A33 antibody compositions and methods of using the same in radioimmunotherapy

Abstract

To provide immunoglobulin-related compositions (e.g., antibodies or antigen binding fragments thereof) that can bind to and neutralize the activity of A33 protein, wherein the antibodies of the present technology are useful in methods for detecting and treating an A33-positive cancer in a subject animal in need thereof.SOLUTION: The present disclosure generally provides immunoglobulin-related compositions (e.g., antibodies or antigen binding fragments thereof). The compositions can specifically bind to and neutralize the biological activity of A33 polypeptide.SELECTED DRAWING: None

Description

[Technical field]

[0001] (Cross-reference of related applications) This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 562,373, filed September 23, 2017, and U.S. Provisional Patent Application No. 62 / 562,374, filed September 23, 2017, the contents of which are incorporated herein by reference in their entireties. (Technical field) The present technology relates generally to the preparation and use of immunoglobulin-related constructs (e.g., antibodies or antigen-binding fragments thereof) that specifically bind to A33 protein. In particular, the present technology relates to the preparation of A33-neutralizing antibodies and their use in the detection and treatment of A33-related cancers. [Background technology]

[0002] The following description of the background of the present technology is provided merely as an aid in the understanding of the present technology and is not intended to describe or constitute prior art to the present technology. Colorectal cancer (CRC) constitutes the third leading cause of cancer death in the United States (Siegel RL, Miller KD, Jemal A, CA: A Cancer Journal for Clinicians 67:7-30, 2017) and accounts for 10% of all cancers in men and 9.2% of all cancers in women worldwide (Ferlay J, Soerjomataram I, Dikshit R, et al., International Journal of Cancer 136:E359-E386, 2015). Despite a steady declining trend of 3% per year from 2004 to 2013, 135,000 new cases are expected in 2017 in the United States alone. Although localized and regional disease is curable, metastatic CRC (mCRC) has a poor prognosis with a 5-year survival rate of only 14% (Siegel RL, Miller KD, Jemal A, CA: A Cancer Journal for Clinicians 67:7-30, 2017). Standard treatment for mCRC consists of chemotherapy combined with monoclonal antibodies that block tumor signaling or angiogenesis. Currently, four monoclonal antibody drugs are FDA approved for the treatment of CRC. Bevacizumab and ramucirumab target the VEGF-VEGFR angiogenesis pathway, while cetuximab and panitumumab target the EGFR pathway. However, cetuximab and panitumumab provide no clinical benefit in mCRC with RAS mutations (Van Cutsem E, Cervantes A, Adam R, et al., Annals of Oncology 27:1386-1422, 2016), which are found in 40% of all mCRC (Bencsikova B, Bortlicek Z, Halamkova J, et al., BMC Gastroenterology 15:37, 2015). Furthermore, the improvement in survival with these antibodies is generally modest (Peeters M et al., Journal of Clinical Oncology 28:4706-4713, 2010; Cutsem EV et al., Journal of Clinical Oncology 29:2011-2019, 2011; Saltz LB et al., Journal of Clinical Oncology 26:2013-2019, 2008) and may be accompanied by severe side effects. The benefit of immune checkpoint inhibitors (ICIs) is limited to a subset of CRC patients with microsatellite instability (MSI). However, the majority of patients with mCRC are microsatellite stable (MSS), and therefore the benefit of ICI monotherapy is not expected. Thus far, the use of antibodies that direct toxins to tumors (e.g., radioimmunotherapy using directly conjugated antibodies) has only had limited success, which is partly due to suboptimal tumor dosimetry and therapeutic indices. Furthermore, due to normal tissue bystander toxicity, dose escalation is not feasible and therefore such treatment methods produce limited antitumor efficacy. Summary of the Invention [Problem to be solved by the invention]

[0003] Generally, the technology provides immunoglobulin-related constructs (eg, antibodies or antigen-binding fragments thereof) that can bind to and neutralize the activity of A33 protein. [Means for solving the problem]

[0004] In one aspect, the disclosure provides a method for the production of a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domains (V L (a) an antibody or antigen-binding fragment thereof comprising: H is the V of FTFSTYDMS (SEQ ID NO: 37) H - CDR1 sequence, TISSGGSYTYYLDSVKG (SEQ ID NO: 38) H and / or (b) a VH-CDR2 sequence of TTVVPFAY (SEQ ID NO: 39), and a VH-CDR3 sequence of TTVVPFAY (SEQ ID NO: 39), L is selected from the group consisting of KASQNVRTVVA (SEQ ID NO: 40), LASNRHT (SEQ ID NO: 41) and QYWSYPLT (SEQ ID NO: 42); KASQNVRTVVA (SEQ ID NO: 40), LASDRHT (SEQ ID NO: 43) and QYWSYPLT (SEQ ID NO: 42); KASQNVRTLVA (SEQ ID NO: 44), LASNRHT (SEQ ID NO: 41) and QHWSYPLT (SEQ ID NO: 45); and KASQNVRTLVA (SEQ ID NO: 44), LASNRHT (SEQ ID NO: 41) and QYWSYPLT (SEQ ID NO: 42). L - CDR1 sequence, V L -CDR2 sequence and V L - Includes the CDR3 sequence. The antibody may further comprise an Fc domain of an isotype 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 comprising one or more amino acid substitutions selected from the group consisting of N297A and K322A. Additionally or alternatively, in some embodiments, the antibody comprises an IgG4 constant region comprising an S228P mutation. In certain embodiments, the antigen-binding fragment is selected from the group consisting of Fab, F(ab')2, Fab', scFv and Fv. In some embodiments, the antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, or a bispecific antibody. In certain embodiments, the antibody or antigen-binding fragment binds to an epitope of the A33 protein comprising at least 5 to 8 consecutive amino acid residues of SEQ ID NO:57. In some embodiments, the epitope is a conformational epitope.

[0005] In another aspect, the disclosure provides an antibody, the antibody comprising a heavy chain (HC) amino acid sequence comprising SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:58, SEQ ID NO:62, or a variant thereof having one or more conservative amino acid substitutions, and / or a light chain (LC) amino acid sequence comprising SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:60, SEQ ID NO:63, or a variant thereof having one or more conservative amino acid substitutions. In certain embodiments, the antibody is selected from the group consisting of SEQ ID NO:5 and SEQ ID NO:9 (3A3-H1 / L1); SEQ ID NO:5 and SEQ ID NO:10 (3A3-H1 / L2); SEQ ID NO:6 and SEQ ID NO:9 (3A3-H2 / L1); SEQ ID NO:6 and SEQ ID NO:10 (3A3-H2 / L2); SEQ ID NO:15 and SEQ ID NO:17 (huA33-IgG1(H2L2)); SEQ ID NO:19 and SEQ ID NO:21 (huA33-BsAb); SEQ ID NO:23 and SEQ ID NO:24 (clone 31); SEQ ID NO:25 and SEQ ID NO:26 (clone 32); SEQ ID NO:27 and SEQ ID NO:28 (clone 48); SEQ ID NO:29 and SEQ ID NO:30 (clone 49); SEQ ID NO:31 and SEQ ID NO:32 (clone 53); SEQ ID NO:33 and SEQ ID NO:34 (clone 56); SEQ ID NO:35 and SEQ ID NO:36 (clone 57); SEQ ID NO:58 and SEQ ID NO:60 (huA33-huC825); and SEQ ID NO:62 and SEQ ID NO:63 (huA33-mC825).

[0006] In one aspect, the disclosure provides an antibody comprising: (a) a light chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a light chain immunoglobulin variable domain sequence present in any one of SEQ ID NOs: 9, 10, 17, 21, 24, 26, 28, 30, 32, 34, 36, 60, or 63; and / or (b) a heavy chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a heavy chain immunoglobulin variable domain sequence present in any one of SEQ ID NOs: 5, 6, 15, 19, 23, 25, 27, 29, 31, 33, 35, 58, or 62. In another aspect, the disclosure provides an antibody, the antibody comprising: (a) an LC sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to an LC sequence present in any one of SEQ ID NOs: 9, 10, 17, 21, 24, 26, 28, 30, 32, 34, 36, 60, or 63; and / or (b) an HC sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to an HC sequence present in any one of SEQ ID NOs: 5, 6, 15, 19, 23, 25, 27, 29, 31, 33, 35, 58, or 62. In any of the above embodiments, the antibody is a chimeric antibody, a humanized antibody, or a bispecific antibody. Additionally or alternatively, in some embodiments, the antibody comprises an IgG1 constant region comprising one or more amino acid substitutions selected from the group consisting of N297A and K322A. In certain embodiments, the antibody of the present technology comprises an IgG4 constant region comprising an S228P mutation. In any of the above embodiments, the antibody binds to an epitope of the A33 protein comprising at least 5 to 8 consecutive amino acid residues of SEQ ID NO:57. In some embodiments, the epitope is a conformational epitope. Additionally or alternatively, in some embodiments, the antibody of the present technology lacks α-1,6-fucose modifications.

[0007] In one aspect, the disclosure provides a recombinant nucleic acid sequence encoding any of the antibodies described herein. In some embodiments, the recombinant nucleic acid sequence is selected from the group consisting of SEQ ID NOs:7, 8, 11, 12, 16, 18, 20, 22, 59, and 61. In another aspect, the disclosure provides a host cell or vector comprising any of the recombinant nucleic acid sequences disclosed herein. In one aspect, the disclosure provides a composition comprising an antibody or antigen-binding fragment of the technology and a pharma- ceutically acceptable carrier, wherein the antibody or antigen-binding fragment is optionally conjugated to an agent selected from the group consisting of an isotope, a dye, a chromogen, an imaging agent, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA, or any combination thereof. In some embodiments of the bispecific antibodies of the present technology, the bispecific antibodies bind to T cells, B cells, myeloid cells, plasma cells, or mast cells. Additionally or alternatively, in some embodiments, the bispecific antibodies bind to CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, TCR gamma / delta, NKp46, KIR, or the small molecule DOTA hapten.To connect with DOTA, you can find Facebook picture:DOT A、DOTA-Bn、GIFT-デスフェリオオサミン、GIFT -Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH2、Ac-Lys(HSG)D-Tyr-Lys(HSG)-L ys(Tscg-Cys)-NH2、DOTA-D-Asp-D-Lys(HSG)-D-Asp-D-Lys(HSG)-NH2;DOTA-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2、(-DOTA-D-T-T HSG)-D-Glu-D-Lys(HSG)-NH2、DOTA-D-Ala-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2、DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-PhD-NH2、 -D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-NH2、Ac-D-Phe-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH2、Ac-D-Phe-D-Lys(Bz-DTPA)-D-DTPA(Bz-D-Lys )-NH2、Ac-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH2、DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(TscH2-Cys g-Cys)-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(DOTA)-NH2、Tscg-D-Cys-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2、D-Gly(Ts)c u-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2、Ac-D-Cys-D-Lys(DOTA)-D-Tyr-D-Ala-D-Lys(DOTA)-D-Cys-NH2、Ac-D-Cys-D-Lys(TyrPA-D-D s(DTPA)-NH2、Ac-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-D-Lys(Tscg-Cys)-NH2、およびAc-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA(CTA)-Nc-Lys

[0008] In another aspect, the disclosure provides a method of treating an A33-related cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of any one of the antibodies disclosed herein. In certain embodiments, the antibodies are selected from the group consisting of SEQ ID NO:5 and SEQ ID NO:9 (3A3-H1 / L1); SEQ ID NO:5 and SEQ ID NO:10 (3A3-H1 / L2); SEQ ID NO:6 and SEQ ID NO:9 (3A3-H2 / L1); SEQ ID NO:6 and SEQ ID NO:10 (3A3-H2 / L2); SEQ ID NO:15 and SEQ ID NO:17 (huA33-IgG1(H2L2)); SEQ ID NO:19 and SEQ ID NO:21 (huA33-BsAb); SEQ ID NO:23 and SEQ ID NO:24 (clone 31); SEQ ID NO:25 and SEQ ID NO:26 (clone 32); SEQ ID NO:27 and SEQ ID NO:28 (huA33-IgG1(H2L2)); SEQ ID NO:29 and SEQ ID NO:30 (huA33-IgG1(H2L2)); SEQ ID NO:31 and SEQ ID NO:32 (huA33-IgG1(H2L2)); SEQ ID NO:33 and SEQ ID NO:34 (clone 35); SEQ ID NO:35 and SEQ ID NO:36 (clone 37); SEQ ID NO:37 and SEQ ID NO:38 (huA33-IgG1(H2L2)); SEQ ID NO:39 and SEQ ID NO:40 (huA33-IgG1(H2L2)); SEQ ID NO:41 and SEQ ID NO:42 (huA33-IgG1(H2L2)); SEQ ID NO:42 and SEQ ID NO:43 (huA33-IgG1(H2L2)); S and SEQ ID NO:28 (clone 48); SEQ ID NO:29 and SEQ ID NO:30 (clone 49); SEQ ID NO:31 and SEQ ID NO:32 (clone 53); SEQ ID NO:33 and SEQ ID NO:34 (clone 56); SEQ ID NO:35 and SEQ ID NO:36 (clone 57); SEQ ID NO:58 and SEQ ID NO:60 (huA33-huC825); and SEQ ID NO:62 and SEQ ID NO:63 (huA33-mC825), wherein the antibody specifically binds to and neutralizes A33 activity. In some embodiments, the A33-related cancer is colorectal cancer, pseudomyxoma peritonei, appendix cancer, pancreatic cancer or gastric cancer.The A33-related cancer can be colorectal cancer with MSI genotype or MSS genotype.In addition or alternatively, in some embodiments, the colorectal cancer is associated with KRAS G12D mutation or p53 mutation.

[0009] Additionally or alternatively, in some embodiments of the method, the antibody is administered to the subject separately, sequentially, or simultaneously with an additional therapeutic agent, examples of which include one or more of the following: alkylating agents, platinum agents, taxanes, vinca agents, antiestrogens, aromatase inhibitors, ovarian suppressants, VEGF / VEGFR inhibitors, EGF / EGFR inhibitors, PARP inhibitors, cytostatic alkaloids, cytotoxic antibiotics, antimetabolites, endocrine / hormonal agents, and bisphosphonate therapeutic agents. In another aspect, the present 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 of the present technology, the antibody being configured to localize to tumors expressing A33 and being labeled with a radioisotope; and (b) detecting the presence of tumors in the subject by detecting a radioactivity level emitted by the antibody that is higher than a reference value. In some embodiments, the subject is diagnosed with or suspected of having cancer. The radioactivity level emitted by the antibody can be detected using positron emission tomography or single photon emission computed tomography. Additionally or alternatively, in some embodiments, the method further comprises administering to the subject an effective amount of an immunoconjugate comprising an antibody of the present technology conjugated to a radionuclide. In some embodiments, the radionuclide is an alpha particle emitting isotope, a beta particle emitting isotope, an Auger-emitter, or any combination of the above. Examples of beta particle emitting isotopes include: 86 Y, 90 Y, 89 Sr, 165 Dy, 186 Re, 188 Re, 177 Lu, and 67 In some embodiments of the method, non-specific FcR dependent binding in normal tissues is eliminated or reduced (e.g., by N297A mutation in the Fc region, which results in aglycosylation).

[0010] Also disclosed herein is a kit for detecting and / or treating A33-related cancer, said kit comprising at least one immunoglobulin-related construct of the present technology (e.g., any antibody or antigen-binding fragment described herein), or a functional variant thereof (e.g., a substitution variant), and instructions for use. In certain embodiments, the immunoglobulin-related construct is conjugated with one or more detectable labels. In some embodiments, the one or more detectable labels comprise a radioactive label, a fluorescent label, or a chromogenic label. Additionally or alternatively, in some embodiments, the kit further comprises a secondary antibody that specifically binds to an anti-A33 immunoglobulin-related construct described herein, hi some embodiments, the secondary antibody is conjugated to at least one detectable label selected from the group consisting of a radioactive label, a fluorescent label, or a chromogenic label.

[0011] In one aspect, the present disclosure provides a method for detecting a solid tumor in a subject in need thereof, the method comprising: (a) administering to the subject an effective amount of a conjugate comprising a radiolabeled DOTA hapten and a bispecific antibody of the present technology that binds to the radiolabeled DOTA hapten and the A33 antigen, wherein the conjugate is configured to localize to a solid tumor expressing the A33 antigen that is recognized by the bispecific antibody of the conjugate; and (b) detecting the presence of the solid tumor in the subject by detecting a level of radioactivity emitted by the conjugate that is higher than a reference value. In another aspect, the present disclosure provides a method for selecting a subject for pretargeting radioimmunotherapy, the method includes: (a) administering to the subject an effective amount of a conjugate comprising radiolabeled DOTA hapten and the bispecific antibody of the present technology that binds to radiolabeled DOTA hapten and A33 antigen, the conjugate being configured to localize in solid tumors that express the A33 antigen that is recognized by the bispecific antibody of the conjugate; (b) detecting the radioactivity level emitted by the conjugate; and (c) selecting the subject for pretargeting radioimmunotherapy when the radioactivity level emitted by the conjugate is higher than a reference value.

[0012] In one aspect, the disclosure provides a method of increasing tumor sensitivity to radiation therapy in a subject diagnosed with A33 positive cancer, the method comprising administering to the subject an effective amount of a conjugate comprising a radiolabeled DOTA hapten and a bispecific antibody of the present technology that recognizes and binds to an A33 antigen target, wherein the conjugate is configured to localize to tumors that express the A33 antigen target recognized by the bispecific antibody of the conjugate. In another aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a conjugate comprising a radiolabeled DOTA hapten and a bispecific antibody of the present technology that recognizes and binds to an A33 antigen target, wherein the conjugate is configured to localize to a tumor expressing the A33 antigen target recognized by the bispecific antibody of the conjugate. In any of the above embodiments of the methods disclosed herein, the conjugate is administered intravenously, intramuscularly, intraarterially, intrathecally, intravesically, intraocularly, intradermally, intraperitoneally, subcutaneously, intracerebroventricularly, orally, or intranasally. In some embodiments of the methods disclosed herein, the subject animal is a human. Additionally or alternatively, in any of the above embodiments of the methods disclosed herein, the radiolabeled DOTA hapten is administered intravenously, intramuscularly, intraarterially, intravesicularly, intraocularly, intradermally, intraperitoneally, subcutaneously, intracerebroventricularly, orally, or intranasally. 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 an alpha particle emitting isotope, a beta particle emitting isotope, or an Auger emitter.

[0013] In one aspect, the present disclosure provides a method of increasing tumor sensitivity to radiation therapy in a subject diagnosed with A33 positive cancer, the method comprising: (a) administering to the subject an effective amount of an anti-DOTA bispecific antibody of the present technology, the anti-DOTA bispecific antibody configured to localize to tumors expressing the A33 antigen target; and (b) administering to the subject an effective amount of a radiolabeled DOTA hapten, the radiolabeled DOTA hapten configured to bind to the anti-DOTA bispecific antibody. In another aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising: (a) administering to the subject an effective amount of an anti-DOTA bispecific antibody of the present technology, the anti-DOTA bispecific antibody configured to localize to tumors expressing the A33 antigen target; and (b) administering to the subject an effective amount of a radiolabeled DOTA hapten, the radiolabeled DOTA hapten configured to bind to the anti-DOTA bispecific antibody. In some embodiments, the methods of the present technology further comprise the step of administering an effective amount of a clearing agent to the subject prior to administration of the radiolabeled DOTA hapten. Additionally or alternatively, in any of the above embodiments of the methods disclosed herein, the radiolabeled DOTA hapten is 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 an alpha particle emitting isotope, a beta particle emitting isotope, or an Auger emitter.In any of the above embodiments of the methods disclosed herein, the subject animal is a human. [Brief description of the drawings]

[0014] [Figure 1A-B] (A) shows the design and construction of humanized A33 bispecific antibody (huA33-BsAb). (B) shows accelerated stability study of purified huA33-BsAb at 37° C. for 4 weeks. [Figure 1C-D] (C) Surface plasmon resonance (SPR) analysis of huA33-BsAb at 25°C and 37°C. Data were fitted to a 1:1 binding model. (D) FACS staining of various tumor cell lines and activated T cells. Mean fluorescence intensity (MFI) values ​​were geometric means. [Figure 2A-B](A) Activation of CD25 and CD69 markers of T cells after 24 hours of incubation with Colo205 cells and various antibodies. (B) Quantification of dividing cells based on CFSE dye dilution after 96 hours of incubation with Colo205 cells and various antibodies. [Figure 2C] (C) shows a representative image of FIG. 2(B). [Fig. 2D-E] (D) shows CD45RO staining after 96 h incubation with Colo205 cells and huA33-BsAb in an independent experiment. (E) shows in vivo activation and prolliferation of T cells by huA33-BsAb. Briefly, CFSE-labeled peripheral blood mononuclear cells (PBMCs) were mixed with Colo205 cells, and the mixture was implanted subcutaneously into DKO mice. The following day, huA33-BsAb was injected intravenously, and after another 4 days, tumors were isolated and analyzed by FACS. [Diagram 3] Profile of cytokines and cytotoxic components secreted by huA33-BsAb-activated T cells in the presence of target tumor. Kinetics of cytokine and cytolytic molecule production by T cells in the presence of huA33-BsAb and target cells Colo205 or negative control cells SKMEL5 were determined over a 4-day period. Because SKMEL5 secreted large amounts of IL-6, supernatants from T cells incubated with Col205 cells in the absence of antibody were used as negative controls for IL-6 kinetics experiments. [Figure 4A-B] (A) Cytotoxicity elicited by huA33-BsAb against various target tumor cell lines and control cells. Activated T cells were used as effector cells at an effector to target ratio (E:T) of 10:1. Cells were incubated for 16 hours before readings were taken. (B) Cytotoxicity elicited by huA33-BsAb against Colo205 cells. Freshly sorted T cell subsets were used as effector cells (E:T=5:1). Cells were incubated for 48 hours before readings were taken. EC50 against CD4 memory T cells was 25 pM. [Figure 5A-B] (A) shows a summary of affinity maturation of huA33-BsAb(H2L2) by yeast screening. (B) shows a summary of the KD of parental and affinity matured huA33-BsAbs (top) and a summary of the on-off rate maps of the different huA33-BsAbs obtained from SPR analysis (bottom). [Figure 5C] (C) shows the results of a T cell dependent cytotoxicity (TDCC) assay of parental and affinity matured huA33-BsAb. [Figure 6A-B] (A) shows the growth of subcutaneous LS174T tumors in the treatment and control groups. Tumor size was assessed by volume (p=0.0133 for tumor+scPBMC vs. tumor+scPBMC+huA33-BsAb; p=0.006 for tumor only vs. tumor+scPBMC+huA33-BsAb). (B) shows the growth of intraperitoneal LS174T tumors in the treatment and control groups (top row) (p=0.0125 for tumor+ATC vs. tumor+ATC+huA33-BsAb; p=0.0026 for tumor only vs. tumor+ATC+huA33-BsAb) as well as the survival rate of mice in the treatment and control groups (bottom row). [Figure 6C] (C) shows luminescence images of abdominal LS174T tumors in various groups. There was one mouse in the tumor-only group that did not acquire a tumor after 21 days (#4), and this mouse was excluded from the analysis. [Figure 7A-B] (A) shows luminescence images showing the growth of subcutaneous Colo205 tumors in the various groups. (B) shows quantification of the signal (top) and survival of the mice in the various groups in Fig. 7(A) (bottom). [Figure 7C-D] (C) shows luminescence images of intraperitoneal SW1222 tumors. Mouse #1 in the tumor-only group and mouse #3 in the tumor + ATC group, which did not acquire tumors after 21 days, were excluded from images at later time points and were not included in the survival analysis in Figure 7(D). (D) shows survival of mice bearing intraperitoneal SW1222 tumors. [Figure 8A-B](A) shows the growth of subcutaneous SNU16 tumors in the various groups. (B) shows the engraftment of human cells from the blood of the mice in Figure 8(A). [Figure 9] Figure 1 shows SPR analysis of four versions of humanized A33. All antibodies contain IgG1 constant domain. 3A3-H1L1, 3A3-H1L2, 3A3-H2L1 and 3A3-H2L2 were the four versions of humanized 3A3. 3A3-chA33 was chimeric 3A3. [Figure 10] The results of FACS analysis of various cell lines stained with huA33-BsAb are shown. [Figure 11A-B] (A) shows PD-1 upregulation after 24 hours (left) and 96 hours (right) on T cells activated with huA33-BsAb in the presence of Colo205 cells, and (B) shows the absence of T cell division after 96 hours of incubation with SKMEL5 in the presence of huA33-BsAb. [Figure 11C] (C) Activation of T cell division by huA33-BsAb in the presence of LS174T cells. The same T cell preparations were used in Figures 11(B) and 11(C). [Figure 12A-B] (A) and (B) show staining of CD45RO and CD25 markers on T cells after 48 hours of incubation with Colo205 cells in the presence of various antibodies. Cells were obtained from TDCC assay after using supernatants for LDH measurement. Figure 12(A): Gating based on CD4(+) T cells; Figure 12(B): Gating based on CD8(+) T cells. [Figure 13]A strategy for rapid engineering of scFv into the huA33-BsAb format is presented. The expression vector (1) was linearized with HindIII / ApaI. The promoter fragment (3) was prepared from the SpaI digested promoter-containing vector. Large amounts of both the vector and the promoter fragment could be prepared for higher throughput cloning. The VH (4) and VL (2) were amplified directly from yeast using two 5' primers to add a leader sequence, which was digested with HindIII / SapI (VL) or ApaI / SapI (VH). The four fragments were ligated in a one-step reaction. [Figure 14] The amino acid sequences of the VH and VL domains of the murine A33 antibody and their corresponding homologous human sequences (SEQ ID NO:1-4) are shown. The CDR1, CDR2 and CDR3 regions of the VH and VL domains of the murine A33 antibody are shown in bold underlined font. [Figure 15] 1 shows the amino acid sequences of the humanized heavy chains of huA33-H1 (3A3-H1) (SEQ ID NO: 5) and huA33-H2 (3A3-H2) (SEQ ID NO: 6). CDR1, CDR2 and CDR3 of 3A3-H1 and 3A3-H2 are shown in bold underlined font. [Figure 16] 1 shows the cDNA sequences of the humanized heavy chains of huA33-H1 (3A3-H1) (SEQ ID NO: 7) and huA33-H2 (3A3-H2) (SEQ ID NO: 8). [Figure 17] 1 shows the amino acid sequences of the humanized light chains of huA33-L1 (3A3-L1) (SEQ ID NO: 9) and huA33-L2 (3A3-L2) (SEQ ID NO: 10). The CDR1, CDR2 and CDR3 regions of 3A3-L1 and 3A3-L2 are shown in bold underlined font. [Figure 18] 1 shows the cDNA sequences of the humanized light chains of huA33-L1 (3A3-L1) (SEQ ID NO: 11) and huA33-L2 (3A3-L2) (SEQ ID NO: 12). [Figure 19] An alignment of the original humanized sequence hA33 by King et al. (King et al., 1995, supra) with the newly re-humanized huA33 (3A3) sequence is shown. [Figure 20] Figure 1 shows the binding kinetics of humanized IgG variants of huA33 assayed using SPR (Biacore T100) with GPA33 recombinant protein. All four versions retained the high binding affinity of chimeric A33 (chA33). [Figure 21] Figure 1 shows the binding kinetics of the original humanized hA33 (hA33-mC825 bispecific format as described in literature (Cheal et al, Eur. J. Nucl. Med. Mol. Imaging, 43:925-937, 2016)) and huA33 (3A3-H2L2) assayed using SPR (Biacore T100) on GPA33 recombinant protein. The original humanized hA33 lost significant affinity compared to huA33. [Figure 22] Figure 1 shows the binding kinetics of the original humanized hA33 (hA33-mC825 bispecific format as described in literature (Cheal et al, Eur. J. Nucl. Med. Mol. Imaging, 43:925-937, 2016)) and huA33 (3A3-H2L2) assayed using SPR (Biacore T100) on GPA33 recombinant protein. The original humanized hA33 lost significant affinity compared to huA33. [Figure 23] The humanity analysis of the huA33 heavy and light chain sequences (3A3-H1, 3A3-H2, 3A3-L1, 3A3-L2) as well as the original hA33 sequence is shown. Since all four rehumanized versions retained the high binding affinity of the original chA33, the H2L2 version was selected for further development based on its higher humanity T20 score. [Figure 24] 1 shows the amino acid sequences of the light and heavy chains of chimeric chA33-IgG1, which correspond to SEQ ID NO:13 and SEQ ID NO:14, respectively. [Diagram 25] Figure 2 shows the amino acid and cDNA sequences of the heavy chain of huA33-IgG1(H2L2), which correspond to SEQ ID NO:15 and SEQ ID NO:16, respectively. [Figure 26]Figure 2 shows the amino acid and cDNA sequences of the light chain of huA33-IgG1(H2L2), which correspond to SEQ ID NO:17 and SEQ ID NO:18, respectively. [Figure 27] 1 shows the amino acid and cDNA sequences of the heavy chain of the T cell-engaging huA33-BsAb bispecific antibody, which correspond to SEQ ID NO:19 and SEQ ID NO:20, respectively. [Figure 28] The amino acid and cDNA sequences of the light chain of the T cell-engaging huA33-BsAb bispecific antibody are shown, which correspond to SEQ ID NO:21 and SEQ ID NO:22, respectively. The underlined sequence corresponds to the GS linker sequence. [Figure 29] 1 shows a summary of potential modifications to the T cell-engaging huA33-BsAb bispecific antibody disclosed herein. [Diagram 30] Figure 1 shows the amino acid sequences of the heavy and light chains of affinity matured clone 31 in huA33-BsAb format, which correspond to SEQ ID NO: 23 and SEQ ID NO: 24, respectively. The underlined sequences correspond to the GS linker sequences. [Diagram 31] The amino acid sequences of the heavy and light chains of affinity matured clone 32 in the huA33-BsAb format are shown, which correspond to the sequences SEQ ID NO: 25 and SEQ ID NO: 26, respectively. The underlined sequences correspond to the GS linker sequences. [Diagram 32] Figure 1 shows the amino acid sequences of the heavy and light chains of affinity matured clone 48 in huA33-BsAb format, which correspond to SEQ ID NO: 27 and SEQ ID NO: 28, respectively. The underlined sequences correspond to the GS linker sequences. [Diagram 33] Figure 1 shows the amino acid sequences of the heavy and light chains of affinity matured clone 49 in huA33-BsAb format, which correspond to SEQ ID NO:29 and SEQ ID NO:30, respectively. The underlined sequence corresponds to the GS linker sequence. [Diagram 34] Figure 1 shows the amino acid sequences of the heavy and light chains of affinity matured clone 53 in huA33-BsAb format, which correspond to SEQ ID NO: 31 and SEQ ID NO: 32, respectively. The underlined sequence corresponds to the GS linker sequence. [Diagram 35] Figure 1 shows the amino acid sequences of the heavy and light chains of affinity matured clone 56 in huA33-BsAb format, which correspond to SEQ ID NO: 33 and SEQ ID NO: 34, respectively. The underlined sequence corresponds to the GS linker sequence. [Diagram 36] Figure 1 shows the amino acid sequences of the heavy and light chains of affinity matured clone 57 in huA33-BsAb format, which correspond to SEQ ID NO: 35 and SEQ ID NO: 36, respectively. The underlined sequences correspond to the GS linker sequence. [Figure 37] 1 shows the amino acid and cDNA sequences of the heavy chain of bispecific antibody huA33-huC825 (H2L2), which correspond to SEQ ID NO:58 and SEQ ID NO:59, respectively. [Figure 38] 1 shows the amino acid and cDNA sequences of the light chain of bispecific antibody huA33-huC825 (H2L2), which correspond to SEQ ID NO: 60 and SEQ ID NO: 61, respectively. The underlined sequence corresponds to the GS linker sequence. [Figure 39] 1 shows the amino acid sequences of the heavy and light chains of bispecific antibody huA33-mC825(H2L2), which correspond to SEQ ID NO:62 and SEQ ID NO:63, respectively. The underlined sequences correspond to the GS linker sequence. [Diagram 40] 1 shows ex vivo biodistribution results for targeting to GPA33-positive (GPA33(+)) human colorectal tumor xenografts in mice bearing subcutaneous GPA33(+)SW1222 human colorectal tumor xenografts treated with a rehumanized huA33-DOTA bispecific antibody disclosed herein and a tracer amount of 177Lu-DOTA-biotin. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] It will be appreciated that certain features, modes, embodiments, variations and characteristics of the present methods are described below at various levels of detail in order to provide a substantial understanding of the present technology. The present disclosure generally provides immunoglobulin-related constructs (e.g., antibodies or antigen-binding fragments thereof), which can specifically bind to and neutralize the biological activity of A33 polypeptide. The immunoglobulin-related constructs of the present technology are useful in methods for detecting or treating A33-related cancer in a subject in need thereof. Thus, various features of the present methods relate to the preparation, characterization, and manipulation of anti-A33 antibodies. The immunoglobulin-related constructs of the present technology are useful alone or in combination with additional therapeutic agents for cancer treatment. In some embodiments, the immunoglobulin-related constructs are humanized antibodies, chimeric antibodies, or bispecific antibodies. The practice of the subject methods employs many conventional techniques in molecular biology, protein chemistry, cell biology, immunology, microbiology, and recombinant DNA. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; series; Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; series; Methods in Enzymology (Academic Press, 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; U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Synthesis; 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 (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (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 polypeptide gene expression product levels (i.e., gene translation levels) are well known in the art and include the use of polypeptide detection methods, such as antibody detection and quantification techniques (see also Strachan & Read, Human Molecular Genetics, Second Edition. (John Wiley and Sons, Inc., NY, 1999)).

[0016] definition Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by anyone skilled in the art to which this technology belongs. As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural equivalents unless the context clearly indicates otherwise. For example, "a cell" includes a combination of two or more cells. Generally, the nomenclature used herein and below in the laboratory procedures of cell culture, molecular genetics, organic chemistry, analytical chemistry, and nucleic acid chemistry and hybridization is well known and commonly used in the art. As used herein, the term "about" in reference to a number is intended to include numbers within 1%, 5% or 10% of the number in either direction (higher or lower) unless otherwise indicated or clear from the context (except where such number is less than 0% or greater than 100% of its possible value).

[0017] As used herein, "administration" of an agent or drug to a subject includes any route by which the compound is introduced or delivered to the subject for the performance of its intended function. Administration can be by any suitable route, including, but not limited to, oral, intranasal, parenteral (intravenous, intramuscular, intraperitoneal or subcutaneous), rectal, intrathecal, intratumoral, or topical. Administration includes self-administration or administration by another. "Adjuvant" refers to one or more substances that cause stimulation of the immune system. In the present context, adjuvants are used to enhance the response to one or more vaccine antigens or antibodies. Adjuvants can be administered to the subject prior to, together with, or after administration of the vaccine. Examples of chemical compounds used as adjuvants include aluminum compounds, oils, block polymers, immune stimulating complexes, vitamins and minerals (e.g., vitamin E, vitamin A, selenium, and vitamin B12), Quil A (saponin), bacterial and fungal cell wall components (e.g., lipopolysaccharides, lipid proteins, and glycoproteins), hormones, cytokines, and costimulatory factors. As used herein, the term "antibody" refers generically to an immunoglobulin or immunoglobulin-like molecule. By way of example, it includes, but is not limited to, IgA, IgD, IgE, IgG, and IgM, combinations of the above, and similar molecules generated during an immune response in any vertebrate, e.g., mammals (e.g., humans, goats, rabbits, and mice), as well as non-mammalian species (e.g., shark immunoglobulins). As used herein, "antibodies" (including intact immunoglobulins) and "antigen-binding fragments" refer to any immunoglobulin that specifically binds to a molecule of interest (or a group of highly similar molecules of interest) and substantially excludes binding to other molecules (e.g., antibodies and antibody fragments have a binding constant of at least 10 higher than that for other molecules in a biological sample). 3 M -1 , at least 10 4 M -1 Or at least 10 5 M -1 (Antibody has a binding constant of 0.1 to the molecule of interest). The term "antibody" also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (e.g., bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology, 3 rd Ed., W. H. Freeman & Co., New York, 1997.

[0018] More specifically, an antibody refers to a polypeptide ligand that contains at least one light or heavy chain immunoglobulin variable region that specifically recognizes and binds to an epitope of an antigen. Antibodies are composed of heavy and light chains, each of which contains a variable region, and variable heavy chains (V H ) region and the variable light chain (V L ) region. Together, they form the V H Area and V LThe regions are necessary for binding to the antigen recognized by the antibody. Typically, immunoglobulins interconnect heavy (H) and light (L) chains by disulfide bonds. There are two types of light chains, lambda (λ) and kappa (κ). There are five major heavy chain classes (or isotypes), IgM, IgD, IgG, IgA, and IgE, which determine the functional activity of the antibody molecule. Each heavy and light chain contains a constant region and a variable region (the regions are also known as "domains"). Together, the heavy and light chain variable regions specifically bind to the antigen. The light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions (also called "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, incorporated herein by reference). The Kabat database is currently maintained online. The sequences of the framework regions of various light or heavy chains are relatively conserved within a species. The framework regions of an antibody (i.e. the combined framework regions of the constituent light and heavy chains) adopt a predominantly β-sheet structure, with the CDRs forming loops that connect, and in some cases even forming part of, the β-sheet structure. Thus, the framework regions function to form a scaffold that orients the CDRs by non-covalent interactions between the chains.

[0019] CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2 and CDR3, are numbered consecutively starting from the N-terminus and are typically also identified by the chain in which the individual CDR is located. Thus, V H CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, while V LCDR1 is the CDR1 from the light chain variable domain of the antibody in which it is found. Antibodies that bind to the A33 protein have a specific V H Area and V L Each CDR will have a specific CDR sequence and therefore a specific CDR sequence. Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. Although it is the CDRs that vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly required for antigen binding. These positions within the CDRs are called specificity determining residues (SDRs). As used herein, "immunoglobulin-related constructs" refers to antibodies, including monoclonal, polyclonal, humanized, chimeric, recombinant, multispecific, bispecific, and the like, as well as antibody fragments. An antibody or antigen-binding fragment thereof specifically binds to an antigen.

[0020] As used herein, the term "antibody-related polypeptide" refers to an antigen-binding antibody fragment, including single chain antibodies. Single chain antibodies may contain only the variable region or may be combined with all or part of the following polypeptide components: the hinge region, CH1, CH2, and CH3 domains of an antibody molecule. Any combination of the variable region and the hinge region, CH1, CH2, and CH3 domains is also encompassed by the present technology. Antibody-related molecules useful in the present methods include, for example, Fab, Fab' and F(ab')2, Fd, single chain Fv (scFv), single chain antibodies, disulfide-linked Fv (sdFv), and VFv. L or V H Examples of such fragments include, but are not limited to, Fab fragments, V L , V H , C L and the CH1 domain; (ii) the F(ab')2 fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) the V H and an Fd fragment consisting of the CH1 domain; (iv) a V of a single arm of an antibody. L and VH (v) dAb fragments (Ward et al., Nature 341: 544-546, 1989), which consist of the V H domains; and (vi) an isolated complementarity determining region (CDR). Thus, an "antibody fragment" or "antigen-binding fragment" can include a portion of a full-length antibody, generally the antigen-binding or variable region thereof. Examples of antibody fragments or antigen-binding fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0021] As used herein, a "bispecific antibody" or "BsAb" refers to an antibody that can simultaneously bind to two targets having distinct structures, e.g., two different target antigens, two different epitopes of the same target antigen, or a hapten and a target antigen or an epitope of a target antigen. A variety of different bispecific antibody structures are known in the art. In some embodiments, each antigen-binding portion of a bispecific antibody is V H and / or V L In some such embodiments, the V H and / or V L The V domains are those found in individual monoclonal antibodies. In some embodiments, a bispecific antibody comprises two antigen-binding portions, each derived from a different monoclonal antibody. H and / or V L In some embodiments, a bispecific antibody comprises two antigen-binding moieties, where one of the two antigen-binding moieties comprises a V domain that includes the CDRs from a first monoclonal antibody. H and / or V L The other antigen-binding portion comprises an immunoglobulin molecule having a V domain that includes CDRs derived from a second monoclonal antibody. H and / or V L Antibody fragments having a specific region (e.g., Fab, F(ab'), F(ab')2, Fd, Fv, dAB, scFv). As used herein, a "clearing agent" is an agent that binds to excess bispecific antibodies present in the blood compartment of a subject animal and promotes their rapid clearance by the kidney. The use of a clearing agent (e.g., DOTA) prior to hapten administration promotes better tumor-to-background ratios in pretargeting radioimmunotherapy (PRIT) systems. Examples of clearing agents include 500kD-dextran-DOTA-Bn(Y) (Orcutt et al., Mol Cancer Ther. 11(6): 1365-1372, 2012), 500kD-aminodextran-DOTA conjugates, antibodies against pretargeting antibodies, and the like. As used herein, the term "complexed" refers to the association of two molecules by any method known to those of skill in the art. Suitable types of associations include chemical bonds and physical bonds. Chemical bonds include, for example, covalent bonds and coordinate bonds. Physical bonds include, for example, hydrogen bonds, dipolar interactions, van der Waals forces, electrostatic interactions, hydrophobic interactions, and aromatic ring stacking.

[0022] As used herein, the term "diabody" refers to a small antibody fragment that has two antigen-binding sites, said fragment comprising a light chain variable domain (V L ) connected to a heavy chain variable domain (V H ) in the same polypeptide chain (V H V L ). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains on another chain, creating two antigen-binding sites. Diabodies are more fully described in EP 404,097; WO 93 / 11161; and 30 Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448, 1993. As used herein, a "single chain antibody" or "single chain Fv (scFv)" refers to an antibody that comprises two domains of an Fv fragment (V L and V HA single chain antibody molecule can comprise a polymer containing multiple individual molecules, such as a dimer, trimer, or other polymer. In addition, the two domains of the Fv fragment (V L and V H ) are encoded by separate genes, but they can be joined using recombinant methods or by a synthetic linker, which allows the two domains to be produced as a single protein chain, which is called the V L and V H The pair of domains forms a monovalent molecule known as a single-chain Fv (scFv) (Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad Sci. USA 85:5879-5883). Such single chain antibodies can be prepared by recombinant techniques or by enzymatic or chemical cleavage of intact antibodies. Any of the above-described antibody fragments can be obtained using conventional techniques known to those of skill in the art, and the fragments are screened for binding specificity and neutralizing activity in the same manner as intact antibodies.

[0023] As used herein, "antigen" refers to a molecule to which an antibody (or antigen-binding fragment thereof) can selectively bind. The target antigen can be a protein, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the target antigen can be a polypeptide (e.g., A33 polypeptide). Antigens can also be administered to an animal to generate an immune response in the animal. The term "antigen-binding fragment" refers to a fragment of the entire immunoglobulin structure that retains the portion of the polypeptide necessary to bind to the antigen. Examples of antigen-binding fragments useful in the present technology include, but are not limited to, scFv, (scFv)2, scFvFc, Fab, Fab', and F(ab')2. "Binding affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or antigenic peptide). The affinity of a molecule X for its partner Y is generally measured by the dissociation constant (K d ). Affinity can be measured by standard methods known in the art, including those described herein. Low affinity complexes generally contain antibodies that tend to dissociate easily from the antigen, whereas high affinity complexes generally contain antibodies that tend to remain bound to the antigen for longer periods of time.

[0024] As used herein, the term "biological sample" refers to sample material derived from living cells. Biological samples can include tissues, cells, protein or membrane extracts of cells, and tissues, cells and fluids present within a subject animal, as well as biological fluids isolated from the subject animal (e.g., ascites or cerebrospinal fluid (CSF)). Biological samples of the present technology include, but are not limited to, samples taken from breast tissue, kidney tissue, cervix, endometrium, head and neck, gallbladder, parotid 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 tissue, testicular tissue, tonsils, thymus, blood, hair, cheek, skin, serum, plasma, CSF, semen, prostatic fluid, seminal fluid, urine, feces, sweat, saliva, sputum, mucus, bone marrow, lymph, and tears. Biological samples can also be obtained from biopsies of internal organs or cancers. Biological samples can be obtained from animals of interest for diagnosis or research, or from non-diseased individuals as controls or for basic research. Samples can be obtained by standard methods, including, for example, venipuncture and surgical biopsy. In certain embodiments, the biological sample is a breast, lung, colon, or prostate tissue sample obtained by needle biopsy.

[0025] As used herein, the term "CDR-grafted antibody" refers to an antibody in which at least one CDR of an "acceptor" antibody has been replaced by a CDR "graft" from a "donor" antibody possessing the desired antigen specificity. As used herein, the term "chimeric antibody" refers to an antibody in which the Fc constant region of a monoclonal antibody of one species (e.g., a murine Fc constant region) has been replaced with the Fc constant region of an antibody of another species (e.g., a human Fc constant region) using recombinant DNA techniques. See generally: Robinson et al., PCT / US86 / 02269; Akira et al., European Patent Publication 184,187; Taniguchi, European Patent Publication 171,496; Morrison et al., European Patent Publication 173,494; Neuberger et al., WO 86 / 01533; Cabilly et al. U.S. Patent 4,816,567; Cabilly et al., European Patent Publication 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. 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. As used herein, the term "consensus FR" refers to the framework (FR) antibody region of the consensus immunoglobulin sequence. The FR region of an antibody does not contact the antigen.

[0026] As used herein, a "control" is a substitute sample used in an experiment for comparison purposes. A control can be "positive" or "negative." For example, if the purpose of an experiment is to determine the correlation of the effectiveness of a therapeutic agent for treating a particular disease type, typically a positive control (a compound or composition known to exhibit the desired therapeutic effect) and a negative control (a subject or sample that does not receive treatment or receives a placebo) are used. As used herein, the term "effective amount" refers to an amount sufficient to achieve a desired therapeutic and / or prophylactic effect. This amount is, for example, an amount that results in the prevention or alleviation of a disease or condition described herein, or one or more signs or symptoms associated with a disease or condition described herein. In the context of therapeutic or prophylactic applications, the amount of the composition administered to the subject will vary depending on the composition, the extent, type and severity of the disease, and the individual's characteristics (e.g., general health, age, sex, weight and tolerance to drugs). Those skilled in the art will be able to determine the appropriate dosage depending on these and other considerations. The composition may also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the therapeutic composition may be administered to a subject having one or more signs or symptoms of a disease or condition described herein. As used herein, a "therapeutically effective amount" of a composition refers to a level of the composition that alleviates or eliminates the physiological effects of a disease or condition. A therapeutically effective amount may be provided in one or more administrations.

[0027] As used herein, the term "effector cell" refers to an immune cell that is required in the effector phase of an immune response, as opposed to the cognitive and activation phase of an immune response. Exemplary immune cells include cells of myeloid or lymphoid origin, such as lymphocytes (e.g., B cells, and T cells, including cytotoxic T cells (CTL)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mast cells, and basophils. Effector cells express specific Fc receptors and perform specific immune functions. Effector cells can induce antibody-dependent cell-mediated cytotoxicity (ADCC), e.g., neutrophils can induce ADCC. For example, monocytes, macrophages, neutrophils, eosinophils, and lymphocytes expressing FcαR are required for specific killing of target cells and for antigen presentation to other immune system components, or binding to cells that present antigen.

[0028] As used herein, the term "epitope" refers to a protein determinant capable of specific binding to an antibody. Epitopes usually consist of chemically active surface groupings of molecules (e.g., amino acids or sugar side chains) and usually have specific three-dimensional structural characteristics, together with specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former, but not the latter, is lost in the presence of denaturing solvents. In some embodiments, the "epitope" of the A33 protein is the region of the protein to which the anti-A33 antibody of the present technology specifically binds. In some embodiments, the epitope is a conformational epitope. To screen for anti-A33 antibodies that bind to the epitope, routine cross-blocking assays (e.g., those described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane, 1988) can be performed. The above assay can be used to determine whether the anti-A33 antibody binds to the same site or epitope as the anti-A33 antibody of the present technology.Alternatively or additionally, epitope mapping can be performed by methods known in the art.For example, the antibody sequence can be mutated, for example by alanine scanning, to identify contact residues.In another method, peptides corresponding to different regions of the A33 protein can be used in a competition assay with the test antibody, or with the test antibody and an antibody with a previously characterized or known epitope. As used herein, "expression" includes one or more of the following: transcription of a gene into precursor mRNA; splicing and other processing of the precursor mRNA to produce mature mRNA; mRNA stability; translation of mature mRNA into protein (including codon usage and tRNA availability); and glycosylation and / or other modifications of the translation product (if required for proper expression and function). As used herein, the term "gene" refers to a DNA sequence that contains all the information for the regulated biosynthesis of an RNA product, including promoters, exons, introns, and other untranslated regions that control expression.

[0029] "Homology" or "identity" or "similarity" refers to 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 purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. When a polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of "sequence identity" to another sequence when aligned, it means that that percentage of bases (or amino acids) are the same when the two sequences are compared. This alignment and percent homology or sequence identity can be determined using software programs known in the art. In some embodiments, default parameters are used for the alignment. One alignment program is BLAST, which uses default parameters. In particular, the programs are BLASTN and BLASTP, which use the following default parameters: genetic code=normal; filter=none; strand=both; cutoff=60; expectation=10; matrix=BLOSUM62; hits=50 sequences; sorting criteria=HIGH SCORE; database=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translation+SwissProtein+SPupdate+PIR. Details of these programs can be found at the National Center for Biotechnology Information. Biologically equivalent polynucleotides are those that have a specified percent homology and encode polypeptides that have the same or similar biological activity. Two sequences are considered "unrelated" or "non-homologous" if they share less than 40% or less than 25% identity with each other.

[0030] As used herein, the term "humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins in which recipient hypervariable region residues are replaced by hypervariable region residues from a non-human species (donor antibody) (e.g., mouse, rat, rabbit, or non-human primate) having the desired specificity, affinity, and performance. In some embodiments, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can include residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance (e.g., binding affinity). Generally, humanized antibodies will comprise substantially all of at least one, and typically two, variable domains (e.g., Fab, Fab', F(ab')2, or Fv). In the above, all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the FR regions are of a human immunoglobulin consensus FR sequence, except that the FR regions can contain one or more amino acid substitutions that improve binding affinity. The number of these amino acid substitutions in the FR typically does not exceed six in the H chain and does not exceed three in the L chain. The humanized antibody can also optionally contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. 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, e.g., Ahmed & Cheung, FEBS Letters 588(2):288-297, 2014.

[0031] As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody which are responsible for antigen binding. Hypervariable regions generally consist of amino acid residues from the "complementarity determining regions" or "CDRs" (e.g., V L In the case of H In the present study, the most common residues are approximately 31-35B (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD., 1991)), and / or residues from the "hypervariable loops" (e.g., L In , residues approximately 26-32 (L1), 50-52 (L2), and 91-96 (L3), and V H H1, H2 and H3 comprise approximately residues 26-32 (H1), 52A-55 (H2) and 96-101 (H3) (Chothia and Lesk J. Mol. Biol. 196:901-917, 1987). As used herein, the term "identical" or percent "identity," when used in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that are the same or have a specified percentage of identical amino acid residues or nucleotides (i.e., about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity over a specified region (e.g., nucleic acid sequences encoding an antibody described herein or amino acid sequences of an antibody described herein)), where the two or more sequences are compared and aligned for maximum correspondence over a comparison window or specified region, as measured using the BLAST or BLAST 2.0 sequence comparison algorithm with default parameters as described below, or by manual alignment and visual inspection (e.g., at the NCBI website). Such sequences are then referred to as "substantially identical." The term also refers to, or can be applied to, the complement of a test sequence. The term also includes sequences that have deletions and / or additions, as well as sequences that have substitutions. In some embodiments, identity exists over a region that is at least 25 amino acids or nucleotides in length, or over a region that is 50-100 amino acids or nucleotides in length.

[0032] As used herein, an "intact antibody" or "intact immunoglobulin" refers to an antibody having at least two heavy (H) chain polypeptides and two light (L) chain polypeptides interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable region (referred to herein as HCVR or V H Each light chain comprises a light chain variable region (herein abbreviated as LCVR or VVR) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. ... L The light chain constant region comprises one domain (C L ) is included. V H and V LThe region is further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). H and V L contains three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region 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 (C1q) of the classical complement system. As used herein, an "individual," "patient," or "subject" can be an individual organism, a vertebrate, a mammal, or a human. In some embodiments, the individual, patient, or subject is a human.

[0033] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies. That is, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. For example, a monoclonal antibody can be an antibody derived from a single clone (including any eukaryotic, prokaryotic, or phage clone), regardless of the method by which it is produced. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Monoclonal antibodies are highly specific, being directed against only one antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against only one determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. Monoclonal antibodies can be prepared using a wide variety of techniques known in the art, including, but not limited to, hybridoma, recombinant, and phage display techniques. For example, the monoclonal antibodies used in the present methods may be made by the hybridoma method first described by Kohler et al. (Kohler et al., Nature 256:495, 1975), or may be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries, using, for example, the techniques described in Clackson et al., Nature 352:624-628, 1991; and Marks et al., J. Mol. Biol. 222:581-597, 1991.

[0034] As used herein, the term "pharmaceutical acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal compounds, isotonic and absorption delaying compounds, and the like, which are compatible with pharmaceutical administration. Pharmaceutically acceptable carriers and their formulation are known to those skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences (20 th edition, ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, Pa.). As used herein, the term "polyclonal antibody" refers to a preparation of antibodies derived from at least two different antibody-producing cell lines. Use of this term includes at least two antibody preparations that contain antibodies that specifically bind to different epitopes or antigenic regions.

[0035] As used herein, the term "polynucleotide" or "nucleic acid" refers to any RNA or DNA, which may be unmodified or modified RNA or DNA. Polynucleotides include, but are not limited to, single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, RNA that is a mixture of single-stranded and double-stranded regions, and hybrid molecules containing DNA and RNA, which may be single-stranded, more typically double-stranded, or a mixture of single-stranded and double-stranded regions. In addition, the term polynucleotide refers to triple-stranded regions, which includes 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 backbones modified for stability or other purposes. As used herein, the terms "polypeptide", "peptide" and "protein" are used interchangeably herein and refer to a polymer comprising two or more amino acids joined together by peptide bonds or modified peptide bonds (i.e., peptide isosteres). Polypeptide refers to both short chains, commonly referred to as peptides, glycopeptides or oligomers, and longer chains, commonly referred to as proteins. Polypeptides can contain amino acids other than the 20 genetically encoded amino acids. Polypeptides include amino acid sequences modified by natural processes, such as post-translational processing, or by chemical modification techniques, which are well known in the art. Such modifications are well described in basic textbooks and more specifically in monographs as well as in a voluminous monograph literature. As used herein, "PRIT" or "pretargeted radioimmunotherapy" refers to a multi-step process that achieves gradual blood clearance of tumor-targeting antibodies, which contribute to undesirable toxicity to normal tissues (e.g., bone marrow). In pretargeting, a radionuclide or other diagnostic or therapeutic agent is conjugated to a small hapten. A pretargeting bispecific antibody has binding sites for the target antigen and the hapten and is administered first. Unbound antibodies are then cleared from the circulation, after which the hapten is administered.

[0036] As used herein, the term "recombinant," e.g., when used in reference to a cell or a nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein, or by the alteration of a native nucleic acid or protein, or that the material is derived from a cell so modified. Thus, for example, a recombinant cell may express genes that are not found in the native (non-recombinant) form of the cell, or in a recombinant cell, native genes may be aberrantly expressed, reduced in expression, or not expressed at all. As used herein, the term "separate" therapeutic use refers to the administration of at least two active ingredients at the same or substantially the same time by different routes. As used herein, the term "sequential" therapeutic use refers to the administration of at least two active ingredients at different times, where the administration route is the same or different. More specifically, sequential use refers to the complete administration of one of the active ingredients before the start of administration of one or more other ingredients. Thus, one of the active ingredients can be administered minutes, hours or days before the administration of the other active ingredient or ingredients. In this case, there is no simultaneous treatment. As used herein, "specifically binds" refers to a molecule (e.g., an antibody or antigen-binding fragment thereof) that recognizes and binds to another molecule (e.g., an antigen) but does not substantially recognize or bind to other molecules. As used herein, the terms "specific binding," "specifically binds to," or "specific" for a particular molecule (e.g., a polypeptide or epitope on a polypeptide) refer to, for example, a specific binding activity of about 10% to the molecule to which it binds. -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 K of M d The term "specifically binds" can also refer to binding of a molecule (e.g., an antibody or antigen-binding fragment thereof) to a particular polypeptide (e.g., an A33 polypeptide) or an epitope on a particular polypeptide, and not to substantially bind to any other polypeptides or polypeptide epitopes.

[0037] As used herein, the term "concurrent" therapeutic use refers to the administration of at least two active ingredients by the same route and at the same or substantially the same time. 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 in a subject in need thereof. As used herein, "treating" or "treatment" covers the treatment of a disease or disorder described herein in a subject (e.g., a human), including: (i) preventing the disease or disorder, i.e., arresting its development; (ii) alleviating the disease or disorder, i.e., causing regression of the disorder; (iii) slowing the rate of progression of the disorder; and / or (iv) preventing, alleviating, or slowing the rate of progression of one or more symptoms of the disease or disorder. In some embodiments, treating means that a symptom associated with the disease is, for example, alleviated, relieved, cured, or placed in remission. It will be understood that the various aspects of treating disorders described herein are intended to mean "substantially." Substantially includes complete treatment, but also includes less than complete treatment, where some biologically or medically relevant outcome is achieved. Treatment may be continuous long-term treatment for chronic conditions, or one or several sessions for treating acute symptoms.

[0038] Modifications of the amino acid sequence of the anti-A33 antibody described herein are contemplated. For example, it may be desired to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the anti-A33 antibody are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletion, and / or insertion, and / or substitution of residues within the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be performed to obtain an antibody of interest, so long as the resulting antibody has the desired properties. Modifications also include changes in the glycosylation pattern of the protein. The most attractive sites for substitutional mutagenesis include hypervariable regions, although FR changes are also contemplated. "Conservative substitutions" are shown in the table below.

[0039] TIFF2025061650000001.tif179153

[0040] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody. A convenient method for generating such substitutional variants involves affinity maturation using phage display. Specifically, several hypervariable region sites (e.g., 6-7 sites) are matured to generate all possible amino acid substitutions at each site. The antibody variants thus generated are displayed in a monovalent manner from filamentous phage particles as fusions with the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as disclosed herein. To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis is performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively or additionally, it may be beneficial to analyze a crystal structure of an antigen-antibody complex to identify contact points between the antibody and the antigen. Such contact and nearby residues are candidates for substitution by the refinement techniques herein. Once such variants are generated, the panel of variants can be subjected to screening as described herein and antibodies with similar or superior properties in one or more corresponding assays can be selected for further development.

[0041] CRC and A33 CRC is a heterogeneous disease and can be subdivided into four consensus molecular subtypes (i.e., CMS1-4) based on gene expression analysis. MSI tumors mainly belong to CMS1 (14% of all CRC patients) and are characterized by genomic hypermutation and microsatellite instability (due to DNA repair pathway defects). Presumably, hypermutation generates excessive neoantigens that are present on the cell surface and attract T cells to the tumor. Indeed, CMS1 has a strong molecular signature of immune system activation and evasion. ICIs reactivate suppressed tumor-infiltrating lymphocytes (TILs) to regain tumor-killing capabilities, and thus MSI tumors are the most responsive CRC tumors to ICIs. However, MSI tumors represent less than 5% of mCRCs, and for the majority of mCRCs, the efficacy of ICIs has been disappointing so far. Peritoneal carcinomatosis is typically the terminal phase of incurable CRC. It is usually unresectable, chemotherapy-unresponsive, and causes significant morbidity, unlike liver and lung metastases. Conventional treatments are mostly palliative and consist of cytoreductive surgery (CRS) and hyperthermia (HIPEC), which are effective in only a low percentage of patients with low-volume disease. Human glycoprotein A33 (GPA33 or A33) is a single-pass type I membrane protein that belongs to the CTX family of cell adhesion molecules within the immunoglobulin family. A33 is expressed in 95% of CRC tissues and has very limited expression in normal tissues. A33 contains one Ig-like C2-type domain and one Ig-like V-type domain. The predicted mature protein contains a single transmembrane domain, an extracellular region and an intracellular tail. A33 plays a role in intracellular trafficking, cell-cell recognition / signaling and recycling to the cell surface. The amino acid sequence of the ectodomain of A33 (Ile22-Val235) is provided below: ISVETPQDVLRASQGKSVTLPCTYHTSTSSREGLIQWDKLLLTHTERVVIWPFSNKNYIHGELYKNRVSISNNAEQSDASITIDQLTMADNGTYECSVSLMSDLEGNTKSRVRLLVLVPPSKPECGIEGETIIGNNIQLTCQSKEGSPTPQYSWKRYNILNQEQPLAQPASGQPVSLKNISTDTSGYYICTSSNEEGTQFCNITVAVRSPSMNV (SEQ ID NO:57).

[0042] Immunoglobulin-related constructs of the present technology Existing humanized A33 IgG1 antibodies have been found to be immunogenic in colon cancer patients (see Ritter G et al., Cancer Res 61:6851-9, 2001). The present technology describes methods and compositions for the production and use of anti-A33 immunoglobulin-related constructs (e.g., anti-A33 antibodies or antigen-binding fragments thereof). The anti-A33 immunoglobulin-related constructs of the present disclosure may be useful in the diagnosis or treatment of A33-positive cancer. Anti-A33 immunoglobulin-related constructs within the scope of the present technology include, but are not limited to, monoclonal antibodies, chimeric antibodies, humanized antibodies, and diabodies that specifically bind to a target polypeptide, or a homolog, derivative, or fragment thereof. The present disclosure also provides an antigen-binding fragment of any of the anti-A33 antibodies disclosed herein, wherein the antigen-binding fragment is selected from the group consisting of Fab, F(ab)'2, Fab', scFv, and Fv. In one aspect, the technology provides a method for the production of a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domains (V L (a) an antibody or antigen-binding fragment thereof comprising: H is the V of FTFSTYDMS (SEQ ID NO: 37) H - CDR1 sequence, TISSGGSYTYYLDSVKG (SEQ ID NO: 38) H -CDR2 sequence, and V of TTVVPFAY (SEQ ID NO: 39) Hand / or (b) a V L is selected from the group consisting of KASQNVRTVVA (SEQ ID NO: 40), LASNRHT (SEQ ID NO: 41) and QYWSYPLT (SEQ ID NO: 42); KASQNVRTVVA (SEQ ID NO: 40), LASDRHT (SEQ ID NO: 43) and QYWSYPLT (SEQ ID NO: 42); KASQNVRTLVA (SEQ ID NO: 44), LASNRHT (SEQ ID NO: 41) and QHWSYPLT (SEQ ID NO: 45); and KASQNVRTLVA (SEQ ID NO: 44), LASNRHT (SEQ ID NO: 41) and QYWSYPLT (SEQ ID NO: 42). L - CDR1 sequence, V L -CDR2 sequence and V L -CDR3 sequences. In some embodiments, the antibody further comprises an Fc domain of any isotype, 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:

[0043] Human IgD constant region, Uniprot: P01880 (SEQ ID NO: 46) APTKAPDVFPIISGCRHPKDNSPVVLACLITGYHPTSVTVTWYMGTQSQPQRTFPEIQRRDSYYMTSSQLSTPLQQWRQGEYKCVVQHTASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFV VGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDHGPMK Human IgG1 constant region, Uniprot: P01857 (SEQ ID NO: 47) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human IgG2 constant region, Uniprot: P01859 (SEQ ID NO: 48) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKDTLMISRTPEVTCVVDVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human IgG3 constant region, Uniprot: P01860 (SEQ ID NO: 49) ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK

[0044] Human IgM constant region, Uniprot: P01871 (SEQ ID NO: 50) GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITLSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKV SVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFA IPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPARE QLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY Human IgG4 constant region, Uniprot: P01861 (SEQ ID NO: 51) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Human IgA1 constant region, Uniprot: P01876 (SEQ ID NO: 52) ASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQDASGDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPE RDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY Human IgA2 constant region, Uniprot: P01877 (SEQ ID NO: 53) ASPTSPKVFPLSLDSTPQDGNVVVACLVQGFFPQEPLSVTWSESGQNVTARNFPPSQDASGDLYTTSSQLTLPATQCPDGKSVTCHVKHYTNPSQDVTVPCPVPPPPPCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPERDLCGCY SVSSVLPGCAQPWNHGETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRMAGKPTHVNVSVVMAEVDGTCY Human Ig kappa constant region, Uniprot: P01834 (SEQ ID NO: 54) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0045] In some embodiments, the immunoglobulin-associated constructs of the present technology comprise a heavy chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NOs:46-53. Additionally or alternatively, in some embodiments, the immunoglobulin-associated constructs of the present technology comprise a light chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:54. In some embodiments, the immunoglobulin-associated constructs of the present technology bind to an epitope of the A33 polypeptide that comprises at least 5 to 8 consecutive amino acid residues of SEQ ID NO:57. In some embodiments, the epitope is a conformational epitope. In another aspect, the disclosure provides an isolated immunoglobulin-related construct (e.g., an antibody or antigen-binding fragment thereof), the construct comprising a heavy chain (HC) amino acid sequence comprising SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:58, SEQ ID NO:62, or a variant of said sequence having one or more conservative amino acid substitutions. Additionally or alternatively, in some embodiments, the immunoglobulin-related constructs of the present technology comprise a light chain (LC) amino acid sequence comprising SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:60, SEQ ID NO:63, or a variant of said sequence having one or more conservative amino acid substitutions.

[0046] In some embodiments, the immunoglobulin-related constructs of the present technology include SEQ ID NO:5 and SEQ ID NO:9 (3A3-H1 / L1); SEQ ID NO:5 and SEQ ID NO:10 (3A3-H1 / L2); SEQ ID NO:6 and SEQ ID NO:9 (3A3-H2 / L1); SEQ ID NO:6 and SEQ ID NO:10 (3A3-H2 / L2); SEQ ID NO:15 and SEQ ID NO:17 (huA33-IgG1(H2L2)); SEQ ID NO:19 and SEQ ID NO:21 (huA33-BsAb); SEQ ID NO:23 and SEQ ID NO:24 (clone 31); SEQ ID NO:25 and SEQ ID NO:26 (huA33-IgG1(H2L2)); SEQ ID NO:6 and SEQ ID NO:63 (huA33-mC825); SEQ ID NO:64 and SEQ ID NO:65 (huA33-mC825); SEQ ID NO:66 and SEQ ID NO:67 (clone 32); SEQ ID NO:27 and SEQ ID NO:28 (clone 48); SEQ ID NO:29 and SEQ ID NO:30 (clone 49); SEQ ID NO:31 and SEQ ID NO:32 (clone 53); SEQ ID NO:33 and SEQ ID NO:34 (clone 56); SEQ ID NO:35 and SEQ ID NO:36 (clone 57); SEQ ID NO:58 and SEQ ID NO:60 (huA33-huC825); and SEQ ID NO:62 and SEQ ID NO:63 (huA33-mC825). In any of the above embodiments of the immunoglobulin related constructs, the HC and LC immunoglobulin variable domain sequences form an antigen binding site that binds to an epitope of the A33 polypeptide that comprises at least 5 to 8 consecutive amino acid residues of the A33 ectodomain (SEQ ID NO:57). In some embodiments, the epitope is a conformational epitope. In some embodiments, the HC and LC immunoglobulin variable domain sequences are components of the same polypeptide chain. In other embodiments, the HC and LC immunoglobulin variable domain sequences are components of different polypeptide chains. In certain embodiments, the antibody is a full-length antibody.

[0047] In some embodiments, the immunoglobulin-related constructs of the present technology specifically bind to at least one A33 polypeptide. In some embodiments, the immunoglobulin-related constructs of the present technology specifically bind to at least one A33 polypeptide. -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 Dissociation constant of M (K D ) to at least one A33 polypeptide. In certain embodiments, the immunoglobulin related construct is a monoclonal antibody, a chimeric antibody, a humanized antibody, or a bispecific antibody. In some embodiments, the antibody comprises a framework region of a human antibody. In certain embodiments, the immunoglobulin related construct comprises one or more of the following features: (a) a light chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to a light chain immunoglobulin variable domain sequence present in any one of SEQ ID NOs: 9, 10, 17, 21, 24, 26, 28, 30, 32, 34, 36, 60 or 63; and / or (b) a heavy chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to a heavy chain immunoglobulin variable domain sequence present in any one of SEQ ID NOs: 5, 6, 15, 19, 23, 25, 27, 29, 31, 33, 35, 58 or 62. In another aspect, one or more amino acid residues of the immunoglobulin related constructs provided herein are replaced with another amino acid. The substitution may be a "conservative substitution" as defined herein. In some embodiments, the immunoglobulin related construct comprises: (a) an LC sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to an LC sequence present in any one of SEQ ID NOs: 9, 10, 17, 21, 24, 26, 28, 30, 32, 34, 36, 60 or 63; and / or (b) an HC sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to an HC sequence present in any one of SEQ ID NOs: 5, 6, 15, 19, 23, 25, 27, 29, 31, 33, 35, 58 or 62. In certain embodiments, the immunoglobulin related construct comprises an IgG1 constant region comprising one or more amino acid substitutions selected from the group consisting of N297A and K322A. Additionally or alternatively, in some embodiments, the immunoglobulin related construct comprises an IgG4 constant region comprising an S228P mutation.

[0048] In some aspects, the anti-A33 immunoglobulin-related constructs described herein include structural modifications to promote rapid binding and cellular uptake and / or slow release. In some aspects, the anti-A33 immunoglobulin-related constructs (e.g., antibodies) of the present technology include deletions in the CH2 constant heavy chain region, which can promote rapid binding and cellular uptake and / or slow release. In some aspects, Fab fragments are used to promote rapid binding and cellular uptake and / or slow release. In some aspects, F(ab)'2 fragments are used to promote rapid binding and cellular uptake and / or slow release. In one aspect, the technology provides a nucleic acid sequence encoding a heavy or light chain of an immunoglobulin related construct described herein. Also disclosed are recombinant nucleic acid sequences encoding any of the antibodies described herein. In some embodiments, the nucleic acid sequence is selected from the group consisting of SEQ ID NOs: 7, 8, 11, 12, 16, 18, 20, 22, 59, and 61. In another aspect, the technology provides a host cell expressing any of the nucleic acid sequences encoding a heavy or light chain of an immunoglobulin related construct described herein. The immunoglobulin-related constructs of the present technology (e.g., anti-A33 antibodies) can be monospecific, bispecific, trispecific, or of greater multispecificity. Multispecific antibodies can be specific for different epitopes of one or more A33 polypeptides, or can be specific for both A33 polypeptides and heterologous constructs (e.g., heterologous polypeptides or solid support materials). See, e.g., WO93 / 17715; WO92 / 08802; WO91 / 00360; WO92 / 05793; Tutt et al., J. Immunol. 147: 60-69, 1991; US ​​Pat. 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-related construct is chimeric. In certain embodiments, the immunoglobulin-related construct is humanized. The immunoglobulin-related construct of the present technology can also be recombinantly fused to heterologous polypeptides at N- or C-terminus, or chemically conjugated to polypeptides or other constructs (including covalent and non-covalent conjugation).For example, the immunoglobulin-related construct of the present technology can be recombinantly fused to or conjugated with molecules useful as labels for detection assays and effector molecules (e.g., heterologous polypeptides, drugs, or toxins).See, for example, WO92 / 08495; WO91 / 14438; WO89 / 12624; US Pat. No. 5,314,995; and EP0 396 387.

[0049] In any of the above embodiments of the immunoglobulin-related constructs of the present technology, the antibody or antigen-binding fragment can be optionally conjugated with an agent selected from the group consisting of: a radioisotope, a dye, a chromogen, an imaging agent, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RAN, DNA, or any combination thereof. Chemical or physical conjugation typically involves binding a functional group of the immunoglobulin-related construct to a functional group of the agent. Alternatively, a functional group of the agent is bound to a functional group of the immunoglobulin-related construct. The functional groups of the drug and the immunoglobulin-related construct can be directly linked. For example, a functional group (e.g., a sulfhydryl group) of the drug can be linked to a functional group (e.g., a sulfhydryl group) of the immunoglobulin-related construct to form a disulfide. Alternatively, the functional groups can be linked via a cross-linking agent (i.e., a linker). Some examples of cross-linking agents are described below. The cross-linking agent can be linked to either the drug or the immunoglobulin-related construct. The number of drugs or immunoglobulin-related constructs in the conjugate is also limited by the number of functional groups present on the other. For example, the maximum number of drugs that can be linked to the conjugate depends on the number of functional groups present on the immunoglobulin-related construct. Alternatively, the maximum number of immunoglobulin-related constructs that can be linked to the drug depends on the number of functional groups present on the drug. In yet another embodiment, the complex comprises an immunoglobulin-related construct coupled to an agent. In one embodiment, the complex comprises at least one agent chemically coupled (e.g., conjugated) to at least one immunoglobulin-related construct. The agent can be chemically coupled to the immunoglobulin-related construct by any method known to those skilled in the art. For example, a functional group of the agent can be directly coupled to a functional group of the immunoglobulin-related construct. Some examples of suitable functional groups include, for example, amino, carboxyl, sulfhydryl, maleimide, isocyanate, isothiocyanate, and hydroxyl.

[0050] Agents can also be chemically coupled to immunoglobulin-related constructs by means of cross-linking agents, such as dialdehydes, carbodiimides, dimaleimides, and the like. Cross-linking agents can be obtained, for example, from Pierce Biotechnology, Inc., Rockford, Ill. The supplier's website can provide assistance. Additional cross-linking agents include platinum cross-linkers, as described in U.S. Patents 5,580,990, 5,985,566, and 6,133,038 (Kreatech Biotechnology, BV, Amsterdam, The Netherlands). Alternatively, the functional groups of the drug and the immunoglobulin-related construct may be the same. Homobifunctional crosslinkers are typically used to crosslink the same functional groups. Examples of homobifunctional crosslinkers include: EGS (i.e., ethylene glycol bis[succinimidyl succinate]), DSS (i.e., disuccinimidyl suberate), DMA (i.e., dimethyl adipimidate dimethyl diHCl), DTSSP (i.e., 3,3'-dithiobis[sulfosuccinimidyl propionate]), DPDPB (i.e., 1,4-di-[3'-(2'-pyridyldithio)-propionamido]butane), and BMH (i.e., bis-maleimidohexane). Such homobifunctional crosslinkers are also available from Pierce Biotechnology.

[0051] In other instances, it may be beneficial to cleave the agent from the immunoglobulin-associated construct. The Pierce Biotechnology website referenced above can also provide assistance to the skilled artisan in the selection of suitable cross-linkers that can be cleaved, for example, by intracellular enzymes, thus allowing the agent to be separated from the immunoglobulin-associated construct. Examples of cleavable linkers include: SMPT (i.e., 4-succinimidyloxycarbonyl-methyl-[2-pyridyldithio]toluene), sulfo-LC-SPDP (i.e., sulfosuccinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), LC-SPDP (i.e., succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), sulfo-LC-SPDP (i.e., sulfosuccinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), SPDP (i.e., N-succinimidyl 3-[2-pyridyldithio]-propionamidohexanoate), and AEDP (i.e., 3-[(2-aminoethyl)dithio]propionic acid HCl). In another embodiment, the complex comprises at least one agent physically associated with at least one immunoglobulin-related construct. Any method known to those skilled in the art can be used to physically associate the agent with the immunoglobulin-related construct. For example, the immunoglobulin-related construct and the agent can be mixed together by any method known to those skilled in the art. The order of mixing is not important. For example, the agent can be physically mixed with the immunoglobulin-related construct by any method known to those skilled in the art. For example, the immunoglobulin-related construct and the agent can be placed in a container and agitated, for example by shaking the container, to mix the immunoglobulin-related construct and the agent. The immunoglobulin-related constructs may be modified by any method known to those of skill in the art, for example by means of crosslinking agents or functional groups as described above.

[0052] A. Methods for Preparing the Anti-A33 Antibody of the Present Technology Summary First, a target polypeptide is selected that can generate the antibody of the present technology. For example, the antibody can be generated against the full-length A33 protein or against a portion of the extracellular domain of the A33 protein. The techniques for generating antibodies directed to such target polypeptides are well known to those skilled in the art. Examples of such techniques include, but are not limited to, those that require, for example, display libraries, heterologous or human-mouse hybridomas, etc. Target polypeptides within the scope of the present technology include any polypeptide derived from the A33 protein, including the extracellular domain, that can elicit an immune response. Preparation of antibodies specific to the A33 protein is illustrated in Examples 1, 2, 3 and 5. It will be understood that recombinantly engineered antibodies and antibody fragments, such as antibody-related polypeptides (directed towards the A33 protein and fragments thereof), are suitable for use in accordance with the present disclosure. Anti-A33 antibodies that can be subjected to the techniques presented herein include monoclonal and polyclonal antibodies, as well as antibody fragments, such as Fab, Fab', F(ab')2, Fd, scFv, diabodies, antibody light chains, antibody heavy chains, and / or antibody fragments. Methods useful for high-yield production of antibody Fv-containing polypeptides (e.g., Fab' and F(ab')2 antibody fragments) have been reported. See U.S. Patent No. 5,648,237. Generally, antibody is obtained from original species.More specifically, obtain the nucleic acid or amino acid sequence of the variable part of the light chain, heavy chain or both chains of the original species antibody that has specificity for target polypeptide.Original species can be any species (e.g., rat, mouse, rabbit, chicken, monkey, human, etc.) that is useful for producing the antibody or antibody library of the present technology. Phage or phagemid display technology is a useful technique for inducing the antibodies of the present technology. Techniques for producing and cloning monoclonal antibodies are well known to those skilled in the art. Expression of sequences encoding the antibodies of the present technology can be achieved in Escherichia coli ( E. coli ) can be implemented.

[0053] Due to the degeneracy of nucleic acid coding sequences, other sequences that code for substantially the same sequence as the naturally occurring amino acid sequence can be used in the practice of the present technology. These include, but are not limited to, all or part of the nucleic acid sequences encoding the above polypeptides, which are modified by substitution of different codons that code for functionally equivalent amino acid residues in the sequence (thus resulting in silent changes). It will be understood that the nucleotide sequences of the immunoglobulins of the present technology allow for sequence homology variations of up to 25%, 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 functional antibodies that recognize the A33 protein. For example, one or more amino acid residues in a polypeptide sequence can be replaced by another amino acid of similar polarity that acts as a functional equivalent, resulting in a silent mutation. Substitutions in an amino acid in a sequence can be selected from other amino acids of the class to which the amino acid belongs. For example, non-polar (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. Proteins, fragments or derivatives thereof, which are differentially modified during or after translation, such as by glycosylation, proteolytic cleavage, linkage to antibody molecules or other cellular ligands, are also included within the scope of the present technology.In addition, immunoglobulin-encoding nucleic acid sequences can be mutated in vitro or in vivo to create or destroy translation sequences, initiation and / or termination sequences, or to create variations in the coding region and / or to create new restriction endonuclease sites or destroy existing sites to facilitate further in vitro modifications. Any mutagenesis technique known in the art can be used, including, but not limited to, in vitro site-directed mutagenesis (J. Biol. Chem. 253:6551), the use of Tab linkers (Pharmacia), and the like.

[0054] Preparation of polyclonal antisera and immunogens The method of producing the antibody or antibody fragment of the present technology typically includes immunizing a subject (generally a non-human subject, such as a mouse or rabbit) with purified A33 protein or its fragment, or with a cell expressing A33 protein or its fragment. Suitable immunogenic preparations can include, for example, recombinantly expressed A33 protein or chemically synthesized A33 peptide. The ECM of A33 protein or its part or fragment can be used as an immunogen to produce anti-A33 antibodies that bind to A33 protein or its part or fragment by standard techniques for polyclonal and monoclonal antibody preparation. The full-length A33 protein or fragments thereof are useful as immunogens, as are fragments thereof, in some embodiments, an A33 fragment comprises at least 5 to 8 consecutive amino acid residues of the amino acid sequence of SEQ ID NO:57 and encompasses an epitope of the A33 protein such that antibodies raised against the peptide form specific immune complexes with the A33 protein. In some embodiments, antigenic A33 peptides overlapping the A33 ectodomain (Ile22-Val235) contain at least 5, 8, 10, 15, 20, or 30 amino acid residues. Longer antigenic peptides are sometimes more desirable than shorter antigenic peptides, depending on the application and methods known in the art. Multimers of a given epitope are sometimes more effective than monomers. If necessary, the immunogenicity of the A33 protein (or fragments thereof) can be enhanced by fusion or conjugation with a hapten, such as keyhole limpet hemocyanin (KLH) or ovalbumin (OVA). Many such haptens are known in the art. Additionally, the A33 protein can be combined with a conventional adjuvant, such as Freund's complete or incomplete adjuvant, to enhance the immune response of the subject animal to the polypeptide. Various adjuvants that can be used to enhance the immunological response include Freund's adjuvant (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), human adjuvants (e.g., Bacillus Calmette-Guerin and Corynebacterium parvum ( Corynebacterium parvum )), or similar immune stimulating compounds.

[0055] In the present disclosure, immune responses can be described as "primary" or "secondary" immune responses. A primary immune response (also described as a "protective" immune response) refers to an immune response that occurs in an individual as a result of some initial exposure (e.g., initial "immunization") to a particular antigen (e.g., A33 protein). In some embodiments, immunity can occur as a result of vaccination of an individual with a vaccine that includes the antigen. For example, the vaccine can be an A33 vaccine that includes one or more A33 protein-derived antigens. A primary immune response can weaken or fade over time, even disappear, or be weakened to the point of being undetectable. Thus, the present technology also relates to a "secondary" immune response (also described herein as a "memory immune response"). The term secondary immune response refers to an immune response that is elicited in an individual after a primary immune response has already occurred. Thus, a secondary immune response can be elicited, for example, to strengthen a pre-existing immune response that has weakened or been attenuated, or to recreate a previous immune response that has disappeared or is no longer detectable. A secondary or memory immune response can be a humoral (antibody) response or a cellular response. A secondary or memory humoral response occurs upon stimulation of memory B cells generated upon the first presentation of the antigen. A delayed type hypersensitivity (DTH) reaction is a type of cellular secondary or memory immune response and is mediated by CD4 + It is mediated by T cells. Initial exposure to an antigen primes the immune system and further exposure results in DTH. After appropriate immunization, anti-A33 antibodies can be prepared from the serum of the subject animal. If desired, the antibody molecules directed against the A33 protein can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques (e.g., polypeptide A chromatography) to obtain the IgG fraction.

[0056] Monoclonal antibodiesIn some embodiments of the present technology, the antibody is an anti-A33 monoclonal antibody. For example, in some embodiments, the anti-A33 monoclonal antibody is a human or mouse anti-A33 monoclonal antibody. For the preparation of monoclonal antibodies directed against A33 protein, or derivatives, fragments, analogs or homologs thereof, any technique that provides for the production of antibody molecules by continuous cell line culture can be utilized. Such techniques include, but are not limited to, the hybridoma technique (see, e.g., Kohler & Milstein, 1975. Nature 256: 495-497); the trioma technique; the human B cell hybridoma technique (see, e.g., Kozbor, et al., 1983. Immunol. Today 4: 72); and the EBV hybridoma technique for producing human monoclonal antibodies (see, e.g., Cole, et al., 1985. In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies can be used in the practice of the technology, and can be generated by using human hybridomas (see, e.g., Cote, et al., 1983. Proc. Natl. Acad. Sci. USA 80: 2026-2030) or by in vitro transformation of human B cells with Epstein-Barr virus (see, e.g., Cole, et al., 1985. In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). For example, a population of nucleic acids encoding regions of an antibody can be isolated. Sequences encoding portions of the antibody can be amplified from the population using PCR using primers derived from sequences encoding conserved regions of the antibody, and then DNA encoding the antibody or a fragment thereof (e.g., variable domain) is reconstructed from the amplified sequences.Such amplified sequences can also be fused to other proteins (e.g., bacteriophage coat or bacterial cell surface proteins) and the fusion polypeptides expressed and displayed on phages or bacteria. The amplified sequences can then be expressed and selected or isolated based on the affinity of the expressed antibodies or fragments thereof for, for example, antigens or epitopes present on the A33 protein. Alternatively, hybridomas expressing anti-A33 monoclonal antibodies can be prepared using routine methods by immunizing a subject animal and then isolating hybridomas from the subject animal's spleen. See, e.g., Milstein et al. (Galfre and Milstein, Methods Enzymol (1981) 73: 3-46). Screening of the hybridomas using standard methods will yield monoclonal antibodies of various specificities (i.e., against different epitopes). Selected monoclonal antibodies with the desired properties (e.g., A33 binding) expressed by hybridomas can be used and conjugated with molecules (e.g., polyethylene glycol (PEG)) to modify their properties, or the cDNAs encoding them can be isolated, sequenced, and further manipulated in a variety of ways. Synthetic dendrimer trees can be added to reactive amino acid side chains (e.g., lysine) to enhance the immunogenic properties of the A33 protein. Furthermore, CPG dinucleotide technology can be used to enhance the immunogenic properties of the A33 protein. Other manipulations include substitution or deletion of specific amino acid residues responsible for antibody instability during storage or after administration to a subject animal, and affinity maturation techniques to improve the affinity of the antibody for the A33 protein.

[0057] Hybridoma TechnologyIn some embodiments, the antibody of the present technology is an anti-A33 monoclonal antibody produced by a hybridoma. The hybridoma is obtained from a transgenic non-human animal (e.g., a transgenic mouse) having a genome that includes a human heavy chain transgene and a light chain transgene, and includes a B cell fused to an immortalized cell. Hybridoma technology includes methods known in the art and is taught in: 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 of producing hybridomas and monoclonal antibodies are well known to those skilled in the art.

[0058] Phage Display TechnologyAs noted above, the antibodies of the present technology can be generated by the application of recombinant DNA and phage display technology. For example, anti-A33 antibodies can be prepared using a variety of phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles carrying polynucleotide sequences encoding the domains. Phages with the desired binding properties are selected from repertoire or combinatorial antibody libraries (e.g., human or murine) by direct selection with antigen, typically antigen bound or captured to a solid surface or bead. The phages used in these methods are typically filamentous phages (including fd and M13) with Fab, Fv or disulfide-stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. In addition, these methods have been adapted for the construction of Fab expression libraries (see, e.g., Huse, et al., Science 246: 1275-1281, 1989), allowing the rapid and efficient identification of monoclonal Fab fragments with the desired specificity for an A33 polypeptide (e.g., a polypeptide or a derivative, fragment, analog or homolog thereof).Examples of other phage display methods that can be used to generate antibodies of the present technology include those disclosed in: 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;WO90 / 02809;WO91 / 10737;WO92 / 01047;WO92 / 18619;WO93 / 11236;WO95 / 15982;WO95 / 20401;WO96 / 06213;WO92 / 01047(Medical Research Council et al. al.); WO97 / 08320 (Morphosys); WO92 / 01047 (CAT / MRC); WO91 / 17271 (Affymax); and U.S. Patents 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. A method useful for displaying polypeptides on the surface of bacteriophage particles by attaching the polypeptides through disulfide bonds is described by Lohning in US Pat. No. 6,753,136.As described in the above references, after phage selection, the antibody coding region of the phage can be isolated and used to generate complete antibodies (including human antibodies) or any other antigen-binding fragments desired, and then expressed in any host cell desired, including mammalian cells, insect cells, plant cells, yeast, and bacteria. For example, techniques for recombinantly producing Fab, Fab', and F(ab')2 fragments can also be utilized using methods known in the art, such as those disclosed in WO92 / 22324; Mullinax et al., BioTechniques 12: 864-869, 1992; and Sawai et al., AJRI 34: 26-34, 1995; and Better et al., Science 240: 1041-1043, 1988. Generally, hybrid antibodies or hybrid antibody fragments cloned into a display vector can be screened against the appropriate antigen to identify variants that maintain good binding activity, since the antibody or antibody fragment is presented on the surface of the phage or phagemid particle (see, e.g., Barbas III et al., Phage Display, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001). However, other vector formats could also be used in this process, such as cloning antibody fragment libraries into lytic phage vectors (modified T7 or lambda Zap systems) for selection and / or screening.

[0059] Expression of recombinant anti-A33 antibodyAs noted above, the antibody of the present technology can be produced by applying recombinant DNA technology. The recombinant polynucleotide construct encoding the anti-A33 antibody of the present technology typically includes an expression control sequence (including a naturally associated or heterologous promoter region) operably linked to the coding sequence of the anti-A33 antibody chain. Thus, another feature of the present technology includes a vector containing one or more nucleic acid sequences encoding the anti-A33 antibody of the present technology. For recombinant expression of one or more of the polypeptides of the present technology, a nucleic acid containing all or a portion of the nucleotide sequence encoding the anti-A33 antibody is inserted into an appropriate cloning or expression vector (i.e., a vector containing the elements necessary for the transcription and translation of the inserted polypeptide coding sequence) by recombinant DNA technology well known in the art and described in detail below. Methods for generating a diverse collection of vectors are described in U.S. Patents 6,291,160 and 6,680,192 (Lerner et al.).

[0060] In general, expression vectors useful in recombinant DNA technology are often in the form of plasmids. In this disclosure, "plasmid" and "vector" are used interchangeably since plasmids are the most commonly used form of vector. However, the technology is intended to include other forms of expression vectors that are not technically plasmids, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions. Such viral vectors allow for infection of a subject animal and expression of the construct in the subject animal. In some embodiments, the expression control sequence is a eukaryotic promoter system in a vector capable of transforming or transfecting a eukaryotic host cell. Once the vector is incorporated into a suitable host, the host is maintained under suitable conditions for high level expression of the nucleotide sequence encoding the anti-A33 antibody, collection and purification of the anti-A33 antibody (e.g., cross-reactive anti-A33 antibody). See generally US 2002 / 0199213. These expression vectors are typically capable of replicating in the host organism as episomes or as an integral part of the host chromosomal DNA. Usually, expression vectors contain a selection marker (e.g., ampicillin resistance or hygromycin resistance) to permit detection of cells transformed with the desired DNA sequences. Vectors may also encode a signal peptide (pectate lyase) which is useful for directing the secretion of extracellular antibody fragments. See U.S. Patent No. 5,576,195.

[0061] The recombinant expression vector of the present technology comprises a nucleic acid encoding a protein having A33-binding properties in a suitable form for expression of the nucleic acid in a host cell. By suitable form, it is meant that the recombinant expression vector comprises one or more regulatory sequences selected based on the host cell to be used for expression, i.e., operably linked to the nucleic acid sequence to be expressed. "Operably linked" in a recombinant expression vector means that the nucleotide sequence of interest is linked to a regulatory sequence in a manner that allows expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell). 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 IN ENZYMOLOGY 185, Academic Press, San Diego, Calif., 1990. Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Those skilled in the art will appreciate that the design of an expression vector may depend on factors such as the choice of the host cell to be transformed, the level of expression of the polypeptide desired, and the like. Exemplary regulatory sequences useful as promoters of recombinant polypeptide expression (e.g., anti-A33 antibodies) include, but are not limited to, promoters of 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, inter alia, promoters of alcohol dehydrogenase, isocytochrome C, and enzymes required for maltose and galactose utilization. In one embodiment, a polynucleotide encoding the anti-A33 antibody of the present technology is Ara BThe expression vector of the present technology is operably linked to a promoter and can be expressed in a host cell. See US Patent No. 5,028,530. The expression vector of the present technology can be introduced into a host cell, thereby producing the polypeptide or peptide (such as an anti-A33 antibody) encoded by the nucleic acid described herein, including a fusion polypeptide.

[0062] Another feature of the present technology relates to anti-A33 antibody-expressing host cells, the cells comprising a nucleic acid encoding one or more anti-A33 antibodies. The recombinant expression vectors of the present technology can be designed for expression of anti-A33 antibodies in prokaryotic or eukaryotic cells. For example, anti-A33 antibodies can be expressed in bacterial cells (e.g., E. coli ( Escherichia coli ), insect cells (using baculovirus expression vectors), fungal cells (e.g., yeast), yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif., 1990. Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example, using T7 promoter regulatory sequences and T7 polymerase. Methods useful for preparing and screening polypeptides with predetermined properties (e.g., anti-A33 antibodies) by expression of stochastically generated polynucleotides have been previously 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, and 6,569,641. Expression of polypeptides in prokaryotic cells is most frequently carried out in E. coli, using vectors containing constitutive or inducible promoters directing the expression of fusion or non-fusion polypeptides. Fusion vectors add a number of amino acids to a polypeptide encoded therein, usually to the amino terminus of the recombinant polypeptide. Such fusion vectors typically serve three purposes: (i) to enhance expression of the recombinant polypeptide; (ii) to enhance the solubility of the recombinant polypeptide; and (iii) to aid in the purification of the recombinant polypeptide by acting as a ligand for affinity purification. In fusion expression vectors, a proteolytic cleavage site is often introduced at the interface between the fusion moiety and the recombinant polypeptide to allow separation of the recombinant polypeptide from the fusion moiety after purification of the fusion polypeptide. Such enzymes and their cognate recognition sequences include factor Xa, thrombin and enterokinase. Exemplary 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), in which glutathione S-transferase (GST), maltose E binding polypeptide, or polypeptide A, respectively, are fused to the target recombinant polypeptide.

[0063] 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: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif., 1990, 60-89). Methods for targeted assembly of separate active peptides or protein domains to obtain multifunctional polypeptides via polypeptide fusion are described in U.S. Patents 6,294,353 and 6,692,935 (Pack et al.). One approach to maximizing recombinant polypeptide (e.g., anti-A33 antibody) expression in E. coli is to express the polypeptide in a host bacterium in which the recombinant polypeptide's proteolytic cleavage capacity is impaired. See, for example, Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128. Another approach is to modify the nucleic acid sequence of the nucleic acid to be inserted into the expression vector so that the individual codons of each amino acid are preferentially utilized in the expression host (e.g., E. coli) (see, for example, Wada, et al., 1992. Nucl. Acids Res. 20: 2111-2118). Such modification of the nucleic acid sequence of the present technology can be carried out by standard DNA synthesis techniques. In another embodiment, the anti-A33 antibody expression vector is a yeast expression vector. Saccharomyces cerevisiaeExamples of vectors for expression in Escherichia coli 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, anti-A33 antibodies can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expressing a polypeptide (e.g., an anti-A33 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).

[0064] In yet another embodiment, the nucleic acid encoding the anti-A33 antibody of the present technology 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 polyoma, adenovirus 2, cytomegalovirus and simian virus 40. For other suitable expression systems, both prokaryotic and eukaryotic, that are useful for expressing the anti-A33 antibodies of the present technology, see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid in a particular cell type (e.g., tissue-specific regulatory elements). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al., Genes Dev. 1: 268-277, 1987), lymphoid-specific promoters (Calame and Eaton, Adv. Immunol. 43: 235-275, 1988), promoters of T cell receptors (Winoto and Baltimore, EMBO J. 8: 729-733, 1989) and immunoglobulins (Banerji, et al., 1983. Cell 33: 729-740;Queen and Baltimore, Cell 33: 741-748, 1983), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, Proc. Natl. Acad. Sci. USA 86: 5473-5477, 1989), pancreatic specific promoters (Edlund, et al., 1985. Science 230: 912-916), and mammary gland specific promoters (e.g., whey promoter; U.S. Pat. No. 4,873,316 and European Patent Publication No. 264,166). Developmentally regulated promoters are also encompassed, such as the murine Hox promoters (Kessel and Gruss, Science 249: 374-379, 1990) and the α-fetoprotein promoter (Campes and Tilghman, Genes Dev. 3: 537-546, 1989).

[0065] Another feature of the present invention relates to a host cell into which a recombinant expression vector of the present technology has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It will be understood that such terms refer not only to the individual subject cell, but also to the progeny or potential progeny of such a cell. In reality, such progeny may not be identical to the parent cell, since certain modifications due to mutation or environmental influences may occur in successive generations, but they are still included within the scope of the terms used herein. The host cell can be any prokaryotic or eukaryotic cell. For example, anti-A33 antibodies can be expressed in bacterial cells (e.g., E. coli), insect cells, yeast, or mammalian cells. Mammalian cells are suitable hosts for expressing nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes To Clones (VCH Publishers, NY, 1987). A number of suitable host cell lines capable of secreting intact heterologous proteins have been developed in the art. These include 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. Expression vectors for these cells can include expression control sequences, such as origins of replication, promoters, enhancers, necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription termination sequences (Queen et al., Immunol. Rev. 89: 49, 1986). Exemplary expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papilloma virus, etc. (Co et al., J Immunol. 148: 1149, 1992). Other suitable host cells are known to those of skill in the art.

[0066] Vector DNA can be introduced into prokaryotic or fungal cells by conventional transformation or transfection techniques. As used herein, the terms "transformation" or "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into host cells, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, biolistics, or viral transfection. Other methods used to transform mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection (see generally Sambrook, et al., Molecular Cloning). Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and other laboratory manuals. The vectors containing the DNA segments of interest are transferred into the host cell by well-known methods, depending on the type of cellular host. It is known that in the case of stable transfection of mammalian cells, only a small proportion of cells can integrate foreign DNA into their genome, depending on the expression vector and transfection technique used. In order to identify and select these integrants, a gene encoding a selectable marker (e.g., antibiotic resistance) is generally introduced into the host cell together with the gene of interest. Various selectable markers include those that confer resistance to drugs (e.g., G418, hygromycin, and methotrexate). The nucleic acid encoding the selectable marker can be introduced into the host cell in the same vector as the vector encoding the anti-A33 antibody, or can be introduced in a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene survive, while other cells die).

[0067] A host cell (e.g., a prokaryotic or eukaryotic host cell culture) containing the anti-A33 antibody of the present technology can be used to produce (i.e., express) a recombinant anti-A33 antibody. In one embodiment, the method includes culturing a host cell (into which a recombinant expression vector encoding the anti-A33 antibody has been introduced) in an appropriate medium so that the anti-A33 antibody is produced. In another embodiment, the method further includes isolating the anti-A33 antibody from the medium or the host cell. Once expressed, the anti-A33 antibody (e.g., an anti-A33 antibody or an anti-A33 antibody-related polypeptide) is purified from the culture medium and the host cell. The anti-A33 antibody can be purified according to standard procedures in the art, including HPLC purification, column chromatography, gel electrophoresis, and the like. In one embodiment, the anti-A33 antibody is produced by the method of Boss et al. (U.S. Pat. No. 4,816,397 (Boss et al.)). Typically, the anti-A33 antibody chain is expressed with a signal sequence, so that it is released into the culture medium. However, if the anti-A33 antibody chains are not naturally secreted by the host cells, they can be released by treatment with mild detergent. Purification of recombinant polypeptides is well known in the art and includes ammonium sulfate precipitation, affinity chromatography purification techniques, column chromatography, ion exchange purification techniques, gel electrophoresis, and the like (see generally, Scopes, Protein Purification (Springer-Verlag, NY, 1982)). A polynucleotide encoding an anti-A33 antibody (e.g., an anti-A33 antibody coding sequence) can be incorporated into a transgene, introduced into the genome of a transgenic animal, and then expressed in the milk of the transgenic animal. See, for example, U.S. Patents Nos. 5,741,957, 5,304,489, and 5,849,992. A suitable transgene includes coding sequences for light and / or heavy chains, which are operably linked to promoters and enhancers derived from mammary gland-specific genes (e.g., casein or β-lactoglobin). For the generation of transgenic animals, the transgene can be microinjected into fertilized oocytes or incorporated into the genome of embryonic stem cells, and the nucleus of such cells can be transferred into enucleated oocytes.

[0068] single chain antibody In one embodiment, the anti-A33 antibody of the present technology is a single-chain anti-A33 antibody.According to the present technology, several techniques can be adapted for the production of single-chain antibodies specific to A33 protein (see, for example, US Patent 4,946,778).Examples of techniques that can be used for the production of single-chain Fv and antibodies of the present technology include the techniques described in US Patents 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.

[0069] Chimeric and Humanized Antibodies In some embodiments, the anti-A33 antibody of the present technology is a chimeric anti-A33 antibody. In some embodiments, the anti-A33 antibody of the present technology is a humanized anti-A33 antibody. In some embodiments of the present technology, the donor and acceptor antibodies are monoclonal antibodies derived from different species. For example, the acceptor antibody is a human antibody (to minimize its antigenicity in humans), and in said case, the resulting CDR-grafted antibody is called a "humanized" antibody.Recombinant anti-A33 antibodies (e.g., chimeric and humanized monoclonal antibodies), which contain both human and non-human portions, can be produced using standard recombinant DNA techniques and are within the scope of the present technology. For some uses (including the in vivo use of the anti-A33 antibodies of the present technology in humans as well as the use of these agents in in vitro detection assays), chimeric or humanized anti-A33 antibodies can be used. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art.Such useful methods include, but are not limited to, those described in, for example, International Application No. PCT / US86 / 02269; U.S. Patent No. 5,225,539; European Patent No. 184187; European Patent No. 171496; European Patent No. 173494; PCT International Publication No. WO86 / 01533; U.S. Patents Nos. 4,816,567 and 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, antibodies can be humanized using a variety of techniques, including CDR grafting (EP 0 239 400; WO 91 / 09967; U.S. Patents Nos. 5,530,101, 5,585,089, 5,859,205, 6,248,516; EP 460167), overlaying or resurfacing (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 shuffling (U.S. Patent No. 5,565,332).In one embodiment, the cDNA encoding the murine anti-A33 monoclonal antibody is digested with restriction enzymes selected to specifically remove sequences encoding the Fc constant region and replaced with the equivalent portion of cDNA encoding a human Fc (see, for example, 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. U.S. 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 Nos. 6,300,064, 6,696,248, 6,706,484, and 6,828,422. In one embodiment, the present technology provides for the construction of humanized anti-A33 antibodies, which are less likely to elicit a human anti-mouse antibody (referred to herein as "HAMA") response, but still have efficient antibody effector functions. As used herein, the terms "human" and "humanized" in reference to antibodies refer to any antibody that is expected to elicit a therapeutically acceptable weak immunogenic response in a human subject. In one embodiment, the present technology provides humanized anti-A33 antibodies, heavy and light chain immunoglobulins.

[0070] CDR antibody In some embodiments, the anti-A33 antibody of the present technology is an anti-A33 CDR antibody. In general, the donor and acceptor antibodies used to generate the anti-A33 CDR antibody are monoclonal antibodies derived from different species. Typically, the acceptor antibody is a human antibody (to minimize its antigenicity in humans), and in such cases, the resulting CDR-grafted antibody is referred to as a "humanized" antibody. The graft is performed by removing only one V of the acceptor antibody. H or V L It is the only CDR (or even part of the only CDR) inside, or V H and V L There may be multiple CDRs (or portions thereof) within one or both of the variable domains of the acceptor antibody. Often, all three CDRs of all variable domains of the acceptor antibody will be replaced with the corresponding donor CDRs, but only as many as are required to allow proper binding of the resulting CDR-grafted antibody to the A33 protein. Methods for making CDR-grafted and humanized antibodies are taught by Queen et al. US Pat. No. 5,585,089; US Pat. No. 5,693,761; US ​​Pat. No. 5,693,762; and US Pat. No. 5,225,539 (Winter); and EP 0682040. H and V L Useful methods for preparing polypeptides are taught by the following: Winter et al., U.S. Patents 4,816,397, 6,291,158, 6,291,159, 6,291,161, 6,545,142, EP0368684, EP0451216, and EP0120694.

[0071] After selecting suitable framework region candidates from the same family and / or from the same family members, one or both of the heavy and light chain variable regions are generated by grafting the CDRs from the original species into the hybrid framework regions. The assembly of hybrid antibodies or hybrid antibody fragments having hybrid variable chains with either of the above characteristics can be achieved using conventional methods known in the art. For example, DNA sequences encoding the hybrid variable domains described herein (i.e., frameworks based on the target species and CDRs from the original species) can be generated by oligonucleotide synthesis and / or PCR. Nucleic acids encoding the CDR regions can also be isolated from the original species antibody using appropriate restriction enzymes and further ligated to the target species framework by ligation with appropriate ligation enzymes. Alternatively, the framework regions of the variable chains of the original species antibody can be altered by site-directed mutagenesis. Since hybrids are constructed by selecting from multiple candidates corresponding to each framework region, there are many sequence combinations, which can be easily constructed according to the principles described herein. Thus, hybrid libraries can be assembled that contain members with various combinations of the individual framework regions. Such libraries can be electronic databases of sequence collections or physical collections of hybrids.

[0072] This process typically does not change the FRs of the acceptor antibody that flank the grafted CDRs. However, one skilled in the art can sometimes improve the antigen binding affinity of the resulting anti-A33 CDR-grafted antibody by replacing certain residues of a given FR to make it more similar to the corresponding FR of the donor antibody. Suitable substitution locations include amino acid residues adjacent to the CDRs or that can interact with the CDRs (see, e.g., US 5,585,089, especially columns 12-16). Alternatively, one skilled in the art can start with the donor FRs and modify them to be more similar to the acceptor FRs or human consensus FRs. Techniques for making these modifications are known in the art. In particular, if the resulting FRs match the human consensus FRs for the positions or are at least 90% or more identical to such consensus FRs, such modifications may not significantly enhance the antigenicity of the resulting modified anti-A33 CDR-grafted antibody compared to the same antibody with fully human FRs.

[0073] Bispecific antibodies (BsAb) : A bispecific antibody is an antibody that can simultaneously bind two targets with distinct structures, such as two different target antigens, two different epitopes of the same target antigen, or a hapten and a target antigen or an epitope of a target antigen. BsAbs can be generated, for example, by combining heavy and / or light chains that recognize different episomes of the same or different antigens. In some embodiments, the molecular function of a bispecific binding agent is that it binds to one antigen (or epitope) with one of its two binding arms (one VH / VL pair) and to a different antigen (or epitope) with its second arm (a different VH / VL pair). According to this definition, a bispecific binding agent has two distinct antigen binding arms (both in specificity and CDR sequence) and is monovalent for each antigen it binds. The bispecific antibodies (BsAb) and bispecific antibody fragments (BsFab) of the present technology have at least one arm that specifically binds, for example, A33, and at least one other arm that specifically binds 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. Additionally or alternatively, in certain 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, TCR gamma / delta, NKp46, and KIR. In certain embodiments, the BsAb has the ability to bind to tumor cells that express the A33 antigen on the cell surface. In some embodiments, the BsAb is engineered to promote tumor cell killing by directing (recruiting) cytotoxic T cells to the tumor site. Other exemplary BsAbs include those having a first antigen binding site specific for A33 and a second antigen binding site specific for a small molecule hapten such as DTP A, IMP288, DOTA, DOTA-Bn, DOTA-desferrioxamine, other DOTA-chelates described herein, biotin, fluorescein, or those disclosed in Goodwin et al. (Goodwin, D A. et al, 1994, Cancer Res. 54(22):5937-5946).

[0074] A variety of bispecific fusion proteins can be made using molecular engineering. For example, BsAbs have been constructed that utilize the complete immunoglobulin framework (e.g., IgG), single chain variable fragments (scFvs), or a combination of the above. In some embodiments, the bispecific fusion protein is bivalent, e.g., comprises an scFv with only one binding site for one antigen and a Fab with only one binding site for a second antigen. In other embodiments, the bispecific fusion protein is tetravalent, e.g., comprises an immunoglobulin (e.g., IgG) with two binding sites for one antigen and two identical scFvs for a second antigen. BsAbs, which contain two scFv units in tandem, have been shown to be a clinically successful bispecific antibody format. In some embodiments, BsAbs contain two single chain variable fragments (scFvs) in tandem, designed such that an scFv that binds a tumor antigen (e.g., A33) is linked to an scFv that engages T cells (e.g., by binding to CD3). In this embodiment, T cells can be recruited to the tumor site and mediate cytotoxic killing of tumor cells. See, e.g., Dreier et al., J. Immunol., 170:4397-4402, 2003; Bargou et al., Science 321:974-977, 2008. Recent methods for generating BsAbs include engineered recombinant monoclonal antibodies that have additional cysteine ​​residues and therefore exhibit stronger cross-linking than more conventional immunoglobulin isotypes. See, e.g., FitzGerald et al., Protein Eng. 10(10):1221-1225, 1997. Another approach is to engineer recombinant fusion proteins in which two or more different single chain antibodies or antibody fragments with the desired dual specificities are linked. See, e.g., Coloma et al., Nature Biotech. 15:159-163, 1997. Using molecular engineering, a variety of bispecific fusion proteins can be generated.

[0075] Bispecific proteins linking two or more different single chain antibodies or antibody fragments are produced in a similar manner. Using recombinant methods, a variety of fusion proteins can be produced. In certain embodiments, the BsAb of the present technology comprises an immunoglobulin, which comprises a heavy and light chain and an scFv. In certain embodiments, the scFv is linked to the C-terminus of the heavy chain of any A33 immunoglobulin disclosed herein. In certain embodiments, the scFv is linked to the C-terminus of the light chain of any A33 immunoglobulin disclosed herein. In various embodiments, the scFv is linked to the heavy or light chain via a linker sequence. The appropriate linker sequence required for the in-frame connection of the heavy chain Fd and the scFv can be generated by PCR reaction using the V L and V kappa The DNA fragment encoding the scFv is then ligated into a staging vector containing a DNA sequence encoding the CH1 domain. The resulting scFv-CH1 construct is excised and transformed into the V domain of the A33 antibody. H The resulting vector is used to transfect an appropriate host cell (e.g., a mammalian cell) for expression of the bispecific fusion protein. In some embodiments, 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 than 100 amino acids in length. In some embodiments, the linker is characterized by not attempting to adopt a rigid three-dimensional structure, but rather providing flexibility to the polypeptide (e.g., the first and / or second antigen binding site). In some embodiments, the linker is used in the BsAbs described herein based on the specific properties (e.g., enhanced stability) that it imparts to the BsAb. In some embodiments, the BsAb of the present technology comprises a G4S linker. In certain embodiments, the BsAb of the present technology is (G4S) nand wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more than 15.

[0076] Fc modification In some embodiments, the anti-A33 antibody of the present technology comprises a variant Fc region, which comprises at least one amino acid modification relative to a wild-type Fc region (or parent 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 locations that make direct contact with an Fc receptor based on crystallographic and structural analysis of Fc-Fc receptor interactions (e.g., as disclosed by Sondermann et al., Nature, 406:267-273, 2000). Examples of locations within the Fc region that make direct contact with an Fc receptor (e.g., FcγR) include amino acids 234-239 (hinge region), amino acids 265-269 (B / C loop), amino acids 297-299 (C7E loop), and amino acids 327-332 (F / G) loop. In some embodiments, the anti-A33 antibodies of the present technology have altered affinity for activating and / or inhibitory receptors and have a variant Fc region comprising one or more amino acid modifications, where the one or more amino acid modifications are substitutions of N297 with alanine or K322 with alanine.

[0077] Glycosylation Modifications In some embodiments, the anti-A33 antibodies of the present technology have an Fc region that comprises variant glycosylation compared to the parent Fc region, in some embodiments, the variant glycosylation comprises a lack of fucose, and in some embodiments, the variant glycosylation results from expression in GnT1-deficient CHO cells. In some embodiments, the antibodies of the present technology can have glycosylation sites that are altered relative to a suitable reference antibody that binds to an antigen of interest (e.g., A33) without altering the functionality (e.g., antigen binding activity) of the antibody. As used herein, a "glycosylation site" includes any specific amino acid sequence within an antibody to which an oligosaccharide (i.e., a carbohydrate comprising two or more monosaccharides linked together) can be specifically and covalently attached. Oligosaccharide side chains are typically linked to the backbone of the antibody via N- or O-linkages. N-linked glycosylation refers to the attachment of an oligosaccharide moiety to the side chain of an asparagine residue. O-linked glycosylation refers to the attachment of an oligosaccharide moiety to a hydroxyamino acid (e.g., serine, threonine). For example, Fc-glycoforms (huA33-IgGln) lacking certain oligosaccharides (including fucose) and terminal N-acetylglucosamine can be produced in specialized CHO cells and exhibit enhanced ADCC effector functions. In some embodiments, the carbohydrate content of the immunoglobulin-related constructs disclosed herein is modified by adding or deleting glycosylation sites. Methods for modifying carbohydrate moieties of antibodies are well known in the art and are included within the present technology. See, for example, U.S. Patent No. 6,218,149; EP 0359096B1; U.S. Patent Publication No. US 2002 / 0028486; International Patent Application Publication No. 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 its corresponding portion or component) is modified by deleting one or more endogenous carbohydrate moieties of the antibody. In certain embodiments, the present technology includes deleting a glycosylation site in the Fc region of an antibody by modifying position 297 from asparagine to alanine.

[0078] Engineered glycoforms may be useful for a variety of purposes, including, but not limited to, enhancing or decreasing effector function. Engineered glycoforms can be made by any method known to those of skill in the art, such as by using engineered or variant expression strains, co-expression with one or more enzymes, such as DIN-acetylglucosamine transferase III (GnTIII), expression of molecules containing an Fc region in various organisms or cell lines derived from various organisms, or carbohydrate modification following expression of the Fc region-containing molecule. Methods for producing engineered glycoforms are known in the art and include, but are not limited to, those described in 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 No. 10 / 277,370; U.S. Patent Application No. 10 / 113,929; International Patent Publication Nos. WO 00 / 61739A1, WO 01 / 292246A1, WO 02 / 311140A1, WO 02 / 30954A1;POTILLEGENT TM Technology (Biowa, Inc. Princeton, NJ); GLYCOMAB TM Glycosylation Engineering Technology (GLYCART biotechnology AG, Zurich, Switzerland), each of which is incorporated herein by reference in its entirety. See, e.g., International Patent Application Publication No. WO 00 / 061739; U.S. Patent Application Publication No. 2003 / 0115614; Okazaki et al., 2004, JMB, 336: 1239-49.

[0079] Fusion proteinsIn one embodiment, the anti-A33 antibody of the present technology is a fusion protein. The anti-A33 antibody of the present technology can be used as an antigenic tag when fused to a second protein. Examples of domains that can be fused to a polypeptide include heterologous signal sequences as well as other functional heterologous regions. The fusion does not necessarily have to be direct, but can occur via a linker sequence. Furthermore, the fusion protein of the present technology can also be engineered to improve the characteristics of the anti-A33 antibody. For example, a region of additional amino acids (especially charged amino acids) can be added to the N-terminus of the anti-A33 antibody to improve stability and durability during purification from the host cell or during subsequent handling and storage. Furthermore, a peptide moiety can be added to the anti-A33 antibody to facilitate purification. Such a region can be removed before the final preparation of the anti-A33 antibody. Adding a peptide moiety to facilitate handling of a polypeptide is well known and routine in the art. The anti-A33 antibody of the present technology can be fused to a marker sequence (e.g., a peptide that facilitates purification of the fusion polypeptide). In selected embodiments, the marker amino acid sequence is a hexahistidine peptide, inter alia, such as the tag provided in the pQE vector (QIAGEN, Inc., Chatsworth, Calif), many of which are commercially available. Hexahistidine provides for convenient purification of the fusion protein, as described, for example, in (Gentz ​​et al., Proc. Natl. Acad. Sci. USA 86: 821-824, 1989). Another peptide tag useful for purification, the "HA" tag, corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37: 767, 1984).

[0080] Thus, any of these above fusion proteins can be engineered using the polynucleotides or polypeptides of the present technology.Furthermore, in some embodiments, the fusion proteins described herein exhibit extended half-life in vivo. Fusion proteins with disulfide-bonded dimeric structures (as occurs with IgG) can be more efficient at binding and neutralizing other molecules compared to monomeric secreted proteins or protein fragments alone (Fountoulakis et al., J. Biochem. 270: 3958-3964, 1995). Similarly, EP-AO 464 533 (Canadian Patent Publication No. 2045869) discloses fusion proteins comprising various parts of the constant region of an immunoglobulin molecule together with another human protein or a fragment thereof. In many cases, the Fc part of the fusion protein is beneficial in therapy and diagnosis and can therefore provide, for example, improved pharmacokinetic properties (see EP-A 0232 262). Alternatively, after expression, detection and purification of the fusion protein, it may be desirable to delete or modify the Fc part. For example, the Fc part may interfere with therapy and diagnosis when the fusion protein is used as an antigen for immunization. In drug discovery, human proteins (e.g., hIL-5) have been fused with Fc portions to identify antagonists of hIL-5 for the purpose of high-throughput screening assays (Bennett et al., J. Molecular Recognition 8: 52-58, 1995; Johanson et al., J. Biol. Chem., 270: 9459-9471, 1995).

[0081] Labeled anti-A33 antibodyIn one embodiment, the anti-A33 antibody of the present technology is conjugated with a label moiety (i.e., a detectable group). The particular label or detectable group conjugated to the anti-A33 antibody is not a critical feature of the present technology, so long as it does not significantly interfere with the specific binding of the anti-A33 antibody of the present technology to the A33 protein. The detectable group can be any substance that has a detectable physical or chemical property. Such detectable labels have been well developed in the field of immunoassays and imaging. In general, almost any label useful in such methods can be applied to the present technology. Thus, a label is any construct that can be detected by spectroscopic, photochemical, immunochemical, electrical, optical, or chemical means. Labels useful in the practice of the present technology include magnetic beads (e.g., Dynabeads, TM ), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas Red, rhodamine, etc.), radioactive labels (e.g. 3 H, 14 C. 35 S, 125 I, 121 I, 131 I, 112 In, 99 mTc), other imaging agents such as microbubbles (for ultrasound imaging), 18 F, 11 C. 15 O (for positron emission tomography), 99m T.C., 111 In (for single photon emission computed tomography), enzymes (horseradish peroxidase, alkaline phosphatase, and other enzymes commonly used in ELISA), and colorimetric labels, such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. Patents describing the use of such labels include U.S. Patents 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 for all purposes). Also see: Handbook of Fluorescent Probes and Research Chemicals (6th Ed., Molecular Probes, Inc., Eugene OR.). Labels can be attached directly or indirectly to the desired assay components according to methods known in the art. As noted above, a wide variety of labels can be used, and the choice of label will depend on several factors, such as the sensitivity required, ease of conjugation with the compound, stability requirements, available equipment, and disposal regulations. Non-radioactive labels are often added by indirect means. Typically, a ligand molecule (e.g., biotin) is covalently attached to the molecule. The ligand is then bound to an antiligand (e.g., streptavidin) molecule, which is either inherently detectable or covalently attached to a signal system (e.g., a detectable enzyme, a fluorescent compound, or a chemiluminescent compound). A large number of ligands and antiligands can be used. Where a ligand has a natural antiligand (e.g., biotin, thyroxine, and cortisol), the ligand can be used in conjunction with a labeled, naturally occurring antiligand. Alternatively, any haptenic or antigenic compound can be used in combination with an antibody (e.g., anti-A33 antibody).

[0082] Molecules can also be directly conjugated to signal generating compounds (e.g., by conjugation to enzymes or fluorophores). Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidoreductases, particularly peroxidases. Fluorescent compounds useful as labeling moieties include, but are not limited to, fluorescein compounds and their derivatives, rhodamine and its derivatives, dansyl, umbelliferone, and the like. Chemiluminescent compounds useful as labeling moieties include, but are not limited to, for example, luciferin and 2,3-dihydrophthalazinediones, such as luminol. See U.S. Pat. No. 4,391,904 for a review of the various labeling or signal generating systems that can be used. Means for detecting labels are well known to those skilled in the art. Thus, for example, if the label is a radioactive label, detection means include a scintillation counting device, as in autoradiography, or photographic film. If the label is a fluorescent label, the label can be detected by exciting the fluorescent dye with the appropriate wavelength of light and detecting the resulting fluorescence. Fluorescence can be detected visually, by photographic film, by electronic detection devices (such as charge-coupled devices (CCDs) or photomultiplier tubes). Similarly, enzyme labels can be detected by providing the enzyme with the appropriate substrate and detecting the resulting reaction product. Finally, simple colorimetric labels can be easily detected by observing the color associated with the label. Thus, in various dipstick assays, conjugated gold often appears pink, while various conjugated beads appear the color of the bead. Some assay formats do not require the use of labeled components. For example, an agglutination assay can be used to detect the presence of a target antibody (e.g., anti-A33 antibody). In this case, antigen-coated particles are agglutinated by a sample containing the target antibody. In this format, none of the components need to be labeled, and the presence of the labeled antibody is detected by simple visual inspection.

[0083] B. Identification and Characterization of Anti-A33 Antibodies of the Present Technology Methods for identifying and screening anti-A33 antibodies of the present technologyMethods useful for identifying and screening antibodies against A33 polypeptides for those possessing the desired specificity for the A33 protein include any immunologically mediated technique known in the art. Immune response components can be detected in vitro by a variety of methods well known to those of skill 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 cells can be incubated with antigen and antigen-presenting cells and cytokine synthesis and secretion 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 antigen and the presentation of the antigen on 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 agents that crosslink their Fc-epsilon 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).

[0084] Similarly, the products of an immune response in a model organism (e.g., mouse) or human subject can also be detected by a variety of methods well known to those of skill in the art. For example, (1) antibody production in response to vaccination can be readily detected by standard methods currently used in clinical laboratories (e.g., ELISA); (2) migration of immune cells to sites of inflammation can be detected by scratching the surface of the skin and placing it in a sterile container to capture cells migrating through the scratch site (Peters et al., Blood, 72: 1310-5, 1988); (3) proliferation of peripheral blood mononuclear cells (PBMCs) in response to mitogens or mixed lymphocyte reactions can be detected by ELISA (Peters et al., Blood, 72: 1310-5, 1988); (4) proliferation of peripheral blood mononuclear cells (PBMCs) in response to mitogens or mixed lymphocyte reactions can be detected by ELISA (Peters et al., Blood, 72: 1310-5, 1988); (5) proliferation of peripheral blood mononuclear cells (PBMCs) in response to mitogens or mixed lymphocyte reactions can be detected by ELISA (Peters et al., Blood, 72: 1310-5, 1988); (6) proliferation of peripheral blood mononuclear cells (PBMCs) in response to mitogens or mixed lymphocyte reactions can be detected by ELISA (Peters et al., Blood, 72: 1310-5, 1988); (7) proliferation of peripheral blood mononuclear cells (PBMCs) in response to mitogens or mixed lymphocyte reactions can be detected by ELISA (Peters et al., Blood, 72: 1310-5, 1988); (8) proliferation of peripheral blood mononuclear cells (PBMCs) in response to mitogens or mixed lymphocyte reactions can be detected by ELISA (Peters et al., Blood, 72: 13 3H-thymidine; (4) the phagocytic ability of granulocytes, macrophages and other phagocytes of PBMCs can be measured by placing PBMCs in wells with labeled particles (Peters et al., Blood, 72: 1310-5, 1988); and (5) differentiation of immune system cells can be determined by labeling PBMCs with antibodies against CD molecules (e.g., CD4 and CD8) and measuring the proportion of PBMCs expressing these markers.

[0085] In one embodiment, the anti-A33 antibodies of the present technology are selected using the display of A33 peptides on the surface of replicable genetic packages.See, for example, U.S. Patents 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 585287; EP 605522; EP 616640; EP 1024191; EP 589877; EP 774511; EP 844306.Useful methods have been described for generating / selecting filamentous bacteriophage particles that contain phagemid genomes that encode binding molecules with desired specificities. See, for example, EP774511; US5871907; US5969108; US6225447; US6291650; US6492160. In some embodiments, the anti-A33 antibodies of the present technology are selected using display of the A33 peptide on the surface of a yeast host cell. A method useful for isolating scFv polypeptides by yeast surface display is described by Kieke et al. (Kieke et al., Protein Eng. 1997 Nov; 10(11): 1303-10). In some embodiments, the anti-A33 antibodies of the present technology are selected using ribosome display. A method useful for identifying ligands in peptide libraries using ribosome display is described by Mattheakis et al. (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). In certain embodiments, the anti-A33 antibodies of the present technology are selected using tRNA display of the A33 peptide. A method useful for in vitro selection of ligands using tRNA display is described by Merryman et al. (Merryman et al., Chem. Biol., 9: 741-46, 2002).

[0086] In one embodiment, the anti-A33 antibody of the present technology is selected using RNA display.A method useful for selecting peptides and proteins using RNA display libraries is described in the following documents (Roberts et al. Proc. Natl. Acad. Sci. USA, 94: 12297-302, 1997; and Nemoto et al., FEBS Lett., 414: 405-8, 1997).A method useful for selecting peptides and proteins using non-natural RNA display libraries is described by Frankel et al. (Frankel et al., Curr. Opin. Struct. Biol., 13: 506-12, 2003). In some embodiments, the anti-A33 antibody of the present technology is expressed in the periplasm of Gram-negative bacteria and mixed with labeled A33 protein (see WO02 / 34886). Clones expressing recombinant polypeptides with affinity to A33 protein will increase the concentration of labeled A33 protein that binds to the anti-A33 antibody, allowing the cells to be isolated from the remaining cells 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. After selection of the desired anti-A33 antibody, the antibody can be produced in large volume by any technique known to those skilled in the art, such as prokaryotic or eukaryotic expression. Anti-A33 antibodies, such as but not limited to anti-A33 hybrid antibodies or fragments, can be produced by using conventional techniques to construct an expression vector encoding an antibody heavy chain, in which the CDRs and, if necessary, a minimal portion of the variable region framework (which is necessary to maintain the antibody binding specificity of the original species and is engineered according to the techniques described herein) are derived from the original species antibody, and the remaining portion of the antibody is derived from the target species immunoglobulin, which can be engineered as described herein, thereby generating a vector for hybrid antibody heavy chain expression.

[0087] Measurement of A33 bindingIn some embodiments, A33 binding assay refers to an assay format in which A33 protein and anti-A33 antibody are mixed under suitable conditions for binding between the protein and the antibody, and the amount of binding between the protein and the antibody is evaluated. The amount of binding is compared to a suitable control. The control can be the amount of binding in the absence of A33 protein, the amount of binding in the presence of a non-specific immunoglobulin construct, or both. The amount of binding can be evaluated 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 direct measurement of A33 protein binding with anti-A33 antibody include, 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 assay, ELISA, FRET, immunoprecipitation, SPR, NMR (2D-NMR), mass spectrometry, etc. If the specific binding of a candidate anti-A33 antibody is at least 1% greater than the binding observed in the absence of the candidate anti-A33 antibody, the candidate anti-A33 antibody is useful as an anti-A33 antibody of the present technology. Measurement of A33 neutralization As used herein, "A33 neutralization" refers to the reduction of A33 protein activity and / or expression by binding of an anti-A33 antibody. The ability of the anti-A33 antibodies of the present technology to neutralize A33M activity / expression can be assessed in vitro or in vivo using methods known in the art.

[0088] Use of the anti-A33 antibody of the present technology OverviewThe anti-A33 antibodies of the present technology are useful in methods known in the art for localizing and / or quantifying A33 protein (e.g., for use in measuring the levels of A33 protein in appropriate physiological samples, for use in diagnostic methods, for use in imaging the polypeptide, etc.). The antibodies of the present technology are useful for isolating A33 protein by standard techniques (e.g., affinity chromatography or immunoprecipitation). The anti-A33 antibodies of the present technology facilitate the purification of native immunoreactive A33 protein from biological samples (e.g., mammalian serum or cells), as well as recombinantly produced immunoreactive A33 protein expressed in a host system. Furthermore, the anti-A33 antibodies can be used to detect immunoreactive A33 protein (e.g., in plasma, cell lysates, or cell suspensions) to assess the abundance and expression pattern of the immunoreactive polypeptide. The anti-A33 antibodies of the present technology can be used diagnostically to monitor tissue immunoreactive A33 protein levels as part of a clinical testing procedure, for example, to determine the effectiveness of a given therapeutic regimen. As noted above, detection can be facilitated by conjugating (ie, physically linking) the anti-A33 antibodies of the subject technology to a detectable substance.

[0089] Detection of A33 protein An exemplary method for detecting the presence or absence of immunoreactive A33 protein in a biological sample includes obtaining a biological sample from a test animal and contacting the biological sample with an anti-A33 antibody of the present technology capable of detecting immunoreactive A33 protein to detect immunoreactive A33 protein in the biological sample. Detection may be accomplished by means of a detectable label attached to the antibody. The term "labeled" with respect to an anti-A33 antibody is intended to encompass direct labeling of the antibody by conjugating (i.e., physically linking) a detectable substance to the antibody, as well as indirect labeling of the antibody by reactivity with another compound (e.g., a secondary antibody) that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody, and end-labeling of a DNA probe with biotin so that the DNA probe can be detected with fluorescently labeled streptavidin. In some embodiments, the anti-A33 antibodies disclosed herein are conjugated to one or more detectable labels. For such uses, the anti-A33 antibodies are detectably labeled by covalent or non-covalent attachment of a chromogenic, enzymatic, radioisotopic, isotopic, fluorescent, toxic, chemiluminescent agent, nuclear magnetic resonance contrast agent or other label. Examples of suitable chromogenic labels include diaminobenzidine and 4-hydroxyazo-benzene-2-carboxylic acid. Examples of suitable enzyme labels include malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase.

[0090] Examples of suitable radioisotope labels 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 and others are included. 111 In is an exemplary isotope when in vivo imaging is used because: 125 I or 131This is because it avoids the problem of dehalogenation of I-labeled A33-binding antibodies by the liver. In addition, this isotope has 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, 1-(P-isothiocyanatobenzyl)-DPTA conjugated to monoclonal antibodies 111 In shows little uptake in non-tumor tissues (especially the liver), enhancing the specificity of tumor localization (Esteban et al., J. Nucl. Med. 28:861-870, 1987). Examples of suitable non-radioactive isotope labels include 157 Gd, 55 Mn, 162 Dy, 52 Tr, and 56 Contains Fe. Examples of suitable fluorescent labels include: 152 These include Eu labels, fluorescein labels, isothiocyanate labels, rhodamine labels, phycoerythrin labels, phycocyanin labels, allophycocyanin labels, green fluorescent protein (GFP) labels, o-phthaldehyde labels, and fluorescamine labels. Suitable toxin labels include diphtheria toxin, ricin, and cholera toxin. Examples of chemiluminescent labels include luminol labels, isoluminol labels, aromatic acridinium ester labels, imidazole labels, acridinium salt labels, oxalate ester labels, luciferin labels, luciferase labels, and aequorin labels. Examples of nuclear magnetic resonance imaging agents include heavy metal nuclei such as Gd, Mn, and iron. Using the detection method of the present technology, immunoreactive A33 protein can be detected in biological samples in vitro as well as in vivo. In vitro detection techniques for immunoreactive A33 protein include enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation, radioimmunoassay, and immunofluorescence. Furthermore, in vivo detection techniques for immunoreactive A33 protein include introducing labeled anti-A33 antibody into a subject. For example, anti-A33 antibody can be labeled with a radioactive marker, and its presence and location in a subject can be detected by standard techniques. In some embodiments, the biological sample contains A33 protein molecules from a test subject.

[0091] Immunoassays and imaging : The anti-A33 antibody of the present technology can be used to assay the immunoreactive A33 protein level of biological samples (e.g., human plasma) using antibody-based techniques. For example, protein expression in tissues can be examined 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 useful for detecting protein gene expression include immunoassays, such as enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs). Suitable antibody assay labels are known in the art and include enzyme labels (e.g., glucose oxidase), and radioisotopes or other radiopharmaceuticals (e.g., iodine ( 125 I, 121 I, 131 I), Carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), Indium ( 112 In) and technetium ( 99 mTc), as well as fluorescent labels such as fluorescein, rhodamine, and green fluorescent protein (GFP) along with biotin. In addition to assaying immunoreactive A33 protein levels in biological samples, the anti-A33 antibody of the present technology can be used for in vivo imaging of A33. Antibodies useful in this method include antibodies that can be detected by X-radiography, NMR or ESR. For X-radiography, suitable labels include radioisotopes, such as barium or cesium, which emit detectable radiation but are not obviously harmful to the subject animal. Suitable markers for NMR and ESR include those with detectable characteristic spins (e.g. deuterium), which can be incorporated into anti-A33 antibodies by nutritional labeling for the corresponding scFv clone. An anti-A33 antibody labeled with a suitable detectable imaging moiety is introduced (e.g., orally, subcutaneously, or intraperitoneally) into the subject animal. The imaging moiety can be, for example, a radioisotope (e.g., 131 I, 112 In, 99 mTc), a radiopaque substance, or a material detectable by nuclear magnetic resonance. It will be understood in the art that the size of the subject and the imaging system used will determine the amount of imaging moiety required to produce a diagnostic image. In the case of radioisotopes, for human subjects, the amount of radioactivity injected is usually in the range of about 5 to 20 millicuries. 99 mTc.The labeled anti-A33 antibody will then accumulate in the location of cells that contain specific target polypeptide.For example, the labeled anti-A33 antibody of the present technology will accumulate in the cells and tissues in which A33 protein is localized in the subject animal. Thus, the present technology provides a method for diagnosing a medical condition, the method comprising the steps of: (a) assaying expression of immunoreactive A33 protein in an individual's cells or body fluids by measuring binding of an anti-A33 antibody of the present technology; (b) comparing the amount of immunoreactive A33 protein present in the sample with a standard reference, wherein an increase or decrease in immunoreactive A33 protein level compared to the standard is indicative of the condition.

[0092] affinity purificationthe anti-A33 antibody of the present technology can be used to purify immunoreactive A33 protein from a sample. In some embodiments, the antibody is immobilized on a solid support. Examples of such solid supports include plastics (e.g., polycarbonate), complex carbohydrates (e.g., agarose and sepharose), acrylic resins, and e.g., polyacrylamide and latex beads. The techniques for binding 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). The simplest way to bind antigen to an antibody-support matrix is ​​to collect beads in a column and pass an antigen solution through 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 slow flow rate. The immobilized antibody captures the antigen as it passes through. Alternatively, the antigen can be contacted with the antibody-support matrix by mixing the antigen solution with the support (e.g., beads) and rotating or rocking the slurry to allow maximum contact between the antigen and the immobilized antibody. After the binding reaction is complete, the slurry is passed through a column for collection of the beads. The beads are washed with an appropriate wash buffer, followed by elution of the pure or substantially pure antigen. The antibody or polypeptide of interest can be conjugated to a solid support (e.g., a bead). In addition, the first solid support (e.g., a bead) can also be conjugated to a second solid support (which can be a second bead or other support) if desired. The conjugation is carried out by any suitable means, including those disclosed herein for conjugating a polypeptide to a support. Thus, any of the conjugation methods and means disclosed herein for conjugating a polypeptide to a solid support can also be applied for conjugating a first support to a second support, whether the first and second solid supports are the same or different.

[0093] Suitable linkers (which may be crosslinkers) used to complex a polypeptide to a solid support include a variety of agents that can react with functional groups present on the surface of the support, or with the polypeptide, or with both. Reagents useful as crosslinkers include homobifunctional and especially heterobifunctional reagents. Useful bifunctional crosslinkers include, but are not limited to, N-SIAB, dimaleimide, DTNB, N-SATA, N-SPDP, SMCC and 6-HYNIC. Crosslinkers can be selected to provide selective cleavage of the bond between the polypeptide and the solid support. For example, photolabile crosslinkers such as 3-amino-(2-nitrophenyl)propionic acid can be utilized as a means to cleave a polypeptide 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. Pat. No. 5,643,722). Other cross-linking agents are known in the art (see, for example, Wong, 1991, supra; and Hermanson, 1996, supra). The antibody or polypeptide can be immobilized on a solid support (e.g., beads). The immobilization is via a covalent amide bond formed between a carboxyl-functionalized bead and the amino terminus of the polypeptide, or conversely, via a covalent amide bond formed between an amino-functionalized bead and the carboxyl terminus of the polypeptide. In addition, a bifunctional trityl linker can be attached to the support, for example, via an amino resin through an amino or carboxyl group on the resin to a 4-nitrophenyl active ester on the resin (e.g., Wang resin). When using the bifunctional trityl approach, the solid support may require treatment with volatile acid (e.g., formic acid or trifluoroacetic acid) to ensure that the polypeptide can be cleaved and removed. In such cases, the polypeptide can be deposited as a bead-free patch on the bottom of a well of the solid support or on the flat surface of the solid support. After addition of the matrix solution, the polypeptide can be released into the MS.

[0094] Hydrophobic trityl linkers can also be used as acid-sensitive linkers, and amino-linked trityl groups can be cleaved from polypeptides by using volatile acid or a suitable matrix solution (e.g., a matrix solution containing 3-HPA). Acid sensitivity can also be changed. For example, trityl, monomethoxytrityl, dimethoxytrityl or trimethoxytrityl can be changed to a suitable p-substituted derivative of the polypeptide or a more acid-sensitive tritylamine derivative. That is, trityl ether and tritylamine bonds can be generated in the polypeptide. Thus, the polypeptide can be removed from the hydrophobic linker, for example, by disrupting the hydrophobic attraction or by cleaving the trityl ether or tritylamine bond under acidic conditions (including typical MS conditions, if desired, in which case the matrix (e.g., 3-HPA) acts as the acid). Orthogonal cleavable linkers may also be useful for binding a first solid support (e.g., beads) to a second solid support or for binding a polypeptide of interest to a solid support. Such linkers may be used to selectively cleave a first solid support (e.g., beads) from a second solid support without cleaving the polypeptide from the support, and then the polypeptide may be cleaved from the beads. For example, a disulfide linker (cleavable with a reducing agent, e.g., DTT) may be utilized to bind the beads to the second solid support, and an acid-cleavable bifunctional trityl group may be used to immobilize the polypeptide to the support. If desired, the bond between the polypeptide and the solid support may be cleaved first, e.g., leaving the bond between the first support and the second support intact. The trityl linker may provide for covalent or hydrophobic conjugation, regardless of the nature of the conjugation, and the trityl group is easily cleaved under acidic conditions. For example, beads can be attached to a second support via a linking group, which can be selected to have a length and chemical nature that facilitates high density binding of the solid support to the beads or the beads to the polypeptide. Such linking groups can have, for example, a "tree-like" structure, thereby providing multiple functional groups per binding site on the solid support. Examples of such linking groups include polylysine, polyglutamic acid, pentaerythrol, and tris-hydroxy-aminomethane.

[0095] non-covalent association :Antibodies or polypeptides can be complexed to a solid support or a first support can be complexed to a second support via non-covalent interactions. For example, magnetic beads (which can be magnetized) made of ferromagnetic materials can be attracted to a magnetic solid support and released from the support by removing the magnetic field. Alternatively, a solid support can be provided with ionic or hydrophobic moieties, allowing the ionic or hydrophobic moieties to interact with a polypeptide (e.g., a polypeptide containing an attached trityl group) or a second solid support having hydrophobic characteristics, respectively. A solid support can also be provided with a member of a specific binding pair and thus complexed with a polypeptide or a second solid support that contains a complementary binding moiety, for example, avidin or streptavidin coated beads can be bound to a polypeptide that has incorporated therein a biotin moiety or a second solid support coated with biotin or a biotin derivative (e.g., iminobiotin). It will be understood that any of the binding members disclosed herein or known in the art can be reversed. Thus, for example, biotin can be incorporated into a polypeptide or solid support, and conversely, avidin or other biotin-binding moiety could be incorporated into the support or polypeptide, respectively. Other specific binding pairs contemplated for use herein include, but are not limited to, hormones and their receptors, enzymes and their substrates, nucleotide sequences and their complementary sequences, antibodies and the antigens with which they specifically interact, and other pairs known to those of skill in the art.

[0096] A. Diagnostic Uses of the Anti-A33 Antibody of the Present Technology Overview The anti-A33 antibodies of the present technology are useful in diagnostic methods. Thus, the present technology provides a method of using the antibodies to diagnose A33 activity in a subject animal. The anti-A33 antibodies of the present technology can be selected to have any level of epitope binding specificity and very high binding affinity for the A33 protein. In general, the higher the binding affinity of the antibody, the more stringent washing conditions can be performed in the immunoassay to remove non-specifically bound materials without removing the target polypeptide. Thus, the anti-A33 antibodies of the present technology useful in diagnostic assays are typically about 10 8 M -1 , 10 9 M -1 , 10 10 M -1 , 10 11 M -1 or 10 12 M -1Furthermore, anti-A33 antibodies for use as diagnostic reagents have a sufficient onset rate to reach equilibrium in at least 12 hours, at least 5 hours, or at least 1 hour under standard conditions. Anti-A33 antibodies can be used to detect immunoreactive A33 protein in a variety of standard assay formats, including immunoprecipitation, Western blot formation, ELISA, radioimmunoassay, and immunometric assays. See, Harlow & Lane, Antibodies, A Laboratory Manual (Cold Spring Harbor Publications, New York, 1999). 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 fluid of the subject animal. In some embodiments, the subject is at an early stage of cancer. In some embodiments, the early stage of cancer is determined by the level or expression pattern of A33 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 biopsy tissue.

[0097] Immunoassay or sandwich assay is one type of diagnostic method of this technology. See US Patent Nos. 4,376,110, 4,486,530, 5,914,241, and 5,965,375. Such assay uses one antibody (e.g., anti-A33 antibody or population of anti-A33 antibodies) immobilized on a solid phase, and another anti-A33 antibody or population of anti-A33 antibodies in solution. Typically, the solution anti-A33 antibody or population of anti-A33 antibodies is labeled. When an antibody population is used, the population can include antibodies that bind to different epitope specificities in the target polypeptide. Thus, the same population can be used for both solid phase and solution antibodies. When an anti-A33 monoclonal antibody is used, first and second A33 monoclonal antibodies with different binding specificities are used for the solid phase and solution phase. 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 A33 protein with the anti-A33 antibody, the sample is incubated for a period of time, which typically varies from about 10 minutes to about 24 hours, and is usually about 1 hour. A washing step is then performed to remove sample components that are non-specifically bound to the anti-A33 antibody used as a diagnostic reagent. When the solid phase and solution antibodies are bound in separate steps, washing can be performed after one or both binding steps. After washing, binding is typically quantified by detecting a label that is linked to the solid phase via binding of a labeled solution antibody. Typically, for a given antibody or antibody group pair and given reaction conditions, a calibration curve is prepared with samples containing known concentrations of target antigen.Then, the concentration of immunoreactive A33 protein in the test sample is read by interpolation from the calibration curve (i.e., standard curve).The analyte can be determined from the amount of labeled solution antibody bound at equilibrium, or by kinetic measurement of the amount of labeled solution antibody bound at a series of time points before equilibrium is reached.The slope of such a curve is a measure of the concentration of A33 protein in the sample.

[0098] Suitable supports for use in the above methods include, for example, nitrocellulose membranes, nylon membranes, and derivatized nylon membranes, as well as particles, such as agarose, dextran-based gels, dipsticks, microparticles, microspheres, magnetic particles, test tubes, microtiter wells, SEPHADEX TM (Amersham Pharmacia Biotech, Piscataway NJ), and the like. Immobilization is by adsorption or covalent attachment. Optionally, the anti-A33 antibody can be conjugated to a linker molecule (e.g., biotin) for attachment to a surface-bound linker (e.g., avidin). In some embodiments, the present disclosure provides an anti-A33 antibody of the present technology conjugated to a diagnostic agent. The diagnostic agent can include a radioactive or non-radioactive label, an imaging agent (e.g., for magnetic resonance imaging, computer-assisted tomography, or ultrasound), and the radioactive label can be a gamma-, beta-, alpha-, Auger electron-, or positron-emitting isotope. A diagnostic agent is a molecule administered conjugated to an antibody moiety (i.e., an antibody or antibody fragment or subfragment), which is useful in diagnosing or detecting disease by indicating the location of cells containing the antigen.

[0099] Useful diagnostic agents include, but are not limited to, radioisotopes, dyes (e.g., with biotin-streptavidin complexes), contrast agents, fluorescent compounds or molecules, and enhancement agents for magnetic resonance imaging (MRI) (e.g., paramagnetic ions). U.S. Patent 6,331,175 describes MRI techniques and the preparation of antibodies conjugated with MRI enhancement agents, which is incorporated herein by reference in its entirety. In some embodiments, the diagnostic agent is selected from the group consisting of radioisotopes, enhancement agents used in magnetic resonance imaging, and fluorescent compounds. To load the antibody component with radiometals or paramagnetic ions, the antibody may have to be reacted with a reagent having a long tail to which multiple chelating groups are added to bind the ions. Such tails can be polymers (e.g., polylysine, polysaccharides), or other derivatized or derivatizable chains with pendant groups. Attached to the pendant group is a chelating group, such as ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DPTA), porphyrins, polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and similar groups that have been found to be useful for this purpose. The chelates can be attached to the antibodies of the present technology using standard chemical techniques. The chelates are usually linked to the antibody by a group that allows for the formation of 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, which are used with diagnostic isotopes for radioimaging. The same chelates, when complexed with non-radioactive metals (e.g., magnesium, iron, and gadolinium), are useful in MRI when used with the A33 antibodies of the present technology. Macrocyclic chelates such as NOTA (1,4,7-triaza-cyclononane-N,N',N"-triacetic acid), DOTA, and TETA (p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid), have been used with a variety of metals and radiometals, such as radionuclides of gallium, yttrium, and copper, respectively. Such metal-chelate complexes can be stabilized by tailoring the ring size to the metal of interest. Other examples of DOTA chelates include: (i) DOTA-Phe-Lys(HSG)-D-T yr-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(DT PA)-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- (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. ; Other ring-type chelates, such as macrocyclic polyethers, are also contemplated, which may be used to regulate the nuclide (e.g., for RAIT). 223 It is useful for stably binding the carboxyl group (Ra).

[0100] B. Therapeutic Uses of Anti-A33 Antibodies of the Present Technology The immunoglobulin-related constructs (e.g., antibodies or antigen-binding fragments thereof) of the present technology are useful for treating A33-associated cancer. Such treatment can be used in patients identified as having pathologically elevated levels of A33 (e.g., patients diagnosed by the methods described herein) or patients diagnosed as having a disease known to be associated with such pathological levels. In one aspect, the present disclosure provides a method for treating A33-associated cancer in a subject in need thereof, comprising administering to the subject an effective amount of an antibody (or antigen-binding fragment thereof) of the present technology. Examples of cancers that can be treated by the antibodies of the present technology include, but are not limited to, colorectal cancer, pseudomyxoma peritonei, appendix cancer, pancreatic cancer, and gastric cancer. The A33-associated cancer can be colorectal cancer with MSI or MSS genotype. Additionally or alternatively, in some embodiments, the colorectal cancer is associated with KRAS G12D mutation or p53 mutation.

[0101] The composition of the present technology can be used together with other therapeutic agents useful in the treatment of A33-related cancer. For example, the antibody of the present technology can be administered separately, sequentially or simultaneously with at least one additional therapeutic agent selected from the group consisting of: alkylating agents, platinum agents, taxanes, vinca agents, antiestrogens, aromatase inhibitors, ovarian suppressants, VEGF / VEGFR inhibitors, EGF / EGFR inhibitors, PARP inhibitors, cytostatic 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, the following: cyclophosphamide, fluorouracil (or 5-fluorouracil or 5-FU), methotrexate, edatrexate (10-ethyl-10-deaza-aminopterin), thiotepa, carboplatin, cisplatin, taxanes, paclitaxel, protein-bound paclitaxel, docetaxel, vinorelbine, tamoxifen, raloxifene, toremifene, fulvestrant, gemcitabine, irinotecan, ixabepilone, temozolamide, topotecan, vincristine, bile acid, ramblastine, eribulin, mutamycin, capecitabine, anastrozole, exemestane, letrozole, leuprolide, abarelix, buserelin, goserelin, megestrol acetate, risedronate, pamidronate, ibandronate, alendronate, denosumab, zoledronate, trastuzumab, tykerb, anthracyclines (e.g., daunorubicin and doxorubicin), bevacizumab, oxaliplatin, melphalan, etoposide, mechlorethamine, bleomycin, microtubule poisons, annonaceae acetogenins, or combinations thereof.

[0102] The compositions of the present technology can optionally be administered as a single bolus to a subject in need thereof. Alternatively, the administration regimen can include multiple administrations administered at various times after the appearance of the tumor. Administration can be by any suitable route, including oral, intranasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), rectal, intracranial, intrathecal, or topical. Administration includes self-administration and administration by another. It will also be understood that various aspects of the treatment of the described conditions are intended to mean "substantial." "Substantial" includes complete treatment, but also includes less than complete treatment, in which some corresponding biological or medical outcome is achieved. In some embodiments, the antibodies of the present technology comprise a pharmaceutical formulation that can be administered in one or more doses to a subject in need thereof. Dosage regimens can be adjusted to provide the desired response (e.g., a therapeutic response).

[0103] Typically, an effective amount (sufficient to achieve a therapeutic effect) of the antibody composition of the present technology ranges from about 0.000001 mg per kilogram of body weight per day to about 10,000 mg per kilogram of body weight per day. Typically, the dosage range is from about 0.0001 mg per kilogram of body weight per day to about 100 mg per kilogram of body weight per day. For administration of anti-A33 antibodies, the dosage ranges from about 0.0001 to 100 mg per kg of subject body weight every week, every two weeks, or every three weeks, more usually 0.01 to 5 mg. For example, the dosage can be 1 mg / kg body weight or 10 mg / kg body weight every week, every two weeks, or every three weeks, or within the range of 1-10 mg / kg every week, every two weeks, or every three weeks. In some embodiments, a single dosage of the antibody ranges from 0.1-10,000 micrograms per kg of body weight. In some embodiments, the antibody concentration in the carrier ranges from 0.2 to 2000 micrograms per milliliter delivered. Exemplary treatment regimens involve administration once every two weeks or once a month or once every three to six months. The anti-A33 antibody may be administered multiple times. The interval between single doses may be one hour, one day, one week, one month, or one year. The interval may also be irregular as indicated by measuring the blood level of the antibody in the subject. In some methods, the dosage is adjusted to achieve a serum antibody concentration in the subject of about 75 μg / mL to about 125 μg / mL, 100 μg / mL to about 150 μg / mL, about 125 μg / mL to about 175 μg / mL, or about 150 μg / mL to about 200 μg / mL. Alternatively, the anti-A33 antibody may be administered as a sustained release formulation, in which case less frequent administration may be required. Dosage and frequency vary according to the half-life of the antibody in the subject.Dosage and frequency vary according to whether the treatment is preventive or therapeutic.In preventive applications, relatively low dosages are administered at relatively infrequent intervals over a long period of time.In therapeutic applications, relatively high dosages are often required at relatively short intervals until the progression of the disease slows or stops, or until the subject shows partial or complete alleviation of the symptoms of the disease.The patient can then be administered a preventive regimen.

[0104] In another aspect, the present 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 antigen-binding fragment thereof) of the present technology, the antibody being designed to localize to tumors expressing A33 and labeled with a radioisotope; and (b) detecting the presence of tumors 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 dose injected per gram (%ID / g). The reference value can be calculated by measuring the level of radioactivity present in non-tumor (normal) tissues and calculating the average level of radioactivity present in non-tumor (normal) tissues ± standard deviation. In some embodiments, the ratio of radioactivity levels between tumor and normal tissue is 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. 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 or single photon emission computed tomography. Additionally or alternatively, in some embodiments, the method further comprises administering to the subject an effective amount of an immunoconjugate comprising an antibody of the present technology conjugated to a radionuclide. In some embodiments, the radionuclide is an alpha particle emitting isotope, a beta particle emitting isotope, an Auger-emitter, or any combination of the above. Examples of beta particle emitting isotopes include: 86 Y, 90 Y, 89 Sr, 165 Dy, 186 Re, 188 Re, 177 Lu, and 67 Examples of alpha particle emitting 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 emitters include 111 In, 67 Ga, 51 Cr, 58 Co, 99m Tc, 103m Rh, 195m Pt, 119 Sb, 161 Ho, 189m Os, 192 Ir, 201 Tl, and 203 Pb. In some embodiments of the method, non-specific FcR dependent binding of normal tissues is eliminated or reduced (e.g., via the N297A mutation in the Fc region, which results in aglycosylation). The therapeutic efficacy of such immunoconjugates can be determined by calculating the tumor area under the curve (AUC):normal tissue AUC ratio. In some embodiments, the immunoconjugates have a tumor AUC:normal tissue AUC 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.

[0105] PRITIn one aspect, the present disclosure provides a method for detecting a solid tumor in a subject in need thereof, comprising: (a) administering to the subject an effective amount of a conjugate comprising a radiolabeled DOTA hapten and a bispecific antibody of the present technology that binds to the radiolabeled DOTA hapten and the A33 antigen, the conjugate configured to localize to a solid tumor expressing the A33 antigen that is recognized by the bispecific antibody of the conjugate; and (b) detecting the presence of a solid tumor in the subject by detecting a level of radioactivity emitted by the conjugate that is higher than a reference value. In some embodiments, the subject is a human. In another aspect, the present disclosure provides a method for selecting a subject for pretargeting radioimmunotherapy, the method comprising: (a) administering to the subject an effective amount of a conjugate comprising a radiolabeled DOTA hapten and a bispecific antibody of the present technology that binds to the radiolabeled DOTA hapten and the A33 antigen, the conjugate configured to localize to a solid tumor that expresses the A33 antigen recognized by the bispecific antibody of the conjugate; (b) detecting the level of radioactivity emitted by the complex; and (c) selecting the subject for pretargeting radioimmunotherapy when the level of radioactivity emitted by the complex is higher than the reference value. In some embodiments, the subject is a human.

[0106] DOTA does not 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 s(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 e-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 s(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(LysG)-D (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-GHSG) u-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-TAD-Lys -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-NH2;Cys)(xix) Ac-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-D-Lys(Tscg-Cys)-NH2 and (xx) DOTA. The radiolabel can be an alpha particle emitting isotope, a beta particle emitting isotope, or an Auger emitter. Examples of radiolabels 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.

[0107] In some embodiments of the methods disclosed herein, the level of radioactivity emitted by the conjugate is detected using positron emission tomography or single photon emission computed tomography. Additionally or alternatively, in some embodiments of the methods disclosed herein, the subject is diagnosed with or suspected of having an A33-positive cancer (e.g., colorectal cancer, pseudomyxoma peritonei, appendix cancer, pancreatic cancer, and gastric cancer). Additionally or alternatively, in some embodiments of the methods disclosed herein, the complex is administered intravenously, intramuscularly, intraarterially, intrathecally, intravesically, intraocularly, intradermally, intraperitoneally, subcutaneously, intracerebroventricularly, orally, or intranasally, hi certain embodiments, the complex is administered into the cerebrospinal fluid or blood of the subject animal. In some embodiments of the methods disclosed herein, the level of radioactivity emitted by the conjugate is detected between 2 and 120 hours after administration of the conjugate. In certain embodiments of the methods disclosed herein, the level of radioactivity emitted by the conjugate is expressed as a percentage of the injected dose per gram of tissue (%ID / g). The reference value can be calculated by measuring the level of radioactivity present in non-tumor (normal) tissue and calculating the average level of radioactivity present in the non-tumor (normal) tissue ± standard deviation. In some embodiments, the reference value is the 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 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.

[0108] In another aspect, the present disclosure provides a method of increasing tumor sensitivity to radiation therapy in a subject diagnosed with A33-positive cancer, the method comprising: (a) administering to the subject an effective amount of an anti-DOTA bispecific antibody of the present technology, the anti-DOTA bispecific antibody configured to localize to tumors expressing the A33 antigen target; and (b) administering to the subject an effective amount of a radiolabeled DOTA hapten, the radiolabeled DOTA hapten configured to bind to the anti-DOTA bispecific antibody. In some embodiments, the subject is a human. The anti-DOTA bispecific antibody is administered (e.g., according to a dosing regimen) under conditions and for a time sufficient for it to saturate the tumor cells. In some embodiments, unbound anti-DOTA bispecific antibody is cleared from the bloodstream after administration of the anti-DOTA bispecific antibody. In some embodiments, a radiolabeled DOTA hapten is administered after a time that may be sufficient to allow for the clearance of unbound anti-DOTA bispecific antibody. The radiolabeled DOTA hapten can be administered any time between 1 minute to 4 days or more after administration of the anti-DOTA bispecific antibody. For example, in some embodiments, the radiolabeled DOTA hapten is administered 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 therebetween, after administration of the anti-DOTA bispecific antibody. Alternatively, the radiolabeled DOTA hapten can be administered any time 4 days or more after administration of the anti-DOTA bispecific antibody.

[0109] Additionally or alternatively, in some embodiments, the method further comprises administering an effective amount of a clearing agent to the subject prior to administration of the radiolabeled DOTA hapten. The clearing agent can be any molecule (dextran or dendrimer) that can be conjugated to the C825 hapten. In some embodiments, the clearing agent does not exceed 2000kD, 1500kD, 1000kD, 900kD, 800kD, 700kD, 600kD, 500kD, 400kD, 300kD, 200kD, 100kD, 90kD, 80kD, 70kD, 60kD, 50kD, 40kD, 30kD, 20kD, 10kD, 5kD. In some embodiments, the clearing agent is a 500 kD aminodextran-DOTA conjugate (such as 500 kD dextran-DOTA-Bn(Y), 500 kD dextran-DOTA-Bn(Lu), or 500 kD dextran-DOTA-Bn(In)). In some embodiments, the clearing agent and the radiolabeled DOTA hapten are administered without further administration of the anti-DOTA bispecific antibody of the present technology.For example, in some embodiments, the anti-DOTA bispecific antibody of the present technology is administered according to a regimen that includes at least one cycle of: (i) administration of the anti-DOTA bispecific antibody of the present technology (optionally administered to saturate corresponding tumor cells); (ii) administration of the radiolabeled DOTA hapten and optionally the clearing agent; (iii) optionally administration of additional radiolabeled DOTA hapten and / or clearing agent, but without additional administration of the anti-DOTA bispecific antibody.In some embodiments, the method can include multiple such cycles (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 or more cycles). Additionally or alternatively, in some embodiments of the methods, the anti-DOTA bispecific antibody and / or radiolabeled DOTA hapten is administered intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intraocularly, intradermally, intraperitoneally, subcutaneously, intracerebroventricularly, orally or intranasally.

[0110] In one aspect, the present disclosure provides a method for increasing tumor sensitivity to radiation therapy in a subject diagnosed with A33-positive cancer, comprising administering to the subject an effective amount of a conjugate comprising a radiolabeled DOTA hapten and a bispecific antibody of the present technology that recognizes and binds to an A33 antigen target, the conjugate being configured to localize to tumors expressing the A33 antigen target recognized by the bispecific antibody of the conjugate. The conjugate is administered intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intraocularly, intradermally, intraperitoneally, transtracheally, subcutaneously, intracerebroventricularly, orally, or intranasally. In some embodiments, the subject is a human. In another aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising: (a) administering to the subject an effective amount of an anti-DOTA bispecific antibody of the present technology, the anti-DOTA bispecific antibody being configured to localize to a tumor expressing an A33 antigen target; and (b) administering to the subject an effective amount of a radiolabeled DOTA hapten, the anti-DOTA bispecific antibody being configured to bind to the anti-DOTA bispecific antibody. The anti-DOTA bispecific antibody is administered under conditions and for a time (e.g., according to a dosing regimen) sufficient for it to saturate tumor cells. In some embodiments, unbound anti-DOTA bispecific antibody is removed from the bloodstream after administration of the anti-DOTA bispecific antibody. In some embodiments, the radiolabeled DOTA hapten is administered after a time that may be sufficient to allow removal of unbound anti-DOTA bispecific antibody. In some embodiments, the subject is a human. Thus, in some embodiments, the method further comprises administering to the subject an effective amount of a clearing agent prior to administration of the radiolabeled DOTA hapten, which may be administered any time between 1 minute to 4 days or more after administration of the anti-DOTA bispecific antibody. For example, in some embodiments, the radiolabeled DOTA hapten is administered 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 therebetween, after administration of the anti-DOTA bispecific antibody. Alternatively, the radiolabeled DOTA hapten can be administered any time 4 days or more after administration of the anti-DOTA bispecific antibody.

[0111] The clearing agent can be a 500 kD aminodextran-DOTA conjugate (e.g., 500 kD dextran-DOTA-Bn(Y), 500 kD dextran-DOTA-Bn(Lu), or 500 kD dextran-DOTA-Bn(In), etc.). In some embodiments, the clearing agent and radiolabeled DOTA hapten are administered without further administration of an anti-DOTA bispecific antibody. For example, in some embodiments, the anti-DOTA bispecific antibody is administered according to a regimen comprising at least one cycle of: (i) administration of an anti-DOTA bispecific antibody of the present technology (optionally administered to saturate the corresponding tumor cells); (ii) administration of a radiolabeled DOTA hapten and optionally a clearing agent; (iii) optionally administration of additional radiolabeled DOTA hapten and / or clearing agent, but without additional administration of an anti-DOTA bispecific antibody. In some embodiments, the method can include multiple such cycles (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more cycles). Also provided herein is a method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a conjugate comprising a radiolabeled DOTA hapten and a bispecific antibody of the present technology that recognizes and binds to an A33 antigen target, wherein the conjugate is configured to localize to a tumor expressing the A33 antigen target recognized by the bispecific antibody of the conjugate. The therapeutic efficacy of such a conjugate can be determined by calculating the tumor area under the curve (AUC):normal tissue AUC ratio. In some embodiments, the complex has a tumor AUC:normal tissue AUC 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.

[0112] toxicityOptimally, an effective amount (e.g., dose) of the anti-A33 antibodies described herein will provide therapeutic benefit without causing substantial toxicity to the subject animal. Toxicity of the anti-A33 antibodies described herein can be determined by standard pharmaceutical procedures in cell culture or experimental animals, e.g., LD 50 (the dose lethal to 50% of the population) or LD 100 The dose ratio between toxic and therapeutic effects is the therapeutic index. The data obtained from these cell culture assays and animal studies can be used in formulating a non-toxic dosage range for use in humans. The dosage of anti-A33 antibodies described herein lies within a range of circulating concentrations that include the effective dose with little or no toxicity. Dosages may vary within said ranges depending on the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be selected by the individual physician in view of the condition of the subject animal. See, for example, Fingl et al., In: The Pharmacological Basis of Therapeutics, Ch.1, 1975.

[0113] Formulation of pharmaceutical compositions According to the methods of the present technology, the anti-A33 antibody can be incorporated into a pharmaceutical composition suitable for administration. The pharmaceutical composition generally comprises a recombinant or substantially purified antibody and a pharma- ceutical carrier in a form suitable for administration to a subject animal. The pharma- ceutical acceptable carrier is determined, in part, by the particular composition being administered as well as the particular method used to administer the composition. Thus, there are a wide variety of formulations of pharmaceutical compositions that are suitable for administration of antibody compositions (see, for example, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA 18). thed., 1990). Generally, pharmaceutical compositions are formulated as sterile, substantially isotonic, and in full compliance with Good Manufacturing Practice (GMP) regulations of the US Food and Drug Administration. The terms "pharmaceutical acceptable", "physiologically acceptable" and grammatical variations thereof are used interchangeably when referring to compositions, carriers, diluents and reagents, and refer to a substance that can be administered to a subject animal without producing undesirable physiological effects to an extent that would prohibit administration of the composition. For example, a "pharmaceutical acceptable excipient" refers to an excipient that is useful in the preparation of pharmaceutical compositions and is generally safe, non-toxic and desirable, and is acceptable for veterinary use as well as for human pharmaceutical use. Such excipients can be solid, liquid, semi-solid, and in the case of aerosol compositions, gaseous. "Pharmaceutical acceptable salts and esters" refers to salts and esters that are pharmaceutical acceptable and have desirable pharmacological properties. Such salts include salts formed when acidic protons present in the composition are capable of reacting with inorganic or organic bases. Suitable inorganic salts include those formed with alkali metals (e.g., sodium and potassium), magnesium, calcium and aluminum. Suitable organic salts include those formed with organic bases, such as the amine bases (e.g., ethanolamine, diethanolamine, triethanolamine, N-methylglucamine, and the like). Such salts also include the 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 arene-sulfonic acids (e.g., methanesulfonic acid and benzenesulfonic acid)). Pharmaceutically acceptable esters include esters formed from carboxy, sulfonyloxy, and phosphonoxy groups present on the anti-A33 antibody, such as C 1-6The term "anti-A33 antibody" includes alkyl esters. When two acidic groups are present, the pharma- ceutically acceptable salts or esters can be monoacid-monosalts or esters or disalts or esters; similarly, when three or more acidic groups are present, some or all of such groups can be salified or esterified. The anti-A33 antibody described in the present technology can exist in unsalted or unesterified form, or salified and / or esterified form, and reference to the anti-A33 antibody is intended to include the parent (unsalted and unesterified) compound and its pharma- ceutically acceptable salts and esters. Furthermore, certain embodiments of the present technology can exist in two or more stereoisomeric forms, and reference to the anti-A33 antibody is intended to include all single stereoisomers and all mixtures of such stereoisomers, racemic or otherwise. A person skilled in the art would have no difficulty in determining the appropriate timing, sequence and dosages for administration of the individual drugs and compositions of the present technology.

[0114] Examples of such carriers or diluents include, but are not limited to, water, physiological saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles (such as non-volatile oils) can also be used. The use of such media and compounds for pharmaceutically active substances is well known in the art. Except where conventional media or compounds are incompatible with anti-A33 antibody, their use in the composition is intended. Supplementary active compounds can also be incorporated into the composition. The pharmaceutical composition of the present technology is formulated to suit its intended route of administration. The anti-A33 antibody composition of the present technology is administered by parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intradermal, transdermal, rectal, intracranial, intrathecal, intraperitoneal, intranasal, or intramuscular route, or as an inhalant. The anti-A33 antibody can be administered in some cases in combination with other drugs that are at least partially effective in treating various A33-related cancers. Solutions or suspensions used for oral, intradermal or subcutaneous application can contain the following components: a sterile diluent, such as water for injection, saline solution, fixed oils, polyethylene glycol, glycerin, 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 acetate, citric acid, or phosphate; and compounds for adjusting tonicity, such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in ampoules, disposable syringes, or glass or plastic multi-dose vials.

[0115] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or suspensions and sterile powders for the extemporaneous preparation of sterile injectable solutions or suspensions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL TM(BASF, Parsippany, NJ), or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. The composition must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms (e.g., bacteria and fungi). The carrier can be a solvent or dispersion medium, including, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and appropriate mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating (e.g., lecithin), by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal compounds, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be desirable to include isotonic compounds, for example, sugars, polyalcohols (e.g., mannitol, sorbitol), sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition a compound which delays absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating the anti-A33 antibody of the present technology in the required amount into a suitable solvent with one or a combination of the ingredients listed above as necessary, followed by filtration sterilization.Generally, dispersions are prepared by incorporating the anti-A33 antibody into a sterile vehicle, which vehicle contains a basic dispersion medium and other ingredients required from those listed above.In the case of sterile powders for preparing sterile injectable solutions, the preparation method is vacuum drying and freeze-drying, which produces a powder containing the active ingredient plus any additional desired ingredients from the solution previously sterilized by filtration.The antibody of the present technology can be administered in the form of depot injection or implant preparations (which can be formulated in a manner that allows sustained or pulsatile release of the active ingredient).

[0116] Oral compositions generally include an inert diluent or an edible carrier. They are either encapsulated in gelatin capsules or compressed into tablets. For oral therapeutic administration, anti-A33 antibodies are incorporated with excipients and used in the form of tablets, troches or capsules. Oral compositions can also be prepared using a liquid carrier for use as a mouthwash, in which case the compound in the liquid carrier is applied to the mouth, swished around the mouth, and expectorated or swallowed. Pharmaceutically compatible binding agents and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like may contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, primogel or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening compound such as sucrose or saccharin; or a flavoring compound such as peppermint, methyl salicylate or orange flavor. For administration by inhalation, the anti-A33 antibodies are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, such as a gas, eg, carbon dioxide, or nebulizer.

[0117] Systemic administration can also be via transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant appropriate for permeating the barrier is used in the formulation. Such penetrants are generally known in the art, and include, for example, detergents, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be achieved by the use of nasal sprays or suppositories. For transdermal administration, the anti-A33 antibody is formulated into ointments, salves, gels, or creams as generally known in the art. Anti-A33 antibodies can also be prepared as pharmaceutical compositions in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery. In one embodiment, the anti-A33 antibody is prepared with a carrier that protects the anti-A33 antibody against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparing such formulations will be apparent to those skilled in the art. Such materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions, including liposomes targeted to infected cells using monoclonal antibodies against viral antigens, can also be used as pharma- ceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, such as those described in U.S. Pat. No. 4,522,811.

[0118] C.Kit The present technology provides a kit for detecting and / or treating A33-related cancer, the kit comprising at least one immunoglobulin-related component of the present technology (any antibody or antigen-binding fragment described herein), or a functional variant thereof (e.g., a substitution variant). Optionally, the above-described components of the kit of the present technology are packaged in a suitable container and labeled for the diagnosis and / or treatment of A33-related cancer. The above-described components may be stored in a unit or multi-dose container (e.g., sealed ampoules, vials, bottles, syringes, and test tubes) as an aqueous (preferably sterile) solution or as a lyophilized (preferably sterile) formulation for reconstitution. The kit may further comprise a second container, the container holding a suitable diluent for diluting the pharmaceutical composition to a larger volume. Suitable diluents include, but are not limited to, pharma- ceutical acceptable excipients and saline solutions of the pharmaceutical composition. Furthermore, the kit may comprise instructions for diluting the pharmaceutical composition and / or instructions for administering the pharmaceutical composition (whether diluted or not). The containers can be formed from a variety of materials, such as glass or plastic, and can have a sterile access port (for example, the container can be an intravenous solution bag or a vial with a stopper, the stopper being pierceable by a hypodermic injection needle). The kits can also include more containers, including pharma- ceutically acceptable buffers, such as phosphate-buffered saline, Ringer's solution, and dextrose solution. The kits can also include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, culture media for one or more suitable hosts. The kits can optionally include instructions customarily included in commercial packaging of therapeutic or diagnostic products, said instructions including, for example, information on the indications, usage, dosage, manufacture, administration, contraindications, and / or warnings concerning the use of such therapeutic or diagnostic products.

[0119] The kit is useful for detecting the presence of immunoreactive A33 protein in a biological sample, including, for example, any bodily fluid (e.g., serum, plasma, lymph, cyst fluid, urine, feces, cerebrospinal fluid, ascites, or blood) and a biopsy sample of a body tissue. For example, the kit may include: one or more humanized, chimeric, or bispecific anti-A33 antibodies (or antigen-binding fragments thereof) of the present technology capable of binding to A33 protein in a biological sample; a means for determining the amount of A33 protein in the sample; and a means for comparing the amount of immunoreactive A33 protein in the sample to a standard. One or more of the anti-A33 antibodies may be labeled. The components (e.g., reagents) of the kit may be packaged in a suitable container. The kit may further include instructions for using the kit to detect immunoreactive A33 protein. In the case of an antibody-based kit, the kit includes, for example: 1) a first antibody bound to a solid support that binds to the A33 protein, such as a humanized, chimeric or bispecific A33 antibody (or antigen-binding fragment thereof) of the present technology; 2) a second, different antibody that binds to the A33 protein or the first antibody, conjugated to a detectable label. The kit may also include, for example, a buffer, a preservative, or a protein stabilizing agent. The kit may further include components (e.g., an enzyme or a substrate) necessary for detecting the detectable label. The kit may also include a control sample or a series of control samples that may be assayed and compared to the test sample. Each component of the kit may be enclosed in an individual container, and all of the various containers may be present in a single package, together with instructions for interpreting the results of the assays performed using the kit. The kit of the present technology may include a description on or within the kit container. The description may explain how to use the reagents contained in the kit, for example, for in vitro or in vivo detection of A33 protein, or for treating A33-related cancer in a subject in need thereof. In certain embodiments, the use of the reagents may follow the methods of the present technology. Working Example The present technology is further illustrated by the following examples, which should not be construed as limiting in any manner. The following examples show the preparation, characterization and use of an exemplary A33 antibody of the present technology. The following examples show the production of chimeric, humanized and bispecific antibodies of the present technology, and the characterization of their binding specificity and in vivo biological activity. EXAMPLES

[0120] Materials and methods for generating and characterizing the anti-A33 antibodies of the present technology Cell lines and human leukocytes Cell lines LS174T, Colo205, SNU-16 and 293T cells were purchased from ATCC (Manassas, Virginia), and SW1222 was purchased from ECACC (Salisbury, United Kingdom). All cells were confirmed by STR typing. Cells were maintained in RPMI medium supplemented with 10% FBS (Sigma, St. Louis, MO), 0.03% L-glutamine (Gibco Laboratories, Gaithersburg, MD) and Pen / Strep (Gibco Laboratories, Gaithersburg, MD). Buffy coats from healthy donors were purchased from the New York Blood Center (New York City, NY), and human PBMCs were isolated by Ficoll gradient of buffy coats. Establishment of luciferase-expressing cell linesFirst, 293T cells were transfected with a retroviral construct containing luciferase and GFP genes using PolyJet transfection reagent (SignaGen, Rockville, MD) according to the manufacturer's instructions. After 36 hours, the virus-containing supernatant was collected and filtered through a 0.45 μm filter. An aliquot of 2 mL of the filtered supernatant was dispensed into each well of a 12-well plate pre-coated with Retronectin (Clontech Laboratories, Mountain View, CA). The plate was spun at 3500 rpm for 45 min at 4°C. The process was repeated 2 to 3 times. After spinning, the plate was washed once with PBS and then 0.2x10 6 Tumor cells were plated and incubated at 37°C. Spinnoculation was repeated once more after 24 h. Cells were then incubated for at least another 48 h before sorting for GFP-expressing cells. For cell sorting, transduced cells were sorted into at least 4 plates of 96-well plates at a density of 1 cell / well and incubated for 2 weeks before colony pick-up. Picked colonies were scored and selected for luciferase, GFP and GPA33 expression compared to the parental cell line. SEC-HPLC analysis The size and purity of huA33-BsAb were analyzed using an HPLC system (Shimadzu Scientific Instruments Inc., Columbia, MD). Monomeric species were identified using molecular weight standards (Bio-Rad Laboratories, Hercules, CA), and the percent monomer was calculated based on the relative areas under the curve (AUC) of the various non-buffer peaks.

[0121] Humanization of mouse A33: Mouse A33 was humanized as an IgG1 using CDR grafting. Two different VH and VL sequences were combined to generate four different humanized A33 antibodies. Surface plasmon resonance (SPR) analysis was used to compare the binding kinetics to that of the chimeric antibody chA33. The heavy chain sequence is SEQ ID NO:6 (3A3-H2) and the light chain sequence is SEQ ID NO:10 (3A3-H2). The heavy chain sequence, including the leader sequence, is MGWSCIILFLVATATG and the light chain sequence, including the leader sequence, is: EVQLVESGGGLVKPGGSLRLSCAASGFAFSTYDMSWVRQAPGKRLEWVATISSGGSYTYYLDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAPTTVVPFAYWGQGTLVTVSS (SEQ ID NO:55). MGWSCIILFLVATATG DIQMTQSQSSLSTSVGDRVTITCKASQNVRTVVAWYQQKPGKSPKTLIYLASNRHTGVPSRFSGSGSGTEFTLTISNVQPEDFADYFCLQHWSYPLTFGSGTKLEIKR (SEQ ID NO:56). The underlined sequence corresponds to the leader sequence. SPR analysis : Human GPA33 (Novoprotein, Summit, NJ) was immobilized on a CM5 chip. Five concentrations (starting at 20 nM) of 2-fold serially diluted huA33 IgG1 or huA33-BsAb were run over the chip using a Biacore™ T100 system (GE Healthcare, Chicago, IL). Binding kinetics of huA33 were measured at 25°C, and binding kinetics of huA33-BsAb were measured at both 25°C and 37°C. Sensorgrams were fitted to a 1:1 binding model for both to obtain kinetic parameters. Generation of huA33-BsAb bispecific antibody: huA33-BsAb was constructed by fusing humanized OKT3 scFv to the C-terminus of the huA33 antibody light chain via a (G4S)3 linker as previously described by Xu et al. and Lopez-Albaitero et al. (Xu H et al., Cancer Immunology Research 3:266-277, 2015; and Lopez-Albaitero A et al., OncoImmunology 6:e1267891, 2017). N297A and K322A mutations were introduced into the Fc region of the antibody to eliminate FcR and complement binding activities, respectively (Shields RL et al., Journal of Biological Chemistry 276:6591-6604, 2001; Idusogie EE et al., Journal of Immunology 164:4178-4184, 2000). The DNA constructs were then transfected into CHO-S cells and stable clones were selected for high-level antibody production. Selected stable clones were grown in shake flasks for large-scale antibody purification. Bispecific antibodies were purified from the supernatant using one-step Protein A affinity chromatography. T cell dependent cytotoxicity (TDCC) assay : 51 Cytotoxicity assays were performed using both Cr release assays and Pierce LDH release assays (Thermo Fisher Scientific, Cambridge, MA). For both assays, T cells activated by exposure to anti-CD3 / anti-CD28 Dynabeads for 14 days were subsequently used as effector cells, with the exception of cells sorted from PBMCs, which were used in the TDCC assay without prestimulation. 51 Cr assays were performed as previously described by Cheng et al. (Cheng M et al., International Journal of Cancer 136:476-486, 2015). LDH assays were performed according to the manufacturer's instructions with the following modifications. Briefly, 1.5x10 IgG was added to each assay well of a 96-well round-bottom plate. 41000 target cells were incubated with various numbers of effector cells depending on the desired E:T ratio. Various dilutions of antibody were then added and the plates were incubated for 16 hours at 37°C. Each condition was performed in triplicate. The supernatants were then transferred to flat-bottom plates with reaction substrate and incubated for 30 minutes before being read at 490 nm. 680 nm was used as the reference wavelength. EC 50 was calculated by curve fitting to a four-parameter nonlinear regression model using GraphPad Prism.

[0122] Cytokine and cytolytic molecule release assays T cells were purified from PBMCs using a pan T cell isolation kit (Miltenyi Biotec, Cambridge, MA). Target and control tumor cells were cultured at 2x10 cells / well in a 5:1 effector to target ratio in a 24-well plate. 6 Cells / well were incubated in triplicate with a total volume of 2 mL per well. Culture supernatants were collected at 24, 48, 72, and 96 hours and incubated with LEGENDplex. TM Cytokine levels were measured by flow cytometry using a human CD8 / NK panel (Biolegend, San Diego, Calif.) according to the manufacturer's protocol. T cell proliferation assay Fresh PBMCs from healthy donors were labeled with 2.5 nM CFSE (Life Technologies Corp., Carlsbad, CA) for 5 min at room temperature, followed by neutralization with PBS containing 5% FBS. Target cells were then incubated with the labeled PBMCs under various conditions at 37°C, and surface activation marker expression and T cell proliferation were analyzed for 24 and 96 h. In vivo tumor treatment For subcutaneous (sc) tumor models, LS174T, Colo205 or SNU16 cells were combined with fresh PBMCs at a 1:1 ratio and mixed with Matrigel (cell:gel volume = 1:2) in Balb / c Rag2 - / - IL2Rγ - / -The tumors were implanted (100 μL / mouse) into the flanks of (DKO) mice (currently commercially available as CIEA BRG mice from Taconic). After confirming the presence of tumors, treatment was initiated at 100 μg / mouse on a BIW schedule and continued for 3-4 weeks. Tumor growth was monitored by measuring tumor volume weekly using calipers or a digital Peira TM900 scanner (Peira Scientific Instruments, Turnhout, Belgium). For the intraperitoneal (ip) tumor model, luciferase-expressing LS174T-luc or SW1222-luc cells were resuspended in RPMI medium and injected into the peritoneal cavity of DKO mice. Human effector cells were provided as activated T cells and injected intravenously. Mice were treated with 2-3 week cycles of antibody-T cell-antibody regimen (each component separated by 3-4 days). Tumor growth was followed weekly by measuring luminescence signal after injection of 3.3 mg / mouse luciferin on an IVIS Spectrum in vivo imaging system (PerkinElmer Inc., Waltham, MA). Luminescence was analyzed and quantified using in vivo imaging software (PerkinElmer Inc., Waltham, MA).

[0123] Antibodies and flow cytometryAntibodies (anti-hCD25-PE, anti-CD69-PE, anti-hCD8-APC, anti-hCD45-PECy7, anti-hCD4-PE, anti-hCD4-BV421, anti-hCD62L-PercpCy5.5, anti-hPD1-BV421, anti-hCD45RO-PECy7) were purchased from Biolegend (San Diego, CA). Goat anti-human IgG-PE was purchased from SouthernBiotech (Birmingham, AL). Streptavidin-PE, anti-hCD4-APC, and anti-hCD25-APC were purchased from BD Biosciences (San Jose, CA). All FACS analyses were performed using a FACSCalibur or LSRII system (BD Biosciences, San Jose, Calif.) and analyzed using FlowJo (FLOWJO, Ashland, Oreg.). Affinity maturation using yeast displayThe parental huA33 was converted to scFv format using a :20 amino acid (G4S)4 linker and cloned into a yeast display vector. The huA33 scFv was randomly mutated using the GeneMorph II mutagenesis kit (Agilent Technologies, Santa Clara, CA). The PCR products were electroporated into yeast along with the linearized vector, and the library was subjected to four rounds of panning with biotinylated GPA33. Individual clones from the final round were PCR amplified and sequenced to analyze mutation patterns. Conversion of selected scFv clones to the huA33-BsAb format was performed using a one-step four-fragment ligation method, using 50 ng of linearized vector and a 1:3 vector to insert molar ratio for the other three components. Ligation was performed using the Rapid DNA ligation kit (Thermo Fisher Scientific, Cambridge, MA) for 1 h at room temperature. The type II restriction enzyme SapI (New England Biolabs, Ipswich, MA) was used to ensure seamless ligation between the different components (Figure 13). Selected clones were transiently expressed using the Expi293 Expression System (Thermo Fisher Scientific, Cambridge, MA) according to the manufacturer's instructions. Antibodies were purified with MabSelect SuRe (GE Healthcare, Chicago, IL) from Expi293 cell supernatants after 4-5 days of culture in shake flasks and dialyzed against citrate buffer, pH 8.0, through a dialysis membrane (Spectrum Laboratories, Inc., Rancho Dominguez, CA). statistical analysis Significance (p<0.05) was tested using the Student's t test. EXAMPLES

[0124] Structure and binding affinity of the humanized anti-A33 antibody of the present technology Figure 14 shows the V H and V LThe amino acid sequences of the domains and their corresponding homologous human sequences are shown in SEQ ID NOs: 1-4. H and V L The amino acid sequences of the domains are shown in Figures 15 and 17. The V domains of the 3A3-H1 / L1, 3A3-H1 / L2, 3A3-H2 / L1 and 3A3-H2 / L2 humanized A33 antibodies are H and V L The cDNA sequences of the domains are shown in Figure 16 and Figure 18. Figure 19 shows an alignment of the original humanized amino acid sequence hA33 of King et al. (Ibid. (1995)) with the newly re-humanized huA33 (3A3) amino acid sequence. Figure 24 shows the amino acid sequences of the light and heavy chains of chimeric chA33-IgG1, which correspond to SEQ ID NO: 13 and SEQ ID NO: 14, respectively. Figure 25 shows the amino acid and cDNA sequences of the heavy chain of huA33-IgG1(H2L2), which correspond to SEQ ID NO: 15 and SEQ ID NO: 16, respectively. Figure 26 shows the amino acid and cDNA sequences of the light chain of huA33-IgG1(H2L2), which correspond to SEQ ID NO: 17 and SEQ ID NO: 18, respectively. All four novel humanized A33 antibodies showed slightly improved binding to immobilized GPA33 in SPR analysis when compared to chimeric A33. off (FIGS. 9 and 20). D Based on 37°C stability and T20 humanity score, the H2L2 huA33 clone was selected for further development (see Figure 23). Figures 21 and 22 show that the original humanized hA33 (hA33-mC825 BsAb) described supra (Cheal et al., 2016) showed a substantial decrease in binding affinity compared to huA33-H2L2. These results show that the antibody or binding fragment of the present technology specifically binds to A33 antigen with high binding affinity. Therefore, the immunoglobulin-related constructs disclosed herein are useful for detecting A33 protein in a sample. EXAMPLES

[0125] Structure and binding affinity of the T cell-engaging huA33-BsAb antibody of the present technology Reformatting of the huA33 antibody into a bispecific format was performed by fusing the scFv of humanized OKT3 to the C-terminus of the light chain via a flexible GS linker (Figure 1(A)). The DNA construct was used to establish a CHO-S stable cell line and huA33-BsAb was purified from the supernatant. Protein yields using protein A chromatography were 50 mg / L to 100 mg / L without extensive optimization. Similar yields were observed using the Expi293 transient expression system (data not shown). One-step protein A purification routinely yielded proteins with purity of >90% as measured by SEC-HPLC. Four freeze-thaw cycles caused no notable changes in the SEC-HPLC profile (data not shown). After incubating the molecule at 37°C for 4 weeks, there was only a slight decrease in the monomer percentage as shown in Figure 1(B). These data indicate that the huA33-BsAb had good solubility, purity and thermal stability, all of which are important characteristics for subsequent development. Figure 27 shows the amino acid and cDNA sequences of the heavy chain of the T cell engaging huA33-BsAb bispecific antibody, which correspond to SEQ ID NO: 19 and SEQ ID NO: 20, respectively. Figure 28 shows the amino acid and cDNA sequences of the light chain of the T cell engaging huA33-BsAb bispecific antibody, which correspond to SEQ ID NO: 21 and SEQ ID NO: 22, respectively. Figure 29 shows a summary of potential modifications to the T cell engaging huA33-BsAb bispecific antibody disclosed herein. The avidity of huA33-BsAb for GPA33 at both 25°C and 37°C was measured using a GPA33-immobilized CM5 chip. As shown in Figure 1(C), huA33-BsAb bound GPA33 with a high apparent affinity of approximately 0.2 nM, slightly lower than the 0.13 nM value observed with the parental huA33. FACS analysis of a panel of cell lines derived from various cancers showed that huA33-BsAb stained multiple colon cancer cell lines and one gastric cancer cell line, but not the GPA33(-) neuroblastoma cell line IMR32, osteosarcoma cell line TC32, or melanoma cell line SKMEL5 (Figure 1(D) and Figure 10), indicating that huA33-BsAb retains the specificity of the parental antibody A33 to bind to its target antigen on colon cancer cells and a subset of gastric cancer cells. Staining of activated T cells also demonstrated that huA33-BsAb bound to CD3 on the surface of T cells, see Figure 1(D). These results show that the antibody or binding fragment of the present technology specifically binds to A33 antigen with high binding affinity. Therefore, the immunoglobulin-related composition disclosed herein is useful for detecting A33 protein in a sample. EXAMPLES

[0126] Biological activity of the T cell-engaging huA33-BsAb antibody of this technology huA33-BsAb activated fresh T cells and induced their cell cycle entry To test the ability of huA33-BsAb to activate naive T cells, CFSE-labeled PBMCs were mixed with Colo205 cells at an E:T ratio of 5 and cultured in the presence of huA33-BsAb (1 μg / mL). An irrelevant T cell-engaging-BsAb antibody L1CAM-BsAb (against the antigen L1CAM (L1CAMxCD3) and does not bind to L1CAM(-) Colo205 by FACS) was used as a negative control, along with huA33-C825 (Cheal SM et al., Eur J Nucl Med Mol Imaging 43:925-37, 2016), which carries an irrelevant scFv instead of the anti-CD3 scFv. After 24 and 96 hours, cells were stained with various T cell activation markers to assess T cell activation status and proliferation. As early as 24 hours, huA33-BsAb caused activation of both CD4(+) and CD8(+) T cells, as indicated by upregulation of CD25 and CD69 markers at the cell surface (Figure 2(A)). In contrast, huA33-C825 and L1CAM-BsAb only caused minimal CD25 upregulation. L1CAM-BsAb did not increase CD69 expression, especially in CD4(+) T cells (presumably due to expression of L1CAM on T cells). Similarly, PD-1 upregulation was observed after 24 hours and persisted until 96 hours (Figure 11(A)). Cell division (measured by CFSE dye dilution) was observed in CD4(+) and CD8(+) T cells after 96 hours (Figure 2(B)). CD8(+) T cells exhibited a faster cell division cycle, suggesting that CD8(+) T cells divide faster than CD4(+) T cells (Figure 2(C)). The control antibody huA33-C825 did not stimulate a significant amount of cell division in either T cell subset, confirming the requirement of CD3 activation for T cell division. Low levels of cell division were induced by L1CAM-BsAb, consistent with the low level of activation observed above. When using GPA33(-) SKMEL5 target cells, huA33-BsAb did not activate T cells (Figure 1(B)). These results indicate that T cell activation by huA33-BsAb is dependent on the presence of cognate antigen on tumor cells. We also observed that cell division was accompanied by the expansion of CD45RO(+) effector / memory cells (Figure 2(D)), suggesting the importance of this subset in mediating T-BsAb activity (confirmed below). These assays were repeated using a different colon cancer cell line, LS174T, with similar results (Figure 11(C)). To determine whether T cells could be activated in vivo, an in vivo proliferation assay was performed by mixing CFSE-labeled PBMCs with Colo205 cells and implanting the mixture subcutaneously into DKO mice. The following day, huA33-BsAb was injected intravenously, and after another 4 days, tumors were isolated and analyzed by FACS. As shown in Figure 2(E), approximately 25% of CD4(+) and CD8(+) T cells upregulated CD25 expression during cell division (CFSE fluorescence was progressively halved), suggesting that huA33-BsAb could also stimulate T cell activation and proliferation in vivo.

[0127] huA33-BsAb induced the secretion of inflammatory cytokines and cytolytic molecules : We investigated the cytokine secretion profile of T cells activated by huA33-BsAb in the presence of target tumor. Total T cells were purified from PBMC and cultured in the presence of Colo205 tumor cells at an effector:target ratio of 5:1. SKMEL5 cells were used as a negative control to estimate non-specific activation. Cell culture supernatants were collected daily for 4 days, and the levels of cytokines and cytotoxic molecules were measured in a multiplex manner by flow cytometry. As shown in Figure 3, both Th1 cytokines (IL-2, IFNγ, TNFα) and Th2 cytokines (IL-4, IL-10) were secreted by activated T cells, although IL-4 was secreted at a much lower level than the other cytokines. Similarly, significant amounts of IL-6 were secreted by activated T cells. The Th17 cytokine IL-17α was also secreted at high levels. The cytotoxic components sFas, granzyme A, granzyme B, perforin and granulysin were all released into the supernatant.

[0128] huA33-BsAb redirected T cells to specifically kill colon and gastric cancerWe tested the ability of huA33-BsAb to redirect T cells to kill cancer cells. Based on the survey of GPA33 expression in multiple human cancer cell lines (Figure 10), three colon cancer cell lines (LS174T, SW1222 and Colo205) and one gastric cancer cell line (SNU16) were selected for cytotoxicity experiments. These cell lines were classified as MSI (LS174T) or MSS (SW1222, Colo205, SNU16) subtypes based on their microsatellite instability profiles (Williams DS et al., PLOS ONE 5:e16012, 2011;Yoon K et al., Genome Research 23:1109-1117, 2013;Suter CM et al., Br J Cancer 88:413-419, 2003). Furthermore, LS174T cells harbored a KRAS G12D mutation, while the other cells harbored p53 mutations or deletions (Ahmed D et al., Oncogenesis 2:e71, 2013; Liu Y & Bodmer WF, Proceedings of the National Academy of Sciences of the United States of America 103:976-981, 2006; Ku JL & Park JG: Cancer Research and Treatment : Official Journal of Korean Cancer Association 37:1-19, 2005; Ikediobi ON, Davies H, Bignell G, et al., Molecular cancer therapeutics 5:2606-2612, 2006). Cancer cells were incubated with activated T cells in the presence of 10-fold serial dilutions of huA33-BsAb at an effector-to-target ratio of 10:1. GPA33(-) melanoma cell line SKMEL5 and osteosarcoma cell line TC32 were used as negative controls. As shown in Figure 4(A), huA33-BsAb redirected T cells to specifically kill all GPA33-expressing cancer cells regardless of their genetic background, whereas SKMEL5 and TC32 did not, confirming the antigen specificity of huA33-BsAb-mediated TDCC. The maximum level of cytotoxicity appeared to correlate with the FACS staining level (Figure 1(D) and Figure 4(A)). Furthermore, TDCC induced by huA33-BsAb was potent and EC 50 Values ​​were in the pM range, see Figure 4(A). To determine which T cell subsets were recruited by huA33-BsAb, we sorted CD45RA(+)CD62L(+) and CD45RA(-)CD45RO(+)CD62L(-) memory subsets from both CD4(+) and CD8(+) T cells. Sorted cells were cultured in the presence of Colo205 tumor cells at an E:T ratio of 5:1 for 48 hours, and then cytotoxicity was measured using an LDH assay. As shown in Figure 4(B), both CD4(+) and CD8(+) memory T cell subsets were able to induce cytotoxicity, but CD8(+) memory T cells mediated more effective killing at higher concentrations. Both CD45RA(+)CD62L(+) subsets of the CD4(+) and CD8(+) populations (which are mostly naive T cells) were able to induce cytotoxicity after 48 hours, although with a reduced capacity compared to memory T cells. When T cells in the TDCC assay were stained for CD45RO and CD25, CD25 expression was found to be upregulated in the presence of huA33-BsAb in both the CD45RO+ and CD45RO- fractions (Figure 12(A) and Figure 12(B)), confirming that both naive and memory T cells can be activated by huA33-BsAb. Although the majority of CD45RA(+)CD62L(+) T cells remained CD45RO(-) after incubation, a small but significant population increased their CD45RO expression, especially CD8(+) cells. See Figure 12(B). However, this population could be derived from maturation of naive T cells or from the expansion of rare CD45RO+ cells that were present in the initial culture. Taken together, these data demonstrated that huA33-BsAb could induce potent cytotoxicity against colon and gastric cancer cells in a GPA33-dependent manner by recruiting both CD4 and CD8 T cells, especially those with memory phenotype. These results indicate that the antibody or antigen-binding fragment of the present technology specifically binds to the A33 antigen with high binding affinity. Thus, the immunoglobulin-related constructs disclosed herein are useful for detecting A33 protein in a sample. EXAMPLES

[0129] Affinity maturation of huA33-BsAb by yeast display To attempt to further improve the performance of huA33-BsAb, a yeast display method was used for affinity maturation of the huA33-derived scFv. Because scFvs tend to aggregate easily, we developed a method to rapidly reformat the scFv into T cell-engaging huA33-BsAb, a more suitable format that could be rapidly produced with high yield and purity (Figure 13). The method is based on a one-step four-fragment ligation approach that utilizes the type II enzyme SapI to allow seamless ligation of different fragments, and could be easily applied to other vectors and scaled up to a high-yield workflow.Multi-fragment In-Fusion™ cloning (Clontech Laboratories, Mountain View, CA) was also tried with success, but was not very robust (data not shown), possibly due to the specific sequences of the expression vector. From sequence analysis of 60 single clones, seven clones (Figure 5(A)) were selected for further characterization by SPR and TDCC assays. All seven clones showed increased binding affinity ranging from 4.3 to 51 fold compared to the parent antibody (Figure 5(B)). The amino acid sequences of the heavy and light chains of the seven affinity matured clones are shown in Figures 30-36. Most of the improvement was due to slower off rates. All clones showed slight improvements in maximum killing in the TDCC assay (Figure 5(C)). These results indicate that the antibody or antigen-binding fragment of the present technology specifically binds to the A33 antigen with high binding affinity. Therefore, the immunoglobulin-related constructs disclosed herein are useful for detecting A33 protein in a sample. EXAMPLES

[0130] In vivo therapeutic trial using huA33-BsAb The efficacy of huA33-BsAb was tested in vivo in humanized DKO mice using two different tumor models: (1) subcutaneous tumors + subcutaneous effectors and (2) intraperitoneal tumors + intravenous effectors with the following xenografts. huA33-BsAb cured MSI tumor LS174T in a subcutaneous xenograft model and suppressed tumor growth in an abdominal modelLS174T cells were mixed with PBMCs at a 1:1 ratio and subcutaneously implanted into DKO mice. As shown in Figure 6(A), in the absence of antibody treatment, the tumor alone group and the tumor + PBMC group showed rapid tumor growth. Mice in the tumor + PBMC group showed tumor ulceration and had to be euthanized. In contrast, 6 doses of huA33-BsAb in 3 weeks effectively cured the mice, and the mice remained tumor-free for at least 120 days. To mimic malignant ascites (commonly seen in colon cancer), luciferase-expressing LS174T cells were implanted into the peritoneal cavity of DKO mice. Upon confirmation of tumor growth by luminescence, mice were randomized into different treatment groups: no antibody group (tumor alone), tumor + T cell alone group (tumor + ATC), or intravenous injection of huA33-BsAb (7 doses over 3 weeks) plus weekly injections of T cells for 3 weeks. Treatment began 5 days after tumor implantation. All T cells were injected via the retro-orbital route. As shown in Figure 6(B) and Figure 6(C), all control groups (no antibody group or tumor + T cell alone group) showed exponential growth of tumors in the abdominal area. By day 28, 3 out of 4 mice in the no antibody group and 3 out of 5 mice in the tumor + T cell group, respectively, succumbed to tumors. In contrast, huA33-BsAb significantly inhibited the metastatic growth of LS174T tumor cells in the abdominal region of treated mice. All mice maintained survival for at least 60 days without further treatment. These results indicated that huA33-BsAb was effective against CRC tumors with MSI genotype. However, as previously described, MSI tumors account for only a minority of CRC cancer patients. The majority of CRC patients are MSS. Therefore, the efficacy of huA33-BsAb was further tested using MSS tumors.

[0131] huA33-BsAb cured the MSS tumor COLO205 in a subcutaneous xenograft model and inhibited tumor growth of the metastatic MSS tumor SW1222Two MSS colon cancer cell lines, Colo205 and SW1222, were tested. For Colo205 cells, tumors were implanted subcutaneously with PBMCs as effector cells. In this model, four doses of huA33-BsAb were sufficient for complete eradication of Colo205 tumors, and mice remained tumor-free for at least four months after a total of six doses of antibody treatment (Figure 7(A) and Figure 7(B)). For the SW1222 cell line, tumors were implanted intraperitoneally and treated with six doses of intravenous antibody for three weeks and weekly intravenous injections of T cells for two weeks. As shown in Figure 7(C) and Figure 7(D), the two control groups spread tumors throughout the entire abdominal cavity area, while huA33-BsAb inhibited tumor growth and significantly extended mouse survival (p=0.0125). These results indicate that the efficacy of huA33-BsAb in treating MSS tumors may be comparable to that observed when treating MSI tumors. It is expected that additional treatment cycles will completely eradicate tumor growth as in the case of the subcutaneous model. huA33-BsAb inhibited the growth of gastric cancer in a subcutaneous xenograft model : GPA33 is expressed in a subset of gastric cancer cells. FACS analysis and TDCC assays showed that SNU16 expressed GPA33 and were sensitive to huA33-BsAb redirected T cell killing. SNU16 cells were mixed with human PBMCs and then xenografted subcutaneously. huA33-BsAb effectively cured mice bearing subcutaneous tumors (Figure 8(A)) and significantly extended survival. Unlike NSG mice, DKO mice were more resistant to human PBMC engraftment. In the subcutaneous SNU16 model at day 58 (Figure 8(B)), most mice showed low levels of chimerism, although with high variability. As shown above, the efficacy of huA33-BsAb is not dependent on the genetic background or mutational status of the CRC tumor, which is an important biological property if application to the majority of CRCs is desired. Unlike CAR-modified T cells, huA33-BsAb-redirected T cells did not exhibit cell exhaustion and demonstrated sustained and quantitative tumor homing properties for over 3 weeks. Tumors were cured despite the presence of PD1 and PD-L1, and the addition of subtherapeutic doses of huA33-BsAb to block checkpoints further enhanced these antitumor properties. The A33 immunoglobulin-related constructs disclosed herein provide both FcR-dependent and T cell-dependent immunotherapeutic approaches to the diagnosis and treatment of A33-related cancers. Taken together, these results indicate that the antibodies or antigen-binding fragments of the present technology can detect tumors and inhibit tumor growth progression and / or metastasis. Thus, the immunoglobulin-related constructs disclosed herein are useful for detecting and treating A33-positive cancers in subjects in need thereof. EXAMPLES

[0132] Use of the huA33-C825 antibody of the present technology in pretargeting radioimmunotherapy The major drawbacks of developing antibody drugs for pretargeting radioimmunotherapy (PRIT) are radiation overexposure in normal tissues, immunogenicity, suboptimal tumor dose and low therapeutic index. This example shows that the huA33-DOTA bispecific antibody of the present technology is useful in PRIT to treat cancers expressing human A33 antigen (e.g., colorectal cancer). The A33-DOTA bispecific antibody of the present technology comprises a first antigen-binding site based on the humanized A33 antibody described herein and a second antigen-binding site that binds a small molecule hapten (e.g., benzyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid [DOTA-Bn]). An anti-DOTA-Bn single-chain Fv fragment (scFv) based on the affinity-matured 2D 12.5 antibody will be linked to the carboxyl terminus of the humanized A33 light chain. Figure 37 shows the amino acid and cDNA sequences of the heavy chain of the bispecific antibody huA33-huC825(H2L2), which correspond to SEQ ID NO:58 and SEQ ID NO:59, respectively. Figure 38 shows the amino acid and cDNA sequences of the light chain of the bispecific antibody huA33-huC825(H2L2), which correspond to SEQ ID NO:60 and SEQ ID NO:61, respectively. FIG. 39 shows the amino acid sequences of the heavy and light chains of bispecific antibody huA33-mC825(H2L2), which sequences correspond to SEQ ID NO:62 and SEQ ID NO:63, respectively. Tumor cell lines and cell culture reagents : The human colorectal cancer cell line SW1222 will be maintained by serial passage. Cells will be cultured at 37°C with 5% CO2 in minimal essential medium supplemented with the following (heat-inactivated 10% fetal bovine serum, 2.0 mM glutamine, 100 units / mL penicillin and 100 units / mL streptomycin). Cultures will be established and stored frozen in small aliquots to limit passage to less than 3 months, and will be tested periodically for mycoplasma using a commercial kit (Lonza, Portsmouth, NH) according to the manufacturer's instructions. For passaging and trypsinization at cell harvest, a 0.25% trypsin / 0.53 mM EDTA solution in Hank's buffered salt solution (calcium and magnesium free) will be used. huA33-huC825 and huA33-mC825 will be produced in mammalian expression vectors in CHO cells and purified by protein A affinity chromatography. Surface plasmon resonance testBiacore T100 biosensors, CM5 sensor chips, and associated reagents will be purchased from a commercial supplier (GE Healthcare, Chicago, IL). Recombinant human A33 protein will be purchased from a commercial supplier (Novoprotein Scientific, Inc., Summit, NJ). BSA-(Y)-DOTA-Bn conjugates will be prepared as described in Cheal et al., Mol. Cancer Ther. 13(7), 2014. A33 and DOTA antigens will be immobilized using an Amino Coupling kit (GE Healthcare, Chicago, IL). Purified bispecific and control antibodies will be analyzed and data will be fitted to a bivalent analyte model using the Biacore T100 evaluation software as described in Cheal et al. (Cheal et al., 2014).

[0133] PRIT Reagents, Protocols and Xenograft Studies : All animal experiments will be approved by the Institutional Animal Care and Use Committee of Memorial Sloan Kettering Cancer Center and will follow the Cancer Center guidelines for appropriate humane animal use during research. Athymic nu / nu female mice (6-8 weeks old; Harlan Sprague Dawley) will be acclimated to the vivarium for at least 1 week. Five hundred and ten (5x10) of A33-positive SW1222 formulated in a 1:1 ratio of Matrigel (BD Biosciences, San Jose, CA) will be cultured in a 20-well plate. 6 Tumor cells were injected subcutaneously into the left flank of groups of animals and established tumors (100-900 mm) were created using the formula for elliptical volume (V=4 / 3π(length / 2xwidth / 2xheight / 2). 2 ) and observed for 7-10 days. All reagents will be administered intravenously (i.v.) via the lateral tail vein. The PRIT protocol consists of an injection of huA33-huC825 or huA33-mC825 [t=-28h], followed by the removal agent [t=-4h] and 4 hours later [t=0h]. 177The injection of Lu-DOTA-Bn was included. The removal agent is a 500 kDa dextran-(Y)-DOTA-Bn conjugate, prepared according to the literature (Orcutt et al., Nucl. Med. Biol. 38:223-233, 2011) and formulated in saline for injection 24 hours later. The substitution ratio of moles of (Y)-DOTA-Bn to moles of dextran is 61(Y)-DOTA-Bn / dextran. 177 Lu-DOTA-Bn was synthesized from aminobenzyl-DOTA (p-NH2-Bn-DOTA) from Macrocyclics as previously described. 177 LuCl3 (specific activity-30 Ci / mg (Perkin Elmer)) and formulated in saline for injection. In addition, huA33-C825 was assayed using I 131 It is radioactively trace-labeled with Using the IODOGEN method (Cheal, S. et al., Mol. Cancer Ther. 13(7): 1-10, 2014), 131 I-huA33-mC825 or 131 I-huA33-huC825 will be prepared and in vitro cell-binding immunoreactivity will be assessed using SW1222 cells essentially as described (Lindmo, T. et al., J. Immunol. Meth. 126(2): 183-189, 1990). 131 I-huA33-mC825 or 131 The final specific activity of I-huA33-huC825 is 95.5 MBq / mg (cold huA33-huC825 or huA33-mC825 is added to achieve the desired mg dose) and the radiochemical purity is >98% using size-exclusion high-pressure liquid chromatography. For PRIT using nonspecific IgG-C825, an equivalent mg ​​dose of GD2-targeting bispecific antibody (hu3F8-C825) is used in place of huA33-mC825 or huA33-huC825. For ex vivo biodistribution analysis, mice were euthanized by CO2(g) asphyxiation, tumors and selected organs were harvested, washed with water, air-dried, weighed, and radioassays were performed by gamma scintillation counting (Perkin Elmer Wallac Wizard 3", PerkinElmer Inc., Waltham, MA). Count rates were background and decay corrected, converted to activity using system calibration factors, normalized to administered activity, and expressed as percent injected dose per gram (%ID / g). 177 Differences in Lu-activity concentrations will be analyzed by unpaired Student's t-test where appropriate.

[0134] Estimation of absorbed dose Groups of A33-positive SW1222 tumor-bearing mice (n=4-5) were treated with 0.25 mg of huA33-C825 (either huA33-huC825 or huA33-mC825), ablative agent (62.5 μg; 25% (w / w)) and 1.85-2.0 MBq (-10 pmol) of 177 Lu-DOTA-B will be administered and sacrificed at 2, 24 and 120 hours post-injection. For each tissue, the non-decay corrected time activity concentration data will be fitted to one-component, two-component or more complex exponential functions as appropriate using Excel, and further analytical integration will be performed to obtain the cumulative activity concentration per administered activity unit (MBq-h / g per MBq). 177 Tumor-to-tumor and selected organ-to-organ self-absorbed doses will be estimated using the Lu equilibrium dose constant (8.49 g-cGy / MBq-h) (where: 177 (Assume complete local absorption of Lu beta radiation only, ignoring gamma and non-self-dose contributions) of the tumor and selected tissues with the highest absorbed dose (e.g. blood, liver, spleen and kidneys). 177 In the relative uptake of Lu-DOTA-Bn 177To determine the effect of Lu-DOTA-Bn dose, administer the following to groups (n=5 / group) of SW1222 tumor-bearing female athymic nude mice: 0.25 mg (1.19 nmol) of huA33-C825 (either huA33-huC825 or huA33-mC825) at t=-28h and 62.5 µg of ablative agent at t=-4h, followed by one of the following: 11.1 MBq (11.14-11.40 MBq), 55.5 MBq (54.61-55.06 MBq), or 111 MBq (109.52-112.5 MBq). 177 For biodistribution analysis of Lu activity, all groups were 177 Sacrifice occurs 24 hours after Lu-DOTA-Bn injection (ie, time of maximal tumor uptake).

[0135] PRIT+ 86 PET imaging of Y-DOTA-Bn A group of mice (n=5) bearing A33-positive SW1222 tumors in the shoulder was treated with 0.25 mg of huA33-C825 (huA33-huC825 or huA33-mC825), ablative agent (62.5 μg 25% (w / w)) and 8.6-8.8 MBq (-50 pmol) of 86 Y-DOTA-Bn is administered and imaged non-invasively with a microPET Focus 120 (CTI Molecular Imaging, Inc. Knoxville, TN) at approximately 2 and 20 hours post-administration. The following image acquisition parameters are used: energy width 350-750 keV, coincidence time width 6 nsec, and acquisition time 20 min. The resulting list-mode data is sorted into 2D histograms by Fourier rebinning, and transverse images are reconstructed into a 128x128x95 matrix by filtered backprojection (reconstructed spatial resolution is 2.6 mm full width at half maximum (FWHM)). Scanner response non-homogeneity, dead time count loss, physical decay (relative to injection time), and 86Image data are corrected for the Y positron decay branching ratio. No attenuation, scatter, or partial volume averaging corrections may be applied. A system calibration factor empirically determined for the mouse (i.e., μCi / mL / cps / voxel) is used to convert voxel count rates to activity concentrations. The resulting image data are then normalized for administered activity, and the percent of injected dose per gram of tissue (%ID / g), corrected for radioactive decay versus time of injection, is determined by region of interest analysis. Imaging and region of interest (ROI) analysis are performed using AsiPRO VM 5.0 software (Concorde Microsystems, Knoxville, TN) (%ID / g at ROI maximum). Animals are sacrificed 24 hours post-dose for ex vivo biodistribution analysis.

[0136] Autoradiography and immunohistochemistry : huA33-C825 (huA33-huC825 or huA33-mC825) PRIT followed by 11.1 (11.14-11.40 MBq), 55.5 (54.61-55.06 MBq), or 111 MBq (109.52-112.5 MBq) of ESRD. 177 Frozen and OCT-embedded tumors and kidneys of selected mice administered Lu-DOTA-Bn (sacrificed 24 hours after injection) are sectioned at 10 μm using a cryostat (Avantik, Springfield, NJ), immediately exposed to an imaging plate (Fuji Photo Film, Kanagawa, Japan) for 72 hours, and then scanned using a Typhoon FLA 7000 scanner (GE, Pittsburg, PA). The same sections are subjected to hematoxylin-eosin staining and scanned under an Olympus BX60 microscope equipped with a movement-controlled stage (Olympus, Central Valley, PA). Both autoradiograms and microscopic images are processed and analyzed using ImageJ (NIH, Bethesda, MD).

[0137] Treatment methods and scintigraphic studiesGroups of mice bearing established subcutaneous A33-positive SW1222 xenografts were treated with huA33-C825 (huA33-huC825 or huA33-mC825), or with nonspecific (ns) IgG-C825 PRIT (i.e., one treatment, 7 days after tumor inoculation). 177 Lu-DOTA-Bn injection), or 2 PRIT (i.e., 2 treatments, 10 and 17 days after tumor inoculation) 177 Lu-DOTA-Bn injection). For two-treatment studies, tumor volumes 10 days after tumor inoculation (TV10) are reported and, when appropriate, expressed as mean ± SD. The following definitions are used to describe response to treatment: complete response (CR) is <100 mm 3 A long-term response (DR) is defined as survival 140 days after treatment. Excess tumor burden is >2000mm 3 For scintigraphy studies, a select group of treated A33-positive SW1222 tumor-bearing mice were anesthetized by gas inhalation and then imaged for 30 min (approximately 10 images per image) on a nanoSPECT (Bioscan, Washington DC) 20 h after injection. 5 Scan using a low energy high resolution collimator and window settings of 208 keV. Images are reconstructed into a 256x256 matrix using Bioscan HiSPECT software and uploaded to ASIPro VM for analysis. result : One dose of huA33-C825 (huA33-huC825 or huA33-mC825) 177 A biodistribution pilot study in SW1222 tumor-bearing mice 24 hours after injection of Lu-DOTA-Bn (0.1-0.6 mg of huA33-C825 (0.48-2.86 nmol) and a fixed ratio of ablation agents and 177Lu-DOTA-Bn (5.6MBq) is used). Additional biodistribution experiments are then performed to optimize the dose of ablative agent during PRIT. Groups of tumor-bearing mice (n=3-4 / group) are injected with huA33-C825 (huA33-huC825 or huA33-mC825) followed 24 hours later with either saline (i.e., vehicle), 2.4% (w / w), 5% (w / w), 10% (w / w), or 25% (w / w) of ablative agent (0-62.5μg / mouse) (relative to the preselected huA33-C825 dose). After an additional 4 hours, the mice are injected with 177 Inject 5.6 MBq of Lu-DOTA-Bn and sacrifice 24 hours later for biodistribution analysis. The ablation dose had no significant effect on circulating (i.e. blood) 177 Lu activity and subsequent tumor 177 It is expected that this may reduce the capacity for Lu-DOTA-Bn uptake. Next, using the optimized doses of huA33-C825 (huA33-huC825 or huA33-mC825) and the clearing agent, 177 Conduct Lu-DOTA-Bn dose titration studies. For Lu-DOTA-Bn dose titration studies, 177 Lu activity biodistribution data were presented in terms of both %ID / g and absolute uptake (kBq / g). 177 Comparisons will be made between Lu-DOTA-Bn dose groups. 131 Single time point biodistribution experiments 24 h after injection of I-trace labeled huA33-C825 (0.39-0.40 MBq along with 1.19 nmol of added cold huA33-C825) were performed in SW1222 tumor-bearing mice to estimate the absolute antibody uptake (as pmol / g) of huA33-C825 (huA33-huC825 or huA33-mC825) in tumors during PRIT.

[0138] Determine the biodistribution and absorbed dose estimates of radiolabeled DOTA-Bn in SW1222 tumor cell-implanted mice. Optimal doses of huA33-C825 and ablation agent were administered, followed by 2.0MBq (-10pmol) of 177 PRIT was performed in the A33-positive SW1222 tumor-bearing mouse group using Lu-DOTA-Bn. 177 Biodistribution studies were performed 2 to 120 hours after injection of Lu-DOTA-Bn to determine the distribution of the Lu-DOTA-Bn in tumors and various normal tissues. 177 Briefly, the Lu activity residence time was determined in tumors and various normal tissues. 177 Lu activity was evaluated by the optimal dose of PRIT (A33-C825 (huA33-huC825 or huA33-mC825) and dextran-removal agent, as well as 177 Groups of SW1222 tumor-bearing mice (n=4 to 5) were administered huA33-C825, followed by dextran-removal agent 24 hours later and then 4 hours later. 177 2.0MBq (-10pmol) of Lu-DOTA-Bn was administered to one group of animals. 177 The animals were sacrificed 2, 24 and 120 hours after injection of Lu-DOTA-Bn. These data were used as described herein to 177 Estimate the absorbed dose for radioimmunotherapy with Lu-DOTA-Bn. 177 Tumor uptake of Lu is expected to occur very rapidly after administration and decline over the next 96 hours up to 120 hours after injection. For each target area, the absorbed dose is 177 Lu equilibrium dose constant and target area 177 Lu is estimated as the product of cumulative activity (where 177(Assuming complete local absorption of Lu beta radiation and ignoring gamma and non-self dose contributions.) It is expected that a high therapeutic index of tumor to blood will be observed during single cycle treatment and that little or no toxicity will be observed over time in subjects with long-term responses. Similar results are expected for tumor uptake after PRIT when assessed using PET imaging. This example shows the efficacy of PRIT using the A33-DOTA bispecific antibody of the present technology against A33-positive tumors.It is expected that a progressive increase in therapeutic index and a decrease in absolute tumor uptake will be observed with increasing dose.Therefore, the huA33-DOTA bispecific antibody of the present technology is useful in the treatment of cancers expressing human A33 antigen (e.g., colorectal cancer). EXAMPLES

[0139] In vivo therapeutic effect of the huA33-C825 antibody of this technology This example demonstrates the in vivo efficacy of huA33-C825 bispecific antibody in PRIT to mediate tumor burden reduction in A33-positive cancer cell-bearing mice. In particular, this example describes the effect of one- and two-cycle regimens on tumor burden in SW1222 tumor-bearing mice. During the one-cycle therapy study, five groups of tumor-bearing mice (n=6-8 / group) are treated with either: vehicle (i.e., no treatment, n=8; TV7: 76±15 mm 3 ), 33.3MBq 177 Lu-DOTA-Bn alone (bispecific antibody and vehicle administered at the time of clearing agent injection, n = 6, TV7: 116 ± 23 mm 3 ), 1 cycle IgG-C825 PRIT+33.3MBq 177 Lu-DOTA-Bn (ns IgG-C825 administered instead of huA33-C825, n = 8, TV7: 100 ± 10 mm 3 ), or 1 cycle of huA33-C825 (huA33-huC825 or huA33-mC825) PRIT+11.1MBq or 33.3MBq177 Lu-DOTA-Bn (both n = 8, TV7: 103 ± 17 mm 3 and TV7: 93±15mm 3 During the procedure 177 As the amount of Lu-DOTA-Bn increases, the relative tumor uptake is expected to decrease. 177 Lu-DOTA-Bn only or 1 cycle IgG-C825 PRIT+33.3MBq 177 It is expected that the group of tumor-bearing mice receiving 1Lu-DOTA-Bn) will not demonstrate a tumor response. 177 Scintigraphy of the latter two groups receiving Lu-DOTA-Bn is expected to show minimal activity in the tumor area. In contrast, 1 cycle of huA33-C825 PRIT (huA33-huC825 or huA33-mC825) + 11.1 MBq or 33.3 MBq 177 It is expected that the group treated with Lu-DOTA-Bn will show a delay in tumor growth following treatment. The second treatment trial will investigate two cycles of huA33-C825 PRIT (huA33-huC825 or huA33-mC825) treatment when mice receive either no treatment (n=5, TV 10 :314±77mm 3 ), all mice will be sacrificed within 30 days due to excessive tumor burden. 2 cycles of PRIT+11.1MBq 177 Lu-DOTA-Bn(total 177 Lu-DOTA-Bn dose 22.2MBq) (n=5, TV 10 :462±179mm 3 ) will result in complete tumor response and / or tumor growth delay in treated animals.

[0140] Toxicity testing In simple terms, 2 cycles of PRIT + 11.1MBq 177 Lu-DOTA-Bn (n=3) or 2 cycles of PRIT+1.5mCi 177A total of 6 mice treated with either Lu-DOTA-Bn (n=3) will be subjected to autopsy of kidney, bone marrow, liver and spleen up to 9 weeks after treatment. Kidney, bone marrow, liver and spleen will be normal in treated animals, and it is expected that PRIT treatment will not be associated with radiation-induced toxicity. Theranostic PRIT for healing Nude mice bearing established SW1222 subcutaneous xenografts (n=20; tumor volume=102±40 mm3 (mean±standard deviation (SD)) were subjected to one of the following treatments (n=5-10 / group): no treatment (n=5); 177 Lu-DOTA-Bn alone (n=5) or 3-cycle PRIT regimen (huA33-C825 PRIT(huA33-huC825 or huA33-mC825) + 55MBq 177 Lu-DOTA-Bn) (n=10, total 165 MBq). Sequential nanoSPECT / CT imaging is performed on 5 randomly selected mice (the mice are 177 (Subject to DPRIT for dosimetry calculations up to 160 hours after injection of the first cycle of Lu-DOTA-Bn). It is expected that DPRIT will induce complete tumor responses in all treated mice and will not induce overt toxicity in the kidney, liver, spleen, and bone marrow. Lutetium-177 nanoSPECT / CT imaging of animals treated with the three-cycle PRIT regimen is expected to show high contrast with visible uptake in the tumor and minimal tissue background. These results indicate that the huA33-DOTA bispecific antibody of the present technology is useful in reducing tumor burden in vivo and that PRIT-based theranostics may have a curative effect and / or be useful for tumor detection in target animals. Theranostic "real-time" simultaneous treatment and image-guided dosimetry :For "real-time" dose measurement 177 NanoSPECT / CT was used for high-resolution quantitative imaging of mice undergoing Lu-DPRIT treatment. SW1222 tumor-bearing nude mice (volume: 100 mm by caliper measurement) 3 ) to huA33-C825 PRIT(huA33-huC825 or huA33-mC825)+55MBq 177Treated with one cycle of Lu-DOTA-Bn 177 Injection of Lu-DOTA-Bn is followed by imaging by nanoSPECT / CT three times: 1, 24, and 160 hours post-injection. Images are decay-corrected to the time of injection and further calibrated using known activity standards. Region-of-interest analysis of the calibrated images is used to determine the activity concentration in the tumor. Thus, the huA33-DOTA bispecific antibody of the present technology is useful in treating A33-positive cancers and in detecting tumors in subject animals. EXAMPLES

[0141] Ex vivo biodistribution study of the huA33-DOTA bispecific antibody of this technology GPA33(+) human colorectal cancer cell line SW1222 was obtained from the Ludwig Institute for Cancer Immunotherapy (New York, NY). SW1222 cells were cultured in minimal essential medium supplemented with: 10% heat-inactivated fetal bovine serum, 2.0 mM glutamine, 100 units / mL penicillin, and 100 μg / mL streptomycin. All cells were maintained in a 37°C environment with 5% CO2(g). Cultures were established upon receipt of cell lines, stored frozen in small aliquots to limit passage to less than 3 months, and routinely tested for mycoplasma negativity using a commercial kit (Lonza, Portsmouth, NH). For cell passaging and trypsinization at harvest, a 0.25% trypsin / 0.53 mM EDTA solution in Hank's buffered salt solution (calcium and magnesium free) was used. For SW1222 tumor establishment, groups of mice were seeded with 5x10 cells in 200 μL cell suspension. 6 Cells were inoculated by subcutaneous (sc) injection into the lower flank (the cell suspension is a 1:1 mixture of medium with reconstituted basement membrane (BD Matrigel, Collaborative Biomedical Products Inc., Bedford, MA)) and allowed to grow into established tumors (100-300 mm 3 ) was observed within 7-10 days. For all intravenous injections, mice were gently warmed with a heat lamp and placed in a restrainer. Mouse tails were cleaned with an alcohol swab and injected into the lateral tail vein. For biodistribution analysis following administration of the radiotracer, mice were euthanized by CO2(g) asphyxiation and tumors and selected organs were harvested, washed with water, air-dried, weighed, and assayed for radioactivity by gamma scintillation counting (Wallac Wizard 3", PerkinElmer Inc., Waltham, MA). Count rates were corrected for background and decay, converted to activity using system calibration coefficients specific for the radioisotope, normalized to administered activity, and expressed as mean ± SD %ID / g unless otherwise stated. To determine the biodistribution of huA33-DOTA BsAb, SW1222 tumor-bearing mice (group size: n=2 for a given clearing agent (CA) step, n=1 for saline control; ex vivo tumor mass ranged from 225-571 mg at the time of biodistribution) were administered three separate reagents intravenously via the lateral tail vein: 0.25 mg (1.19 nmol) of huA33-C825 (BC155-3 clone 2G7) at t=-28 h, followed by 500 kD dextran-DOTA(Y) clearing agent at t=-4 h, and then [ 177 Lu]Lu-DOTA-biotin (50 μCi) at t = 0 h. Biodistribution studies were performed using [ 177 The study was performed 24 hours after injection of the 500 kD dextran-DOTA(Y) remover and [ 177 [Lu]Lu-DOTA-biotin was prepared using the method described in the literature (Orcutt, KD et al., Mol Cancer Ther 11:1365-1372, 2012), and [ 177 The same radiation chemistry protocol described for [Lu]Lu-DOTA-benzene was used. 177 Lu]Lu-DOTA-biotin. %ID / gm = percent injected dose per gram of tissue. As shown in Figure 40, animals undergoing PRIT using the huA33-DOTA bispecific antibody of the present technology showed a high degree of tumor penetration (approximately 20-25% ID / g localized to GPA33(+) tumors) with or without the administration step of an ablative agent, thus demonstrating that the huA33-DOTA bispecific antibody can effectively target tumors in vivo. Thus, the huA33-DOTA bispecific antibody of the present technology is useful in treating A33-positive cancers and in detecting tumors in subject animals.

[0142] Equivalent The present technology should not be limited to the specific embodiments described in this application, which are intended as only one illustration of individual features of the present technology. As will be apparent to those skilled in the art, many modifications and variations of the present technology can be made without departing from its spirit and scope. In addition to those listed herein, functionally equivalent methods and devices within the scope of the present technology will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to be included within the scope of the present technology. It should be understood that the present technology is not limited to specific methods, reagents, compounds, compositions, or biological systems, which may of course vary. It should also be understood that the terms used herein are intended to describe specific embodiments and are not intended to be limiting. In addition, where a feature or characteristic of the present disclosure is described in terms of a Markush group, it will be understood by those skilled in the art that the disclosure is also thereby described in terms of individual members or subgroups of members of the Markush group. As will be understood by those of skill in the art, for any and all purposes, especially when a written description is provided, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges. Any range recited is fully described, and it will be readily understood that the same range can be broken down into at least the same half, 1 / 3, 1 / 4, 1 / 5, 1 / 10, etc. As a non-limiting example, each range discussed herein can be easily broken down into a lower 1 / 3, a middle 1 / 3, an upper 1 / 3, etc. As will also be understood by those of skill in the art, for example, terms such as "up to," "at least," "greater than," "less than," etc., all refer to ranges that are inclusive of the recited numbers and can be further broken down into subranges as discussed above. Finally, as will be understood by those of skill in the art, a range includes each of its individual members. Thus, for example, a group containing 1-3 cells refers to a group containing 1, 2, or 3 cells. Similarly, a group containing 1-5 cells refers to a group containing 1, 2, 3, 4, or 5 cells, and so on. All patents, patent applications, provisional patent applications, and publications referenced or cited in this specification are hereby incorporated by reference in their entirety (including all figures and tables) to the extent not inconsistent with the explicit teachings of this application.

Claims

1. Heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domains (V L ) , (a) V H but, FTFSTYDMS (SEQ ID NO:37) V H - CDR1 sequence, TISSGGSYTYYLDSVKG (SEQ ID NO: 38) H -CDR2 sequence, and V of TTVVPFAY (SEQ ID NO: 39) H - comprising a CDR3 sequence, and / or (b) V L but, KASQNVRTVVA (SEQ ID NO:40), LASNRHT (SEQ ID NO:41) and QYWSYPLT (SEQ ID NO:42); KASQNVRTVVA (SEQ ID NO:40), LASDRHT (SEQ ID NO:43) and QYWSYPLT (SEQ ID NO:42); KASQNVRTLVA (SEQ ID NO:44), LASNRHT (SEQ ID NO:41) and QHWSYPLT (SEQ ID NO:45); and KASQNVRTLVA (SEQ ID NO: 44), LASNRHT (SEQ ID NO: 41) and QYWSYPLT (SEQ ID NO: 42), V is selected from the group consisting of L -CDR1 sequence, V L -CDR2 sequence and V L - comprising a CDR3 sequence, The antibody or antigen-binding fragment thereof.

2. 2. The antibody or antigen-binding fragment of claim 1, further comprising an Fc domain of an isotype selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD and IgE.

3. The antibody of claim 2, comprising an IgG1 constant region comprising one or more amino acid substitutions selected from the group consisting of N297A and K322A.

4. The antibody of claim 2, comprising an IgG4 constant region comprising an S228P mutation.

5. Fab, F(ab') 2 2. The antigen-binding fragment of claim 1, selected from the group consisting of Fab', scFv and Fv.

6. The antibody or antigen-binding fragment of any one of claims 1 to 5, which binds to an epitope of the A33 protein that contains at least 5 to 8 consecutive amino acid residues of SEQ ID NO:

57.

7. The antibody of any one of claims 1-4 or 6, which is a monoclonal antibody, a chimeric antibody, a humanized antibody, or a bispecific antibody.

8. 23. An antibody comprising a heavy chain (HC) amino acid sequence comprising SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:58, SEQ ID NO:62, or a variant of said sequence having one or more conservative amino acid substitutions, and / or a light chain (LC) amino acid sequence comprising SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:60, SEQ ID NO:63, or a variant of said sequence having one or more conservative amino acid substitutions.

9. The antibody of claim 8, comprising an HC amino acid sequence and an LC amino acid sequence, each of which is selected from the group consisting of: SEQ ID NO:5 and SEQ ID NO:9 (3A3-H1 / L1); SEQ ID NO:5 and SEQ ID NO:10 (3A3-H1 / L2); SEQ ID NO:6 and SEQ ID NO:9 (3A3-H2 / L1); SEQ ID NO:6 and SEQ ID NO:10 (3A3-H2 / L2); SEQ ID NO:15 and SEQ ID NO:17 (huA33-IgG1(H2L2)); SEQ ID NO:19 and SEQ ID NO:21 (huA33-BsAb); SEQ ID NO:23 and SEQ ID NO:24 (clone 31); SEQ ID NO:25 and SEQ ID NO:26 (clone 32); SEQ ID NO:27 and SEQ ID NO:28 (clone 48); SEQ ID NO:29 and SEQ ID NO:30 (clone 49); SEQ ID NO:31 and SEQ ID NO:32 (clone 53); SEQ ID NO:33 and SEQ ID NO:34 (clone 56); SEQ ID NO:35 and SEQ ID NO:36 (clone 57); SEQ ID NO:58 and SEQ ID NO:60 (huA33-huC825); and SEQ ID NO:62 and SEQ ID NO:63 (huA33-mC825).

10. An antibody, comprising: (a) a light chain immunoglobulin variable domain sequence that is at least 95% identical to a light chain immunoglobulin variable domain sequence present in any one of SEQ ID NOs: 9, 10, 17, 21, 24, 26, 28, 30, 32, 34, 36, 60, or 63; and / or (b) a heavy chain immunoglobulin variable domain sequence that is at least 95% identical to a heavy chain immunoglobulin variable domain sequence present in any one of SEQ ID NOs: 5, 6, 15, 19, 23, 25, 27, 29, 31, 33, 35, 58 or 62.

11. An antibody, comprising: (a) an LC sequence that is at least 95% identical to an LC sequence present in any one of SEQ ID NOs: 9, 10, 17, 21, 24, 26, 28, 30, 32, 34, 36, 60, or 63; and / or (b) an HC sequence that is at least 95% identical to an HC sequence present in any one of SEQ ID NOs: 5, 6, 15, 19, 23, 25, 27, 29, 31, 33, 35, 58 or 62.

12. The antibody of any one of claims 8 to 11, which is a chimeric antibody, a humanized antibody or a bispecific antibody.

13. An antibody described in any one of claims 8 to 12, which binds to an epitope of A33 protein comprising at least 5 to 8 consecutive amino acid residues of SEQ ID NO:

57.

14. The antibody of any one of claims 8-13, comprising an IgG1 constant region comprising one or more amino acid substitutions selected from the group consisting of N297A and K322A.

15. An antibody described in any one of claims 8-13, comprising an IgG4 constant region comprising an S228P mutation.

16. A recombinant nucleic acid sequence encoding the antibody of any one of claims 8-15.

17. A recombinant nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7, 8, 11, 12, 16, 18, 20, 22, 59 and 61.

18. A host cell or vector comprising a recombinant nucleic acid sequence according to claim 16 or claim 17.

19. 8. A composition comprising the antibody or antigen-binding fragment of any one of claims 1-7 and a pharma- ceutically acceptable carrier, wherein the antibody or antigen-binding fragment is optionally conjugated to an agent selected from the group consisting of an isotope, a dye, a chromogen, an imaging agent, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA, or any combination thereof.

20. 16. A composition comprising an antibody according to any one of claims 8-15 and a pharma- ceutically acceptable carrier, wherein the antibody is optionally conjugated to an agent selected from the group consisting of an isotope, a dye, a chromogen, an imaging agent, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA or any combination thereof.

21. The antibody of any one of claims 1-4, 6 or 7, wherein the antibody lacks an alpha-1,6-fucose modification.

22. 16. The antibody of any one of claims 8-15, lacking alpha-1,6-fucose modifications.

23. 13. The bispecific antibody of claim 7 or 12, which binds to T cells, B cells, myeloid cells, plasma cells, or mast cells.

24. 13. The bispecific antibody of claim 7 or 12, which binds to CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, TCR gamma / delta, NKp46, KIR, or the small molecule DOTA hapten.

25. 1. A method of treating an A33-related cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of an antibody comprising an HC amino acid sequence and an LC amino acid sequence, each antibody comprising: SEQ ID NO:5 and SEQ ID NO:9 (3A3-H1 / L1); SEQ ID NO:5 and SEQ ID NO:10 (3A3-H1 / L2); SEQ ID NO:6 and SEQ ID NO:9 (3A3-H2 / L1); SEQ ID NO:6 and SEQ ID NO:10 (3A3-H2 / L2); SEQ ID NO:15 and SEQ ID NO:17 (huA33-IgG1(H2L2)); SEQ ID NO:19 and SEQ ID NO:21 (huA33-BsAb); SEQ ID NO:23 and SEQ ID NO:24 (clone 31); SEQ ID NO:25 and SEQ ID NO:26 (clone 32); SEQ ID NO:27 and SEQ ID NO:28 (clone 48); SEQ ID NO:29 and SEQ ID NO:30 (clone 49); SEQ ID NO:31 and SEQ ID NO:32 (clone 53); SEQ ID NO:33 and SEQ ID NO:34 (clone 56); SEQ ID NO:35 and SEQ ID NO:36 (clone 57); SEQ ID NO:58 and SEQ ID NO:60 (huA33-huC825); and SEQ ID NO: 62 and SEQ ID NO: 63 (huA33-mC825), and wherein the antibody specifically binds to and neutralizes A33 activity.

26. 26. The method of claim 25, wherein the A33-associated cancer is colorectal cancer, pseudomyxoma peritonei, appendiceal cancer, pancreatic cancer or gastric cancer.

27. 27. The method of claim 25 or 26, wherein the antibody is administered to the subject separately, sequentially or simultaneously with the additional therapeutic agent.

28. 28. The method of claim 27, wherein the additional therapeutic agent is one or more of an alkylating agent, a platinum agent, a taxane, a vinca agent, an anti-estrogen, an aromatase inhibitor, an ovarian suppressant, a VEGF / VEGFR inhibitor, an EGF / EGFR inhibitor, a PARP inhibitor, a cytostatic alkaloid, a cytotoxic antibiotic, antimetabolite, an endocrine / hormonal agent, or a bisphosphonate therapeutic agent.

29. The method of any one of claims 25-28, wherein the A33-related cancer is colorectal cancer with an MSI genotype.

30. The method of any one of claims 25-28, wherein the A33-associated cancer is colorectal cancer with an MSS genotype.

31. The method of any one of claims 26-30, wherein the colorectal cancer is associated with a KRAS G12D mutation or a p53 mutation.

32. A method for detecting tumors in a subject animal in vivo, comprising the steps of: (a) administering to a subject an effective amount of an antibody according to any one of claims 8-15, the antibody being adapted to localize to tumors expressing A33 and being labeled with a radioisotope; and (b) detecting the presence of a tumor in the subject animal by detecting a level of radioactivity emitted by the antibody that is higher than a reference value.

33. 33. The method of claim 32, wherein the subject is diagnosed with or suspected of having cancer.

34. 34. The method of claim 32 or 33, wherein the level of radioactivity emitted by the antibody is detected using positron emission tomography or single photon emission computed tomography.

35. The method of any one of claims 32-34, further comprising the step of administering to a subject an effective amount of an immunoconjugate comprising an antibody of any one of claims 8-15 conjugated to a radionuclide.

36. 36. The method of claim 35, wherein the radionuclide is an alpha particle emitting isotope, a beta particle emitting isotope, an Auger emitter, or any combination thereof.

37. Beta particle emitting isotopes 86 Y, 90 Y, 89 Sr, 165 Dy, 186 Re, 188 Re, 177 Lu, and 67 37. The method of claim 36, wherein the at least one aryl group is selected from the group consisting of Cu.

38. A kit comprising an antibody according to any one of claims 1-15 and instructions for use.

39. The kit according to claim 38, wherein the antibody according to any one of claims 1 to 15 is conjugated to at least one detectable label selected from the group consisting of a radioactive label, a fluorescent label and a chromogenic label.

40. The kit according to claim 38 or 39, further comprising a secondary antibody which specifically binds to the antibody according to any one of claims 1 to 15.

41. 13. A bispecific antibody according to claim 7 or 12, which binds to a radiolabelled DOTA hapten and an A33 antigen.

42. A method for detecting a solid tumor in a subject in need thereof, comprising the steps of: (a) administering to a subject an effective amount of a conjugate comprising a radiolabeled DOTA hapten and the bispecific antibody of claim 41, said conjugate configured to localize to A33-expressing solid tumors; and (b) detecting the presence of a solid tumor in the subject animal by detecting a level of radioactivity emitted by the conjugate that is higher than a reference value.

43. A method for selecting a subject animal for pretargeting radioimmunotherapy, comprising the steps of: (a) administering to a subject an effective amount of a conjugate comprising a radiolabeled DOTA hapten and the bispecific antibody of claim 41, said conjugate configured to localize to A33-expressing solid tumors; (b) detecting the level of radioactivity emitted by the complex; and (c) selecting the subject animal for pretargeting radioimmunotherapy when the level of radioactivity emitted by the complex is higher than the reference value.

44. 42. A method for increasing tumor sensitivity to radiation therapy in a subject diagnosed with A33-positive cancer, comprising administering to the subject an effective amount of a conjugate comprising a radiolabeled DOTA hapten and the bispecific antibody of claim 41, wherein the conjugate is configured to localize to A33-expressing tumors.

45. 42. A method for treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a conjugate comprising a radiolabeled DOTA hapten and the bispecific antibody of claim 41, wherein the conjugate is configured to localize to A33-expressing tumors.

46. 1. A method for increasing tumor sensitivity to radiation therapy in a subject diagnosed with A33 positive cancer, comprising the steps of: (a) administering an effective amount of the bispecific antibody of claim 41, wherein the bispecific antibody is configured to localize to A33-expressing tumors; and (b) administering to the subject an effective amount of a radiolabeled DOTA hapten, said radiolabeled DOTA hapten configured to bind to the bispecific antibody.

47. 1. A method of treating cancer in an animal subject in need thereof, comprising the steps of: (a) administering an effective amount of the bispecific antibody of claim 41, wherein the bispecific antibody is configured to localize to A33-expressing tumors; and (b) administering to the subject an effective amount of a radiolabeled DOTA hapten, said radiolabeled DOTA hapten configured to bind to the bispecific antibody.

48. 48. The method of claim 46 or 47, further comprising administering to the subject an effective amount of a clearing agent prior to administration of the radiolabeled DOTA hapten.

49. The method of any one of claims 42-48, wherein the subject animal is a human.

50. 49. The method of any one of claims 42-48, wherein the conjugate is administered intravenously, intramuscularly, intraarterially, intrathecally, intravesically, intraocularly, intradermally, intraperitoneally, subcutaneously, intracerebroventricularly, orally, or intranasally.

51. 51. The method of any one of claims 42-50, wherein the radiolabeled DOTA hapten comprises an alpha particle emitting isotope, a beta particle emitting isotope, or an Auger emitter.

52. The radiolabeled DOTA hapten is 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 The method of any one of claims 42-51, comprising Cu.

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