of (v) dAb fragments (Ward et al., Nature 341: 544-546, 1989), which are composed of V HThe structure consists of domains; and (vi) separate complementarity-determining regions (CDRs). Therefore, an "antibody fragment" or "antigen-binding fragment" can include a portion of a full-length antibody, typically its antigen-binding region or variable region. Examples of antibody fragments or antigen-binding fragments include Fab, Fab', F(ab')2, and Fv fragments; biantibodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0068] As used herein, "bispecific antibody" or "BsAb" refers to an antibody that can simultaneously bind to two targets with different structures (e.g., two different target antigens, two different epitopes on the same target antigen, or a hapten and a target antigen or an epitope on a target antigen). Various bispecific antibody structures are well known in the art. In some embodiments, each antigen-binding portion of the bispecific antibody contains V... H and / or V L Zone; in some such embodiments, V H and / or V L The regions are those found in specific monoclonal antibodies. In some embodiments, a bispecific antibody contains two antigen-binding moieties, each containing a V from a different monoclonal antibody. H Zone and / or V L In some embodiments, the bispecific antibody contains two antigen-binding portions, one of which includes an immunoglobulin molecule having a V region containing a CDR from the first monoclonal antibody. H and / or V L The region, and another antigen-binding portion includes an antibody fragment (e.g., Fab, F(ab'), F(ab')2, Fd, Fv, dAB, scFv, etc.), said antibody fragment having a V containing a CDR from a second monoclonal antibody. H and / or V L district.

[0069] As used herein, a “clearant” is an agent that binds to excess bispecific antibodies present in the blood chamber of a subject to promote rapid clearance via the kidneys. Using a clearant prior to hapten administration (e.g., DOTA) promotes a better tumor-to-background ratio in pre-targeted radioimmunotherapy (PRIT) systems. Examples of clearants include 500 kD-glucan-DOTA-Bn(Y) (Orcutt et al., Mol Cancer Ther. 11(6): 1365–1372 (2012)), 500 kD glycosaminoglucan-DOTA conjugates, antibodies against pre-targeted antibodies, etc.

[0070] As used herein, the term "association" refers to the association of two molecules by any method known to those skilled in the art. Suitable types of association include chemical bonds and physical bonds. Chemical bonds include, for example, covalent bonds and coordinate bonds. Physical bonds include, for example, hydrogen bonds, dipole interactions, van der Waals forces, electrostatic interactions, hydrophobic interactions, and aromatic stacking.

[0071] As used herein, the term "dual antibody" refers to a small antibody fragment having two antigen-binding sites, which contains antibodies against the same polypeptide chain (V). H V L The light chain variable structural domain (V) in ) L ) connected heavy chain variable structural domain (V H By using a linker that is too short to allow pairing between two domains on the same strand, the domain is forced to pair with a complementary domain on the other strand, resulting in two antigen-binding sites. Biantibodies are described in more detail in, for example, the following literature: EP 404,097; WO 93 / 11161; and 30 Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).

[0072] As used herein, the terms "single-chain antibody" or "single-chain Fv (scFv)" refer to the two V domains of an Fv fragment. L and V H Antibody fusion molecules. Single-chain antibody molecules can include polymers having many individual molecules, such as dimers, trimers, or other polymers. Furthermore, although F... v The two structural domains V of the fragment L and V H Encoded by separate genes, these two domains can be linked using recombination methods by enabling them to act as synthetic linkers for a single protein chain in which V... L District and V H Pairing occurs between molecules to form monovalent molecules (called single-chain F). v (scF v Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc. Natl. Acad Sci. USA 85:5879-5883. These single-chain antibodies can be prepared using recombinant techniques or by enzymatic or chemical cleavage of intact antibodies.

[0073] Any of the above-described antibody fragments were obtained using conventional techniques known to those skilled in the art, and the fragments were screened for binding specificity and neutralizing activity in the same manner as for intact antibodies.

[0074] As used herein, “antigen” refers to a molecule that an antibody (or an antigen-binding fragment thereof) can selectively bind to. Target antigens can be proteins, carbohydrates, nucleic acids, lipids, haptens, or other naturally occurring or synthetic compounds. In some embodiments, the target antigen can be a polypeptide (e.g., the GPA33 polypeptide). Antigens can also be administered to animals to elicit an immune response.

[0075] The term "antigen-binding fragment" refers to a segment of the entire immunoglobulin structure that contains a portion of a polypeptide responsible for binding to an antigen. Examples of antigen-binding fragments that can be used in the present invention include, but are not limited to, scFv, (scFv)2, scFvFc, Fab, Fab', and F(ab')2.

[0076] "Binding affinity" refers to the strength of the total non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or antigenic peptide). The affinity of molecule X for its partner Y can generally be expressed by the dissociation constant (K0). D Affinity is expressed as a definite affinity. It can be measured using standard methods known in the art, including those described herein. Low-affinity complexes contain antibodies that generally tend to dissociate easily from the antigen, while high-affinity complexes contain antibodies that generally tend to remain bound to the antigen for a longer period.

[0077] As used herein, the term "biological sample" means sample material derived from living cells. Biological samples can include tissues, cells, cellular protein or membrane extracts, and biological fluids (e.g., ascites or cerebrospinal fluid (CSF)) isolated from a subject, as well as tissues, cells, and fluids present within the subject. Biological samples of the technology of this invention include, but are not limited to, samples taken from: breast tissue, kidney tissue, cervix, endometrium, head or neck, gallbladder, parotid gland tissue, prostate, brain, pituitary gland, kidney tissue, muscle, esophagus, stomach, small intestine, colon, liver, spleen, pancreas, thyroid tissue, heart tissue, lung tissue, bladder, adipose tissue, lymph node tissue, uterus, ovarian tissue, adrenal gland tissue, testicular tissue, tonsils, thymus, blood, hair, oral cavity, skin, serum, plasma, CSF, sperm, prostatic fluid, semen, urine, feces, sweat, saliva, sputum, mucus, bone marrow, lymph, and tears. Biological samples can also be obtained from biopsies of internal organs or cancer. Biological samples can be obtained from subjects for diagnostic or research purposes, or from non-disease-prone individuals as controls or for basic research. Samples can be obtained using standard methods, including, for example, venipuncture and surgical biopsy. In some embodiments, biological samples are breast, lung, colon, or prostate tissue samples obtained via venipuncture biopsy.

[0078] As used herein, the term “CDR-transplanted antibody” means an antibody in which at least one CDR of a “recipient” antibody is replaced by a CDR “graft” from a “donor” antibody having the desired antigen specificity.

[0079] As used herein, the term "chimeric antibody" refers to an antibody in which the Fc constant region of a monoclonal antibody from one species (e.g., the mouse Fc constant region) is replaced by the Fc constant region of an antibody from another species (e.g., the human Fc constant region) using recombinant DNA technology. See also Robinson et al., PCT / US86 / 02269; Akira et al., European Patent Application No. 184,187; Taniguchi, European Patent Application No. 171,496; Morrison et al., European Patent Application No. 173,494; Neuberger et al., WO 86 / 01533; Cabilly et al., US Patent No. 4,816,567; Cabilly et al., European Patent Application No. 0125,023; Better et al., Science 240: 1041-1043, 1988; Liu et al., Proc. Natl. Acad. Sci. USA 84: 3439-3443, 1987; Liu et al., J. Immunol 139: 3521-3526, 1987; Sun et al., Proc. Natl. Acad. Sci. USA 84: 214-218. 1987; Nishimura et al., Cancer Res 47:999-1005, 1987; Wood et al., Nature 314: 446-449, 1885; and Shaw et al., J. Natl. Cancer Inst. 80: 1553-1559, 1988.

[0080] As used herein, the term "shared FR" refers to a shared frame (FR) antibody region within an immunoglobulin sequence. The FR region of an antibody does not contact the antigen.

[0081] As used herein, a “control” is an alternative sample used in an experiment for comparative purposes. A control can be “positive” or “negative.” For example, when the purpose of an experiment is to determine the relevance of a therapeutic agent to the treatment efficacy of a particular type of disease, a positive control (a known compound or composition that exhibits the desired therapeutic effect) and a negative control (a subject or sample that has not received the therapy or has received a placebo) are typically used.

[0082] As used herein, the term "effective amount" means an amount sufficient to achieve the desired therapeutic and / or preventive effect, for example, an amount that prevents or alleviates the disease or condition described herein or one or more signs or symptoms associated with the disease or condition described herein. In the context of therapeutic or preventive application, the amount of composition administered to a subject will vary depending on the composition, the degree, type, and severity of the disease, and individual characteristics such as general health status, age, sex, weight, and drug tolerance. Those skilled in the art will be able to determine an appropriate dosage based on these and other factors. The composition may also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, a therapeutic composition may be administered to a subject having one or more signs or symptoms of the disease or condition described herein. As used herein, a "therapeutic effective amount" of a composition means a level of composition that improves or eliminates the physiological effects of the disease or condition. A therapeutic effective amount may be given by one or more administrations.

[0083] As used herein, the term "effective cell" refers to an immune cell involved in the effector phase of an immune response, which is opposite to the cognitive and activation phases of the immune response. Exemplary immune cells include cells of bone marrow or lymphoid origin, such as lymphocytes (e.g., B cells and T cells, including cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mast cells, and basophils. Effective cells express specific Fc receptors and perform specific immune functions. Effective cells can induce antibody-dependent cell-mediated cytotoxicity (ADCC), for example, neutrophils capable of inducing ADCC. For example, monocytes, macrophages, neutrophils, eosinophils, and lymphocytes expressing FcαR are involved in specifically killing target cells and presenting antigens to other components of the immune system or binding to antigen-presenting cells.

[0084] As used herein, the term "epitope" refers to a protein determinant that can specifically bind to an antibody. Epitopes typically consist of chemically active surface groups of molecules such as amino acids or sugar side chains and generally possess specific three-dimensional structural characteristics and specific charge characteristics. The difference between conformational and non-conformational epitopes is that binding to the former, but not the latter, is lost in the presence of denaturing solvents. In some embodiments, the "epitope" of the GPA33 protein is a region of the protein that specifically binds to the anti-GPA33 antibody of the present invention. In some embodiments, the epitope is a conformational epitope. To screen for anti-GPA33 antibodies that bind to epitopes, conventional cross-blocking assays, such as those described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow, and David Lane (1988), can be performed. This assay can be used to determine whether the anti-GPA33 antibody binds to the same site or epitope as the anti-GPA33 antibody of the present invention. Alternatively or additionally, epitope mapping can be performed by methods known in the art. For example, the antibody sequence can be mutagenized, such as by alanine scanning, to identify contact residues. In different methods, peptides corresponding to different regions of the GPA33 protein can be used in competitive assays with the test antibody or test antibodies having characterized or known epitopes.

[0085] As used herein, “expression” includes one or more of the following: transcription of a gene into a precursor mRNA; splicing and other processing of the precursor mRNA to produce a mature mRNA; mRNA stability; translation of the mature mRNA into a protein (including codon usage and tRNA availability); and glycosylation and / or other modifications of the translation product if appropriate expression and function are desired.

[0086] As used herein, the term "gene" refers to a segment of DNA containing all the information about the regulation of biosynthesis of RNA products, including promoters, exons, introns, and other untranslated regions that control expression.

[0087] The terms "homology," "identity," or "similarity" refer to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing positions in each sequence that can be aligned for comparative purposes. Molecules are homologous at that position when a position in the compared sequences is occupied by the same base or amino acid. The degree of homology between sequences is a function of the number of shared matching or homologous positions. "Sequence identity" of a polynucleotide or polynucleotide region (or polypeptide or polypeptide region) with another sequence at a certain percentage (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) means that, when aligned, a percentage of the bases (or amino acids) are the same in the comparison of the two sequences. This alignment and percentage homology or sequence identity can be determined using software programs known in the art. In some embodiments, default parameters are used for alignment. One alignment program is BLAST using default parameters. Specifically, the procedures use BLASTN and BLASTP with the following default parameters: Genetic Code = Standard; Filter = None; Chain = Both; Cutoff = 60; Expected Value = 10; Matrix = BLOSUM62; Description = 50 sequences; Sort by = High Score; Database = Non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS Translation+SwissProtein+SPupdate+PIR. Detailed information about these procedures can be found at the National Center for Biotechnology Information. Biologically equivalent polynucleotides are those that have a specified percentage of homology and encode polypeptides with the same or similar biological activities. Two sequences are considered "unrelated" or "non-homologous" if they share less than 40% or less than 25% identity with each other.

[0088] As used herein, the term "humanized" for nonhuman (e.g., mouse) antibodies refers to chimeric antibodies containing a minimal sequence derived from a nonhuman immunoglobulin. In most cases, humanized antibodies are human immunoglobulins in which hypervariable residues of the receptor are replaced by hypervariable residues from a nonhuman species (donor antibody) possessing the desired specificity, affinity, and capability, such as mice, rats, rabbits, or nonhuman primates. In some embodiments, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding nonhuman residues. Furthermore, humanized antibodies may include residues not found in the receptor or donor antibody. These modifications are made to further improve antibody properties, such as binding affinity. Typically, humanized antibodies will contain substantially all of at least one and usually two variable domains (e.g., Fab, Fab', F(ab')2, or Fv), wherein all or substantially all of the hypervariable loops correspond to those loops of the nonhuman immunoglobulin, and all or substantially all of the FR regions are those regions of the common FR sequence of the human immunoglobulin, although the FR regions may contain one or more amino acid substitutions that improve binding affinity. The number of these amino acid substitutions in the FR is generally no more than 6 in the H chain and no more than 3 in the L chain. Humanized antibodies may optionally also include at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the human immunoglobulin constant region. For further details, see Jones et al., Nature 321:522-525 (1986); Reichmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See, for example, Ahmed and Cheung, FEBS Letters 588(2):288-297 (2014).

[0089] As used herein, the term "hypervariate region" refers to the amino acid residues of an antibody responsible for antigen binding. Hypervariate regions typically contain amino acid residues from the "complementarity-determining region" or "CDR" (e.g., V...). L Residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the relevant residues, as well as V HResidues 31-35B (H1), 50-65 (H2), and 95-102 (H3) in (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)), and / or those residues from the “hypervariant ring” (e.g., V L Residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the group, as well as V H Residues 26-32 (H1), 52A-55 (H2), and 96-101 (H3) in (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).

[0090] As used herein, when used in the context of two or more nucleic acid or polypeptide sequences, the term "identical" or "percentage of identity" refers to two or more sequences or subsequences or identical nucleotides (i.e., approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher of identity over a specified region (e.g., the nucleotide sequence encoding the antibody described herein or the amino acid sequence encoding the antibody described herein)). Such sequences are then referred to as "substantially identical." This term also refers to or can be applied to complement in the test sequence. The term also includes sequences with deletions and / or additions, as well as sequences with substitutions. In some embodiments, identity exists within a region of at least about 25 amino acids or nucleotides or of 50-100 amino acids or nucleotides in length.

[0091] As used herein, the term "intact antibody" or "intact immunoglobulin" refers to an antibody having at least two heavy (H) chain polypeptides and two light (L) chain polypeptides linked by disulfide bonds. Each heavy chain contains a heavy chain variable region (abbreviated herein as HCVR or V). H The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated as LCVR or V in this paper). L) and the light chain constant region. The light chain constant region contains a structural domain, C L V H and V L The region can be further subdivided into highly variable regions known as complementarity-determining regions (CDRs), interspersed with more conservative regions known as framing regions (FRs). Each V H and V L It consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0092] As used herein, the terms “individual,” “patient,” or “subject” can refer to an individual organism, a vertebrate, a mammal, or a human. In some embodiments, an individual, patient, or subject is a human.

[0093] As used herein, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous population of antibodies, meaning that individual antibodies comprising said population are identical except for possibly naturally occurring mutations that may be present in small amounts. For example, a monoclonal antibody can be derived from a single clone, comprising any eukaryotic, prokaryotic, or phage clone, and is not a method of antibody production. Monoclonal antibody compositions exhibit single binding specificity and affinity for a specific epitope. Monoclonal antibodies are highly specific, targeting a single antigenic site. Furthermore, each monoclonal antibody targets a single determinant on an antigen, unlike conventional (polyclonal) antibody formulations which typically comprise different antibodies targeting different determinants (epitaxes). The modifier "monoclonal" indicates that the antibody is derived from a substantially homogeneous population of antibodies and should not be construed as requiring antibody production via any particular method. Monoclonal antibodies can be prepared using a variety of techniques known in the art, including, but not limited to, hybridoma, recombinant, and phage display technologies. For example, the monoclonal antibody to be used according to the method of the present invention can be prepared by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or by the recombinant DNA method (see, for example, U.S. Patent No. 4,816,567). The “monoclonal” can also be isolated from the phage antibody library, for example, using the techniques described in Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991).

[0094] As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal compounds, isotonic compounds, and absorption-delaying compounds compatible with drug administration. Pharmaceutically acceptable carriers and formulations thereof are known to those skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences (20th edition, edited by A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, Pa.).

[0095] As used herein, the term "polyclonal antibody" means the preparation of an antibody derived from at least two (2) different antibody-producing cell lines. The use of this term encompasses the preparation of at least two (2) antibodies containing antibodies that specifically bind to different epitopes or regions of an antigen.

[0096] As used herein, the term "polynucleotide" or "nucleic acid" means any RNA or DNA, which may be unmodified or modified. Polynucleotides include, but are not limited to, single-stranded and double-stranded DNA, DNA as a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, RNA as a mixture of single-stranded and double-stranded regions, and hybrid molecules comprising DNA and RNA that may be single-stranded or more typically double-stranded or a mixture of single-stranded and double-stranded regions. Additionally, a polynucleotide refers to a triple-stranded region comprising RNA or DNA, or both RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases, and DNA or RNA with a backbone modified for stability or other reasons.

[0097] As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably to refer to polymers containing two or more amino acids linked together by peptide bonds or modified peptide bonds, i.e., isosteric peptides. A polypeptide refers to both short chains commonly referred to as peptides, glycopeptides, or oligomers, and longer chains commonly referred to as proteins. Polypeptides may contain amino acids in addition to the 20 amino acids encoded by genes. Polypeptides contain amino acid sequences modified through natural processes such as post-translational processing or through chemical modification techniques well known in the art. Such modifications are described in detail in basic texts and more detailed monographs, as well as in a large body of research literature.

[0098] As used herein, “PRIT” or “pre-targeted radioimmunotherapy” refers to a multi-step process that addresses the slow blood clearance of tumor-targeting antibodies, leading to undesirable toxicity to normal tissues such as bone marrow. In pre-targeting, a radionuclide or other diagnostic or therapeutic agent is conjugated to a small hapten. A pre-targeted bispecific antibody with binding sites for both the hapten and the target antigen is first administered. Unbound antibodies are then allowed to be cleared from circulation, and the hapten is subsequently administered.

[0099] As used herein, when referring to, for example, cells or nucleic acids, proteins or vectors, the term “recombinant” indicates that the cell, nucleic acid, protein or vector has been modified by introducing a heterologous nucleic acid or protein or by altering the native nucleic acid or protein, or that the material is derived from such modified cells. Thus, for example, recombinant cells express genes not found in the native (non-recombinant) form of the cell, or express native genes that are otherwise abnormally expressed, underexpressed or not expressed at all.

[0100] As used herein, the term “single” therapeutic use refers to the simultaneous or substantially simultaneous administration of at least two active ingredients via different routes.

[0101] As used herein, the term "sequential" therapeutic use refers to the administration of at least two active ingredients at different times, via the same or different routes of administration. More specifically, sequential use means the complete administration of one active ingredient before the administration of one or more other active ingredients. Therefore, one active ingredient may be administered minutes, hours, or days before the administration of one or more other active ingredients. In this case, there is no simultaneous treatment.

[0102] As used herein, "specific binding" refers to a molecule that recognizes and binds to another molecule (e.g., an antigen), but substantially does not recognize and bind to other molecules (e.g., an antibody or its antigen-binding fragment). As used herein, the terms "specific binding" to a particular molecule (e.g., a polypeptide or an epitope on a polypeptide), "specifically binding to," or "specific to" can be understood, for example, by having approximately 10 [unclear meaning - possibly related to the molecule it binds to]. -4 M, 10 −5 M, 10 −6 M, 10 −7 M, 10 −8 M, 10 −9 M, 10 −10 M, 10 −11 M or 10 −12 M of K DThe term "specific binding" can also refer to a binding in which a molecule (e.g., an antibody or its antigen-binding fragment) binds to a specific polypeptide (e.g., the GPA33 polypeptide) or an epitope on a specific polypeptide, while substantially not binding to any other polypeptide or polypeptide epitope.

[0103] As used herein, the term “simultaneous” therapeutic use refers to the simultaneous or substantially simultaneous administration of at least two active ingredients via the same route.

[0104] As used herein, the term "therapeutic agent" is intended to mean a compound that, when present in an effective amount, produces a desired therapeutic effect on a subject in need.

[0105] As used herein, “treating” or “treatment” encompasses the treatment of a subject (such as a person) of a disease or condition as described herein, and includes: (i) suppressing the disease or condition, i.e., preventing its development; (ii) alleviating the disease or condition, i.e., causing the symptom to subside; (iii) slowing the progression of the condition; and / or (iv) suppressing, alleviating, or slowing the progression of one or more symptoms of the disease or condition. In some embodiments, treatment means, for example, that symptoms associated with the disease are alleviated, reduced, cured, or in remission.

[0106] It should also be understood that the various treatment modalities described in this article are intended to represent "basic," encompassing both complete and sub-treatments, in which some biological or medically relevant outcome is achieved. Treatment can be continuous, long-term therapy for chronic diseases or single or several applications for the treatment of acute symptoms.

[0107] Amino acid sequence modifications of the anti-GPA33 antibodies described herein are envisioned. For example, improvements in antibody binding affinity and / or other biological properties may be desired. Amino acid sequence variants of anti-GPA33 antibodies are prepared by introducing appropriate nucleotide alterations into the antibody nucleic acid or through peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can yield the antibody of interest, provided the resulting antibody possesses the desired properties. Modifications also include alterations to the protein's glycosylation pattern. The most desirable sites for substitution mutagenesis include hypervariable regions, but FR alterations are also envisioned. “Conserved substitutions” are shown in the table below.

[0108]

[0109] One type of substitution variant involves replacing one or more hypervariable residues of the parent antibody. A convenient method for generating such substitution variants involves affinity maturation using phage display. Specifically, several hypervariable sites (e.g., 6 to 7 sites) are mutated to generate all possible amino acid substitutions at each site. The resulting antibody variant is displayed monovalently from filamentous phage particles as a fusion with the gene III product of M13 packaged within each particle. The variant is then screened for biological activity (e.g., binding affinity) against the phage-displayed variant, as disclosed herein. To identify candidate hypervariable sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable residues that significantly contribute to antigen binding. Alternatively or additionally, it may be advantageous to analyze the crystal structure of the antigen-antibody complex to identify contact sites between the antibody and antigen. Such contact residues and adjacent residues are candidates for substitution, according to the techniques detailed herein. Once such variants are generated, they are screened as described herein, and antibodies that have similar or superior properties in one or more relevant assays can be selected for further development.

[0110] GPA33

[0111] Human glycoprotein GPA33 (GPA33) is a single-pass type I membrane protein belonging to the CTX family of cell adhesion molecules within the immunoglobulin family. GPA33 is expressed in 95% of CRC tissues, but its expression in normal tissues is very limited. GPA33 is expressed in the initial T cells... regs And expressed in T-ALL.

[0112] GPA33 contains an Ig-like C2 domain and an Ig-like V domain. The predicted mature protein comprises a single transmembrane domain, an extracellular domain, and an intracellular tail. GPA33 functions in intracellular transport, cell-cell recognition / signaling, and recycling to the cell surface. The amino acid sequence of the extracellular domain (Ile22–Val235) of GPA33 is provided below:

[0113] ISVETPQDVLRASQGKSVTLPCTYHTSTSSREGLIQWDKLLLTHTERVVIWPFSNKNYIHGELYKNRVSISNNAEQSDASITIDQLTMADNGTYECSVSLMSDLEGN TKSRVRLLVLVPPSKPECGIEGETIIGNNIQLTCQSKEGSPTPQYSWKRYNILNQEQPLAQPASGQPVSLKNISTDTSGYYICTSSNEEGTQFCNITVAVRSPSMNV (SEQ ID NO: 53).

[0114] The immunoglobulin-related compositions of the present invention

[0115] This invention describes methods and compositions for generating and using anti-GPA33 immunoglobulin-associated compositions (e.g., anti-GPA33 antibodies or antigen-binding fragments thereof). The anti-GPA33 immunoglobulin-associated compositions disclosed herein can be used for the diagnosis or treatment of GPA33-positive cancers. Anti-GPA33 immunoglobulin-associated compositions within the scope of this invention include, for example, but not limited to, monoclonal, chimeric, humanized, and biantibodies, homologs, derivatives, or fragments thereof that specifically bind to target polypeptides. This disclosure also provides antigen-binding fragments of any of the anti-GPA33 antibodies disclosed herein, wherein the antigen-binding fragments are selected from the group consisting of: Fab, F(ab')2, Fab', scF. v and F v . Figure 5 Up to Figure 6 and Figures 9 to 14 The representative amino acid sequence of the anti-GPA33 immunoglobulin-related composition of the present invention is described in the document.

[0116] On one hand, this disclosure provides an antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising a heavy chain immunoglobulin variable domain (V). H ) and light chain immunoglobulin variable domain (V L ), wherein (a) V H V containing SEQ ID NO: 2 or SEQ ID NO: 7 H -CDR1 sequence, V of SEQ ID NO: 3 or SEQ ID NO: 8 H -CDR2 sequence and V of SEQ ID NO: 4 or SEQ ID NO: 9 H -CDR3 sequence; and (b) the V L V contains groups consisting of the following items L -CDR1 sequence, V L -CDR2 sequence and V L -CDR3 sequences: SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14; and SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19. Alternatively or additionally, in some embodiments, (a) the V H Contains an amino acid sequence comprising either SEQ ID NO: 1 or SEQ ID NO: 6; and / or (b) the V LContains the amino acid sequence of either SEQ ID NO: 11 or SEQ ID NO: 16. In some embodiments, (a) the V H Contains the amino acid sequence of SEQ ID NO: 1, and the V L The amino acid sequence comprising SEQ ID NO:16; or (b) the V H Contains the amino acid sequence of SEQ ID NO: 6, and the V L It contains the amino acid sequence of SEQ ID NO: 11.

[0117] In some embodiments, the antibody further comprises any isotype of Fc domain, such as, but not limited to, IgG (including IgG1, IgG2, IgG3, and IgG4), IgA (including IgA1 and IgA2), IgD, IgE, or IgM and IgY. Non-limiting examples of constant region sequences include:

[0118] Human IgD constant region, Uniprot: P01880 (SEQ ID NO: 54)

[0119] APTKAPDVFPIISGCRHPKDNSPVVLACLITGYHPTSVTVTWYMGTQSQPQRTFPEIQRRDSYYMTSSQLSTPLQQWRQGEYKCVVQHTASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFV VGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDHGPMK

[0120] Human IgG1 constant region, Uniprot: P01857 (SEQ ID NO: 55)

[0121] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0122] Human IgG2 constant region, Uniprot: P01859 (SEQ ID NO: 56)

[0123] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0124] Human IgG3 constant region, Uniprot: P01860 (SEQ ID NO: 57)

[0125] ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK

[0126] Human IgM constant region, Uniprot: P01871 (SEQ ID NO: 58)

[0127] GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITLSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY

[0128] Human IgG4 constant region, Uniprot: P01861 (SEQ ID NO: 59)

[0129] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0130] Human IgA1 constant region, Uniprot: P01876 (SEQ ID NO: 60)

[0131] ASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQDASGDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY

[0132] Human IgA2 constant region, Uniprot: P01877 (SEQ ID NO: 61)

[0133] ASPTSPKVFPLSLDSTPQDGNVVVACLVQGFFPQEPLSVTWSESGQNVTARNFPPSQDASGDLYTTSSQLTLPATQCPDGKSVTCHVKHYTNPSQDVTVPCPVPPPPPCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPERDLCGCY SVSSVLPGCAQPWNHGETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRMAGKPTHVNVSVVMAEVDGTCY

[0134] Human Igκ constant region, Uniprot: P01834 (SEQ ID NO: 62)

[0135] TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0136] In some embodiments, the immunoglobulin-associated compositions of the present invention comprise at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same heavy chain constant region as SEQ ID NO: 54 to 61. Additionally or alternatively, in some embodiments, the immunoglobulin-associated compositions of the present invention comprise at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same light chain constant region as SEQ ID NO: 62. In some embodiments, the immunoglobulin-associated compositions of the present invention bind to an epitope of a GPA33 polypeptide comprising at least five to eight consecutive amino acid residues of SEQ ID NO: 53. In some embodiments, the epitope is a conformational epitope.

[0137] On the other hand, this disclosure provides an antibody comprising a heavy chain (HC) amino acid sequence and a light chain (LC) amino acid sequence selected from the group consisting of: SEQ ID NO: 35 and SEQ ID NO: 36; SEQ ID NO: 35 and SEQ ID NO: 37; SEQ ID NO: 35 and SEQ ID NO: 38; SEQ ID NO: 39 and SEQ ID NO: 40; SEQ ID NO: 39 and SEQ ID NO: 41; SEQ ID NO: 39 and SEQ ID NO: 42; SEQ ID NO: 47 and SEQ ID NO: 48; SEQ ID NO: 47 and SEQ ID NO: 49; SEQ ID NO: 50 and SEQ ID NO: 51; and SEQ ID NO: 50 and SEQ ID NO: 52.

[0138] In any of the above embodiments of the immunoglobulin-related composition, the HC immunoglobulin variable domain sequence and the LC immunoglobulin variable domain sequence form an antigen-binding site, which binds to an epitope of a GPA33 polypeptide comprising at least five to eight consecutive amino acid residues of the extracellular domain of GPA33 (SEQ ID NO: 53). In some embodiments, the epitope is a conformational epitope.

[0139] In some embodiments, the HC immunoglobulin variable domain sequence and the LC immunoglobulin variable domain sequence are components of the same polypeptide chain. In other embodiments, the HC immunoglobulin variable domain sequence and the LC immunoglobulin variable domain sequence are components of different polypeptide chains. In some embodiments, the antibody is a full-length antibody.

[0140] In some embodiments, the immunoglobulin-associated composition of the present invention specifically binds to at least one GPA33 peptide. In some embodiments, the immunoglobulin-associated composition of the present invention is at about 10 −3 M, 10 −4 M, 10 −5 M, 10 −6 M, 10 −7 M, 10 −8 M, 10 −9 M, 10 −10 M, 10 −11 M or 10 −12 The dissociation constant of M (K) DIt combines with at least one GPA33 peptide. In some embodiments, the immunoglobulin-associated composition is a monoclonal antibody, a chimeric antibody, a humanized antibody, or a bispecific antibody. In some embodiments, the antibody comprises a human antibody framework region.

[0141] On one hand, this disclosure provides a multispecific antigen-binding fragment comprising a first polypeptide chain, wherein: the first polypeptide chain comprises, in the direction from the N-terminus to the C-terminus: a heavy chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope; a flexible peptide linker comprising the amino acid sequence (GGGGS)6; a light chain variable domain of the first immunoglobulin; a flexible peptide linker comprising the amino acid sequence (GGGGS)4; a heavy chain variable domain of a second immunoglobulin capable of specifically binding to a second epitope; a flexible peptide linker comprising the amino acid sequence (GGGGS)6; a light chain variable domain of the second immunoglobulin; a flexible peptide linker sequence comprising the amino acid sequence TPLDTTHT; and a self-assembly and disassembly (SADA) polypeptide; wherein the heavy chain variable domain of the first immunoglobulin or the heavy chain variable domain of the second immunoglobulin is selected from any one of the following: SEQ ID NO: 1 and SEQ ID NO: 6, and / or the light chain variable domain of the first immunoglobulin or the light chain variable domain of the second immunoglobulin is selected from any one of the following: SEQ ID NO: 11 and SEQ ID NO: 6. ID NO: 16. On the other hand, this disclosure provides a multispecific antigen-binding fragment comprising a first polypeptide chain, wherein: the first polypeptide chain comprises, in the direction from the N-terminus to the C-terminus: a light chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope; a flexible peptide linker comprising the amino acid sequence (GGGGS)6; a heavy chain variable domain of the first immunoglobulin; a flexible peptide linker comprising the amino acid sequence (GGGGS)4; a heavy chain variable domain of a second immunoglobulin capable of specifically binding to a second epitope; a flexible peptide linker comprising the amino acid sequence (GGGGS)6; a light chain variable domain of the second immunoglobulin; a flexible peptide linker sequence comprising the amino acid sequence TPLDTTHT; and a self-assembly and disassembly (SADA) polypeptide; wherein the heavy chain variable domain of the first immunoglobulin or the heavy chain variable domain of the second immunoglobulin is selected from any one of the following: SEQ ID NO: 1 and SEQ ID NO: 6, and / or the light chain variable domain of the first immunoglobulin or the light chain variable domain of the second immunoglobulin is selected from any one of the following: SEQ ID NO: 11 and SEQ ID NO: 6. NO: 16. The SADA polypeptide may comprise a tetramerizing, pentamerizing, or hexamerizing domain. In some embodiments, the SADA polypeptide comprises a tetramerizing domain of any of the following: p53, p63, p73, hnRNPC, SNA-23, StefinB, KCNQ4, or CBFA2T1. Additionally or alternatively, in some embodiments, the antigen-binding fragment comprises an amino acid sequence selected from the following: SEQ ID NO: 43 to 46.

[0142] In any SADA polypeptide containing a multispecific antibody or antigen-binding fragment disclosed herein, when the antibody or antigen-binding fragment is administered intravenously or intraperitoneally to a subject, the antibody or antigen-binding fragment does not cross the intestinal epithelium or intestinal lumen.

[0143] On one hand, this disclosure provides a multispecific antibody comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain are covalently bonded to each other, the second polypeptide chain and the third polypeptide chain are covalently bonded to each other, and the third polypeptide chain and the fourth polypeptide chain are covalently bonded to each other, and wherein: each of the first polypeptide chain and the fourth polypeptide chain comprises, in the direction from the N-terminus to the C-terminus,: a light chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope; a light chain constant domain of the first immunoglobulin; a flexible peptide linker comprising the amino acid sequence (GGGGS)3; and a second immunoglobulin linked to a complementary heavy chain variable domain of the second immunoglobulin. A light chain variable domain, or a heavy chain variable domain of the second immunoglobulin linked to a complementary light chain variable domain of the second immunoglobulin, wherein the light chain variable domain and the heavy chain variable domain of the second immunoglobulin are specifically capable of binding to a second epitope and are linked together via a flexible peptide linker comprising the amino acid sequence (GGGGS)6 to form a single-chain variable fragment; and each of the second polypeptide chain and the third polypeptide chain comprises, in the direction from the N-terminus to the C-terminus: a heavy chain variable domain of the first immunoglobulin capable of specifically binding to the first epitope; and a heavy chain constant domain of the first immunoglobulin; and wherein the heavy chain variable domain of the first immunoglobulin is selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 6, and / or the light chain variable domain of the first immunoglobulin is selected from the group consisting of SEQ ID NO: 11 and SEQ ID NO: 16.

[0144] In any and all embodiments of the multispecific antibody or multispecific antigen-binding fragment disclosed herein, the antibody or antigen-binding fragment binds to the following: CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, CD32, CD64, TCR γ / δ, NKp46, KIR, PD-1, PD-L1, LAG3, CD28, B7H3, STEAP1, HER2, EGFR, CEA, CECAM5, transferrin receptor, FAP, NKG2D-ligand, TRAIL, FasL, cathepsin G, granzyme, carboxypeptidase, β-lactamase, DOTA (metal) complex, benzyl-DOTA (metal) complex, Proteus spp.-DOTA (metal) complex, NOGADA-Proteus spp.-DOTA (metal) complex, astr-DFO (metal) complex, DFO (metal) complex, or small molecule DOTA hapten. Additionally or alternatively, in some embodiments, the multispecific antibody or multispecific antigen-binding fragment binds to T cells, B cells, myeloid cells, plasma cells, or mast cells.

[0145] On the other hand, this disclosure provides a heterodimeric multispecific antibody comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain are covalently bonded to each other, the second polypeptide chain and the third polypeptide chain are covalently bonded to each other, and the third polypeptide chain and the fourth polypeptide chain, wherein: (a) the first polypeptide chain comprises, in the direction from the N-terminus to the C-terminus,: (i) a light chain variable domain (VL-1) of a first immunoglobulin capable of specifically binding to a first epitope; (ii) a light chain constant domain (CL-1) of the first immunoglobulin; (iii) a flexible peptide linker comprising the amino acid sequence (GGGGS)3; and ( iv) a light chain variable domain (VL-2) of the second immunoglobulin linked to a complementary heavy chain variable domain (VH-2) of the second immunoglobulin, or a heavy chain variable domain (VH-2) of the second immunoglobulin linked to a complementary light chain variable domain (VL-2) of the second immunoglobulin, wherein VL-2 and VH-2 are capable of specifically binding to the second epitope and are linked together via a flexible peptide linker comprising an amino acid sequence (GGGGS)6 to form a single-chain variable fragment; (b) the second polypeptide comprises, in the direction from the N-terminus to the C-terminus: (i) a heavy chain variable domain (VH-1) of the first immunoglobulin capable of specifically binding to the first epitope; (ii) the... The first immunoglobulin comprises: (i) a first CH1 domain (CH1-1); and (iii) a first heterodimerizing domain of the first immunoglobulin, wherein the first heterodimerizing domain cannot form a stable homodimer with another first heterodimerizing domain; (c) the third polypeptide comprises, in the N-terminal to C-terminal direction: (i) a heavy chain variable domain (VH-3) of the third immunoglobulin capable of specifically binding to a third epitope; (ii) a second CH1 domain (CH1-3) of the third immunoglobulin; and (iii) a second heterodimerizing domain of the third immunoglobulin, wherein the second heterodimerizing domain comprises a domain different from that of the first immunoglobulin. The amino acid sequence or nucleic acid sequence of the first heterodimerizing domain, wherein the second heterodimerizing domain cannot form a stable homodimer with another second heterodimerizing domain, and wherein the second heterodimerizing domain of the third immunoglobulin is configured to form a heterodimer with the first heterodimerizing domain of the first immunoglobulin; (d) the fourth polypeptide comprises, in the direction from the N-terminus to the C-terminus: (i) a light chain variable domain (VL-3) of the third immunoglobulin capable of specifically binding to the third epitope; (ii) a light chain constant domain (CL-3) of the third immunoglobulin; and (iii) a flexible peptide linker comprising the amino acid sequence (GGGGS)3.And (iv) the light chain variable domain (VL-4) of the fourth immunoglobulin linked to the complementary heavy chain variable domain (VH-4) of the fourth immunoglobulin, or the heavy chain variable domain (VH-4) of the fourth immunoglobulin linked to the complementary light chain variable domain (VL-4) of the fourth immunoglobulin, wherein VL-4 and VH-4 are capable of specifically binding to the fourth epitope and are linked together via a flexible peptide linker comprising an amino acid sequence (GGGGS)6 to form a single-chain variable fragment; wherein VL-1 and / or VL-3 contain V selected from any of the following: L Amino acid sequences: SEQ ID NO: 11 and SEQ ID NO: 16, wherein VH-1 and / or VH-3 contain V selected from any of the following. H Amino acid sequences: SEQ ID NO: 1 and SEQ ID NO: 6. Additionally or alternatively, in some embodiments, the multispecific antibody binds to T cells, B cells, myeloid cells, plasma cells, or mast cells. In some embodiments, the second epitope and the fourth epitope may be the same or different.

[0146] In any and all embodiments of the multispecific antibodies disclosed herein, the multispecific antibodies bind to one or more of the following: CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, CD32, CD64, TCR γ / δ, NKp46, KIR, PD-1, PD-L1, LAG3, CD28, B7H3, STEAP1, HER2, EGFR, CEA, CECAM5, transferrin receptor, FAP, NKG2D-ligand, TRAIL, FasL, cathepsin G, granzyme, carboxypeptidase, β-lactamase, DOTA (metal) complex, benzyl-DOTA (metal) complex, Proteus spp.-DOTA (metal) complex, NOGADA-Proteus spp.-DOTA (metal) complex, astr-DFO (metal) complex, DFO (metal) complex, or small molecule DOTA hapten. In some embodiments of the multispecific antibodies or multispecific antigen-binding fragments described herein, the antibodies or antigen-binding fragments include catalytic antibodies, immune checkpoint inhibitors, or immune checkpoint activators.

[0147] In some embodiments, the immunoglobulin-associated composition contains an IgG1 constant region comprising one or more amino acid substitutions selected from the group consisting of N297A, K322A, L234A, and L235A. Alternatively or additionally, in some embodiments, the immunoglobulin-associated composition contains an IgG4 constant region comprising an S228P mutation.

[0148] In some aspects, the anti-GPA33 immunoglobulin-associated compositions described herein contain structural modifications to promote rapid binding and cellular uptake and / or slow release. In some aspects, the anti-GPA33 immunoglobulin-associated compositions (e.g., antibodies) of the present invention may contain a deletion in the CH2 constant heavy chain region to promote rapid binding and cellular uptake and / or slow release. In some aspects, the Fab fragment is used to promote rapid binding and cellular uptake and / or slow release. In some aspects, the F(ab)'2 fragment is used to promote rapid binding and cellular uptake and / or slow release.

[0149] On the one hand, the present invention provides a nucleic acid sequence encoding the heavy or light chain of the immunoglobulin-associated compositions described herein. Recombinant nucleic acid sequences encoding any antibodies described herein are also disclosed herein. In some embodiments, the nucleic acid sequence is selected from the group consisting of SEQ ID NOs: 5, 10, 15, and 20. On the other hand, the present invention provides a host cell or vector expressing any nucleic acid sequence encoding the heavy or light chain of the immunoglobulin-associated compositions described herein.

[0150] On the other hand, the present invention provides a cell (e.g., an immune cell, such as a T cell) coated with any and all embodiments of the multispecific antibody disclosed herein.

[0151] The immunoglobulin-associated compositions of the present invention (e.g., anti-GPA33 antibodies) can be monospecific, bispecific, trispecific, or have higher multispecificity. Multispecific antibodies can be specific to different epitopes of one or more GPA33 peptides, or specific to both the GPA33 peptide and the heterologous composition (such as a heterologous peptide or a solid support material). See, for example, WO 93 / 17715; WO 92 / 08802; WO 91 / 00360; WO 92 / 05793; Tutt et al., J. Immunol. 147: 60-69 (1991); U.S. Patent Nos. 5,573,920, 4,474,893, 5,601,819, 4,714,681, 4,925,648; 6,106,835; Kostelny et al., J. Immunol. 148: 1547-1553 (1992). In some embodiments, the immunoglobulin-associated composition is chimeric. In some embodiments, the immunoglobulin-associated composition is humanized.

[0152] The immunoglobulin-associated compositions of the present invention can be further recombinantly fused with heterologous peptides at the N-terminus or C-terminus, or chemically conjugated (including covalent and non-covalent conjugation) with peptides or other compositions. For example, the immunoglobulin-associated compositions of the present invention can be recombinantly fused or conjugated with molecules and effector molecules used as markers in detection assays, such as heterologous peptides, drugs, or toxins. See, for example, WO 92 / 08495; WO 91 / 14438; WO 89 / 12624; U.S. Patent No. 5,314,995; and EP 0 396 387.

[0153] In any of the above embodiments of the immunoglobulin-associated compositions of the present invention, the antibody or antigen-binding fragment may optionally be conjugated with a pharmaceutical agent selected from the group consisting of: isotopes, dyes, chromophores, contrast agents, drugs, toxins, cytokines, enzymes, enzyme inhibitors, hormones, hormone antagonists, growth factors, radionuclides, metals, liposomes, nanoparticles, RNA, DNA, or any combination thereof. For chemical or physical bonds, the functional groups on the immunoglobulin-associated composition are generally associated with functional groups on the pharmaceutical agent. Alternatively, functional groups on the pharmaceutical agent may be associated with functional groups on the immunoglobulin-associated composition.

[0154] Functional groups on pharmaceuticals and immunoglobulin-associated compositions can directly associate. For example, a functional group on a pharmaceutical (e.g., a thiol group) can associate with a functional group on an immunoglobulin-associated composition to form a disulfide. Alternatively, functional groups can associate via a crosslinking agent (i.e., a linker). Some examples of crosslinking agents are described below. Crosslinking agents can be linked to pharmaceuticals or immunoglobulin-associated compositions. The amount of pharmaceutical or immunoglobulin-associated composition in a conjugate is also limited by the number of functional groups present on the other conjugate. For example, the maximum number of agents associated with a conjugate depends on the number of functional groups present on the immunoglobulin-associated composition. Alternatively, the maximum number of immunoglobulin-associated compositions associated with a pharmaceutical depends on the number of functional groups present on the pharmaceutical.

[0155] In yet another embodiment, the conjugate comprises an immunoglobulin-associated composition associated with a pharmaceutical agent. In one embodiment, the conjugate comprises at least one pharmaceutical agent chemically bonded (e.g., conjugated) to at least one immunoglobulin-associated composition. The pharmaceutical agent can be chemically bonded to the immunoglobulin-associated composition by any method known to those skilled in the art. For example, functional groups on the pharmaceutical agent can be directly linked to functional groups on the immunoglobulin-associated composition. Some examples of suitable functional groups include, for example, amino, carboxyl, thiol, maleimide, isocyanate, isothiocyanate, and hydroxyl groups.

[0156] Pharmaceutical agents can also be chemically bonded to immunoglobulin-related compositions using cross-linking agents such as dialdehyde, carbodiimide, and diamaleimide. Cross-linking agents can be obtained, for example, from Pierce Biotechnology, Inc., Rockford, Ill. The Pierce Biotechnology, Inc. website can provide assistance. Other cross-linking agents containing platinum cross-linking agents are described in U.S. Patent Nos. 5,580,990, 5,985,566, and 6,133,038 of Kreatech Biotechnology, BV, Amsterdam, Netherlands.

[0157] Alternatively, the functional groups on the pharmaceutical and immunoglobulin-related compositions can be identical. Homodifunctional crosslinkers are typically used to crosslink the same functional groups. Examples of homodifunctional crosslinkers include EGS (i.e., ethylene glycol bis[succinimide succinate]), DSS (i.e., disuccinimide octanoate), DMA (i.e., dimethyl adipamide .2HCl), DTSSP (i.e., 3,3'-dithiobis[sulfosuccinimide propionate]), DDPPB (i.e., 1,4-di-[3'-(2'-pyridyldithio)-propamido]butane), and BMH (i.e., bismaleimide hexane). Such homodifunctional crosslinkers are also available from Pierce Biotechnology, Inc.

[0158] In other cases, cleaving the drug from the immunoglobulin-associated composition may be beneficial. The website of Pierce Biotechnology, Inc., described above, can also assist those skilled in the art in selecting suitable cross-linking agents, which can be cleaved, for example, by enzymes in cells. Therefore, the drug can be isolated from the immunoglobulin-associated composition. Examples of cleavable connectors include SMPT (i.e., 4-succinimideoxycarbonyl-methyl-a-[2-pyridyldithio]toluene), sulfonyl-LC-SPDP (i.e., sulfosuccinimide-6-(3-[2-pyridyldithio]propamido)hexanoate), LC-SPDP (i.e., succinimide-6-(3-[2-pyridyldithio]propamido)hexanoate), sulfonyl-LC-SPDP (i.e., sulfosuccinimide-6-(3-[2-pyridyldithio]propamido)hexanoate), SPDP (i.e., N-succinimide-3-[2-pyridyldithio]propamidoaminohexanoate), and AEDP (i.e., 3-[(2-aminoethyl)dithio]propionic acid HCl).

[0159] In another embodiment, the conjugate comprises at least one pharmaceutical agent physically bonded to at least one immunoglobulin-associated composition. The pharmaceutical agent can be physically bonded to the immunoglobulin-associated composition using any method known to those skilled in the art. For example, the immunoglobulin-associated composition and the pharmaceutical agent can be mixed together using any method known to those skilled in the art. The order of mixing is not important. For example, the pharmaceutical agent can be physically mixed with the immunoglobulin-associated composition using any method known to those skilled in the art. For example, the immunoglobulin-associated composition and the pharmaceutical agent can be placed in a container and stirred, for example, by shaking the container, to mix the immunoglobulin-associated composition and the pharmaceutical agent.

[0160] Immunoglobulin-related compositions can be modified by any method known to those skilled in the art. For example, immunoglobulin-related compositions can be modified by cross-linking agents or functional groups, as described above.

[0161] A. Method for preparing the anti-GPA33 antibody of the present invention

[0162] General Overview. Initially, antibodies selected using the techniques of this invention can target the resulting target peptides. For example, antibodies can be generated targeting the full-length GPA33 protein or a portion of the extracellular domain of the GPA33 protein. Techniques for generating antibodies involving such target peptides are well known to those skilled in the art. Examples of such techniques include, but are not limited to, techniques involving display libraries, xenogeneic or human-mouse hybridomas, etc. Target peptides within the scope of this invention comprise any peptide derived from the GPA33 protein, which contains an extracellular domain capable of evoking an immune response. In some embodiments, the extracellular domain comprises the amino acid sequence of SEQ ID NO: 53.

[0163] It should be understood that recombinant engineered antibodies and antibody fragments (e.g., antibody-associated peptides) targeting the GPA33 protein and its fragments are suitable for use in accordance with this disclosure.

[0164] Anti-GPA33 antibodies that can be used in the techniques described herein include monoclonal and polyclonal antibodies, as well as antibody fragments such as Fab, Fab', F(ab')2, Fd, scFv, biantibodies, antibody light chains, antibody heavy chains, and / or antibody fragments. Methods for producing polypeptides containing antibody Fv (e.g., Fab' and F(ab')2 antibody fragments) in high yields have been described. See U.S. Patent No. 5,648,237.

[0165] Typically, antibodies are obtained from a source species. More specifically, a nucleic acid or amino acid sequence of the light chain, heavy chain, or a variable portion of both of the source species antibody specific to the target polypeptide antigen is obtained. The source species is any species that can be used to generate the antibody or antibody library of the present invention, such as rat, mouse, rabbit, chicken, monkey, human, etc.

[0166] Phage display technology is a useful technique for obtaining antibodies according to the present invention. Techniques for generating and cloning monoclonal antibodies are well known to those skilled in the art. Expression of sequences encoding antibodies according to the present invention can be performed in *Escherichia coli*.

[0167] Due to the degeneracy of nucleic acid coding sequences, in the practice of this invention, other sequences encoding amino acid sequences substantially identical to those of naturally occurring proteins can be used. These include, but are not limited to, nucleic acid sequences containing all or part of the nucleic acid sequence encoding the aforementioned polypeptide, which is altered by substituting different codons for functionally equivalent amino acid residues within the coding sequence, thereby producing a silencing change. It should be understood that the nucleotide sequences of immunoglobulins according to the invention are permissible with up to 25% sequence homology variation calculated by standard methods (“Current Methods in Sequence Comparison and Analysis,” Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149, 1998, Alan R. Liss, Inc.), provided that such variants form effective antibodies recognizing the GPA33 protein. For example, one or more amino acid residues within the polypeptide sequence can be substituted by another amino acid of similar polarity, which, as a functional equivalent, results in a silencing change. The substitutes for the amino acids within the sequence can be selected from other members of the class to which the amino acid belongs. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine, and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. The scope of this invention also includes proteins or fragments or derivatives thereof that undergo differential modification during or after translation, for example, through glycosylation, proteolytic cleavage, or linkage with antibody molecules or other cellular ligands. Additionally, immunoglobulin-encoding nucleic acid sequences can be mutated in vitro or in vivo to produce and / or disrupt translation, initiation, and / or termination sequences, or to alter and / or form new restriction endonuclease sites or disrupt pre-existing restriction endonuclease sites to facilitate further in vitro modification. Any mutagenesis technique known in the art may be used, including but not limited to in vitro site-directed mutagenesis, J. Biol. Chem. 253:6551, the use of tag adapters (Pharmacia), etc.

[0168] Preparation of polyclonal antiserum and immunogen. Methods for generating antibodies or antibody fragments according to the present invention generally involve immunizing a subject (typically a non-human subject, such as a mouse or rabbit) with purified GPA33 protein or a fragment thereof, or with cells expressing GPA33 protein or a fragment thereof. Suitable immunogenic preparations may contain, for example, recombinantly expressed GPA33 protein or chemically synthesized GPA33 peptides. Extracellular domains of the GPA33 protein, or portions or fragments thereof, can be used as immunogens to generate anti-GPA33 antibodies that bind to the GPA33 protein or portions or fragments thereof using standard techniques for the preparation of polyclonal and monoclonal antibodies. In some embodiments, the extracellular domain comprises the amino acid sequence of SEQ ID NO: 53. In some embodiments, the antigenic GPA33 peptide comprises at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 amino acid residues. Depending on the application and according to methods well known to those skilled in the art, longer antigenic peptides are sometimes preferred over shorter antigenic peptides. Polymers with a given epitope are sometimes more effective than monomers.

[0169] Full-length GPA33 protein or a fragment thereof can be used as a fragment as an immunogen. In some embodiments, the GPA33 fragment comprises at least five to eight consecutive amino acid residues of the amino acid sequence of SEQ ID NO: 53 and covers the epitope of the GPA33 protein, such that antibodies generated against the peptide form specific immune complexes with the GPA33 protein.

[0170] Suitable immunogenic agents may contain, for example, recombinantly expressed GPA33 protein containing the amino acid sequence of SEQ ID NO: 53 or chemically synthesized GPA33 peptide. The extracellular domain of the GPA33 protein, or a portion or fragment thereof, may be used as an immunogen to generate anti-GPA33 antibodies that bind to the extracellular domain of the GPA33 protein.

[0171] If desired, the immunogenicity of GPA33 protein (or fragments thereof) can be enhanced by fusion or conjugation with haptens, such as keyfora hemocyanin (KLH) or ovalbumin (OVA). Many such carrier proteins are known in the art. Synthetic dendromeric trees can be added to reactive amino acid side chains, such as lysine, to enhance the immunogenic properties of GPA33 protein. Furthermore, C can be added... PG-dinucleotide motifs can be used to enhance the immunogenic properties of the GPA33 protein. The GPA33 protein can also be combined with conventional adjuvants, such as Freund's complete or incomplete adjuvants, to increase the subject's immune response to the peptide. Various adjuvants used to enhance the immune response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surfactants (e.g., lysophosphatidylcholine, Pluronic acid polyols, polyanionic peptides, oil emulsions, dinitrophenols, etc.), human adjuvants such as BCG and Corynebacterium pumilus, or similar immunostimulatory compounds. These techniques are standard in the art.

[0172] Alternatively, nanoparticles, such as virus-like particles (VLPs), can be used to present antigens, such as the GPA33 protein, to host animals. Virus-like particles are multi-protein structures that mimic the organization and conformation of true natural viruses without being infectious, as they do not carry any viral genetic material (Urakami A et al., Clin Vaccine Immunol 24:e00090-17 (2017)). When introduced into the host immune system, VLPs can elicit an effective immune response, making them attractive carriers of exogenous antigens. A significant advantage of VLP-based antigen presentation platforms is their ability to display antigens in a dense, repetitive manner. Therefore, antigen-carrying VLPs can induce strong B-cell responses by effectively cross-linking B-cell receptors (BCRs). VLPs can be genetically manipulated to improve their properties, such as immunogenicity. These techniques are standard practice in the field.

[0173] Isolating sufficiently purified proteins or peptides for antibody production can be time-consuming and sometimes technically challenging. Additional challenges associated with conventional protein-based immunization include concerns about the safety, stability, scalability, and consistency of protein antigens. Nucleic acid-based immunization (DNA and RNA) has emerged as a promising alternative. DNA vaccines are typically based on bacterial plasmids encoding candidate antigens, such as peptide sequences of GPA33. Utilizing strong eukaryotic promoters, the encoded antigen can be expressed to produce sufficient levels of transgenic expression once the host is inoculated with the plasmid (Galvin TA et al., Vaccine 2000, 18:2566-2583). Modern DNA vaccine production relies on DNA synthesis, potentially involving a one-step cloning into a plasmid vector followed by plasmid isolation, which significantly reduces preparation time and cost. The resulting plasmid DNA is also highly stable at room temperature, avoiding cold transport and significantly extending shelf life. These techniques are standard in the field.

[0174] Alternatively, a nucleic acid sequence encoding the antigen of interest, such as GPA33, can be synthesized and introduced into an mRNA molecule. The mRNA is then delivered to a host animal, whose cells recognize the mRNA sequence and translate it into a polypeptide sequence of the candidate antigen (e.g., GPA33), thereby triggering an immune response to the exogenous antigen. An attractive feature of mRNA antigens or mRNA vaccines is that mRNA is a non-infectious, non-integrating platform. There is no potential risk of infection or insertional mutagenesis associated with DNA vaccines. Furthermore, mRNA is degraded through normal cellular processes and has a controllable in vivo half-life through modification design and delivery methods (Kariko, K. et al., Mol Ther 16: 1833–1840 (2008); Kauffman, KJ et al., J Control Release 240, 227–234 (2016); Guan, S. and Rosenecker, J., GeneTher 24, 133–143 (2017); Thess, A. et al., Mol Ther 23, 1456–1464 (2015)). These techniques are standard in the art.

[0175] In describing the techniques of this invention, an immune response may be described as a “primary” or “secondary” immune response. A primary immune response, also described as a “protective” immune response, refers to an immune response that arises in an individual due to some initial exposure to a specific antigen, such as the GPA33 protein (e.g., initial “immunity”). In some embodiments, immunization may occur as a result of vaccination of an individual with a vaccine containing the antigen. For example, the vaccine may be a GPA33 vaccine containing one or more GPA33 protein-derived antigens. Over time, a primary immune response may weaken or diminish and may even disappear or at least diminish to the point of being undetectable. Therefore, the techniques of this invention also relate to a “secondary” immune response, also referred to herein as a “memory immune response.” The term secondary immune response refers to an immune response that arises in an individual after a primary immune response has already been generated.

[0176] Therefore, secondary immune responses can be triggered, such as to enhance existing immune responses that have weakened or diminished, or to reconstruct previous immune responses that have disappeared or are no longer detectable. Secondary or memory immune responses can be humoral (antibody) responses or cellular responses. Secondary or memory humoral responses occur when memory B cells, generated during the first presentation of an antigen, are stimulated. Delayed-type hypersensitivity (DTH) reactions are mediated by CD4+. + T cell-mediated secondary or memory immune responses. The first exposure to an antigen triggers the immune system, and subsequent exposures lead to DTH.

[0177] Following appropriate immunization, anti-GPA33 antibodies can be prepared from the serum of the subject. If desired, antibody molecules targeting the GPA33 protein can be isolated from mammals (e.g., from blood) and further purified using well-known techniques such as peptide A chromatography to obtain IgG fractions.

[0178] Monoclonal antibody. In one embodiment of the present invention, the antibody is an anti-GPA33 monoclonal antibody. For example, in some embodiments, the anti-GPA33 monoclonal antibody may be a human or mouse anti-GPA33 monoclonal antibody. To prepare a monoclonal antibody against the GPA33 protein or its derivatives, fragments, analogs, or homologs, any technique that provides antibody molecule production through continuous cell line culture can be utilized. Such techniques include, but are not limited to, hybridoma techniques (see, for example, Kohler and Milstein, 1975. Nature 256: 495-497); three-source hybridoma techniques; human B-cell hybridoma techniques (see, for example, Kozbor et al., 1983. Immunol. Today 4: 72); and EBV hybridoma techniques to produce human monoclonal antibodies (see, for example, Cole et al., 1985. MONCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies can be used in the practice of the present invention and can be produced using human hybridomas (see, for example, Cote et al., 1983. Proc. Natl. Acad. Sci. USA 80: 2026-2030) or by transforming human B cells in vitro with Epstein-Barr virus (see, for example, Cole et al., 1985. In: MONCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). For example, a population of nucleic acids encoding regions of antibodies can be isolated. PCR using primers derived from sequences encoding conserved regions of antibodies is used to amplify sequences encoding portions of antibodies from the population and then reconstruct DNA encoding antibodies or fragments thereof, such as variable domains, from the amplified sequences. Such amplified sequences can also be fused with DNA encoding other proteins—such as phage coats or bacterial cell surface proteins—for expression and display of fusion polypeptides on phages or bacteria. The amplified sequence can then be expressed and further selected or isolated based on, for example, the affinity of the expressed antibody or fragment thereof for the antigen or epitope present on the GPA33 protein. Alternatively, hybridomas expressing anti-GPA33 monoclonal antibodies can be prepared by immunizing a subject and then isolating the hybridoma from the subject's spleen using standard methods. See, for example, Milstein et al. (Galfre and Milstein, Methods Enzymol (1981) 73: 3-46). Screening hybridomas using standard methods will yield monoclonal antibodies with different specificities (i.e., for different epitopes) and affinities.Selected monoclonal antibodies with desired properties (e.g., GPA33 binding) expressed by hybridomas can be used. These antibodies can be bound to molecules such as polyethylene glycol (PEG) to alter their properties, or the cDNA encoding them can be isolated, sequenced, and manipulated in various ways. Synthetic dendromeric trees can be added to reactive amino acid side chains, such as lysine, to enhance the immunogenic properties of the GPA33 protein. Furthermore, CPG-dinucleotide technology can be used to enhance the immunogenic properties of the GPA33 protein. Other manipulations include substituting or deleting specific aminoacyl residues that cause instability of the antibody during storage or after administration to a subject, and affinity maturation techniques to improve the affinity of antibodies against the GPA33 protein.

[0179] Hybridoma technology. In some embodiments, the antibody of the present invention is an anti-GPA33 monoclonal antibody produced by a hybridoma comprising B cells obtained from a transgenic nonhuman animal, such as a transgenic mouse, thereby having a genome comprising human heavy chain transgenes and light chain transgenes fused with immortalized cells. Hybridoma technology includes those techniques known in the art and taught in the following literature: Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 349 (1988); Hammerling et al., Monoclonal Antibodies And T-Cell Hybridomas, 563-681 (1981). Other methods for producing hybridomas and monoclonal antibodies are well known to those skilled in the art.

[0180] Phage display technology. As described above, the antibodies of the present invention can be generated by applying recombinant DNA and phage display technology. For example, anti-GPA33 antibodies can be prepared using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles carrying a polynucleotide sequence encoding said functional antibody domain. Phages with desired binding properties are selected from a complete or combined antibody library (e.g., human or mouse) by directly selecting antigens, typically antigens that bind to or capture solid surfaces or beads. The phages used in these methods are typically filamentous phages, including fd and M13, having Fab, Fv, or disulfide bond-stabilized Fv antibody domains that are recombinantly fused to phage gene III or gene VIII proteins. In addition, the method can be applied to constructing Fab expression libraries (see, for example, Huse et al., Science 246: 1275-1281, 1989) to allow for rapid and efficient identification of monoclonal Fab fragments with desired specificity for GPA33 peptides, such as peptides or their derivatives, fragments, analogs or homologs.Other examples of phage display methods that can be used to prepare antibodies according to the present invention include those methods disclosed in the following literature: Huston et al., Proc. Natl. Acad. Sci USA, 85: 5879-5883, 1988; Chaudhary et al., Proc. Natl. Acad. Sci USA, 87: 1066-1070, 1990; Brinkman et al., J. Immunol. Methods 182: 41-50, 1995; Ames et al., J. Immunol. Methods 184: 177-186, 1995; Kettleborough et al., Eur. J. Immunol. 24: 952-958, 1994; Persic et al., Gene 187: 9-18, 1997; Burton et al., Advances in Immunology 57: 191-280, 1994; PCT / GB91 / 01134; WO 90 / 02809; WO 91 / 10737; WO 92 / 01047; WO 92 / 18619; WO 93 / 11236; WO 95 / 15982; WO 95 / 20401; WO 96 / 06213; WO 92 / 01047 (Medical Research Council et al); WO 97 / 08320 (Morphosys); WO 92 / 01047 (CAT / MRC); WO 91 / 17271 (Affymax); and U.S. Patent Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, and 5,733,743. Methods for displaying peptides on the surface of phage particles by linking peptides via disulfide bonds have been described in the following literature: Lohning, U.S. Patent No. 6,753,136. As described in the above references, after phage selection, antibody-coding regions from phages can be isolated and used to generate complete antibodies (containing human antibodies or any other desired antigen-binding fragments) and expressed in any desired host (including mammalian cells, insect cells, plant cells, yeast, and bacteria).For example, techniques for recombining Fab, Fab', and F(ab')2 fragments using methods known in the art can also be employed, such as those disclosed in the following documents: WO 92 / 22324; Mullinax et al., BioTechniques 12: 864-869, 1992; Sawai et al., AJRI 34: 26-34, 1995; and Better et al., Science 240: 1041-1043, 1988.

[0181] Typically, hybrid antibodies or hybrid antibody fragments cloned into a display vector can be selected against appropriate antigens to identify variants that maintain good binding activity, as the antibody or antibody fragment will be present on the surface of the phage or phage particle. See, for example, Barbas III et al., Phage Display, A Laboratory Manual (ColdSpring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001). However, other vector forms can also be used for this process, such as cloning antibody fragment libraries into lytic phage vectors (modified T7 or λZap systems) for selection and / or screening.

[0182] Expression of Recombinant Anti-GPA33 Antibodies. As described above, the antibodies of the present invention can be generated by applying recombinant DNA technology. Recombinant polynucleotide constructs encoding the anti-GPA33 antibodies of the present invention typically contain an expression control sequence operatively linked to the coding sequence of the anti-GPA33 antibody chain, which includes a naturally associated or heterologous promoter region. Therefore, another aspect of the present invention comprises a vector containing one or more nucleic acid sequences encoding the anti-GPA33 antibodies of the present invention. For recombinant expression of one or more of the polypeptides of the present invention, a nucleic acid containing all or part of the nucleotide sequence encoding the anti-GPA33 antibody is inserted into a suitable cloning vector or expression vector (i.e., a vector containing the necessary elements of the polypeptide coding sequence for transcription and translation insertion) using recombinant DNA technology well known in the art and detailed below. Methods for generating different vector populations have been described in the following documents: U.S. Patents 6,291,160 and 6,680,192, Lerner et al.

[0183] Typically, expression vectors used in recombinant DNA technologies are in plasmid form. In this disclosure, "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used vector form. However, the present invention is intended to include other forms of expression vectors that are not technically plasmids but provide equivalent functionality, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses). Such viral vectors allow a subject to be infected and to express the construct within that subject. In some embodiments, the expression control sequence is a eukaryotic promoter system in the vector capable of transforming or transfecting eukaryotic host cells. Once the vector has been incorporated into a suitable host, the host is maintained under conditions suitable for high-level expression of the nucleotide sequence encoding an anti-GPA33 antibody and for the collection and purification of the anti-GPA33 antibody (e.g., cross-reactive anti-GPA33 antibody). See generally US 2002 / 0199213. These expression vectors can typically replicate in a host organism as an episome or as part of the host chromosomal DNA. Typically, expression vectors contain selection markers, such as ampicillin resistance or hygromycin resistance, to allow detection of those cells transformed with the desired DNA sequence. Vectors may also encode secretory signal peptides, such as pectin lyase, that can be used to guide extracellular antibody fragments. See U.S. Patent No. 5,576,195.

[0184] The recombinant expression vectors of this invention comprise nucleic acids in a form suitable for expression in host cells, encoding proteins having GPA33-binding properties. This means that the recombinant expression vector contains one or more regulatory sequences selected based on the host cell for expression, which are operatively linked to the nucleic acid sequence to be expressed. In the recombinant expression vector, "operatively linked" is intended to mean that the nucleotide sequence of interest (e.g., in an in vitro transcription / translation system or in the host cell when the vector is introduced into the host cell) is linked to the regulatory sequence in a manner that allows the expression of the nucleotide sequence. The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS INENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those sequences that guide constitutive expression of the nucleotide sequence in many types of host cells and those sequences that guide expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Those skilled in the art will understand that the design of expression vectors can depend on factors such as the choice of host cells to be transformed and the desired expression level of the peptide. Typical regulatory sequences that can be used as promoters for recombinant peptide expression (e.g., anti-GPA33 antibody) include, for example, but not limited to, promoters of 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for the utilization of maltose and galactose. In one embodiment, a polynucleotide encoding the anti-GPA33 antibody of the present invention is operatively linked to an ara B promoter and can be expressed in a host cell. See U.S. Patent 5,028,530. Expression vectors of the present invention can be introduced into host cells, thereby producing peptides or polypeptides (e.g., anti-GPA33 antibody, etc.) encoded by nucleic acids as described herein.

[0185] Another aspect of the present invention relates to host cells expressing anti-GPA33 antibodies, said host cells containing nucleic acids encoding one or more anti-GPA33 antibodies. The recombinant expression vectors of the present invention can be designed for expression of anti-GPA33 antibodies in prokaryotic or eukaryotic cells. For example, anti-GPA33 antibodies can be expressed in bacterial cells such as *Escherichia coli*, insect cells (using baculovirus expression vectors), fungal cells such as yeast, yeast cells, or mammalian cells. Suitable host cells are further discussed in *Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185*, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vectors can be transcribed and translated in vitro, for example, using a T7 promoter regulatory sequence and a T7 polymerase. Methods for preparing and screening polypeptides (e.g., anti-GPA33 antibodies) with predetermined properties via the expression of randomly generated polynucleotide sequences have previously been described. See U.S. Patent Nos. 5,763,192; 5,723,323; 5,814,476; 5,817,483; 5,824,514; 5,976,862; 6,492,107; 6569641.

[0186] In prokaryotes, peptide expression is most frequently performed in *E. coli* using vectors containing constitutive or inducible promoters that guide the expression of fusion or non-fusion peptides. Fusion vectors add numerous amino acids to the peptide they encode, typically to the N-terminus of the recombinant peptide. Such fusion vectors generally serve three purposes: (i) to increase the expression of the recombinant peptide; (ii) to increase the solubility of the recombinant peptide; and (iii) to aid in the purification of the recombinant peptide by acting as a ligand in affinity purification. Typically, in fusion expression vectors, proteolytic cleavage sites are introduced at the junction of the fusion moiety and the recombinant peptide to allow for the separation of the recombinant peptide from the fusion moiety after purification. Such enzymes and their homologous recognition sequences include factor Xa, thrombin, and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.), and pRIT5 (Pharmacia, Piscataway, NJ), which fuse glutathione S-transferase (GST), maltose E-binding peptide, or peptide A with a targeted recombinant peptide, respectively.

[0187] Examples of suitable inducible non-fusion *E. coli* expression vectors include pTrc (Amrann et al., (1988) Gene 69: 301-315) and pET 11d (Studier et al., *GENE EXPRESSION TECHNOLOGY: METHODSIN ENZYMOLOGY 185*, Academic Press, San Diego, Calif. (1990) 60-89). Methods for targeting the assembly of different bioactive peptides or protein domains via peptide fusion to generate multifunctional peptides have been described in the following literature: Pack et al., US Patent Nos. 6,294,353; 6,692,935. One strategy to maximize the expression of recombinant peptides, such as anti-GPA33 antibodies, in *E. coli* is to express the peptide in host bacteria with impaired proteolytic cleavage capabilities of the recombinant peptide. See, for example, Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to modify the nucleic acid sequence of the nucleic acid to be inserted into the expression vector so that the individual codons for each amino acid are those preferentially used in the expression host, such as *E. coli* (see, for example, Wada et al., 1992. *Nucl. Acids Res.* 20: 2111-2118). Such modifications to the nucleic acid sequence in this invention can be performed using standard DNA synthesis techniques.

[0188] In another embodiment, the anti-GPA33 antibody expression vector is a yeast expression vector. Examples of vectors used for expression in *Saccharomyces cerevisiae* include pYepSec1 (Baldari et al., 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, Cell 30: 933-943, 1982), pJRY88 (Schultz et al., Gene 54:113-123, 1987), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (Invitrogen Corp., San Diego, Calif.). Alternatively, the anti-GPA33 antibody can be expressed in insect cells using a baculovirus expression vector. Baculovirus vectors that can be used to express peptides (e.g., anti-GPA33 antibody) in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith et al., Mol. Cell. Biol. 3: 2156-2165, 1983) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).

[0189] In yet another embodiment, the nucleic acid encoding the anti-GPA33 antibody of the present invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include, but are not limited to, pCDM8 (Seed, Nature 329: 840, 1987) and pMT2PC (Kaufman et al., EMBO J. 6: 187-195, 1987). When used in mammalian cells, the control function of the expression vector is often provided by viral regulatory elements. For example, commonly used promoters are derived from polyomavirus, adenovirus 2, cytomegalovirus, and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells that can be used to express the anti-GPA33 antibody of the present invention, see, for example, Chapters 16 and 17 of Sambrook et al., MOLECULARCLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

[0190] In another embodiment, the recombinant mammalian expression vector is capable of guiding nucleic acid expression in specific cell types (e.g., tissue-specific regulatory elements). Tissue-specific regulatory elements are known in the art. Non-restrictive examples of suitable tissue-specific promoters include albumin promoters (liver-specific; Pinkert et al., Genes Dev. 1:268-277, 1987), lymphocyte-specific promoters (Calame and Eaton, Adv. Immunol. 43: 235-275, 1988), T-cell receptor promoters (Winoto and Baltimore, EMBO J. 8: 729-733, 1989) and immunoglobulin promoters (Banerji et al., 1983. Cell 33: 729-740; Queen and Baltimore, Cell 33: 741-748, 1983.), neuron-specific promoters (e.g., neurofilament promoters; Byrne and Ruddle, Proc. Natl. Acad. Sci. USA 86: 5473-5477, 1989), pancreas-specific promoters (Edlund et al., 1985. Science 230:912-916) and mammary gland-specific promoters (e.g., whey promoter; US Patent No. 4,873,316 and European Application Publication No. 264,166). Developmental regulatory promoters are also covered, such as the mouse hox promoter (Kessel and Gruss, Science 249:374-379, 1990) and the alpha-fetoprotein promoter (Campes and Tilghman, Genes Dev.3: 537-546, 1989).

[0191] Another aspect of the method of the present invention relates to host cells in which the recombinant expression vector of the present invention has been incorporated. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It should be understood that such terms refer not only to specific subject cells but also to the progeny or potential progeny of such cells. Because certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may not actually be identical to the parent cells, but are still included within the scope of the terminology used herein.

[0192] The host cell can be any prokaryotic or eukaryotic cell. For example, the anti-GPA33 antibody peptide can be expressed in bacterial cells, such as *E. coli*, insect cells, yeast, or mammalian cells. Mammalian cells are a suitable host for expressing nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, *From Genes To Clones*, (VCH Publishers, NY, 1987). Several suitable host cell lines capable of secreting intact heterologous proteins have been developed in the art, including Chinese hamster ovary (CHO) cell lines, various COS cell lines, HeLa cells, L cells, and myeloma cell lines. In some embodiments, the cells are non-human cells. Expression vectors targeting these cells may contain expression control sequences such as origin of replication, promoters, and enhancers, and essential processing information sites such as ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcription terminator sequences. Queen et al., *Immunol. Rev. 89: 49*, 1986. The illustrative expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papillomavirus, etc. (Co et al., J Immunol. 148: 1149, 1992). Other suitable host cells are known to those skilled in the art.

[0193] Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of field-recognized techniques for introducing exogenous nucleic acids (e.g., DNA) into host cells, including calcium phosphate or calcium chloride coprecipitation, DEAE-dextran-mediated transfection, lipid transfection, electroporation, gene gun transfection, or virus-based transfection. Other methods for transforming mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection (see Sambrook et al., Molecular Cloning for general information). Appropriate methods for transforming or transfecting host cells can be found in Sambrook et al. (MOLECULAR CLONING: ALABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and other laboratory manuals. Depending on the type of cell host, vectors containing the DNA segment of interest can be transferred into host cells using well-known methods.

[0194] Non-limiting examples of suitable vectors include those designed for proliferation and amplification, or for expression, or both. For example, cloning vectors can be selected from the group consisting of: the pUC series, the pBluescript series (Stratagene, LaJolla, Calif.), the pET series (Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, Calif.). Phage vectors such as λ-GT10, λ-GT11, λ-ZapII (Stratagene), λ-EMBL4, and λ-NM1149 can also be used. Non-limiting examples of plant expression vectors include pBI110, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Non-limiting examples of animal expression vectors include pEUK-C1, pMAM, and pMAMneo (Clontech). The TOPO cloning system (Invitrogen, Calsbad, CA, Carlsbad, CA) can also be used according to the manufacturer's recommendations.

[0195] In some embodiments, the vector is a mammalian vector. In some embodiments, the mammalian vector contains at least one promoter element that mediates the initiation of mRNA transcription, antibody-coding sequences, and signals required for transcription termination and transcript polyadenylation. In some embodiments, the mammalian vector contains additional elements, such as, for example, enhancers, Kozak sequences, and intercalation sequences at donor and recipient sites for lateral RNA splicing. In some embodiments, efficient transcription can be achieved using, for example, early and late promoters from SV40, long terminal repeat sequences (LTRS) from retroviruses such as RSV, HTLVI, and HIVI, and early promoters from cytomegalovirus (CMV). Cellular elements (e.g., human actin promoters) can also be used. Non-limiting examples of mammalian expression vectors include vectors such as pIRESlneo, pRetro-Off, pRetro-On, PLXSN or pLNCX (Clonetech Labs, Palo Alto, Calif.), pcDNA3.1 (+ / -), pcDNA / Zeo (+ / -) or pcDNA3.1 / Hygro (+ / -) (Invitrogen, Calsbad, CA), PSVL and PMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109). Non-limiting examples of mammalian host cells that can be used in combination with such mammalian vectors include human Hela 293, HEK 293, H9 and Jurkat cells, mouse 3T3, NIH3T3 and C127 cells, Cos 1, Cos 7 and CV 1, quail QC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.

[0196] In some embodiments, the vector is a viral vector, such as a retroviral vector, a parvovirus-based vector, such as an adeno-associated virus (AAV)-based vector, an AAV-adenovirus chimeric vector, and an adenovirus-based vector, as well as a lentiviral vector, such as a herpes simplex virus (HSV)-based vector. In some embodiments, the viral vector is manipulated to render viral replication defective. In some embodiments, the viral vector is manipulated to eliminate toxicity to the host. These viral vectors can be prepared using standard recombinant DNA techniques described, for example, in the following literature: Sambrook et al., Molecular Cloning, a Laboratory Manual, 2nd edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989); and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, NY (1994).

[0197] In some embodiments, the vectors or polynucleotides described herein can be transferred to cells (e.g., ex vivo cells) using conventional techniques, and the resulting cells can be cultured using conventional techniques to produce the anti-GPA33 antibody or antigen-binding fragment described herein. Therefore, this document provides cells comprising a polynucleotide encoding an anti-GPA33 antibody or an antigen-binding fragment thereof, said polynucleotide being operatively linked to a regulatory expression element (e.g., a promoter) for expressing such a sequence in a host cell. In some embodiments, a vector encoding a heavy chain operatively linked to a promoter and a vector encoding a light chain operatively linked to a promoter can be co-expressed in cells to express the complete anti-GPA33 antibody or antigen-binding fragment. In some embodiments, the cell comprises a vector comprising a polynucleotide encoding both a heavy chain and a light chain of the anti-GPA33 antibody or antigen-binding fragment described herein, said polynucleotide being operatively linked to a promoter. In some embodiments, the cell comprises two different vectors: a first vector comprising a polynucleotide encoding a heavy chain operatively linked to a promoter; and a second vector comprising a polynucleotide encoding a light chain operatively linked to a promoter. In some embodiments, a first cell comprises a first vector containing a heavy chain polynucleotide encoding a heavy chain of an anti-GPA33 antibody or antigen-binding fragment as described herein, and a second cell comprises a second vector containing a light chain polynucleotide encoding a light chain of an anti-GPA33 antibody or antigen-binding fragment as described herein. In some embodiments, a mixture of cells comprising the first cell and the second cell is provided herein. Examples of cells include, but are not limited to, human cells, human cell lines, *Escherichia coli* (e.g., *E. coli* TB-1, TG-2, DH5a, XL-Blue MRF' (Stratagene), SA2821, and Y1090), *Bacillus subtilis*, *Pseudomonas aeruginosa*, *Saccharomyces cerevisiae*, *Neurospora crassa*, insect cells (e.g., Sf9, Ea4), etc.

[0198] To stably transfect mammalian cells, it is known that, depending on the expression vector and transfection technique used, only a small fraction of cells can incorporate foreign DNA into their genome. To identify and select these integrators, genes encoding selectable markers (e.g., antibiotic resistance) are typically introduced into host cells along with the gene of interest. Various selectable markers include those that confer drug resistance, such as G418, hygromycin, and methotrexate. The nucleic acid encoding the selectable marker can be introduced into host cells on the same vector as the one encoding the anti-GPA33 antibody, or it can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells with the selectable marker gene will survive, while other cells will die).

[0199] Host cells (such as prokaryotic or eukaryotic host cells in a culture) incorporating the anti-GPA33 antibody of the present invention can be used to generate (i.e., express) recombinant anti-GPA33 antibodies. In one embodiment, the method includes culturing host cells in a suitable culture medium (in which a recombinant expression vector encoding the anti-GPA33 antibody has been introduced) to generate the anti-GPA33 antibody. In another embodiment, the method further includes the step of isolating the anti-GPA33 antibody from the culture medium or host cells. Once expressed, an assembly of anti-GPA33 antibodies, such as anti-GPA33 antibodies or anti-GPA33 antibody-associated peptides, is purified from the culture medium and host cells. The anti-GPA33 antibody can be purified according to standard procedures in the art, including HPLC purification, column chromatography, gel electrophoresis, etc. In one embodiment, the anti-GPA33 antibody is generated in a host organism by the method described in U.S. Patent No. 4,816,397 to Boss et al. Typically, the anti-GPA33 antibody chain is expressed with a signal sequence and thus released into the culture medium. However, if the anti-GPA33 antibody chain is not naturally secreted by the host cell, it can be released by treatment with a mild detergent. Purification of recombinant peptides is well-known in the art and includes ammonium sulfate precipitation, affinity chromatography, column chromatography, ion exchange purification, gel electrophoresis, etc. (see Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

[0200] Polynucleotides encoding anti-GPA33 antibodies, such as anti-GPA33 antibody coding sequences, can be incorporated into transgenes to be introduced into the genome of transgenic animals and subsequently expressed in their milk. See, for example, U.S. Patent Nos. 5,741,957, 5,304,489, and 5,849,992. Suitable transgenes include coding sequences of light and / or heavy chains operatively linked to promoters and enhancers from mammary gland-specific genes such as casein or β-lactoglobulin. To produce transgenic animals, the transgene can be microinjected into fertilized oocytes, or it can be incorporated into the genome of embryonic stem cells, with the nucleus of such cells transferred into enucleated oocytes.

[0201] Single-chain antibody. In one embodiment, the anti-GPA33 antibody of the present invention is a single-chain anti-GPA33 antibody. According to the present invention, the technique can be adapted to generate single-chain antibodies specific to the GPA33 protein (see, for example, U.S. Patent No. 4,946,778). Examples of techniques that can be used to generate single-chain Fv and antibodies of the present invention include those described in the following: U.S. Patent Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology, 203:46-88, 1991; Shu, L. et al., Proc. Natl. Acad. Sci. USA, 90: 7995-7999, 1993; and Skerra et al., Science 240: 1038-1040, 1988.

[0202] Chimeric and humanized antibodies. In one embodiment, the anti-GPA33 antibody of the present invention is a chimeric anti-GPA33 antibody. In another embodiment, the anti-GPA33 antibody of the present invention is a humanized anti-GPA33 antibody. In one embodiment of the present invention, the donor and recipient antibodies are monoclonal antibodies from different species. For example, the recipient antibody is a human antibody (to minimize its antigenicity in the human body), in which case the resulting CDR transplanted antibody is referred to as a "humanized" antibody.

[0203] Recombinant anti-GPA33 antibodies, such as chimeric and humanized monoclonal antibodies comprising both human and non-human portions, can be prepared using standard recombinant DNA techniques and are within the scope of this invention. For some applications, including the in vivo use of the anti-GPA33 antibodies of this invention in humans and the use of these agents in in vitro detection assays, it is possible to use chimeric or humanized GPA33 antibodies. Such chimeric and humanized monoclonal antibodies can be produced using recombinant DNA techniques known in the art. Such useful methods include, for example, but not limited to, those described in the following documents: International Application No. PCT / US86 / 02269; US Patent No. 5,225,539; European Patent No. 184187; European Patent No. 171496; European Patent No. 173494; PCT International Publication No. WO 86 / 01533; US Patent Nos. 4,816,567; 5,225,539; European Patent No. 125023; Better et al., 1988. Science 240: 1041-1043; Liu et al., 1987. Proc. Natl. Acad. Sci. USA 84: 3439-3443; Liu et al., 1987. J. Immunol. 139: 3521-3526; Sun et al., 1987. Proc. Natl. Acad. Sci. USA 84: 214-218; Nishimura et al., 1987. Cancer Res. 47: 999-1005; Wood et al., 1985. Nature 314: 446-449; Shaw et al., 1988. J. Natl. Cancer Inst. 80: 1553-1559; Morrison (1985) Science 229: 1202-1207; Oi et al. (1986) BioTechniques 4: 214; Jones et al., 1986. Nature 321: 552-525; Verhoeyan et al., 1988. Science 239: 1534; Morrison, Science 229: 1202, 1985; Oi et al., BioTechniques 4: 214, 1986; Gillies et al., J. Immunol. Methods, 125: 191-202, 1989; U.S. Patent No. 5,807,715; and Beidler et al., 1988. J. Immunol. 141: 4053-4060.For example, a variety of techniques can be used to humanize antibodies, including CDR transplantation (EP 0 239 400; WO 91 / 09967; US Patent Nos. 5,530,101; 5,585,089; 5,859,205; 6,248,516; EP460167), trimming or remodeling (EP 0 592 106; EP 0 519 596; Padlan EA, Molecular Immunology, 28: 489-498, 1991; Studnicka et al., Protein Engineering 7: 805-814, 1994; Roguska et al., PNAS 91:969-973, 1994), and chain tampering (US Patent No. 5,565,332). In one embodiment, the cDNA encoding a mouse anti-GPA33 monoclonal antibody is digested with a specially selected restriction enzyme to remove the sequence encoding the Fc constant region, and an equivalent portion of the cDNA encoding the human Fc constant region is replaced (see Robinson et al. PCT / US86 / 02269; Akira et al. European Patent Application 184,187; Taniguchi, European Patent Application 171,496; Morrison et al. European Patent Application 173,494; Neuberger et al. WO 86 / 01533; Cabilly et al. US Patent No. 4,816,567; Cabilly et al. European Patent Application 125,023; Better et al. (1988) Science 240: 1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84: 3439-3443; Liu et al. (1987) J Immunol 139: 3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84: 214-218; Nishimura et al. (1987) Cancer Res 47: 999-1005; Wood et al. (1985) Nature 314: 446-449; and Shaw et al. (1988) J. Natl. Cancer Inst. 80: 1553-1559; U.S. Patent No. 6,180,370; U.S. Patent No. 6,300,064; 6,696,248; 6,706,484; 6,828,422.

[0204] In one embodiment, the present invention provides the construction of a humanized anti-GPA33 antibody that is unlikely to induce a human anti-mouse antibody (hereinafter referred to as "HAMA") response while still possessing effective antibody effector function. As used herein, the antibody-related terms "human" and "humanized" refer to any antibody expected to elicit a therapeutically tolerable, weakly immunogenic response in a human subject. In one embodiment, the present invention provides a humanized anti-GPA33 antibody, heavy chain immunoglobulin, and light chain immunoglobulin.

[0205] CDR antibodies. In some embodiments, the anti-GPA33 antibody of the present invention is an anti-GPA33 CDR antibody. Generally, the donor and recipient antibodies used to generate anti-GPA33 CDR antibodies are monoclonal antibodies from different species; typically, the recipient antibody is a human antibody (to minimize its antigenicity in the human body), in which case the resulting CDR transplanted antibody is referred to as a "humanized" antibody. The graft can belong to a single V of the recipient antibody. H or V L A single CDR (or even a part of a single CDR) within or that can belong to V H and V L Multiple CDRs (or portions thereof) within one or both of the receptor antibody's variable domains are typically replaced with corresponding donor CDRs, although only the required number need to be replaced to allow the resulting CDR-grafted antibody to bind adequately to the GPA33 protein. The following literature teaches methods for generating CDR-grafted and humanized antibodies: Queen et al., US Patent No. 5,585,089; US Patent No. 5,693,761; US ​​Patent No. 5,693,762; and Winter US 5,225,539; and EP 0682040. The following references teach methods that can be used to prepare V H and V L Methods for peptides: Winter et al., U.S. Patent Nos. 4,816,397; 6,291,158; 6,291,159; 6,291,161; 6,545,142; EP 0368684; EP0451216; and EP0120694.

[0206] After selecting suitable frame region candidates from the same family and / or members of the same family, one or both of the heavy chain variable regions and light chain variable regions are generated by transplanting a CDR from the source species into the hybridization frame region. The assembly of hybrid antibodies or hybrid antibody fragments with hybridization variable chain regions for any of the above aspects can be accomplished using conventional methods known to those skilled in the art. For example, DNA sequences encoding the hybridization variable domains described herein (i.e., frames based on the target species and CDRs from the source species) can be generated by oligonucleotide synthesis and / or PCR. Nucleic acids encoding the CDR regions can also be isolated from the source species antibody using suitable restriction endonucleases and ligated to the target species frame by ligation with suitable ligases. Alternatively, the frame regions of the variable chain of the source species antibody can be altered by site-directed mutagenesis.

[0207] Since hybrids are constructed from multiple candidates corresponding to each frame region, numerous sequence combinations exist, which can be constructed according to the principles described herein. Therefore, libraries of hybrids with different combinations of members having a single frame region can be assembled. Such libraries can be collections of electronic databases of sequences or physical collections of hybrids.

[0208] This process typically does not alter the receptor FR of the recipient antibody for the laterally transplanted CDR. However, those skilled in the art can sometimes improve the antigen-binding affinity of the resulting anti-GPA33CDR transplanted antibody by replacing certain residues of a given FR to make the FR more similar to the corresponding FR of the donor antibody. Suitable positions for substitution include amino acid residues adjacent to the CDR, or amino acid residues capable of interacting with the CDR (see, for example, US 5,585,089, especially columns 12-16). Alternatively, those skilled in the art can start with the donor FR and modify it to be more similar to the recipient FR or a human common FR. Techniques for making these modifications are known in the art. In particular, if the resulting FR conforms to a human common FR at that position, or is at least 90% or more identical to such a common FR, doing so does not significantly increase the antigenicity of the resulting modified anti-GPA33CDR transplanted antibody compared to the same antibody having a fully human FR.

[0209] Multispecific fusion proteins. Multispecific fusion proteins, such as bispecific antibodies (BsAb) and bispecific antibody fragments (BsFab), have at least one arm that specifically binds to, for example, GPA33 and at least one other arm that specifically binds to a second target antigen.

[0210] A bispecific antibody is an antibody that can simultaneously bind to two targets with different structures (e.g., two different target antigens, two different epitopes on the same target antigen, or a hapten and a target antigen or an epitope on a target antigen). BsAbs can be prepared, for example, by combining heavy and / or light chains that recognize different epitopes of the same or different antigens. In some embodiments, by molecular function, a bispecific binder binds to one antigen (or epitope) on one of its two binding arms (a VH / VL pair) and to a different antigen (or epitope) on its second arm (a different VH / VL pair). By this definition, a bispecific binder has two distinct antigen-binding arms (in both specificity and CDR sequence) and is monovalent for each antigen it binds to.

[0211] The bispecific antibody (BsAb) and bispecific antibody fragment (BsFab) of the present invention have at least one arm that specifically binds to, for example, GPA33 and at least one other arm that specifically binds to a second target antigen. In some embodiments, the second target antigen is an antigen or epitope of B cells, T cells, myeloid cells, plasma cells, or mast cells. Alternatively or concurrently, in some embodiments, the second target antigen is selected from the group consisting of: CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, CD32, CD64, TCRγ / δ, NKp46, KIR, PD-1, PD-L1, LAG3, CD28, B7H3, STEAP1, HER2, EGFR, CEA, CECAM5, transferrin receptor, FAP, NKG2D-ligand, TRAIL, FasL, cathepsin G, granzyme, carboxypeptidase, and β-lactamase. In some embodiments, BsAb is capable of binding to tumor cells expressing the GPA33 antigen on their cell surface. In some embodiments, BsAb has been engineered to promote tumor cell killing by directing (or recruiting) cytotoxic T cells to tumor sites. Other exemplary BsAbs include those having a first antigen-binding site specific to GPA33 and a second antigen-binding site specific to a small molecule hapten (e.g., DTP A, IMP288, DOTA, DOTA-Bn, DOTA deferoxamine, DOTA (metal) complex, benzyl-DOTA (metal) complex, Proteus spp.-DOTA (metal) complex, NOGADA-Proteus spp.-DOTA (metal) complex, astr-DFO (metal) complex, DFO (metal) complex, other DOTA chelates described herein, biotin, fluorescein, or those disclosed in Goodwin, D A. et al., 1994, Cancer Res. 54(22):5937-5946). In some embodiments, the bispecific antibody or bispecific antigen-binding fragment includes a catalytic antibody, an immune checkpoint inhibitor, or an immune checkpoint activator.

[0212] Molecular engineering can be used to generate a variety of multispecific fusion proteins. For example, BsAbs have been constructed utilizing a whole immunoglobulin framework (e.g., IgG), single-chain variable fragments (scFvs), or combinations thereof. In some embodiments, the bispecific fusion protein is bivalent, comprising, for example, an scFv having a single binding site for one antigen and a Fab fragment having a single binding site for a second antigen. In some embodiments, the bispecific fusion protein is bivalent, comprising, for example, an scFv having a single binding site for one antigen and another scFv fragment having a single binding site for a second antigen. In other embodiments, the bispecific fusion protein is tetravalent, comprising, for example, an immunoglobulin (e.g., IgG) having two binding sites for one antigen and two identical scFvs for a second antigen. BsAbs consisting of two scFv units tandemly have proven to be a clinically successful form of bispecific antibody. In some embodiments, BsAbs have been engineered to include two single-chain variable fragments (scFvs) tandemly, such that an scFv binding to a tumor antigen (e.g., GPA33) is linked to an scFv binding to T cells (e.g., by binding to CD3). In this way, T cells are recruited to tumor sites, enabling them to mediate cytotoxic killing of tumor cells. See, for example, Dreier et al., J. Immunol. 170: 4397-4402 (2003); Bargou et al., Science 321: 974-977 (2008). In some embodiments, BsAbs have been designed to contain two single-stranded variable fragments (scFvs) in tandem, such that the scFv binding to a tumor antigen (e.g., GPA33) is linked to the scFv binding to the small molecule DOTA hapten.

[0213] Recent methods for generating BsAbs include engineered recombinant monoclonal antibodies with additional cysteine ​​residues, resulting in stronger cross-linking than more common immunoglobulin isotypes. See, for example, FitzGerald et al., Protein Eng. 10(10):1221-1225 (1997). Another approach is to engineer recombinant fusion proteins that link two or more different single-chain antibodies or antibody fragments to the desired bispecificity. See, for example, Coloma et al., Nature Biotech. 15:159-163 (1997). A variety of bispecific fusion proteins can be generated using molecular engineering.

[0214] Multispecific fusion proteins linking two or more different single-chain antibodies or antibody fragments can be generated in a similar manner. Recombinant methods can be used to generate a variety of fusion proteins. In some embodiments, the BsAb according to the invention comprises an immunoglobulin and a scFv, said immunoglobulin comprising a heavy chain and a light chain. In some embodiments, the scFv is linked to the C-terminus of the heavy chain of any GPA33 immunoglobulin disclosed herein. In some embodiments, the scFv is linked to the C-terminus of the light chain of any GPA33 immunoglobulin disclosed herein.

[0215] In various embodiments, scFv is ligated to the heavy or light chain via an adapter sequence. The appropriate adapter sequence necessary for ligation of the heavy chain Fd to the scFv frame is introduced into the V via a PCR reaction. L and V κ The DNA fragment encoding scFv was then ligated into a hierarchical vector containing a DNA sequence encoding the CH1 domain. The resulting scFv-CH1 construct was excised and ligated into a V vector containing an antibody encoding the GPA33 domain. H The resulting vector contains the DNA sequence of the region. This vector can then be used to transfect appropriate host cells, such as mammalian cells, to express multispecific fusion proteins.

[0216] In some embodiments, the length of the linker is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids. In some embodiments, the linker is characterized by its tendency to not adopt a rigid three-dimensional structure, but rather to provide flexibility for the polypeptide (e.g., the first and / or second antigen binding site). In some embodiments, the linker is employed in the multispecific fusion proteins described herein based on specific properties conferred upon the multispecific fusion protein, such as, for example, increased stability. In some embodiments, the multispecific fusion protein of the present invention comprises a G4S linker. In some other embodiments, the multispecific fusion protein of the present invention comprises (G4S). n Connector, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or greater.

[0217] Self-Assembled Disassembly (SADA) Conjugates. In some embodiments, the anti-GPA33 antibody of the present invention comprises one or more SADA domains. SADA domains can be designed and / or customized to achieve environment-dependent polymerization with beneficial kinetic, thermodynamic, and / or pharmacological properties. For example, it should be appreciated that the SADA domain can be part of a conjugate that allows efficient delivery of the payload to the target site of interest while minimizing the risk of off-target interactions. The anti-GPA33 antibody of the present invention may include a SADA domain linked to one or more binding domains. In some embodiments, such conjugates are characterized by polymerizing under relevant conditions (e.g., in solution, where the concentration or pH of the conjugate is above a threshold and / or when present at a target site characterized by a relevant level or density of the receptor for the payload) to form a complex of a desired size, and disassembling into a smaller form under other conditions (e.g., without relevant environmental polymerization triggering).

[0218] Compared to conjugates without the SADA domain, SADA conjugates can exhibit improved properties. In some embodiments, the improved properties of the multimeric conjugate include: increased affinity / binding to the target, increased specificity to target cells or tissues, and / or prolonged initial serum half-life. In some embodiments, the improved properties include reduced nonspecific binding, decreased toxicity, and / or improved renal clearance by dissociating into smaller states (e.g., dimers or monomers). In some embodiments, the SADA conjugate comprises a SADA polypeptide having an amino acid sequence showing at least 75% identity with the amino acid sequence of a human isomeric polypeptide, and is characterized by one or more multimerization dissociation constants (K0). D ).

[0219] In some embodiments, the SADA conjugate is constructed and arranged such that it adopts a first polymerized state and one or more higher-order polymerized states. In some embodiments, the size of the first polymerized state is less than about 70 kDa. In some embodiments, the first polymerized state is a non-polymerized state (e.g., monomer or dimer). In some embodiments, the first polymerized state is a monomer. In some embodiments, the first polymerized state is a dimer. In some embodiments, the first polymerized state is a polymerized state (e.g., trimer or tetramer). In some embodiments, the higher-order polymerized state is a homotetramer or a higher-order homopolymer with a size greater than 150 kDa. In some embodiments, when the conjugate adopts a size greater than that of the SADA peptide K D In the presence of certain concentrations, higher-order homopolymer conjugates are stable in aqueous solution. In some embodiments, when the concentration of the conjugate is below that of the SADA peptide K... DUnder physiological conditions, SADA conjugates transition from a higher-order polymerization state to the first polymerization state.

[0220] In some embodiments, the SADA peptide is covalently linked to the binding domain via a linker. Any suitable linker known in the art can be used. In some embodiments, the SADA peptide is linked to the binding domain via a peptide linker. In some embodiments, the peptide linker is a Gly-Ser linker. In some embodiments, the peptide linker is or contains a sequence of (GGGGS)n, where n represents the number of repeating GGGGS units and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more. In some embodiments, the binding domain is directly fused to the SADA peptide.

[0221] In some embodiments, the SADA domain is a human polypeptide or a fragment and / or derivative thereof. In some embodiments, the SADA domain is substantially non-immunogenic in the human body. In some embodiments, the SADA polypeptide is stable as a polymer. In some embodiments, the SADA polypeptide lacks unpaired cysteine ​​residues. In some embodiments, the SADA polypeptide does not have a large exposed hydrophobic surface. In some embodiments, the SADA domain has or is predicted to have a structure comprising helical bundles that can associate in parallel or antiparallel orientations. In some embodiments, the SADA polypeptide is capable of reversible polymerization. In some embodiments, the SADA domain is a tetramerizing domain, a heptamerizing domain, a hexamerizing domain, or an octamerizing domain. In some embodiments, the SADA domain is a tetramerizing domain. In some embodiments, the SADA domain is composed of polymerizing domains, each polymerizing domain consisting of helical bundles associated in parallel or antiparallel orientations. In some embodiments, the SADA domain is selected from the group consisting of one of the following human proteins: p53, p63, p73, heteronuclear ribonucleoprotein C (hnRNPC), the N-terminal domain of synaptosome-associated protein 23 (SNAP-23), Stefin B (cystatin B), potassium voltage-gated channel subfamily KQT member 4 (KCNQ4), or the tetramerized domain of cyclin-D-associated protein (CBFA2T1). Examples of suitable SADA domains are described in PCT / US2018 / 031235, which is hereby incorporated herein by reference in its entirety. The polypeptide sequences of exemplary SADA domains are provided below.

[0222] Human p53 tetramerization domain amino acid sequence (321-359): KPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEP (SEQ ID NO: 63)

[0223] Human p63 tetramerization domain amino acid sequence (396-450): RSPDDELLYLPVRGRETYEMLLKIKESLELMQYLPQHTIETYRQQQQQQHQHLLQKQ (SEQ ID NO: 64)

[0224] The amino acid sequence of the human p73 tetramerization domain (348-399) is RHGDEDTYYLQVRGRENFEILMKLKESLELMELVPQPLVDSYRQQQQLLQRP (SEQ ID NO: 65).

[0225] The amino acid sequence of the human HNRNPC tetramerization domain (194-220) is QAIKKELTQIKQKVDSLLENLEKIEKE (SEQ ID NO: 66).

[0226] Human SNAP-23 tetramerization domain amino acid sequence (23-76) STRRILGLAIESQDAGIKTITMLDEQKEQLNRIEEGLDQINKDMRETEKTLTEL (SEQ ID NO: 67)

[0227] Human Stefin B tetramerization domain amino acid sequence (2-98) MCGAPSATQPATAETQHIADQVRSQLEEKENKKFPVFKAVSFKSQVVAGTNYFIKVHVGDEDFVHLRVFQSLPHENKPLTLSNYQTNKAKHDELTYF (SEQ ID NO: 68)

[0228] The amino acid sequence of the KCNQ4 tetramerization domain (611-640) is DEISMMGRVVKVEKQVQSIEHKLDLLLGFY (SEQ ID NO: 69).

[0229] The amino acid sequence of the CBFA2T1 tetramerization domain (462-521) is: TVAEAKRQAAEDALAVINQQEDSSESCWNCGRKASETCSGCNTARYCGSFCQHKDWEKHH (SEQ ID NO: 70)

[0230] In some embodiments, the SADA polypeptide is or comprises the N-terminal domain of p53, p63, p73, heterologous nucleoribonucleoprotein C (hnRNPC), synaptosome-associated protein 23 (SNAP-23), Stefin B (cystatin B), potassium voltage-gated channel subfamily KQT member 4 (KCNQ4), or cyclin-D-associated protein (CBFA2T1). In some embodiments, the SADA polypeptide is or comprises at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in any of SEQ ID NO: 63 to 70.

[0231] Fc modification. In some embodiments, the anti-GPA33 antibody of the present invention comprises a variant Fc region, wherein the variant Fc region comprises at least one amino acid modification relative to the wild-type Fc region (or parental Fc region), such that the molecule has an altered affinity for an Fc receptor (e.g., FcγR), provided that the variant Fc region does not have substitutions at sites directly in contact with the Fc receptor, based on crystallographic and structural analyses of Fc-Fc receptor interactions, such as those disclosed by Sondermann et al., Nature, 406:267-273 (2000). Examples of sites within the Fc region directly in contact with an Fc receptor (such as FcγR) include amino acids 234-239 (hinge region), amino acids 265-269 (B / C ring), amino acids 297-299 (C7E ring), and amino acids 327-332 (F / G) ring.

[0232] In some embodiments, the anti-GPA33 antibody of the present invention has altered affinity for activating and / or inhibiting a receptor having a variant Fc region with one or more amino acid modifications, wherein the one or more amino acid modifications are substitutions of N297 for alanine or K322 for alanine. Alternatively or additionally, in some embodiments, the Fc region of the GPA33 antibody disclosed herein contains two amino acid substitutions, Leu234Ala and Leu235Ala (the so-called LALA mutation), to eliminate FcγRIIa binding. LALA mutations are commonly used to reduce cytokine induction from T cells, thereby reducing antibody toxicity (Wines BD et al., J Immunol 164:5313–5318 (2000)).

[0233] Glycosylation modification. In some embodiments, the anti-GPA33 antibody of the present invention has an Fc region with variant glycosylation compared to the parental Fc region. In some embodiments, variant glycosylation includes the absence of fucose; in some embodiments, variant glycosylation is induced by expression in GnT1-deficient CHO cells.

[0234] In some embodiments, the antibodies of the present invention may have modified glycosylation sites relative to a suitable reference antibody that binds to the antigen of interest (e.g., GPA33) without altering the antibody’s functionality, such as its binding activity to the antigen. As used herein, a “glycosylation site” includes any specific amino acid sequence in the antibody that the oligosaccharide (i.e., a carbohydrate containing two or more monosaccharides linked together) will be specifically and covalently linked.

[0235] Oligosaccharide side chains are typically linked to the antibody backbone via N- or O-bonds. N-linked glycosylation refers to the linking of the oligosaccharide moiety to the asparagine residue side chain. O-linked glycosylation refers to the linking of the oligosaccharide moiety to a hydroxy amino acid, such as serine or threonine. For example, the Fc-glycoform (huGPA33-IgGln) lacking certain oligosaccharides, including fucose and terminal N-acetylglucosamine, can be produced in specific CHO cells and exhibit enhanced ADCC effector function.

[0236] In some embodiments, the carbohydrate content of the immunoglobulin-related compositions disclosed herein is modified by adding or deleting glycosylation sites. Methods for modifying the carbohydrate content of antibodies are well known in the art and are included in the present invention, see, for example, U.S. Patent No. 6,218,149; EP 0359096B1; U.S. Patent Publication No. US 2002 / 0028486; International Patent Application Publication WO 03 / 035835; U.S. Patent Publication No. 2003 / 0115614; U.S. Patent No. 6,218,149; U.S. Patent No. 6,472,511; all of which are incorporated herein by reference in their entirety. In some embodiments, the carbohydrate content of an antibody (or an associated portion or component thereof) is modified by deleting one or more endogenous carbohydrate portions of the antibody. In some embodiments, the present invention includes deleting the glycosylation site in the Fc region of the antibody by modifying position 297 from asparagine to alanine.

[0237] Engineered glycoforms can be used for a variety of purposes, including but not limited to enhancing or reducing effector function. Engineered glycoforms can be generated by any method known to those skilled in the art, such as by using engineered or variant expression strains, by co-expression with one or more enzymes such as DI N-acetylglucosamine transferase III (GnTIII), by expressing molecules containing the Fc region in various organisms or cell lines from various organisms, or by modifying carbohydrates after expression of molecules containing the Fc region. Methods for generating engineered glycoforms are known in the art and include, but are not limited to, those described in the following literature: Umana et al., 1999, Nat. Biotechnol. 17: 176-180; Davies et al., 2001, Biotechnol. Bioeng. 74: 288-294; Shields et al., 2002, J. Biol. Chem. 277: 26733-26740; Shinkawa et al., 2003, J. Biol. Chem. 278: 3466-3473; U.S. Patent No. 6,602,684; U.S. Patent Application Serial No. 10 / 277,370; U.S. Patent Application Serial No. 10 / 113,929; International Patent Application Publication WO 00 / 61739A1; WO 01 / 292246A1; WO 02 / 311140A1; WO 02 / 30954A1; POTILLEGENT™ technology (Biowa, Inc. Princeton, NJ); GLYCOMAB™ glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland); each of these patents is incorporated herein by reference in its entirety. See, for example, International Patent Application Publication WO 00 / 061739; U.S. Patent Application Publication No. 2003 / 0115614; Okazaki et al., 2004, JMB, 336: 1239-49.

[0238] Fusion Protein. In one embodiment, the anti-GPA33 antibody of the present invention is a fusion protein. When fused with a second protein, the anti-GPA33 antibody of the present invention can be used as an antigen tag. Examples of domains that can be fused with peptides include not only heterologous signal sequences but also other heterologous functional regions. Fusion need not be direct but can occur through linker sequences. Furthermore, the fusion protein of the present invention can also be engineered to improve the properties of the anti-GPA33 antibody. For example, regions of additional amino acids (especially charged amino acids) can be added to the N-terminus of the anti-GPA33 antibody to improve stability and durability during purification from host cells or subsequent processing and storage. Similarly, peptide moieties can be added to the anti-GPA33 antibody to facilitate purification. Such regions can be removed prior to the final preparation of the anti-GPA33 antibody. Adding peptide moieties to facilitate peptide processing is a well-known and conventional technique in the art. The anti-GPA33 antibody of the present invention can be fused with marker sequences, such as peptides that facilitate the purification of the fused peptide. In selected embodiments, the marker amino acid sequence is a hexahistine peptide, such as the tags provided in pQE vectors (QIAGEN, Inc., Chatsworth, Calif), many of which are commercially available. As described by Gentz ​​et al., Proc. Natl. Acad. Sci. USA 86: 821-824, 1989, for example, hexahistine facilitates the purification of fusion proteins. Another peptide tag that can be used for purification is the “HA” tag, which corresponds to an epitope derived from influenza hemagglutinin proteins. Wilson et al., Cell 37: 767, 1984.

[0239] Therefore, any of these fusion proteins described above can be engineered using the polynucleotides or peptides of the present invention. Furthermore, in some embodiments, the fusion proteins described herein exhibit an increased in vivo half-life.

[0240] Fusion proteins with disulfide-linked dimer structures (due to IgG) are more efficient at binding and neutralizing other molecules than individual monomeric secreted proteins or protein fragments. Fountoulakis et al., J. Biochem. 270:3958-3964, 1995.

[0241] Similarly, EP-AO 464 533 (Canadian equivalent 2045869) discloses fusion proteins containing different portions of the constant region of an immunoglobulin molecule and another human protein or fragment thereof. In many cases, the Fc portion of the fusion protein is beneficial for therapy and diagnosis and can therefore cause, for example, improved pharmacokinetic properties. See EP-A 0232 262. Alternatively, it may be necessary to delete or modify the Fc portion after expression, detection, and purification of the fusion protein. For example, if the fusion protein is used as an antigen for immunity, the Fc portion may hinder therapy and diagnosis. In drug discovery, for example, human proteins such as hIL-5 have been fused with Fc portions for the purpose of high-throughput screening assays to identify hIL-5 antagonists. Bennett et al., J. Molecular Recognition 8: 52-58, 1995; Johanson et al., J. Biol. Chem., 270: 9459-9471, 1995.

[0242] Labeled anti-GPA33 antibody. In one embodiment, the anti-GPA33 antibody of the present invention is coupled to a labeled portion (i.e., a detectable group). The specific label or detectable group conjugated to the anti-GPA33 antibody is not a critical aspect of the present invention, as long as it does not significantly interfere with the specific binding of the anti-GPA33 antibody of the present invention to the GPA33 protein. The detectable group can be any material having detectable physical or chemical properties. Such detectable labels have been well developed in the fields of immunoassay and imaging. Generally, almost any label useful in such methods can be applied to the present invention. Therefore, the label can be any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. Labels that can be used in the practice of the present invention include magnetic beads (e.g., Dynabeads™), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, etc.), and radioactive labels (e.g., 3 H, 14 C 35 S, 125 I, 121 I, 131 I, 112 In、 99 mTc), and other imaging agents such as microbubbles (used for ultrasound imaging). 18 F, 11 C 15 O (used for positron emission tomography) 99m TC 111In (used for single-photon emission computed tomography), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, and other enzymes commonly used in ELISA), and calorimetrically labeled beads such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.). Patents describing the use of such labels include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241, each of which is incorporated herein by reference in its entirety and is used for all purposes. See also Handbook of Fluorescent Probes and Research Chemicals (6th Edition, Molecular Probes, Inc., Eugene OR.).

[0243] According to methods known in the art, a label can be directly or indirectly coupled to the desired component to be determined. As noted above, a variety of labels can be used, and the choice of label depends on a number of factors, such as desired sensitivity, ease of conjugation with the compound, stability requirements, available instruments, and available supplies.

[0244] Non-radioactive labeling is typically achieved through indirect methods. Generally, a ligand molecule (e.g., biotin) covalently binds to the label. The ligand then binds to an anti-ligand molecule (e.g., streptavidin), which can be inherently detectable or covalently bound to a signaling system, such as a detectable enzyme, fluorescent compound, or chemiluminescent compound. Many ligands and anti-ligands can be used. When the ligand has a natural anti-ligand, such as biotin, thyroxine, or cortisol, it can be used in combination with the labeled, naturally occurring anti-ligand. Alternatively, any hapten or antigen compound can be used in combination with an antibody, such as an anti-GPA33 antibody.

[0245] The molecule can also be directly conjugated to the signal-generating compound, for example, by binding to an enzyme or fluorophore. The enzymes of interest for labeling will primarily be hydrolases, specifically phosphatases, esterases, and glycosidases, or oxidoreductases, specifically peroxidases. Fluorescent compounds that can be used as the labeling motif include, but are not limited to, luciferin and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc. Chemiluminescent compounds that can be used as the labeling motif include, but are not limited to, luciferin and 2,3-dihydrophthalazinedione, such as luminol. For a review of the various labeling or signal generation systems that can be used, see U.S. Patent No. 4,391,904.

[0246] Methods for detecting markers are well known to those skilled in the art. Thus, for example, when the marker is radioactive, detection means include scintillation counters or photographic film such as in autoradiography. In the case of a fluorescent marker, it can be detected by exciting a fluorescent dye with an appropriate wavelength of light and detecting the resulting fluorescence. Fluorescence can be visually detected using photographic film or by using electronic detectors such as charge-coupled devices (CCDs) or photomultipliers. Similarly, enzyme markers can be detected by providing an appropriate substrate of the enzyme and detecting the resulting reaction product. Finally, simple colorimetric markers can be easily detected by observing the color associated with the marker. Therefore, in various test strip assays, conjugated gold typically appears pink, while various conjugated beads exhibit the color of the beads.

[0247] Some assays do not require the use of labeled components. For example, agglutination assays can be used to detect the presence of target antibodies, such as anti-GPA33 antibodies. In this case, antigen-coated particles are agglutinated by a sample containing the target antibody. In this form, no labeled components are required, and the presence of the target antibody is detected by simple visual inspection.

[0248] B. Identification and characterization of the anti-GPA33 antibody of the present invention.

[0249] Methods for identifying and / or screening anti-GPA33 antibodies using the techniques of this invention. Methods for identifying and screening antibodies against the GPA33 peptide to find those with desired specificity for the GPA33 protein include any immune-mediated techniques known in the art. Components of the immune response can be detected in vitro using a variety of methods well known to those skilled in the art. For example, (1) cytotoxic T lymphocytes can be incubated with radiolabeled target cells and the lysis of these target cells can be detected by the release of radioactivity; (2) helper T lymphocytes can be incubated with antigens and antigen-presenting cells and the synthesis and secretion of cytokines can be measured by standard methods (Windhagen A et al., Immunity, 2: 373-80, 1995); (3) antigen-presenting cells can be incubated with whole-protein antigens and the presentation of the antigen on the MHC can be detected by T lymphocyte activation assays or biophysical methods (Harding et al., Proc. Natl. Acad. Sci., 86:4230-4, 1989); (4) mast cells can be incubated with reagents cross-linked with their Fc-ε receptors and histamine release can be measured by enzyme immunoassay (Siraganian et al., TIPS, 4: 432-437, 1983); and (5) enzyme-linked immunosorbent assay (ELISA).

[0250] Similarly, products of immune responses in model organisms (e.g., mice) or human subjects can be detected using a variety of methods well known to those skilled in the art. For example, (1) antibody production in response to vaccination can be readily detected using standard methods currently used in clinical laboratories (e.g., ELISA); (2) migration of immune cells to sites of inflammation can be detected by scraping the skin surface and placing a sterile container to capture migrating cells at the scraped site (Peters et al., Blood, 72: 1310-5, 1988); (3) proliferation of peripheral blood mononuclear cells (PBMCs) in response to mitogens or mixed lymphocyte responses can be detected using… 3 (4) The phagocytic capacity of granulocytes, macrophages and other phagocytes in PBMCs can be measured by placing PBMCs together with labeled particles into wells (Peters et al., Blood, 72: 1310-5, 1988); and (5) the differentiation of immune system cells can be measured by labeling PBMCs with antibodies against CD molecules (such as CD4 and CD8) and measuring the fraction of PBMCs expressing these markers.

[0251] In one embodiment, the display of the GPA33 peptide on the surface of a reproducible gene pack is used to select the anti-GPA33 antibody of the present invention. See, for example, U.S. Patent Nos. 5,514,548; 5,837,500; 5,871,907; 5,885,793; 5,969,108; 6,225,447; 6,291,650; 6,492,160; EP 585 287; EP 605522; EP 616640; EP1024191; EP 589 877; EP 774 511; EP 844 306. Methods for generating / selecting filamentous phage particles containing a phage genome encoding a binding molecule with desired specificity have been described. See, for example, EP 774511; US ​​5871907; US 5969108; US 6225447; US 6291650; US 6492160.

[0252] In some embodiments, the display of the GPA33 peptide on the surface of yeast host cells is used to select the anti-GPA33 antibody of the present invention. Methods for isolating scFv peptides by displaying them on yeast surfaces have been described in: Kieke et al., Protein Eng. Nov 1997; 10(11): 1303-10.

[0253] In some embodiments, ribosome display is used to select the anti-GPA33 antibody of the present invention. Methods for identifying ligands in peptide libraries using ribosome display have been described in the following literature: Mattheakis et al., Proc. Natl. Acad. Sci. USA 91: 9022-26, 1994; and Hanes et al., Proc. Natl. Acad. Sci. USA 94: 4937-42, 1997.

[0254] In some embodiments, tRNA display of the GPA33 peptide is used to select the anti-GPA33 antibody of the present invention. Methods for in vitro selection of ligands using tRNA display have been described in: Merryman et al., Chem. Biol., 9: 741-46, 2002.

[0255] In one embodiment, RNA display is used to select the anti-GPA33 antibody using the technique of the present invention. Methods for selecting peptides and proteins using RNA display libraries have been described in: Roberts et al., Proc. Natl. Acad. Sci. USA, 94: 12297-302, 1997; and Nemoto et al., FEBS Lett., 414: 405-8, 1997. Methods for selecting peptides and proteins using non-natural RNA display libraries have been described in: Frankel et al., Curr. Opin. Struct. Biol., 13: 506-12, 2003.

[0256] In some embodiments, the anti-GPA33 antibody of the present invention is expressed in the periplasm of Gram-negative bacteria and mixed with labeled GPA33 protein. See WO 02 / 34886. In clones expressing recombinant polypeptides with affinity for GPA33 protein, the concentration of labeled GPA33 protein bound to the anti-GPA33 antibody is increased, causing the cells to separate from the rest of the library, as described in Harvey et al., Proc. Natl. Acad. Sci. 22: 9193-98 2004 and U.S. Patent Publication No. 2004 / 0058403.

[0257] After selecting the desired anti-GPA33 antibody, it is anticipated that the antibody can be mass-produced using any technique known to those skilled in the art, such as prokaryotic or eukaryotic cell expression. Anti-GPA33 antibodies can be generated by constructing expression vectors encoding the antibody heavy chain using conventional techniques, such as, but not limited to, anti-GPA33 hybrid antibodies or fragments, wherein a minimal portion (if necessary) of the CDR and variable region framework required for the original species antibody binding specificity (e.g., engineered according to the techniques described herein) is derived from the source species antibody, and the remainder of the antibody is derived from a target species immunoglobulin that can be manipulated as described herein, thereby producing a vector for expressing the hybrid antibody heavy chain.

[0258] Measurement of GPA33 binding. In some embodiments, a GPA33 binding assay refers to an assay in which GPA33 protein and anti-GPA33 antibody are mixed under conditions suitable for binding between GPA33 protein and anti-GPA33 antibody and for assessing the amount of binding between GPA33 protein and anti-GPA33 antibody. The amount of binding is compared to a suitable control, which may be the amount of binding in the absence of GPA33 protein, the amount of binding in the presence of a nonspecific immunoglobulin composition, or both. The amount of binding can be assessed by any suitable method. Binding assay methods include, for example, ELISA, radioimmunoassay, scintillation proximity assay, fluorescence energy transfer assay, liquid chromatography, membrane filtration assay, etc. Biophysical assays for directly measuring the binding of GPA33 protein to anti-GPA33 antibody are, for example, nuclear magnetic resonance, fluorescence, fluorescence polarization, surface plasmon resonance (BIACORE chip), etc. Specific binding is determined by standard assays known in the art, such as radioligand binding assays, ELISA, FRET, immunoprecipitation, SPR, NMR (2D-NMR), mass spectrometry, etc. If the specific binding of the candidate anti-GPA33 antibody is at least 1% higher than the binding observed when the candidate anti-GPA33 antibody is not present, then the candidate anti-GPA33 antibody can be used as the anti-GPA33 antibody of the present invention.

[0259] Measurement of GPA33 neutralization. As used herein, "GPA33 neutralization" refers to the reduction of GPA33 protein activity and / or expression by binding to an anti-GPA33 antibody. The ability of the anti-GPA33 antibody of the present invention to neutralize GPA33 activity / expression can be evaluated in vitro or in vivo using methods known in the art.

[0260] Uses of the anti-GPA33 antibody of the present invention

[0261] Overview. The anti-GPA33 antibodies of the present invention can be used in methods known in the art related to the localization and / or quantification of GPA33 protein (e.g., for measuring the level of GPA33 protein in appropriate physiological samples, for diagnostic methods, for imaging peptides, etc.). The antibodies of the present invention can be used to isolate GPA33 protein using standard techniques such as affinity chromatography or immunoprecipitation. The anti-GPA33 antibodies of the present invention can facilitate the purification of innate immunoreactive GPA33 protein from biological samples, such as mammalian serum or cells, as well as recombinant immunoreactive GPA33 protein expressed in host systems. Furthermore, the anti-GPA33 antibodies can be used to detect immunoreactive GPA33 protein (e.g., in plasma, cell lysates, or cell supernatants) to evaluate the abundance and expression patterns of immunoreactive peptides. As part of clinical testing procedures, the anti-GPA33 antibodies of the present invention can be used for diagnostic monitoring of immunoreactive GPA33 protein levels in tissues to, for example, determine the efficacy of a given treatment regimen. As described above, detection can be facilitated by conjugating (i.e., physically linking) the anti-GPA33 antibody of the present invention with a detectable substance.

[0262] Detection of GPA33 protein. An exemplary method for detecting the presence of immunoreactive GPA33 protein in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with an anti-GPA33 antibody of the present invention capable of detecting immunoreactive GPA33 protein, thereby detecting the presence of immunoreactive GPA33 protein in the biological sample. Detection can be performed using a detectable label linked to the antibody.

[0263] The term "labeled" for anti-GPA33 antibodies is intended to encompass both direct labeling of antibodies by conjugating (i.e., physically linking) a detectable substance to the antibody, and indirect labeling of antibodies by their reactivity with another directly labeled compound, such as a secondary antibody. Examples of indirect labeling include the detection of primary antibodies using fluorescently labeled secondary antibodies, and the end-labeling of DNA probes with biotin, which can then be detected using fluorescently labeled streptavidin.

[0264] In some embodiments, the anti-GPA33 antibody disclosed herein is conjugated to one or more detectable labels. For such uses, the anti-GPA33 antibody can be detectably labeled by covalent or non-covalent linkage of chromogenic, enzymatic, radioisotope, isotope, fluorescence, toxic, chemiluminescent, MRI contrast agent, or other labels.

[0265] Examples of suitable chromogenic labels include diaminobenzidine and 4-hydroxyazobenzene-2-carboxylic acid. Examples of suitable enzyme labels include malate dehydrogenase, staphylococcal nuclease, Δ-5-steroid isomerase, yeast alcohol dehydrogenase, α-glycerophosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucosylamylase, and acetylcholinesterase.

[0266] Examples of suitable radioactive isotope labeling include 3 H, 111 In、 125 I, 131 I, 32 P, 35 S, 14 C 51 Cr 57 To 58 Co、 59 Fe、 75 Se、 152 Eu、 90 Y、 67 Cu、 217 Ci、 211 At、 212 Pb, 47 Sc、 109 Pd, etc. 111 In is an exemplary isotope in which in vivo imaging was used because it avoids... 125 I or 131 The issue of liver dehalogenation of I-labeled GPA33-binding antibodies. Additionally, this isotope possesses a more favorable gamma emission energy for imaging (Perkins et al., Eur. J. Nucl. Med. 70:296-301 (1985); Carasquillo et al., J. Nucl. Med. 25:281-287 (1987)). For example, conjugation with monoclonal antibodies containing 1-(p-isothiocyanobenzyl)-DPTA... 111 In is hardly taken up in non-tumor tissues, particularly the liver, and enhances the specificity of tumor localization (Esteban et al., J. Nucl. Med. 28:861-870 (1987)). Examples of suitable non-radioactive isotope labeling include 157 Gd, 55 Mn, 162 Dy、 52 Tr and 56 Fe.

[0267] Examples of suitable fluorescent labels include 152Eu labeling, fluorescein labeling, isothiocyanate labeling, rhodamine labeling, phycoerythrin labeling, phycocyanin labeling, allophycocyanin labeling, green fluorescent protein (GFP) labeling, phthalaldehyde labeling, and fluorescent amine labeling. Examples of suitable toxin labels include diphtheria toxin, ricin, and cholera toxin.

[0268] Examples of chemiluminescent labeling include luminol labeling, isoluminol labeling, aromatic acrylamide labeling, imidazole labeling, acrylamide salt labeling, oxalate ester labeling, luciferin labeling, luciferase labeling, and jellyfish luminescent protein labeling. Examples of nuclear magnetic resonance contrast agents include heavy metal nuclei, such as Gd, Mn, and iron.

[0269] The detection method of this invention can be used for in vitro and in vivo detection of immunoreactive GPA33 protein in biological samples. In vitro techniques for detecting immunoreactive GPA33 protein include enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation, radioimmunoassay, and immunofluorescence. Furthermore, in vivo techniques for detecting immunoreactive GPA33 protein include introducing a labeled anti-GPA33 antibody into a subject. For example, the anti-GPA33 antibody can be labeled with a radiolabel, the presence and location of which in the subject can be detected by standard imaging techniques. In one embodiment, the biological sample contains GPA33 protein molecules from a test subject.

[0270] Immunoassay and Imaging. The anti-GPA33 antibody of the present invention can be used to determine the level of immunoreactive GPA33 protein in biological samples (e.g., human plasma) using antibody-based techniques. For example, protein expression in tissues can be studied using classical immunohistochemical methods. Jalkanen, M. et al., J. Cell. Biol. 101: 976-985, 1985; Jalkanen, M. et al., J. Cell. Biol. 105: 3087-3096, 1987. Other antibody-based methods that can be used to detect protein gene expression include immunoassays such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). Suitable antibody assay markers are known in the art and include enzyme markers (such as glucose oxidase) and radioisotopes or other radioactive agents (such as iodine). 125 I, 121 I, 131 I), carbon ( 14 C), sulfur 35 S), tritium ( 3 H), Indium 112 In) and technetium ( 99 mTc) and fluorescent labels (such as fluorescein, rhodamine and green fluorescent protein (GFP)) and biotin.

[0271] In addition to measuring the level of immunoreactive GPA33 protein in biological samples, the anti-GPA33 antibody of this invention can also be used for in vivo imaging of GPA33. Antibodies suitable for this method include those detectable by X-ray radiography, NMR, or ESR. For X-ray radiography, suitable markers include radioactive isotopes such as barium or cesium, which emit detectable radiation but do not cause significant harm to the subject. Suitable markers for NMR and ESR include markers with detectable spin characteristics, such as deuterium, which can be incorporated into the anti-GPA33 antibody by labeling nutrients of the relevant scFv clone.

[0272] The appropriate detectable imaging components (such as radioactive isotopes) will be used. 131 I, 112 In、 99 Anti-GPA33 antibodies labeled with mTc (a radiopaque substance or a material detectable by nuclear magnetic resonance) are introduced into the subject (e.g., parenteral, subcutaneous, or intraperitoneal). It should be understood in the art that the size of the subject and the imaging system used will determine the number of imaging portions required to produce diagnostic images. In the case of radioisotope portions, for human subjects, the amount of radioactivity injected is typically between approximately 5 and 20 millicuries. 99 Within the range of mTc. The labeled anti-GPA33 antibody will then accumulate at cellular sites containing the specific target peptide. For example, the labeled anti-GPA33 antibody of this invention will accumulate in the cells and tissues where the GPA33 protein is localized in the subject.

[0273] Therefore, the present invention provides a diagnostic method for medical conditions, which involves: (a) determining the expression of immunoreactive GPA33 protein by measuring the binding of the anti-GPA33 antibody of the present invention in the cells or body fluids of an individual; and (b) comparing the amount of immunoreactive GPA33 protein present in a sample with a standard reference, wherein an increase or decrease in the level of immunoreactive GPA33 protein compared with the standard indicates the presence of a medical condition.

[0274] Affinity purification. The anti-GPA33 antibody of the present invention can be used to purify immunoreactive GPA33 protein from a sample. In some embodiments, the antibody is immobilized on a solid support. Examples of such solid supports include plastics such as polycarbonate, complex carbohydrates such as agarose and agarose gel, acrylic resins, and materials such as polyacrylamide and latex beads. Techniques for conjugating antibodies to such solid supports are well known in the art (Weir et al., “Handbook of Experimental Immunology” 4th ed., Blackwell Scientific Publications, Oxford, England, Chapter 10 (1986); Jacoby et al., Meth. Enzym. 34 Academic Press, NY (1974)).

[0275] The simplest method for binding antigen to an antibody-support matrix is ​​to collect beads in a column and allow the antigen solution to flow down the column. The efficiency of this method depends on the contact time between the immobilized antibody and the antigen, which can be extended by using a low flow rate. As the antigen flows through, the immobilized antibody captures it. Alternatively, the antigen can be brought into contact with the antibody-support matrix by mixing the antigen solution with the support (e.g., beads) and rotating or shaking the slurry, thus maximizing contact between the antigen and the immobilized antibody. Once the binding reaction is complete, the slurry is transferred to a column containing the beads. The beads are washed with a suitable washing buffer, and then pure or substantially pure antigen is eluted.

[0276] The antibody or peptide of interest can be conjugated to a solid support such as beads. Additionally, if desired, a first solid support, such as beads, can also be conjugated to a second solid support in any suitable manner. This second solid support can be a second bead or other support, including those disclosed herein for conjugating peptides to supports. Therefore, any conjugation methods and approaches disclosed herein regarding the conjugation of peptides to solid supports can also be applied to the conjugation of a first support to a second support, wherein the first and second solid supports can be the same or different.

[0277] Suitable linkers (which can be crosslinking agents) for conjugating peptides to solid supports include a variety of agents that can react with functional groups present on the surface of the support, or with the peptide, or with both. Reagents that can be used as crosslinking agents include both bifunctional and, specifically, non-bifunctional agents. Useful bifunctional crosslinking agents include, but are not limited to, N-SIAB, dimaleimide, DTNB, N-SATA, N-SPDP, SMCC, and 6-HYNIC. Crosslinking agents can be selected to provide selectively cleavable bonds between the peptide and the solid support. For example, photoinstantaneous crosslinking agents, such as 3-amino-(2-nitrophenyl)propionic acid, can be used as a means of cleaving peptides from a solid support. (Brown et al., Mol. Divers, pp. 4–12 (1995); Rothschild et al., Nucl. Acids Res., 24:351–66 (1996); and U.S. Patent No. 5,643,722). Other crosslinking agents are well known in the art. (See, for example, Wong (1991), ibid.; and Hermanson (1996), ibid.)

[0278] Antibodies or peptides can be immobilized on solid supports such as beads via covalent amide bonds formed between carboxyl-functionalized beads and the amino terminus of the peptide, or conversely, via covalent amide bonds formed between amino-functionalized beads and the carboxyl terminus of the peptide. Alternatively, bifunctional triphenylmethyl linkers can be attached to the support via amino resins through amino or carboxyl groups on the resin, for example, to 4-nitrophenyl active esters attached to resins (such as Wang resin). Using the bifunctional triphenylmethyl method, the solid support may need to be treated with volatile acids such as formic acid or trifluoroacetic acid to ensure that the peptide is cleaved and can be removed. In such cases, the peptide can be deposited as a beadless sheet at the bottom of the pores of the solid support or on a flat surface of the solid support. After the addition of a matrix solution, the peptide can be desorbed into MS.

[0279] Hydrophobic triphenylmethyl linkers can also be used as acid-instable linkers by cleaving the amino-linked triphenylmethyl group from the peptide using a volatile acid or a suitable matrix solution, such as a matrix solution containing 3-HPA. Acid instability can also be modified. For example, triphenylmethyl, monomethoxytriphenylmethyl, dimethoxytriphenylmethyl, or trimethoxytriphenylmethyl can be modified to a suitable para-substituted or more acid-instable triphenylmethylamine derivative of the peptide, i.e., the triphenylmethyl ether and triphenylmethylamine bonds of the peptide can be prepared. Therefore, the peptide can be removed from the hydrophobic linker, for example, by disrupting the hydrophobic attraction under acidic conditions (if desired, including under typical MS conditions where the matrix (such as 3-HPA) acts as the acid) or by cleaving the triphenylmethyl ether or triphenylmethylamine bond.

[0280] Orthogonally cleavable linkers can also be used to bind a first solid support (e.g., beads) to a second solid support, or to bind a peptide of interest to a solid support. Using such linkers, the first solid support (e.g., beads) can be selectively cleaved from the second solid support without cleaving the peptide from the support; the peptide can then be cleaved from the beads later. For example, disulfide linkers that can be cleaved using reducing agents such as DTT can be used to bind beads to a second solid support, and acid-cleavable bifunctional triphenylmethyl groups can be used to immobilize peptides to a support. Depending on the need, the link between the peptide and the solid support can be cleaved first, while, for example, the link between the first and second supports remains intact. Triphenylmethyl linkers can provide covalent or hydrophobic conjugations, and regardless of the nature of the conjugation, triphenylmethyl groups readily cleave under acidic conditions.

[0281] For example, beads can be bound to a second support via a linker group, which can be selected to have length and chemical properties that promote high-density binding of the beads to the solid support, or high-density binding of the peptide to the beads. Such linkers can have, for example, a "dendritic" structure, thereby providing multiple functional groups at each linker site on the solid support. Examples of such linkers include polylysine, polyglutamic acid, pentaerythrole, and trihydroxyaminomethane.

[0282] Non-covalent association. Antibodies or peptides can be conjugated to a solid support through non-covalent interactions, or a first solid support can be conjugated to a second solid support. For example, magnetic beads made of a magnetizable ferromagnetic material can be attracted to a magnetic solid support and released from the support by removing the magnetic field. Alternatively, the solid support can be provided with ionic or hydrophobic portions, which can allow the ionic or hydrophobic portions to interact, respectively, with the peptide (e.g., a peptide containing a linked triphenylmethyl group) or with a second solid support having hydrophobic properties.

[0283] Solid supports can also be provided with members of specific binding pairs, and thus can be conjugated to peptides or second solid supports containing complementary binding moieties. For example, beads coated with avidin or streptavidin can bind to peptides incorporating biotin moieties, or to second solid supports coated with biotin or biotin derivatives such as iminobiotin.

[0284] It should be understood that any binding member disclosed herein or otherwise known in the art is reversible. Thus, biotin can be incorporated, for example, into a polypeptide or a solid support, and conversely, avidin or other biotin-binding moieties will be incorporated into the support or polypeptide, respectively. Other specific binding pairs contemplated herein include, but are not limited to, hormones and their receptors, enzymes and their substrates, nucleotide sequences and their complementary sequences, antibodies and antigens that specifically interact with them, and other such pairs known to those skilled in the art.

[0285] A. Diagnostic applications of the anti-GPA33 antibody of this invention

[0286] Summary. The anti-GPA33 antibody of the present invention can be used in diagnostic methods. Thus, the present invention provides a method for diagnosing GPA33 activity in a subject using an antibody. The anti-GPA33 antibody of the present invention can be selected such that it has any level of epitope binding specificity and very high binding affinity to the GPA33 protein. Generally, the higher the binding affinity of the antibody, the more stringent the washing conditions that can be performed in the immunoassay to remove nonspecifically bound material without removing the target peptide. Therefore, the anti-GPA33 antibody of the present invention that can be used for diagnostic assays typically has about 10 8 M -1 10 9 M -1 10 10 M -1 10 11 M -1 Or 10 12 M -1 The binding affinity. Furthermore, it is desirable for the anti-GPA33 antibody used as a diagnostic reagent to have a sufficient kinetic association rate to reach equilibrium under standard conditions within at least 12 hours, at least five (5) hours, or at least one (1) hour.

[0287] Anti-GPA33 antibodies can be used to detect immunoreactive GPA33 protein in a variety of standard assays. These assays include immunoprecipitation, Western blotting, ELISA, radioimmunoassay, and immunoassay. See Harlow and Lane, Antibodies, A Laboratory Manual (Cold Spring Harbor Publications, New York, 1988); U.S. Patent Nos. 3,791,932; 3,839,153; 3,850,752; 3,879,262; 4,034,074, 3,791,932; 3,817,837; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; and 4,098,876. Biological samples can be obtained from any tissue or bodily fluid of the subject. In some embodiments, the subject is in an early stage of cancer. In one embodiment, the early stage of cancer is determined by the level or expression pattern of the GPA33 protein in a sample obtained from the subject. In some embodiments, the sample is selected from the group consisting of: urine, blood, serum, plasma, saliva, amniotic fluid, cerebrospinal fluid (CSF), and biopsied body tissue.

[0288] Immunoassays or sandwich assays are a form of diagnostic method based on the technology of this invention. See U.S. Patent Nos. 4,376,110, 4,486,530, 5,914,241, and 5,965,375. Such assays use an antibody, such as an anti-GPA33 antibody or a population of anti-GPA33 antibodies immobilized to a solid phase, and another anti-GPA33 antibody or a population of anti-GPA33 antibodies in solution. Typically, the population of anti-GPA33 antibodies in solution is labeled. If a population of antibodies is used, this population may contain antibodies that specifically bind to different epitopes within the target polypeptide. Thus, the same population can be used for both the solid-phase and solution antibodies. If an anti-GPA33 monoclonal antibody is used, first and second GPA33 monoclonal antibodies with different binding specificities are used for both the solid-phase and solution phases. The solid-phase (also called “capture”) and solution (also called “detection”) antibodies can be contacted with the target antigen in either order or simultaneously. If the solid-phase antibody is contacted first, the assay is called a forward assay. Conversely, if the solution antibody is contacted first, the assay is called a reverse assay. If the target is contacted with both antibodies simultaneously, the assay is called a simultaneous assay. After contacting the GPA33 protein with the anti-GPA33 antibody, the sample is incubated for a period typically ranging from about 10 minutes to about 24 hours, and typically about 1 hour. A washing step is then performed to remove components from the sample that do not specifically bind to the anti-GPA33 antibody used as a diagnostic reagent. When the solid phase and solution antibody bind in separate steps, washing can be performed after either or both binding steps. After washing, binding is typically quantified by detecting the label attached to the solid phase by means of the binding of the labeled solution antibody. Typically, a calibration curve is prepared from a sample containing a known concentration of the target antigen for a given antibody pair or antibody population and given reaction conditions. The concentration of immunoreactive GPA33 protein in the test sample is then read by interpolation from the calibration curve (i.e., the standard curve). The analyte can be measured by the amount of labeled solution antibody bound at equilibrium or by kinetic measurements of the bound labeled solution antibody at a series of time points before reaching equilibrium. The slope of this type of curve is a measure of the concentration of GPA33 protein in the sample.

[0289] Suitable supports for the above methods include, for example, nitrocellulose membranes, nylon membranes and derivatized nylon membranes, as well as particles such as agarose, dextran-based gels, test papers, microparticles, microspheres, magnetic particles, test tubes, microburettes, SEPHADEX™ (Amersham Pharmacia Biotech, Piscataway NJ), etc. Immobilization can be achieved by absorption or covalent linkage. Optionally, anti-GPA33 antibodies can be conjugated to linker molecules such as biotin to surface-binding linkers such as avidin.

[0290] In some embodiments, this disclosure provides an anti-GPA33 antibody of the present invention conjugated to a diagnostic agent. The diagnostic agent may comprise a radioactive or non-radioactive label, a contrast agent (such as for magnetic resonance imaging, computed tomography, or ultrasound), and the radioactive label may be a gamma-emitting isotope, a beta-emitting isotope, an alpha-emitting isotope, an Auger electron-emitting isotope, or a positron-emitting isotope. The diagnostic agent is a molecule conjugated to an antibody moiety (i.e., an antibody or an antibody fragment or subfraction) and can be used to diagnose or detect disease by targeting cells containing the antigen.

[0291] Useful diagnostic agents include, but are not limited to, radioisotopes, dyes (such as those having biotin-streptavidin complexes), contrast agents, fluorescent compounds or molecules, and enhancers for magnetic resonance imaging (MRI) (e.g., paramagnetic ions). U.S. Patent No. 6,331,175 describes MRI techniques and the preparation of antibodies conjugated with MRI enhancers, and is incorporated herein by reference in its entirety. In some embodiments, the diagnostic agent is selected from the group consisting of radioisotopes, enhancers for magnetic resonance imaging, and fluorescent compounds. To load the antibody component with a radioactive metal or paramagnetic ion, it may be necessary to react it with a reagent having a long tail having chelating groups for attaching multiple ions to which the binding ions can be attached. Such tails may be polymers, such as polylysine, polysaccharides, or other derivatized or derivatizable chains having side groups to which the chelating groups can bind, such as, for example, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrins, polyamines, crown ethers, bis-thiourea, polyoximes, and similar groups known to be suitable for this purpose. Chelates can be conjugated to antibodies using standard chemical methods. Chelates are typically linked to antibodies via groups that form bonds with the molecule with minimal loss of immunoreactivity and minimal aggregation and / or internal cross-linking. Other methods and reagents for conjugating chelates to antibodies are disclosed in U.S. Patent No. 4,824,659. Particularly useful metal chelate combinations include 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, used in conjunction with diagnostic isotopes for radiographic imaging. The same chelates can be used for MRI when used with the GPA33 antibody of the present invention, in combination with non-radioactive metals such as manganese, iron, and gadolinium.

[0292] Macrocyclic chelates such as NOTA (1,4,7-triazacyclononane-N,N',N''-triacetic acid), DOTA, and TETA (p-bromoacetamyl-benzyl-tetraethylaminetetraacetic acid) can be used for various metal and radioactive metals such as gallium, yttrium, and copper radionuclides. These metal chelates can be stabilized by tailoring the ring size of the metal of interest. Other examples of DOTA chelates include (i) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH2; (ii) Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH2; (iii) DOTA-D-Asp-D-Lys(HSG)-D-Asp-D-Lys(HSG)-NH2; (iv) DOTA-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (v) DOTA-D-Tyr-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (vi) DOTA-D-Ala-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (vii) DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH2; (viii) Ac-D-Phe-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-NH2; (ix) Ac-D-Phe-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH2; (x) Ac-D-Phe-D-Lys(Bz-DTPA)-D-Tyr-D-Lys(Bz-DTPA)-NH2; (xi) Ac-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH2; (xii) DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH2; (xiii) (Tscg-Cys)-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(DOTA)-NH2; (xiv)Tscg-D-Cys-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (xv) (Tscg-Cys)-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (xvi) Ac-D-Cys-D-Lys(DOTA)-D-Tyr-D-Ala-D-Lys(DOTA)-D-Cys-NH2;(xvii) Ac-D-Cys-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH2; (xviii) Ac-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-D-Lys(Tscg-Cys)-NH2; and (xix) Ac-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-D-Lys(Tscg-Cys)-NH2. ;

[0293] Other cyclic chelates of interest for the stable binding of nuclides, such as macrocyclic polyethers, and those used in RAIT, are also envisioned. 223 Ra.

[0294] B. Therapeutic applications of the anti-GPA33 antibody of the present invention

[0295] The immunoglobulin-associated compositions (e.g., antibodies or antigen-binding fragments thereof) of the present invention can be used to treat GPA33-related cancers. Such treatments can be used for patients identified as having pathologically high levels of GPA33 (e.g., those diagnosed by the methods described herein) or patients diagnosed with a disease known to be associated with such pathological levels.

[0296] On one hand, this disclosure provides a method for treating GPA33-related cancers in a subject of need, the method comprising administering to the subject an effective amount of an antibody (or an antigen-binding fragment thereof) of the present invention. Examples of cancers that can be treated with the antibody of the present invention include, but are not limited to, colorectal cancer, T-cell leukemia, pseudomyxoma peritonei, appendiceal cancer, pancreatic cancer, and gastric cancer. The GPA33-related cancer may be colorectal cancer with an MSI genotype or an MSS genotype. Additionally or alternatively, in some embodiments, the colorectal cancer is associated with a KRAS G12D mutation or a p53 mutation.

[0297] The compositions of this invention can be used in combination with other therapeutic agents that can be used to treat GPA33-related cancers. For example, the antibodies of this invention can be administered alone, sequentially, or simultaneously with at least one additional therapeutic agent selected from the group consisting of: alkylating agents, platinum agents, taxanes, vinca extracts, anti-estrogens, aromatase inhibitors, ovarian inhibitors, VEGF / VEGFR inhibitors, EGF / EGFR inhibitors, PARP inhibitors, cell-inhibiting alkaloids, cytotoxic antibiotics, antimetabolites, endocrine / hormonal agents, bisphosphonate therapeutic agents, and targeted biological therapeutic agents (e.g., therapeutic peptides described in US 6306832, WO 2012007137, WO 2005000889, WO 2010096603, etc.). In some embodiments, the at least one additional therapeutic agent is a chemotherapeutic agent.Specific chemotherapeutic agents include, but are not limited to, cyclophosphamide, fluorouracil (or 5-fluorouracil or 5-FU), methotrexate, edaprexate (10-ethyl-10-deoxyaminopterin), thiotepa, carboplatin, cisplatin, taxane, paclitaxel, protein-bound paclitaxel, docetaxel, vinorelbine, tamoxifen, raloxifene, toremifene, fulvestrant, gemcitabine, irinotecan, and isoxazone. ixabepilone, temozolmide, topotecan, vincristine, vinblastine, eribulin, mutamycin, capecitabine, anastrozole, exemestane, letrozole, leuprolide, abarelix, buserlin, goserelin, megestrol acetate acetate, risedronate, pamidronate, ibandronate, alendronate, denosumab, zoledronate, trastuzumab, tykerb, anthracyclines (e.g., daunorubicin and doxorubicin), bevacizumab, oxaliplatin, melphalan, etoposide, mechlorethamine, bleomycin, microtubule poison, annonaceous acetogenin, or combinations thereof.

[0298] Alternatively or alternatively, in some embodiments, the antibody or antigen-binding fragment of the present invention may be administered alone, sequentially or simultaneously with at least one other immunomodulatory / stimulatory antibody, including but not limited to anti-PD-1 antibody, anti-PD-L1 antibody, anti-PD-L2 antibody, anti-CTLA-4 antibody, anti-TIM3 antibody, anti-4-1BB antibody, anti-CD73 antibody, anti-GITR antibody and anti-LAG-3 antibody.

[0299] The compositions of this invention can optionally be administered as a single bolus injection to subjects in need. Alternatively, the dosing regimen may include multiple administrations at different times after tumor onset.

[0300] It can be administered via any suitable route, including oral, intranasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), rectal, intracranial, intrathecal, or local administration. Administration includes self-administration and administration by another person. It should also be understood that the various models of treatment for medical conditions described are intended to represent "basic," which includes complete treatment as well as secondary treatments, and in which some biological or medically relevant outcome is achieved.

[0301] In some embodiments, the antibodies of the present invention comprise pharmaceutical formulations that can be administered to subjects in need at one or more doses. The dosing regimen can be adjusted to provide a desired response (e.g., a therapeutic response).

[0302] Typically, the effective amount of the antibody composition of the present invention sufficient to achieve a therapeutic effect ranges from about 0.000001 mg / kg body weight / day to about 10,000 mg / kg body weight / day. Typically, the dosage ranges from about 0.0001 mg / kg body weight / day to about 100 mg / kg body weight / day. For administering the anti-GPA33 antibody, the dosage ranges from about 0.0001 to 100 mg / kg of the subject's body weight, and more typically from 0.01 to 5 mg / kg weekly, bi-weekly, or bi-weekly. For example, the dosage may be 1 mg / kg body weight or 10 mg / kg body weight weekly, bi-weekly, or bi-weekly, or in the range of 1-10 mg / kg weekly, bi-weekly, or bi-weekly. In one embodiment, a single dose of the antibody ranges from 0.1 to 10,000 micrograms / kg body weight. In one embodiment, the concentration of the antibody in the carrier ranges from 0.2 to 2000 micrograms / delivery mL. Exemplary treatment regimens require administration once every two weeks, once a month, or every 3 to 6 months. Anti-GPA33 antibodies can be administered in a variety of situations. The intervals between single doses can be hourly, daily, weekly, monthly, or yearly. Intervals can also be irregular, as indicated by measuring the blood levels of the antibody in the subject. In some methods, the dose is adjusted to achieve serum antibody concentrations in the subject of approximately 75 μg / mL to approximately 125 μg / mL, 100 μg / mL to approximately 150 μg / mL, approximately 125 μg / mL to approximately 175 μg / mL, or approximately 150 μg / mL to approximately 200 μg / mL. Alternatively, anti-GPA33 antibodies can be administered as a sustained-release formulation, in which case a lower frequency of administration is required. The dose and frequency vary based on the antibody's half-life in the subject. The dose and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic use, relatively low doses are administered at relatively infrequent intervals over a longer period. In therapeutic use, relatively high doses are sometimes required at relatively short intervals until disease progression decreases or ceases, or until the subject shows partial or complete improvement in disease symptoms. After that, preventative measures can be administered to the patient.

[0303] On the other hand, this disclosure provides a method for detecting tumors in a subject in vivo, the method comprising: (a) administering to the subject an effective amount of an antibody (or an antigen-binding fragment thereof) of the present invention, wherein the antibody is configured to target a tumor expressing GPA33 and is labeled with a radioactive isotope; and (b) detecting the presence of a tumor in the subject by detecting a level of radioactivity emitted by the antibody that is higher than a reference value. In some embodiments, the reference value is expressed as an injection dose per gram (ID / g%). The reference value can be calculated by measuring the level of radioactivity present in non-tumor (normal) tissue and calculating the mean level of radioactivity present in non-tumor (normal) tissue ± standard deviation. In some embodiments, the ratio of radioactivity levels between tumor and normal tissue is approximately 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, or 100:1.

[0304] In some embodiments, the subject is diagnosed with or suspected of having cancer. The level of radioactivity emitted by the antibody can be detected using positron emission tomography (PET) or single-photon emission computed tomography (SPECT).

[0305] Alternatively or additionally, in some embodiments, the method further includes administering an effective amount of an immunoconjugate to the subject, the immunoconjugate comprising an antibody of the present invention conjugated to a radionuclide. In some embodiments, the radionuclide is an alpha particle emission isotope, a beta particle emission isotope, an Auger emitter, or any combination thereof. Examples of beta particle emission isotopes include... 86 Y、 90 Y、 89 Sr、 165 Dy、 186 Re、 188 Re、 177 Lu and 67 Examples of Cu alpha particle emission isotopes include 213 Bi、 211 At、 225 Ac、 152 Dy、 212 Bi、 223 Ra、 219 Rn、 215 Po、 211 Bi、 221 Fr、 217 At and 255 Fm. Examples of Auger projectors include 111 In、67 Ga、 51 Cr 58 Co、 99m Tc, 103m Rh、 195m Pt, 119 Sb, 161 Ho、 189m Os、 192 Ir、 201 Tland 203 Pb. In some embodiments of the method, nonspecific FcR-dependent binding in normal tissue is eliminated or reduced (e.g., via an N297A mutation in the Fc region, which leads to glycosylation). The therapeutic effect of such immunoconjugates can be determined by calculating the area under the curve (AUC) tumor:AUC normal tissue ratio. In some embodiments, the immunoconjugates have an AUC tumor:AUC normal tissue ratio of about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, or 100:1.

[0306] PRIT. In one aspect, this disclosure provides a method for detecting tumors in a subject of need, the method comprising: (a) administering to the subject an effective amount of a complex comprising a radiolabeled DOTA hapten and a multispecific antibody of the present invention binding to the radiolabeled DOTA hapten and a GPA33 antigen, wherein the complex is configured to target a tumor expressing the GPA33 antigen recognized by the multispecific antibody of the complex; and (b) detecting the presence of a solid tumor in the subject by detecting a level of radioactivity emitted by the complex above a reference value. In some embodiments, the subject is a human.

[0307] On the other hand, this disclosure provides a method for selecting a subject for pre-targeted radioimmunotherapy, the method comprising: (a) administering to the subject an effective amount of a complex comprising a radiolabeled DOTA hapten and a multispecific antibody of the present invention binding to the radiolabeled DOTA hapten and a GPA33 antigen, wherein the complex is configured to target a tumor expressing the GPA33 antigen recognized by the multispecific antibody of the complex; (b) detecting the level of radioactivity emitted by the complex; and (c) selecting the subject for pre-targeted radioimmunotherapy when the level of radioactivity emitted by the complex is higher than a reference value. In some embodiments, the subject is a human.

[0308] Examples of DOTA haptens include (i) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH2; (ii) Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH2; (iii) DOTA-D-Asp-D-Lys(HSG)-D-Asp-D-Lys(HSG)-NH2; (iv) DOTA-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (v) DOTA-D-Tyr-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (vi) DOTA-D-Ala-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (vii) DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH2; (viii) Ac-D-Phe-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-NH2; (ix) Ac-D-Phe-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH2; (x) Ac-D-Phe-D-Lys(Bz-DTPA)-D-Tyr-D-Lys(Bz-DTPA)-NH2; (xi) Ac-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH2; (xii) DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH2; (xiii) (Tscg-Cys)-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(DOTA)-NH2; (xiv) Tscg-D-Cys-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (xv) (Tscg-Cys)-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (xvi) Ac-D-Cys-D-Lys(DOTA)-D-Tyr-D-Ala-D-Lys(DOTA)-D-Cys-NH2; (xvii) Ac-D-Cys-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH2; (xviii) Ac-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-D-Lys(Tscg-Cys)-NH2;(xix) Ac-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-D-Lys(Tscg-Cys)-NH2; (xx) BnDOTA; (xxi) DOTA; (xxii) BnDOTA-Biotin; and (xxiii) DOTA-Biotin. The radioactive label may be an alpha particle emission isotope, a beta particle emission isotope, or an Auger emitter. Examples of radioactive labels include; 213 Bi、 211 At、 225 Ac、 152 Dy、 212 Bi、 223 Ra、 219 Rn、 215 Po、 211 Bi、 221 Fr、 217 At、 255 Fm、 86 Y、 90 Y、 89 Sr、 165 Dy、 186 Re、 188 Re、 177 Lu、 67 Cu、 111 In、 67 Ga、 51 Cr 58 Co、 99m Tc, 103m Rh、 195m Pt, 119 Sb, 161 Ho、 189m Os、 192 Ir、 201 Tl、 203 Pb, 68 Ga、 227 Th or 64 Cu.

[0309] In some embodiments of the methods disclosed herein, the level of radioactivity emitted by the complex is detected using positron emission tomography (PET) or single-photon emission computed tomography (SPECT). Alternatively or additionally, in some embodiments of the methods disclosed herein, the subject is diagnosed with or suspected of having GPA33-related cancers, such as colorectal cancer, T-cell leukemia, pseudomyxoma peritonei, appendiceal cancer, pancreatic cancer, and gastric cancer.

[0310] Alternatively or concurrently, in some embodiments of the methods disclosed herein, the complex is administered intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intracapsularly, intraorally, intraperitoneally, via the trachea, subcutaneously, intravenously, orally, intratumorally, or intranasally. In some embodiments, the complex is administered into the subject's cerebrospinal fluid or blood.

[0311] In some embodiments of the methods disclosed herein, the level of radioactivity emitted by the complex is detected between 2 and 120 hours after administration of the complex. In some embodiments of the methods disclosed herein, the level of radioactivity emitted by the complex is expressed as a percentage of the injected dose per gram of tissue (ID / g%). A reference value can be calculated by measuring the level of radioactivity present in non-tumor (normal) tissue and calculating the mean level of radioactivity present in non-tumor (normal) tissue ± standard deviation. In some embodiments, the reference value is a standard uptake value (SUV). See Thie JA, J Nucl Med. 45(9):1431-4 (2004). In some embodiments, the ratio of radioactivity levels between tumor and normal tissue is approximately 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, or 100:1.

[0312] On the other hand, this disclosure provides a method for increasing the tumor sensitivity to radiotherapy in a subject diagnosed with GPA33-related cancer, the method comprising: (a) administering to the subject an effective amount of an anti-DOTA multispecific antibody of the present invention, wherein the anti-DOTA multispecific antibody is configured to target a tumor expressing a GPA33 antigen; and (b) administering to the subject an effective amount of a radiolabeled DOTA hapten, wherein the radiolabeled DOTA hapten is configured to bind to the anti-DOTA multispecific antibody. In some embodiments, the subject is a human.

[0313] The anti-DOTA multispecific antibody is administered for a period of time under conditions sufficient to saturate tumor cells (e.g., according to the dosing regimen). In some embodiments, unbound anti-DOTA multispecific antibodies are removed from the bloodstream after administration of the anti-DOTA multispecific antibody. In some embodiments, the radiolabeled DOTA hapten is administered after a period of time sufficient to clear unbound anti-DOTA multispecific antibodies.

[0314] The radiolabeled DOTA hapten can be administered at any time between 1 minute and 4 days or more after administration of the anti-DOTA multispecific antibody. For example, in some embodiments, the radiolabeled DOTA hapten is administered at 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 1.25 hours, 1.5 hours, 1.75 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, or any range thereof, after administration of the anti-DOTA multispecific antibody. Alternatively, the radiolabeled DOTA hapten can be administered at any time 4 days or more after the administration of the anti-DOTA multispecific antibody.

[0315] Additionally or alternatively, in some embodiments, the method further includes administering an effective amount of a scavenger to the subject prior to the administration of the radiolabeled DOTA hapten. The scavenger can be any molecule (dextran or dendritic macromolecule or polymer) that can conjugate with the C825 hapten. In some embodiments, the scavenger is not more than 2000 kD, 1500 kD, 1000 kD, 900 kD, 800 kD, 700 kD, 600 kD, 500 kD, 400 kD, 300 kD, 200 kD, 100 kD, 90 kD, 80 kD, 70 kD, 60 kD, 50 kD, 40 kD, 30 kD, 20 kD, 10 kD, or 5 kD. In some embodiments, the scavenger is a 500 kD glycosaminoglycan-DOTA conjugate (e.g., 500 kD glycosaminoglycan-DOTA-Bn (Y), 500 kD glycosaminoglycan-DOTA-Bn (Lu), or 500 kD glycosaminoglycan-DOTA-Bn (In), etc.).

[0316] In some embodiments, a scavenging agent and a radiolabeled DOTA hapten are administered without further administration of the anti-DOTA multispecific antibody of the present invention. For example, in some embodiments, the anti-DOTA multispecific antibody of the present invention is administered according to a protocol comprising at least one of the following cycles: (i) administration of the anti-DOTA multispecific antibody of the present invention (optionally saturating the relevant tumor cells); (ii) administration of a radiolabeled DOTA hapten and optionally a scavenging agent; (iii) optional additional administration of a radiolabeled DOTA hapten and / or a scavenging agent without additional administration of the anti-DOTA multispecific antibody. In some embodiments, the method may include multiple such cycles (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more cycles).

[0317] Alternatively or alternatively, in some embodiments of the method, the anti-DOTA multispecific antibody and / or the radiolabeled DOTA hapten are administered intravenously, intramuscularly, intra-arterially, intrathecally, intracapsularly, intracapsularly, intraorally, intraperitoneally, via the trachea, subcutaneously, intravenously, intratumorally, or orally, via the mouth.

[0318] On one hand, this disclosure provides a method for increasing the tumor sensitivity to radiotherapy in a subject diagnosed with GPA33-related cancer. The method includes administering an effective amount of a complex to the subject, the complex comprising a radiolabeled DOTA hapten and a multispecific antibody of the present invention that recognizes and binds to the radiolabeled DOTA hapten and a GPA33 antigen target, wherein the complex is configured to target a tumor expressing the GPA33 antigen target recognized by the multispecific antibody of the complex. The complex can be administered intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intracapsularly, intraorally, intraperitoneally, intratracheally, subcutaneously, intravenously, orally, intratumorally, or intranasally. In some embodiments, the subject is a human.

[0319] On the other hand, this disclosure provides a method for treating cancer in a subject of need, the method comprising: (a) administering to the subject an effective amount of an anti-DOTA multispecific antibody of the present invention, wherein the anti-DOTA multispecific antibody is configured to target a tumor expressing a GPA33 antigen; and (b) administering to the subject an effective amount of a radiolabeled DOTA hapten, wherein the radiolabeled DOTA hapten is configured to bind to the anti-DOTA multispecific antibody. The anti-DOTA multispecific antibody is administered for a period of time under conditions sufficient to saturate tumor cells (e.g., according to a dosing regimen). In some embodiments, unbound anti-DOTA multispecific antibodies are removed from the bloodstream after administration of the anti-DOTA multispecific antibody. In some embodiments, the radiolabeled DOTA hapten is administered after a period of time sufficient to clear unbound anti-DOTA multispecific antibodies. In some embodiments, the subject is a human.

[0320] Therefore, in some embodiments, the method further includes administering an effective amount of a scavenger to the subject prior to the administration of the radiolabeled DOTA hapten. The radiolabeled DOTA hapten can be administered at any time between 1 minute and 4 days or more after the administration of the anti-DOTA multispecific antibody. For example, in some embodiments, the radiolabeled DOTA hapten is administered at 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 1.25 hours, 1.5 hours, 1.75 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, or any range thereof, after administration of the anti-DOTA multispecific antibody. Alternatively, the radiolabeled DOTA hapten can be administered at any time 4 days or more after the administration of the anti-DOTA multispecific antibody.

[0321] The scavenger may be a 500 kD glycosaminoglycan-DOTA conjugate (e.g., 500 kD glycosaminoglycan-DOTA-Bn(Y), 500 kD glycosaminoglycan-DOTA-Bn(Lu), or 500 kD glycosaminoglycan-DOTA-Bn(In), etc.). In some embodiments, the scavenger and radiolabeled DOTA hapten are administered without further administration of anti-DOTA multispecific antibody. For example, in some embodiments, the anti-DOTA multispecific antibody is administered according to a protocol comprising at least one of the following cycles: (i) administration of the anti-DOTA multispecific antibody of the present invention (optionally saturating the relevant tumor cells); (ii) administration of radiolabeled DOTA hapten and optionally the scavenger; (iii) optionally additional administration of radiolabeled DOTA hapten and / or the scavenger without additional administration of anti-DOTA multispecific antibody. In some embodiments, the method may include multiple such cycles (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more cycles).

[0322] This article also provides a method for treating cancer in a subject of need, the method comprising administering to the subject an effective amount of a complex comprising a radiolabeled DOTA hapten and a multispecific antibody of the present invention that recognizes and binds to the radiolabeled DOTA hapten and a GPA33 antigen target, wherein the complex is configured to target a tumor expressing the GPA33 antigen target recognized by the multispecific antibody of the complex. The therapeutic effect of such a complex can be determined by calculating the area under the curve (AUC) tumor:AUC normal tissue ratio. In some embodiments, the complex has an AUC tumor:AUC normal tissue ratio of about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, or 100:1.

[0323] Ex vivo armed T cells. In one aspect, this disclosure provides an ex vivo armed T cell coated or conjugated with an effective amount of the present invention's anti-GPA33 multispecific antibody, wherein the anti-GPA33 multispecific antibody contains a CD3-binding domain, and wherein the anti-GPA33 multispecific antibody is an immunoglobulin comprising two heavy chains and two light chains, wherein each of the light chains is fused to a single-chain variable fragment (scFv). In some embodiments, at least one scFv of the anti-GPA33 multispecific antibody contains a CD3-binding domain. Additionally or alternatively, in some embodiments, at least one scFv of the anti-GPA33 multispecific antibody contains a DOTA-binding domain.

[0324] This article also discloses a method for treating GPA33-related cancer in subjects of need, the method comprising administering an effective amount of the ex vivo armed T cells disclosed herein to the subject.

[0325] Toxicity. Optimal, an effective amount (e.g., dose) of the anti-GPA33 antibody described herein will provide a beneficial therapeutic effect without causing substantial toxicity to the subject. The toxicity of the anti-GPA33 antibody described herein can be determined by standard pharmaceutical procedures in cell cultures or laboratory animals, for example by determining the LD50. 50 (50% of the lethal dose) or LD 100 (Dose in the lethal population). The dose-to-toxicity ratio is the therapeutic index. Data from these cell culture assays and animal studies can be used to establish a dose range that is non-toxic for human use. The doses of the anti-GPA33 antibody described herein are within a range of circulating concentrations that contain effective doses with low or no toxicity. The dose may vary within this range depending on the dosage form and route of administration used. Precise dosing, route of administration, and dose can be selected by an individual physician based on the patient's condition. See, for example, Fingl et al., in: The Pharmacological Basis of Therapeutics, Ch. 1 (1975).

[0326] Formulas of pharmaceutical compositions. According to the methods of the present invention, anti-GPA33 antibodies can be incorporated into pharmaceutical compositions suitable for administration. Pharmaceutical compositions typically comprise recombinant or substantially purified antibodies in a form suitable for administration to a subject and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is determined in part by the specific composition being administered and by a specific method used to administer the composition. Therefore, a variety of suitable pharmaceutical composition formulations for administering antibody compositions exist (see, for example, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA 18th edition, 1990). The pharmaceutical compositions are typically formulated to be sterile, substantially isotonic, and fully compliant with all Good Manufacturing Practices (GMP) requirements of the U.S. Food and Drug Administration.

[0327] The terms “pharmaceutically acceptable,” “physiologically tolerable,” and their grammatical variations, used to refer to compositions, carriers, diluents, and reagents, are used interchangeably and indicate that the substance can be administered to or on a subject without producing an undesirable physiological effect to the extent that would prevent the administration of the composition. For example, “pharmaceuticalally acceptable excipients” means excipients that can be used to prepare generally safe, non-toxic, and desirable pharmaceutical compositions, and includes excipients acceptable for veterinary use and for human pharmaceutical use. Such excipients can be solid, liquid, semi-solid, or, in the case of aerosol compositions, gaseous. “Pharmaceuticalally acceptable salts and esters” means salts and esters that are pharmaceutically acceptable and have the desired pharmacological properties. Such salts include those that can be formed when the acidic protons present in the composition can react with inorganic or organic bases. Suitable inorganic salts include those formed with alkali metals, such as sodium and potassium, magnesium, calcium, and aluminum. Suitable organic salts include those formed with organic bases, such as amine bases, such as ethanolamine, diethanolamine, triethanolamine, trimethylamine, N-methylglucosamine, etc. This class of salts also includes acid addition salts formed with inorganic acids (e.g., hydrochloric acid and hydrobromic acid) and organic acids (e.g., acetic acid, citric acid, maleic acid, and alkane and aromatic sulfonic acids, such as methanesulfonic acid and benzenesulfonic acid). Pharmaceutically acceptable esters include esters formed from carboxyl, sulfonyl, and phosphono groups present in anti-GPA33 antibodies, for example, C... 1-6Alkyl esters. When two acidic groups are present, a pharmaceutically acceptable salt or ester can be a monoacid monosalt or ester, or a disalt or ester; and similarly, when more than two acidic groups are present, some or all of such groups may be salted or esterified. Anti-GPA33 antibodies named in this art may be present in an unsalted or unesterified form, or in a salted and / or esterified form, and the naming of such anti-GPA33 antibodies is intended to encompass both the original (unsalted and unesterified) compound and its pharmaceutically acceptable salt and ester. Similarly, in some embodiments of the present invention, they may be present in more than one stereoisomer, and the naming of such anti-GPA33 antibodies is intended to encompass all single stereoisomers and all mixtures of such stereoisomers (whether racemic or otherwise). For specific pharmaceuticals and compositions of the present invention, appropriate timing, sequence, and dosage can be readily determined by those skilled in the art.

[0328] Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextran solution, and 5% human serum albumin. Liposomes and non-aqueous media, such as non-volatile oils, may also be used. The use of such media and compounds for pharmaceutically active substances is well known in the art. The use of said media or compounds in the composition is contemplated, except in cases where any conventional media or compounds are incompatible with anti-GPA33 antibodies. Additional active compounds may also be incorporated into the composition.

[0329] The pharmaceutical compositions of this invention are formulated to be compatible with their intended route of administration. The anti-GPA33 antibody compositions of this invention can be administered via parenteral, topical, intravenous, oral, subcutaneous, intra-arterial, intradermal, transdermal, rectal, intracranial, intrathecal, intraperitoneal, intranasal, or intramuscular routes, or as an inhaler. The anti-GPA33 antibody can optionally be administered in combination with other agents that are at least partially effective in treating various GPA33-related cancers.

[0330] Solutions or suspensions intended for parenteral, intradermal, or subcutaneous application may contain the following components: sterile diluents, such as water for injection, saline solutions, fixative oils, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; antibacterial compounds, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating compounds, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates, or phosphates; and tonic compounds, such as sodium chloride or dextrose. The pH may be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be sealed in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0331] Suitable pharmaceutical compositions for injection include sterile aqueous solutions (water-soluble) or dispersions and sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial aqueous solutions, and Cremophor EL. TM (BASF, Parsippany, NJ) or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and should be an easily injectable fluid. It must be stable under production and storage conditions and must be protected against contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Appropriate flowability can be maintained, for example, by using coatings such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal compounds (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). In many cases, it is desirable to include isotonic compounds in the composition, such as sugars, polyols such as mannitol, sorbitol, or sodium chloride. Extended absorption of injectable compositions can be achieved by including compounds that delay absorption, such as aluminum monostearate and gelatin.

[0332] Sterile injectable solutions can be prepared by incorporating the required amount of the anti-GPA33 antibody of the present invention, as needed, with one or a combination of the components listed above into a suitable solvent, followed by filtration and sterilization. Typically, dispersions are prepared by incorporating the anti-GPA33 antibody into a sterile medium containing a base dispersion medium and any other desired components from those listed above. In the case of sterile powders used to prepare sterile injectable solutions, the preparation method is vacuum drying and freeze-drying, which produces an active ingredient powder from a previously sterile filtered solution plus any other desired components. The antibodies of the present invention can be administered in the form of a reservoir injection or implantation formulation, which can be formulated in such a manner to allow for sustained or pulsatile release of the active ingredient.

[0333] Oral compositions typically contain an inert diluent or an edible carrier. The oral composition may be encapsulated in gelatin capsules or compressed into tablets. For oral therapeutic administration, anti-GPA33 antibodies may be incorporated with excipients and used in tablet, lozenge, or capsule form. Oral compositions may also be prepared using a fluid carrier used as a mouthwash, wherein the compound in the fluid carrier is administered orally and swished and spat out or swallowed. Pharmaceutically compatible binding compounds and / or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, lozenges, etc., may contain any of the following ingredients or compounds with similar properties: binders such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch or lactose; disintegrating compounds such as alginate, Primogel, or corn starch; lubricants such as magnesium stearate or stearates; gliding agents such as colloidal silica; sweeteners such as sucrose or saccharin; or flavoring compounds such as peppermint, methyl salicylate, or orange flavoring.

[0334] For administration by inhalation, anti-GPA33 antibodies are delivered as an aerosol spray from a pressurized container or dispenser containing a suitable propellant, such as a gas or spray agent like carbon dioxide.

[0335] Systemic application can also be performed via mucosal or transdermal routes. For mucosal or transdermal application, a penetrant suitable for the barrier to be penetrated is used in the formulation. Such penetrants are generally known in the art, and for mucosal application, they include, for example, cleansers, bile salts, and clostridial acid derivatives. Mucosal application can be achieved by using nasal sprays or suppositories. For transdermal application, as is generally known in the art, anti-GPA33 antibodies are formulated as ointments, creams, gels, or lotions.

[0336] Anti-GPA33 antibodies can also be formulated into pharmaceutical compositions in the form of suppositories or retention enemas for rectal delivery (e.g., using conventional suppository bases such as cocoa butter and other glycerides).

[0337] In one embodiment, the anti-GPA33 antibody is prepared together with a carrier that protects the anti-GPA33 antibody from rapid elimination from the body, such as a controlled-release formulation, comprising an implant and a microencapsulated delivery system. Biodegradable polymers and biocompatible polymers, such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, can be used. Methods for preparing such formulations will be apparent to those skilled in the art. The materials are also commercially available from Alza and Nova Pharmaceuticals, Inc. Liposome suspensions (containing liposomes with monoclonal antibodies against viral antigens targeting infected cells) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0338] Reagent test kit

[0339] This invention provides a kit for the detection and / or treatment of GPA33-related cancers, the kit comprising at least one immunoglobulin-associated composition of this invention (e.g., any antibody or antigen-binding fragment described herein) or a functional variant thereof (e.g., a substituted variant). Optionally, the components described above in the kit of this invention are packaged in suitable containers and labeled for the diagnosis and / or treatment of GPA33-related cancers. The components may be stored in single or multi-dose containers, such as sealed ampoules, vials, bottles, syringes, and test tubes, in the form of an aqueous solution for reconstitution, preferably a sterile solution, or in lyophilized, preferably sterile formulation. The kit may further include a second container containing a diluent suitable for diluting the pharmaceutical composition to a higher volume. Suitable diluents include, but are not limited to, pharmaceutically acceptable excipients of the pharmaceutical composition and saline solutions. Furthermore, the kit may include instructions for diluting the pharmaceutical composition and / or instructions for administering the pharmaceutical composition (whether diluted or not). The container can be formed of various materials, such as glass or plastic, and can have a sterile access port (e.g., the container can be an intravenous solution bag or a vial with a stopper that can be punctured by a hypodermic needle). The kit may further include additional containers comprising pharmaceutically acceptable buffers, such as phosphate-buffered saline, Ringer's solution, and dextran solution. The kit may further include other materials desirable from a commercial and user perspective, including additional buffers, diluents, filters, needles, syringes, and culture media for one or more suitable hosts. The kit may optionally include instructions for use, typically included in the commercial packaging of a therapeutic or diagnostic product, containing information regarding, for example, indications, usage, dosage, manufacture, administration, contraindications, and / or warnings related to the use of such therapeutic or diagnostic products.

[0340] This kit can be used to detect the presence of immunoreactive GPA33 protein in biological samples, such as any bodily fluid, including but not limited to serum, plasma, lymph, cystic fluid, urine, feces, cerebrospinal fluid, ascites, or blood, and includes biopsy samples of body tissue. For example, the kit may contain: one or more humanized, chimeric, or bispecific anti-GPA33 antibodies (or antigen-binding fragments thereof) of the present invention capable of binding to GPA33 protein in a biological sample; means for determining the amount of GPA33 protein in the sample; and means for comparing the amount of immunoreactive GPA33 protein in the sample with a standard. One or more anti-GPA33 antibodies may be labeled. Kit components (e.g., reagents) may be packaged in suitable containers. The kit may further include instructions for using the kit to detect immunoreactive GPA33 protein.

[0341] For an antibody-based kit, the kit may contain, for example: 1) a first antibody, such as a humanized, chimeric, or bispecific GPA33 antibody (or an antigen-binding fragment thereof) of the present invention, which is linked to a solid support that binds to the GPA33 protein; and optionally; 2) a second different antibody that binds to the GPA33 protein or the first antibody and is conjugated to a detectable label.

[0342] The kit may also contain, for example, buffers, preservatives, or protein stabilizers. The kit may further contain components required for detecting the detectable marker, such as enzymes or substrates. The kit may also contain control samples or a series of control samples that can be measured and compared with the test sample. Each component of the kit may be packaged in a separate container, and all various containers may be in a separate package along with instructions for interpreting the results of assays performed using the kit. The kit of the present invention may contain a written product on or within the kit container. The written product describes how to use the reagents contained in the kit, for example, for detecting GPA33 protein in vitro or in vivo, or for treating GPA33-related cancers in subjects of need. In some embodiments, the use of the reagents may be performed according to the methods of the present invention.

[0343] Example

[0344] The following examples further illustrate the technology of the present invention, and these examples should not be construed as limiting in any way. The following examples demonstrate the preparation, characterization, and use of illustrative GPA33 antibodies based on the technology of the present invention. The following examples demonstrate the generation of chimeric, humanized, and bispecific antibodies based on the technology of the present invention, and the characterization of their binding specificity and in vivo biological activity.

[0345] Example 1: Materials and Methods

[0346] Target antigen quantification

[0347] The Equivalent Soluble Fluorescent Dye Quantum Molecular (MESF) Kit (Bangs Laboratories) was used to quantify GPA33 expression in T-ALL (MOLT16, PF-382, CEM.NRK, and 8402) and CRC (LS274T and SW1222) cell lines using the H1L4 GPA33 IgG1 monoclonal antibody (mAb). These experiments were performed according to the manufacturer's specifications. Briefly, fluorescence intensity was calibrated on an Attune NxT flow cytometer (Invitrogen), and calibration curves were generated using the QuickCal v.3.0 Quantitative Analysis Template (Bangs Laboratories). Cells were incubated with the H1L4 GPA33 IgG antibody, washed, incubated with the secondary fluorescent antibody, washed again, and run on the flow cytometer under the same conditions used to generate the calibration curves.

[0348] Surface plasmon resonance

[0349] Human recombinant GPA33 (GPA33, Novoprotein Scientific Inc.) was immobilized on the S-series CM5Biacore sensor chip. Using Biacore... ™ The T100 system allows five sequentially diluted, two-fold concentrations of GPA33 IgG1 mAb or T-BsAb (starting from 20 nM) to flow through the chip. Binding kinetics of GPA33 IgG1 mAb were measured at 25 °C, and binding kinetics of T-BsAb were measured at both 25 °C and 37 °C. All sensor maps were equipped with a 1:1 binding model to derive kinetic parameters.

[0350] In vivo anti-tumor efficacy experiment

[0351] 6- to 8-week-old females with immunodeficiency NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ (NSG) mice were purchased from Jackson Laboratory and fed Sulfatrim food. Each mouse was injected with 5 x 10 g of the drug via the tail vein. 5 Human leukemia cells transduced with luciferase were used to establish xenograft leukemia. In vivo bioluminescence imaging using an IVIS spectrometer confirmed the transplantation of the leukemia xenograft. Mice received 2 x 10⁻⁶ cells twice weekly. 7Treatment consisted of one EAT (via retroorbital injection) and 1 µg of recombinant human interleukin-15 (IL-15, via intraperitoneal injection), for a total of 4 or 6 treatments. This was achieved by administering 2 x 10 7 Ex vivo armed T cells (EATs) were prepared by incubating activated human T cells with 10 µg T-BsAb in 100 µL of cell culture medium for 20 minutes. After incubation, the EATs were washed twice with phosphate-buffered saline (PBS) to remove unbound T-BsAb. EATs were prepared immediately before injection. All experiments included an untreated group (tumor only) and a group treated with unarmed T cells and IL-15 (T cells only) as a negative control. In vivo bioluminescence imaging was performed weekly to monitor tumor growth. Mice were weighed weekly. Mice were euthanized if they lost more than 10% of their starting body weight, developed hind limb paralysis (a known complication of xenograft tumors in leukemia), or were in a dying state. Mice whose tumors were cured and subsequently developed clinical signs of graft-versus-host disease (GVHD, caused by transplanted human T cells attacking normal mouse tissue) were treated with 2 to 5 doses of CD3 x CD3 T-BsAb (BC276, 3 µg per dose, twice a week) to induce T cell self-killing and reverse GVHD, thereby enabling long-term monitoring of leukemia relapse.

[0352] Example 2: T-cell acute lymphoblastic leukemia cell line expressing cell surface GPA33

[0353] Flow cytometry was performed on T-cell acute lymphoblastic leukemia (T-ALL) (MOLT16, PF-382, CEM.NKR, and 8402) and colorectal cancer (CRC) (LS174T and SW1222) cell lines to quantify GPA33 expression. While CRC cell lines expressed over 200,000 GPA33 molecules per cell, GPA33 expression on T-ALL cell lines was much lower and somewhat variable, ranging from less than 1,000 to over 20,000 molecules per cell. Figure 2 ).

[0354] Example 3: Affinity maturation of low-affinity H3L3 GPA33 antibody improves tumor control and survival in mice.

[0355] Affinity maturation improved the K-type affinity of the low-affinity H3L3 GPA33 antibody. D The multiple is approximately ten times (1.35E-11 compared to 1.68E-10). Figure 3AWhen used in vivo to arm T cells to treat PF-382 leukemia xenografts in mice, the affinity-matured H3L3 GPA33 antibody completely controlled tumor growth compared to the original H3L3 GPA33 antibody, which only delayed tumor growth during treatment but did not eradicate the tumor, leading to relapse after treatment interruption. Figure 3B Although all five mice in the H3L3 group died on day 105 after leukemia engraftment, four out of five mice in the H3L3 affinity maturation group survived for more than 400 days after engraftment without leukemia relapse. Figure 3B ).

[0356] Compared with low-affinity H3L3 GPA33 T-BsAb, affinity-matured GPA33 H3L3 T-BsAb showed improved EC50 TDCC values ​​in CEM.NKR T-ALL cells. Figures 3E to 3F (See top small image). In the more aggressive CEM.NKR T-ALL model, the affinity-matured GPA33 H3L3 T-BsAb was slightly more effective in controlling tumor growth than GPA33 T-BsAb BC123. Figure 3C Even more impressive results were observed in overall survival, with 3 / 5 of the mice in the affinity-mature H3L3 GPA33 T-BsAb group surviving for more than 150 days, while all mice treated with BC123 died by day 80. Figure 3C These results indicate that affinity maturation of low-affinity GPA33 T-BsAb leads to improved antitumor efficacy and survival.

[0357] These results demonstrate that the antibodies or antigen-binding fragments of the present invention specifically bind to the GPA33 antigen with high binding affinity. Therefore, the immunoglobulin-associated compositions disclosed herein can be used for the detection and treatment of GPA33-positive cancers in subjects of need.

[0358] Example 4: High-affinity H1L4 antibody with mature affinity eradicates PF-382 xenograft leukemia and induces small cell lung cancer. Long-term survival of rats

[0359] Compared to the low-affinity H3L3 GPA33 antibody, the high-affinity H1L4 GPA33 antibody exhibits a nearly 100-fold stronger binding affinity to GPA33 (K). D = 9.93E-11 compared to 1.68E-10) Figure 3A The same applies to the affinity-matured H1L4 GPA33 antibody (KD = 8.00E-12 vs. 1.35E-11). Figure 3ABased on previous experiments demonstrating a correlation between binding affinity and in vivo antitumor efficacy, we expected that affinity-matured H1L4 GPA33 T-BsAb would be superior to the original H1L4 GPA33 antibody. We tested T cells armed with these antibodies in mice carrying PF-382 T-ALL xenografts. Indeed, affinity-matured H1L4 GPA33 T-BsAb completely eradicated leukemia in 5 out of 5 mice, resulting in all 5 mice surviving for more than 150 days. Figure 3D The original H1L4 GPA33 T-BsAb inhibited leukemia growth for approximately 40 days, but tumor relapse subsequently occurred, resulting in poor survival outcomes. Figure 3D These results confirm our observations that affinity maturation and the resulting increased binding affinity lead to better tumor control and improved survival in a mouse model of T-ALL xenografts. Compared to low-affinity H1L4 GPA33 T-BsAb, affinity-matured GPA33 H1L4 T-BsAb showed improved EC50 TDCC values ​​in CEM.NKR T-ALL cells. Figure 3E ).

[0360] Therefore, the immunoglobulin-related compositions disclosed herein can be used to detect and treat GPA33-positive cancers in subjects in need.

[0361] Example 5: In vivo distribution of the anti-huGPA33 x DOTA antibody of the present invention.

[0362] Ten days prior to treatment, SW1222 lateral ventral tumors were subcutaneously implanted. Two days prior to payload injection, 250 µg of SADA constructs (TC170, TC386) were injected. 400 pmole of a 40 µCi isotope was then injected. 177 Lu-DOTA was used to evaluate the biodistribution of the SADA construct in tumors and tissues (including the kidneys) at 2, 72, and 120 hours after payload injection. See also Figure 15 .

[0363] like Figure 15 As shown, after payload injection, over time (2 hours, 72 hours, 120 hours), the SADA construct containing the affinity-mature H1L4 sequence (TC386) showed consistently higher tissue uptake than the standard affinity SADA construct (TC170) containing the H1L4 parent clone sequence.

[0364] Therefore, the immunoglobulin-related compositions disclosed herein can be used to detect and treat GPA33-positive cancers in subjects in need.

[0365] Example 6: In vivo therapeutic effect of the huGPA33-C825 antibody of the present invention

[0366] This example illustrates the in vivo efficacy of the huGPA33-C825 bispecific antibody in PRIT in reducing tumor burden in mice carrying GPA33-positive cancer cells. Specifically, this example describes the effects of single-cycle and dual-cycle therapy on tumor burden in mice carrying SW1222 tumors.

[0367] During the single-cycle therapy study, five groups of tumor-bearing mice (n = 6 to 8 per group) received treatment with any of the following: solvent (i.e., untreated, n = 8, TV7: 76 ± 15 mm). 3 ), only 33.3 MBq 177 Lu-DOTA-Bn (the solvent was administered during the bispecific antibody and scavenger injection, n = 6, TV7: 116 ± 23 mm) 3 Single-cycle IgG-C825PRIT + 33.3 MBq 177 Lu-DOTA-Bn (ns replace huGPA33-C825 to administer IgG-C825, n = 8, TV7: 100 ± 10 mm) 3 ), or single-cycle huGPA33-C825 (huGPA33-huC825 or huGPA33-mC825) PRIT + 11.1 MBq or 33.3 MBq 177 Lu-DOTA-Bn (both with n = 8, TV7: 103 ± 17 mm respectively) 3 TV7: 93 ± 15mm 3 It is expected that during the treatment period, as 177 Increased Lu-DOTA-Bn dosage will lead to decreased relative tumor uptake. It is anticipated that untreated patients receiving only 33.3 MBq of this treatment will be more susceptible. 177 Lu-DOTA-Bn or single-cycle IgG-C825 PRIT + 33.3 MBq 177 The group of tumor-bearing mice treated with Lu-DOTA-Bn did not show tumor remission. It was expected that... 177 The latter two groups of Lu-DOTA-Bn showed minimal activity in the tumor region on scintillation scans. In contrast, treatment with single-cycle huGPA33-C825PRIT (huGPA33-huC825 or huGPA33-mC825) + 11.1 MBq or 33.3 MBq was expected. 177 The Lu-DOTA-Bn-treated group showed delayed tumor growth after treatment.

[0368] In the second therapy study, dual-cycle huGPA33-C825 PRIT (huGPA33-huC825 or huGPA33-mC825) treatment was investigated. When mice were not treated (n = 5 / TV) 10 314 ± 77 mm 3 Due to excessive tumor burden, all mice will be euthanized within 30 days. PRIT+ 11.1 MBq is expected to have two cycles. 177 Lu-DOTA-Bn processing (total) 177 Lu-DOTA-Bn dose 22.2 MBq (n = 5 / TV) 10 462 ± 179 mm 3 This will cause complete tumor remission and / or delayed tumor growth in the treated subjects.

[0369] Toxicity studies. In short, a total of six mice received two cycles of PRIT + 11.1 MBq. 177 Lu-DOTA-Bn (n = 3) or two cycles of PRIT + 1.5 mCi 177 Treatment with Lu-DOTA-Bn (n = 3) was performed for anatomical and pathological evaluation of the kidneys, bone marrow, liver, and spleen up to 9 weeks post-treatment. The kidneys, bone marrow, liver, and spleen of the treated animals were expected to be normal, and PRIT treatment was not associated with radiation-induced toxicity.

[0370] Curative treatment diagnosis of PRIT. Nude mice carrying established SW1222 sc xenografts (n = 20; tumor volume = 102 ± 40 mm) 3 The mean ± standard deviation (SD) will be subject to treatment with any of the following (n = 5 to 10 / group): no treatment (n = 5), only 177 Lu-DOTA-Bn (n = 5), or containing huGPA33-C825 PRIT (huGPA33-huC825 or huGPA33-mC825) + 55 MBq 177 A three-cycle PRIT regimen for Lu-DOTA-Bn (n = 10; total: 165 MBq). During the first cycle injection... 177Up to 160 hours after Lu-DOTA-Bn, serial nanoSPECT / CT imaging was performed on five randomly selected mice receiving DPRIT for dose determination calculations. DPRIT was expected to induce complete tumor remission in all treated animals, with no apparent toxicity in the kidneys, liver, spleen, and bone / bone marrow. Lutene-177 nanoSPECT / CT imaging of animals treated with the three-cycle PRIT regimen was expected to show high contrast with visible uptake in the tumor and minimal tissue background. These results demonstrate that the huGPA33-DOTA bispecific antibody of this invention can be used to reduce tumor burden in vivo, and that PRIT-based therapeutic diagnostics may have curative effects and / or be used to detect tumors in subjects.

[0371] Treatment diagnosis: "Real-time" simultaneous treatment and image-guided dosimetry. NanoSPECT / CT is used for... 177 High-resolution quantitative imaging was performed on Lu-DPRIT-treated mice for “real-time” dosimetry. Nude mice carrying SW1222 tumors (volume: 100 mm²) 3 (Based on the vernier caliper measurement results) Use a single-cycle HUGPA33-C825 PRIT (HUGPA33-HUC825 or HUGPA33-MC825) + 55 MBq. 177 Lu-DOTA-Bn was used for treatment, and during injection... 177 Following Lu-DOTA-Bn injection, images were taken three times via nanoSPECT / CT at 1 hour, 24 hours, and 160 hours post-injection. Images were attenuated according to injection time and calibrated using known activity standards. Region-of-interest analysis of the calibrated images was used to determine the active concentration in the tumor.

[0372] Therefore, the huGPA33-DOTA bispecific antibody of the present invention can be used to treat GPA33-positive cancers and to detect tumors in subjects.

[0373] equivalent

[0374] The present invention is not limited to the specific embodiments described herein, which are intended as separate illustrations of a single aspect of the invention. Many modifications and variations can be made to the invention without departing from its spirit and scope, as will be apparent to those skilled in the art. Based on the foregoing description, functionally equivalent methods and apparatuses within the scope of the invention will be apparent to those skilled in the art, in addition to those methods and apparatuses listed herein. Such modifications and variations are intended to fall within the scope of the invention. It should be understood that the present invention is not limited to specific methods, reagents, compounds, compositions, or biological systems, although such specific methods, reagents, compounds, compositions, or biological systems can vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting.

[0375] Furthermore, when features or aspects of this disclosure are described in accordance with the Markush Group, those skilled in the art will recognize that this disclosure is also described in accordance with any individual member or subgroup of members of the Markush Group.

[0376] As those skilled in the art will understand, for any and all purposes, particularly for the purpose of providing a written description, all scopes disclosed herein also encompass any and all possible subscopes and combinations thereof. Any listed scope can be readily considered sufficiently descriptive and such that the same scope can be decomposed into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each scope discussed herein can be readily divided into a lower third, a middle third, and an upper third, etc. As those skilled in the art will also understand, all language, such as “up to,” “at least,” “greater than,” “less than,” etc., includes the listed numbers and refers to a scope that can subsequently be decomposed into subscopes as described above. Finally, as those skilled in the art will understand, a scope includes each individual member. Thus, for example, a group having 1-3 cells means a group having 1, 2, or 3 cells. Similarly, a group having 1-5 cells means a group having 1, 2, 3, 4, or 5 cells, and so on.

[0377] All patents, patent applications, provisional applications and publications mentioned or cited herein are incorporated herein in their entirety by reference to the extent that they are not inconsistent with the explicit teachings of this specification, including all figures and tables.

Claims

1. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising a heavy chain immunoglobulin variable domain (V... H ) and light chain immunoglobulin variable domain (V L ),in: (a) The V H V containing SEQ ID NO: 2 or SEQ ID NO: 7 H -CDR1 sequence, V of SEQ ID NO: 3 or SEQ ID NO: 8 H -CDR2 sequence and V of SEQ ID NO: 4 or SEQ ID NO: 9 H -CDR3 sequence; and (b) The V L V contains groups consisting of the following items L -CDR1 sequence, V L -CDR2 sequence and V L -CDR3 sequence: SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14; and SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO:

19.

2. The antibody or antigen-binding fragment according to claim 1, wherein: (a) The V H Contains an amino acid sequence comprising either SEQ ID NO: 1 or SEQ ID NO: 6; and / or (b) the V L Contains the amino acid sequence of either SEQ ID NO: 11 or SEQ ID NO:

16.

3. The antibody or antigen-binding fragment according to claim 2, wherein: (a) The V H Contains the amino acid sequence of SEQ ID NO: 1, and the V L Contains the amino acid sequence of SEQ ID NO: 16; or (b) The V H Contains the amino acid sequence of SEQ ID NO: 6, and the V L It contains the amino acid sequence of SEQ ID NO:

11.

4. The antibody or antigen-binding fragment according to any one of claims 1 to 3, further comprising an isotype Fc domain selected from the group consisting of: IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, and IgE.

5. The antibody of claim 4, comprising an IgG1 constant region, said constant region comprising one or more amino acid substitutions selected from the group consisting of: N297A, K322A, L234A and L235A.

6. The antibody of claim 4, comprising an IgG4 constant region, wherein the constant region comprises an S228P mutation.

7. The antigen-binding fragment according to any one of claims 1 to 3, wherein the antigen-binding fragment is selected from the group consisting of: Fab, F(ab')2, Fab', scF v or F v .

8. An antibody comprising a heavy chain (HC) amino acid sequence and a light chain (LC) amino acid sequence respectively selected from the group consisting of: SEQ ID NO: 35 and SEQ ID NO: 36; SEQ ID NO: 35 and SEQ ID NO: 37; SEQ ID NO: 35 and SEQ ID NO: 38; SEQ ID NO: 39 and SEQ ID NO: 40; SEQ ID NO: 39 and SEQ ID NO: 41; SEQ ID NO: 39 and SEQ ID NO: 42; SEQ ID NO: 47 and SEQ ID NO: 48; SEQ ID NO: 47 and SEQ ID NO: 49; SEQ ID NO: 50 and SEQ ID NO: 51; and SEQ ID NO: 50 and SEQ ID NO:

52.

9. The antibody according to any one of claims 1 to 8, wherein the antibody is a chimeric antibody, a humanized antibody, a multispecific antibody, or a bispecific antibody.

10. The antibody according to any one of claims 1 to 9, wherein the antibody binds to an epitope of the GPA33 protein comprising at least five to eight consecutive amino acid residues of SEQ ID NO:

53.

11. A multispecific antigen-binding fragment comprising a first polypeptide chain, wherein: The first polypeptide chain comprises, in the direction from the N-terminus to the C-terminus: i. The heavy chain variable domain of the first immunoglobulin that can specifically bind to the first epitope; ii. A flexible peptide linker containing the amino acid sequence (GGGGS)6; iii. The light chain variable domain of the first immunoglobulin; iv. Flexible peptide linkers containing the amino acid sequence (GGGGS)4; v. The heavy chain variable domain of a second immunoglobulin that can specifically bind to the second epitope; vi. A flexible peptide linker containing the amino acid sequence (GGGGS)6; vii. The light chain variable domain of the second immunoglobulin; viii. A flexible peptide linker sequence containing the amino acid sequence TPLGTTHT; and ix. Self-assembly and disassembly (SADA) peptide; The heavy chain variable domain of the first immunoglobulin or the heavy chain variable domain of the second immunoglobulin is selected from any one of the following: SEQ ID NO: 1 and SEQ ID NO: 6, and / or the light chain variable domain of the first immunoglobulin or the light chain variable domain of the second immunoglobulin is selected from any one of the following: SEQ ID NO: 11 and SEQ ID NO:

16.

12. A multispecific antigen-binding fragment comprising a first polypeptide chain, wherein: The first polypeptide chain comprises, in the direction from the N-terminus to the C-terminus: i. The variable domain of the light chain of the first immunoglobulin that can specifically bind to the first epitope; ii. A flexible peptide linker containing the amino acid sequence (GGGGS)6; iii. The heavy chain variable domain of the first immunoglobulin; iv. Flexible peptide linkers containing the amino acid sequence (GGGGS)4; v. The heavy chain variable domain of a second immunoglobulin that can specifically bind to the second epitope; vi. A flexible peptide linker containing the amino acid sequence (GGGGS)6; vii. The light chain variable domain of the second immunoglobulin; viii. A flexible peptide linker sequence containing the amino acid sequence TPLGTTHT; and ix. Self-assembly and disassembly (SADA) peptide; The heavy chain variable domain of the first immunoglobulin or the heavy chain variable domain of the second immunoglobulin is selected from any one of the following: SEQ ID NO: 1 and SEQ ID NO: 6, and / or the light chain variable domain of the first immunoglobulin or the light chain variable domain of the second immunoglobulin is selected from any one of the following: SEQ ID NO: 11 and SEQ ID NO:

16.

13. The multispecific antigen-binding fragment according to claim 11 or 12, wherein the SADA polypeptide comprises a tetramerizing, pentamerizing, or hexamerizing domain.

14. The multispecific antigen-binding fragment of claim 13, wherein the SADA polypeptide comprises a tetramerizing domain of any of the following: p53, p63, p73, hnRNPC, SNA-23, Stefin B, KCNQ4, or CBFA2T1.

15. The multispecific antigen-binding fragment according to any one of claims 11 to 14, wherein the antigen-binding fragment comprises an amino acid sequence selected from SEQ ID NO: 43 to 46.

16. A multispecific antibody comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain are covalently bonded to each other, the second polypeptide chain and the third polypeptide chain are covalently bonded to each other, and the third polypeptide chain and the fourth polypeptide chain are covalently bonded to each other, and wherein: a. Each of the first polypeptide chain and the fourth polypeptide chain comprises, in the direction from the N-terminus to the C-terminus: i. The variable domain of the light chain of the first immunoglobulin that can specifically bind to the first epitope; ii. The light chain constant domain of the first immunoglobulin; iii. A flexible peptide linker containing the amino acid sequence (GGGGS)3; and iv. A light chain variable domain of the second immunoglobulin linked to a complementary heavy chain variable domain of the second immunoglobulin, or a heavy chain variable domain of the second immunoglobulin linked to a complementary light chain variable domain of the second immunoglobulin, wherein the light chain variable domain and the heavy chain variable domain of the second immunoglobulin are capable of specifically binding to a second epitope and are linked together via a flexible peptide linker comprising the amino acid sequence (GGGGS)6 to form a single-chain variable fragment; and b. Each of the second and third polypeptide chains comprises, in the direction from the N-terminus to the C-terminus: i. the heavy chain variable domain of the first immunoglobulin capable of specifically binding to the first epitope; and ii. The heavy chain constant domain of the first immunoglobulin; and The heavy chain variable domain of the first immunoglobulin is selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:6, and / or the light chain variable domain of the first immunoglobulin is selected from the group consisting of SEQ ID NO:11 and SEQ ID NO:

16.

17. The multispecific antibody or multispecific antigen-binding fragment according to any one of claims 9 or 11 to 16, wherein said antibody or antigen-binding fragment binds to: CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, CD32, CD64, TCR γ / δ, NKp46, KIR, PD-1, PD-L1, LAG3, CD28, B7H3, STEAP1, HER2, EGFR, CEA, CECAM5, transferrin receptor, FAP, NKG2D-ligand, TRAIL, FasL, cathepsin G, granzyme, carboxypeptidase, β-lactamase, DOTA (metal) complex, benzyl-DOTA (metal) complex, Proteus spp.-DOTA (metal) complex, NOGADA-Proteus spp.-DOTA (metal) complex, astr-DFO (metal) complex, DFO (metal) complex or small molecule DOTA hapten.

18. The multispecific antibody or multispecific antigen-binding fragment according to any one of claims 9 or 11 to 17, wherein the antibody or antigen-binding fragment binds to T cells, B cells, myeloid cells, plasma cells, or mast cells.

19. A heterodimeric multispecific antibody comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain are covalently bonded to each other, the second polypeptide chain and the third polypeptide chain are covalently bonded to each other, and the third polypeptide chain and the fourth polypeptide chain, wherein: (a) The first polypeptide chain comprises, in the direction from the N-terminus to the C-terminus: (i) The light chain variable domain (VL-1) of the first immunoglobulin that can specifically bind to the first epitope; (ii) The light chain constant domain (CL-1) of the first immunoglobulin; (iii) A flexible peptide linker containing the amino acid sequence (GGGGS)3; and (iv) A light chain variable domain (VL-2) of the second immunoglobulin linked to a complementary heavy chain variable domain (VH-2) of the second immunoglobulin, or a heavy chain variable domain (VH-2) of the second immunoglobulin linked to a complementary light chain variable domain (VL-2) of the second immunoglobulin, wherein VL-2 and VH-2 are capable of specifically binding to a second epitope and are linked together via a flexible peptide linker containing an amino acid sequence (GGGGS)6 to form a single-chain variable fragment; (b) The second polypeptide comprises, in the direction from the N-terminus to the C-terminus: (i) The heavy chain variable domain (VH-1) of the first immunoglobulin that is capable of specifically binding to the first epitope; (ii) the first CH1 domain (CH1-1) of the first immunoglobulin; and (iii) The first heterodimerizing domain of the first immunoglobulin, wherein the first heterodimerizing domain cannot form a stable homodimer with another first heterodimerizing domain; (c) The third polypeptide comprises, in the direction from the N-terminus to the C-terminus: (i) The heavy chain variable domain (VH-3) of the third immunoglobulin that can specifically bind to the third epitope; (ii) the second CH1 domain (CH1-3) of the third immunoglobulin; and (iii) The second heterodimerization domain of the third immunoglobulin, wherein the second heterodimerization domain comprises an amino acid sequence or nucleic acid sequence different from the first heterodimerization domain of the first immunoglobulin, wherein the second heterodimerization domain cannot form a stable homodimer with another second heterodimerization domain, and wherein the second heterodimerization domain of the third immunoglobulin is configured to form a heterodimer with the first heterodimerization domain of the first immunoglobulin; (d) The fourth polypeptide comprises, in the direction from the N-terminus to the C-terminus: (i) The light chain variable domain (VL-3) of the third immunoglobulin that is capable of specifically binding to the third epitope; (ii) the light chain constant domain (CL-3) of the third immunoglobulin; (iii) a flexible peptide linker containing the amino acid sequence (GGGGS)3; and (iv) a light chain variable domain (VL-4) of the fourth immunoglobulin linked to the complementary heavy chain variable domain (VH-4) of the fourth immunoglobulin, or a heavy chain variable domain (VH-4) of the fourth immunoglobulin linked to the complementary light chain variable domain (VL-4) of the fourth immunoglobulin, wherein VL-4 and VH-4 are capable of specifically binding to the fourth epitope and are linked together via a flexible peptide linker containing the amino acid sequence (GGGGS)6 to form a single-chain variable fragment; VL-1 and / or VL-3 contain V selected from any of the following: L Amino acid sequences: SEQ ID NO: 11 and SEQ ID NO: 16, wherein VH-1 and / or VH-3 contain V selected from any of the following. H Amino acid sequences: SEQ ID NO: 1 and SEQ ID NO:

6.

20. The multispecific antibody of claim 19, wherein the multispecific antibody binds to GPA33 and at least one of the following: CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, CD32, CD64, TCR γ / δ, NKp46, KIR, PD-1, PD-L1, LAG3, CD28, B7H3, STEAP1, HER2, EGFR, CEA, CECAM5, transferrin receptor, FAP, NKG2D-ligand, TRAIL, FasL, cathepsin G, granzyme, carboxypeptidase, β-lactamase, DOTA (metal) complex, benzyl-DOTA (metal) complex, Proteus spp.-DOTA (metal) complex, NOGADA-Proteus spp.-DOTA (metal) complex, astr-DFO (metal) complex, DFO (metal) complex or small molecule DOTA hapten.

21. The multispecific antibody according to claim 19 or 20, wherein the multispecific antibody binds to T cells, B cells, myeloid cells, plasma cells or mast cells.

22. The antibody according to any one of claims 1 to 6 or 8 to 21, wherein the antibody lacks α-1,6-fucose modification.

23. A recombinant nucleic acid sequence encoding an antibody according to any one of claims 1 to 22.

24. A recombinant nucleic acid sequence selected from the group consisting of: SEQ ID NO: 5, 10, 15 and 20.

25. A host cell or vector comprising the recombinant nucleic acid sequence according to claim 23 or claim 24.

26. A composition comprising an antibody or antigen-binding fragment according to any one of claims 1 to 22 and a pharmaceutically acceptable carrier, wherein the antibody or antigen-binding fragment is optionally conjugated with a pharmaceutical agent selected from the group consisting of: isotopes, dyes, chromophores, contrast agents, drugs, toxins, cytokines, enzymes, enzyme inhibitors, hormones, hormone antagonists, growth factors, radionuclides, metals, liposomes, nanoparticles, RNA, DNA, or any combination thereof.

27. A method for treating a subject with GPA33-related cancer, the method comprising administering to the subject an effective amount of an antibody or antigen-binding fragment according to any one of claims 1 to 22, wherein the antibody specifically binds to GPA33 and neutralizes its activity.

28. The method of claim 27, wherein the GPA33-related cancer is T-cell acute lymphoblastic leukemia (T-ALL), colorectal cancer, pseudomyxoma peritonei, appendiceal cancer, pancreatic cancer, or gastric cancer.

29. The method of claim 27 or 28, wherein the antibody is administered to the subject alone, sequentially, or simultaneously with another therapeutic agent.

30. The method of claim 29, wherein the additional therapeutic agent is one or more of the following: alkylating agents, platinum agents, taxanes, vinca extract, anti-estrogenic drugs, aromatase inhibitors, ovarian inhibitors, VEGF / VEGFR inhibitors, EGF / EGFR inhibitors, PARP inhibitors, cell-inhibiting alkaloids, cytotoxic antibiotics, antimetabolites, endocrine / hormone agents, and bisphosphonate therapeutic agents.

31. The method according to any one of claims 27 to 30, wherein the GPA33-associated cancer is colorectal cancer with an MSI genotype.

32. The method according to any one of claims 27 to 30, wherein the GPA33-associated cancer is colorectal cancer with the MSS genotype.

33. The method according to any one of claims 28 to 32, wherein the colorectal cancer is associated with a KRAS G12D mutation or a p53 mutation.

34. A method for detecting tumors in a subject in vivo, the method comprising: (a) administering to the subject an effective amount of the antibody according to any one of claims 1 to 22, wherein the antibody is configured to target a tumor expressing GPA33 and is labeled with a radioisotope; and (b) The presence of a tumor in the subject is detected by detecting a level of radioactivity emitted by the antibody that is higher than a reference value.

35. The method of claim 34, wherein the subject is diagnosed with or suspected of having cancer.

36. The method of claim 34 or 35, wherein the level of radioactivity emitted by the antibody is detected using positron emission tomography or single-photon emission computed tomography.

37. The method according to any one of claims 34 to 36, further comprising administering to the subject an effective amount of an immunoconjugate, said immunoconjugate comprising an antibody conjugated to a radionuclide according to any one of claims 8 to 15.

38. The method of claim 37, wherein the radionuclide is an alpha particle emission isotope, a beta particle emission isotope, an Auger emitter, or any combination thereof.

39. The method of claim 38, wherein the β-particle emission isotope is selected from the group consisting of: 86 Y、 90 Y、 89 Sr、 165 Dy、 186 Re、 188 Re、 177 Lu and 67 Cu.

40. A kit comprising the antibody according to any one of claims 1 to 22 and instructions for use.

41. The kit of claim 40, wherein the antibody is coupled to at least one detectable label selected from the group consisting of: radioactive labels, fluorescent labels, and chromogenic labels.

42. The kit according to claim 40 or 41, further comprising a secondary antibody that specifically binds to the antibody according to any one of claims 1 to 22.

43. The multispecific antibody according to claim 17 or 20, wherein the multispecific antibody binds to radiolabeled DOTA hapten and GPA33 antigen.

44. A method for detecting solid tumors in a subject of need, the method comprising: (a) Administering an effective amount of the complex to the subject, the complex comprising a radiolabeled DOTA hapten and a multispecific antibody according to claim 43, wherein the complex is configured to target a solid tumor expressing GPA33; and (b) The presence of a solid tumor in the subject is detected by detecting a level of radioactivity emitted by the complex that is higher than a reference value.

45. A method for selecting subjects for pre-targeted radioimmunotherapy, the method comprising: (a) Administering an effective amount of the complex to the subject, the complex comprising a radiolabeled DOTA hapten and a multispecific antibody according to claim 43, wherein the complex is configured to target solid tumors expressing GPA33. (b) Detecting the level of radioactivity emitted by the complex; and (c) When the level of radioactivity emitted by the complex is higher than a reference value, the subject is selected for pre-targeted radioimmunotherapy.

46. ​​A method for increasing the tumor sensitivity to radiotherapy in a subject diagnosed with GPA33-positive cancer, the method comprising administering to the subject an effective amount of a complex comprising a radiolabeled DOTA hapten and a multispecific antibody according to claim 43, wherein the complex is configured to target a tumor expressing GPA33.

47. A method for treating cancer in a subject in need, the method comprising administering to the subject an effective amount of a complex comprising a radiolabeled DOTA hapten and a multispecific antibody according to claim 43, wherein the complex is configured to target a tumor expressing GPA33.

48. A method for increasing the tumor sensitivity to radiotherapy in a subject diagnosed with GPA33-positive cancer, the method comprising: (a) Administering an effective amount of the multispecific antibody according to claim 43, wherein the multispecific antibody is configured to target a tumor expressing GPA33; and (b) Administering an effective amount of radiolabeled DOTA hapten to the subject, wherein the radiolabeled DOTA hapten is configured to bind to the multispecific antibody.

49. A method for treating cancer in a subject of need, the method comprising: (a) Administering an effective amount of the multispecific antibody according to claim 43, wherein the multispecific antibody is configured to target a tumor expressing GPA33; and (b) Administering an effective amount of radiolabeled DOTA hapten to the subject, wherein the radiolabeled DOTA hapten is configured to bind to the multispecific antibody.

50. The method of claim 48 or 49, further comprising administering an effective amount of a scavenger to the subject prior to the administration of the radiolabeled DOTA hapten.

51. The method according to any one of claims 44 to 50, wherein the subject is a human being.

52. The method according to any one of claims 44 to 47, wherein the complex is administered intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intracapsularly, intraorally, intradermally, intraperitoneally, via the trachea, subcutaneously, intravenously, orally, or intranasally.

53. The method according to any one of claims 44 to 52, wherein the radiolabeled DOTA hapten comprises an alpha particle emission isotope, a beta particle emission isotope, or an Auger emitter.

54. The method according to any one of claims 44 to 53, wherein the radiolabeled DOTA hapten comprises 213 Bi、 211 At、 225 Ac、 152 Dy、 212 Bi、 223 Ra、 219 Rn、 215 Po、 211 Bi、 221 Fr、 217 At、 255 Fm、 86 Y、 90 Y、 89 Sr、 165 Dy、 186 Re、 188 Re、 177 Lu、 67 Cu、 111 In、 67 Ga、 51 Cr 58 Co、 99m Tc, 103m Rh、 195m Pt, 119 Sb, 161 Ho、 189m Os、 192 Ir、 201 Tl、 203 Pb, 68 Ga、 227 Th or 64 Cu.

55. The multispecific antibody or antigen-binding fragment according to any one of claims 17 or 20, wherein the multispecific antibody binds to at least CD3 and GPA33 antigens.

56. An ex vivo armed T cell coated with or compounded with an effective amount of the multispecific antibody according to claim 55, wherein the multispecific antibody comprises a CD3 binding domain.

57. The ex vivo armed T cell of claim 56, wherein the multispecific antibody is an immunoglobulin comprising two heavy chains and two light chains, wherein each of the light chains is fused with a single-chain variable fragment (scFv), and wherein at least one scFv of the multispecific antibody comprises the CD3 binding domain.

58. A method for treating GPA33-related cancer in a subject of need, the method comprising administering to the subject an effective amount of ex vivo armed T cells according to claim 56 or 57.

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