EA046795B1 - COMPOSITIONS BASED ON ANTI-A33 ANTIBODIES AND METHODS OF THEIR APPLICATION IN RADIOIMMUNOTHERAPY - Google Patents

Description

Cross reference to related applications

This application claims the benefit of 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 in their entirety by links.

Field of technology

The technology of the present invention generally relates to the preparation of compositions based on immunoglobulins (eg, antibodies or antigen-binding fragments thereof) that specifically bind to the A33 protein, and to the use of these compositions. In particular, the technology of the present invention relates to the production of A33 neutralizing antibodies and their use for the detection and treatment of A33 associated cancer types.

State of the art

The following description of the prior art is provided solely to facilitate an understanding of the technology of the present invention and is not intended to describe or constitute prior art for the technology of the present invention.

Colorectal cancer (CRC) is the third leading cause of cancer mortality in the United States (Siegel RL, Miller KD, Jemal A, CA: A Cancer Journal for Clinicians 67:7-30 (2017)) and accounts for 10% in men and women. 9.2% in women of all cancer types worldwide (Ferlay J, Soerjomataram I, Dikshit R, et al., International Journal of Cancer 136:E359-E386 (2015)). Despite a steady 3% annual decline in the incidence of this type of cancer from 2004 to 2013, 135,000 new cases are expected in 2017 in the United States alone. Although local and regional disease may be 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 in combination with monoclonal antibodies that block tumor signaling pathways or angiogenesis. To date, four monoclonal antibody drugs have been approved for the treatment of CRC by the US Food and Drug Administration: bevacizumab and ramicirumab target the VEGF-VEGFR angiogenesis pathway, while cetuximab and panitumumab target to the EGFR pathway. However, cetuximab and panitumumab do not provide 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 approximately 40% of all cases mCRC (Bencsikova V, Bortlicek Z, Halamkova J, et al., BMC Gastroenterology 15:37 (2015)). Moreover, the use of these antibodies leads to an overall modest improvement in survival (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 associated with severe side effects. The beneficial effect of immune checkpoint inhibitors (ICIs) was limited to the subgroup of patients with CRC with microsatellite instability (MSI). However, the majority of patients with mCRC have microsatellite stability (MSS) status and are therefore expected to not benefit from ICI monotherapy. To date, the success of using antibodies to deliver toxins to tumors, such as radioimmunotherapy (RIT) with directly conjugated antibodies, is limited in part by the suboptimal dose in tumor tissues and the suboptimal therapeutic index (TI). In addition, the toxic side effect on normal tissue precludes the possibility of increasing the dose, and therefore such therapy leads to limited antitumor effect.

Brief description of the technology according to the present invention

In one aspect, the present invention provides an antibody or antigen binding fragment thereof comprising an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain ( _VL ), wherein (a) the VH comprises the sequence CDR1 VH FTFSTYDMS (SEQ ID NO: 37) , the VH CDR2 sequence TISSGGSYTYYLDSVKG (SEQ ID NO: 38), and the VH CDR3 sequence TTVVPFAY (SEQ ID NO: 39) and/or (b) V _L contains the V _L CDR1 sequence, the V _L CDR2 sequence and the V _L CDR3 sequence 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).

The antibody may further comprise an Fc portion of an immunoglobulin 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 containing one or more amino acid substitutions selected from the group consisting of N297A and K322A. Additionally or alternatively, in some embodiments, the antibody contains an IgG4 constant region containing the S228P mutation. According to specific embodiments of ISO

- 1 046795 bretenie, the antigen-binding fragment is selected from the group consisting of Fab, F(ab')2, Fab', scF _v and F _v . 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 antibody fragment binds to an A33 protein epitope comprising at least five to eight contiguous amino acid residues of SEQ ID NO: 57. In some embodiments, the epitope is a conformational epitope.

According to another aspect, the present invention provides an antibody comprising a heavy chain (HC) amino acid sequence comprising the sequence 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 its a variant containing one or more conservative amino acid substitutions; and/or a light chain (LC) amino acid sequence comprising the sequence 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 containing one or more conservative amino acid substitutions. In specific embodiments, the antibody comprises an HC amino acid sequence and an LC amino acid sequence 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), respectively.

In one aspect, the present invention provides an antibody comprising (a) an immunoglobulin light chain variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or is at least 99% identical to the immunoglobulin light chain variable domain sequence present in any of the sEq ID NO: 9. 10, 17, 21, 24, 26, 28, 30, 32, 34, 36, 60 or 63; And/or (b) an immunoglobulin heavy chain variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the sequence an immunoglobulin heavy chain variable domain present in any of SEQ GO NO: 5, 6, 15, 19, 23, 25, 27, 29, 31, 33, 35, 58 or 62.

According to another aspect, the present invention provides an 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 the LC sequence present in any of SEQ ID NO: 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 the HC sequence present in any of SEQ ID NO: 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 containing one or more amino acid substitutions selected from the group consisting of N297A and K322A. In certain embodiments, an antibody according to the technology of the present invention contains an IgG4 constant region containing the S228P mutation. In any of the above embodiments, the antibody binds to an A33 protein epitope comprising at least five to eight contiguous 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 invention does not contain an α-1,6-fucose modification. According to one aspect, the present invention

- 2 046795 provides a recombinant nucleic acid sequence encoding any of the antibodies described in this application. 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.

According to another aspect, the present invention provides a host cell or vector comprising any of the recombinant nucleic acid sequences described herein.

According to one aspect, the present invention provides a composition comprising an antibody or antigen-binding antibody fragment according to the technology of the present invention and a pharmaceutically acceptable carrier, wherein said antibody or antigen-binding antibody fragment is optionally conjugated to an agent selected from the group consisting of isotopes, dyes , chromogens, contrast agents, drugs, toxins, cytokines, enzymes, enzyme inhibitors, hormones, hormone antagonists, growth factors, radionuclides, metals, liposomes, nanoparticles, RNA, DNA or any combination thereof. In some embodiments of a bispecific antibody according to the technology of the present invention, the bispecific antibody binds to T cells, B cells, myeloid cells, plasma cells, or mast cells. Additionally or alternatively, in some embodiments, the bispecific antibody binds to CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, gamma/delta TCR, NKp46, KIR or low molecular weight DOTA hapten. The low molecular weight DOTA hapten may be selected from the group consisting of

DOTA

DOTA-Bn,

DOTA-desferrioxamine,

DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH2,

Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH ₂ ,

DOTA-D-Asp-D-Lys(HSG)-D-Asp-D-Lys(HSG)-NH ₂ ;

DOTA-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH ₂ ,

DOTA-D-Tyr-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH ₂ ,

DOTA-D-Ala-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH ₂ ,

DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH ₂ ,

Ac-D-Phe-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-NH ₂ ,

Ac-D-Phe-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH2,

Ac-D-Phe-D-Lys(Bz-DTPA)-D-Tyr-D-Lys(Bz-DTPA)-NH ₂ , 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(Tscg-Cys)-NH2, (Tscg- 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, (Tscg-Cys)-D-Glu-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(DTPA)-D-Tyr-D-Lys(DTPA)-NH2,

Ac-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-D-Lys(Tscg-Cys)-NH2 and

Ac-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-D-Lys(Tscg-Cys)-NH2.

According to another aspect, the present invention provides a method of treating A33-associated cancer in a subject in need thereof, comprising administering to said subject an effective amount of any of the antibodies disclosed herein. In specific embodiments, the antibody comprises an HC amino acid sequence and an LC amino acid sequence 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), respectively, wherein the antibody specifically binds to and neutralizes the activity of the A33 protein.

In some embodiments, the A33-associated cancer is colorectal cancer, pseudomyxoma peritonei, appendiceal cancer, pancreatic cancer, or gastric cancer. A33-associated cancer may be colorectal cancer with the MSI genotype or the MSS genotype. Additionally or alternatively, in some embodiments, colorectal cancer is associated with a KRAS G12D mutation or a p53 mutation. Additionally or alternatively, according to some embodiments of the method, the antibody

- 3 046795 is administered to a subject separately, sequentially or simultaneously with an additional therapeutic agent. Examples of additional therapeutic agents include one or more of alkylating agents, platinum-based agents, taxanes, vinca-based agents, anti-estrogen drugs, aromatase inhibitors, ovarian suppressive agents, VEGF/VEGFR inhibitors, EGF/EGFR inhibitors, PARP inhibitors, cytostatic alkaloids, cytotoxic antibiotics, antimetabolites, endocrine/hormonal agents, bisphosphonate therapy agents.

According to another aspect, the present invention provides a method of detecting a tumor in a subject in vivo, comprising (a) administering to said subject an effective amount of an antibody according to the technology of the present invention, wherein said antibody is designed to localize to a tumor expressing A33 and is labeled radioisotope; and (b) detecting the presence of a tumor in the subject by detecting a level of antibody radioactivity greater than a control value. In some embodiments, the subject is diagnosed 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, according to some embodiments of the invention, the method also includes administering to the subject an effective amount of an immunoconjugate comprising an antibody according to the technology of the present invention conjugated to a radionuclide. In some embodiments, the radionuclide is an alpha-emitting isotope, a beta-emitting isotope, an Auger electron-emitting radionuclide, or any combination thereof. Examples of beta-emitting isotopes include ⁸⁶ Y, ⁹⁰ Y, ⁸⁹ Sr, ¹⁶⁵ Dy, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁷⁷ Lu, and ⁶⁷ Cu. In some embodiments of the method, non-specific FcR-dependent binding in normal tissues is eliminated or reduced (eg, due to the N297A mutation in the Fc region, which results in aglycosylation).

In addition, this application describes kits for the detection and/or treatment of A33-associated cancer types containing at least one immunoglobulin composition according to the technology of the present invention (for example, any antibody or antigen-binding antibody fragment described in this application ) or its functional variant (for example, containing a replacement variant) and instructions for use. According to specific embodiments of the invention, the immunoglobulin composition is coupled to one or more detectable tags. According to one embodiment of the invention, the one or more detectable labels include a radioactive label, a fluorescent label, or a chromogenic label.

Additionally or alternatively, in some embodiments, the kit also contains a secondary antibody that specifically binds to the anti-A33 immunoglobulin composition described herein. In some embodiments, the secondary antibody is coupled to at least one detectable label selected from the group consisting of a radioactive label, a fluorescent label, or a chromogenic label.

In one aspect, the present invention provides a method for detecting solid tumors in a subject in need thereof, comprising (a) administering to said subject an effective amount of a complex comprising a radiolabeled DOTA hapten and a bispecific antibody according to the technology of the present invention that binds to said radiolabeled DOTA -hapten and A33 antigen, where the specified complex is designed with the possibility of localization in a solid tumor expressing the A33 antigen, recognized by the bispecific antibody of the complex; and (b) detecting the presence of solid tumors in the subject by detecting a level of radioactivity of the complex in excess of a control value.

According to another aspect, the present invention provides a method of selecting a subject for pretargeted radioimmunotherapy, comprising (a) administering to said subject an effective amount of a complex comprising a radiolabeled DOTA hapten and a bispecific antibody according to the technology of the present invention that binds to said radiolabeled DOTA hapten and the A33 antigen, where the complex is designed to be localized in a solid tumor expressing the A33 antigen, recognized by the bispecific antibody of the complex; (b) detecting the level of radioactivity of the complex; and (c) selecting for pretargeted radioimmunotherapy a subject whose level of radioactivity of the complex exceeds a control value.

In one aspect, the present invention provides a method of sensitizing a tumor to radiation therapy in a subject diagnosed with A33-positive cancer, comprising administering to said subject an effective amount of a complex comprising a radiolabeled DOTA hapten and a bispecific antibody according to the technology of the present invention that recognizes and binds to said radiolabeled DOTA hapten and target A33 antigen, wherein said complex is designed to localize to a tumor expressing the target A33 antigen recognized by the bispecific antibody of the complex.

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According to another aspect, the present invention provides a method of treating cancer in a subject in need thereof, comprising administering to said subject an effective amount of a complex comprising a radiolabeled DOTA hapten and a bispecific antibody according to the technology of the present invention that recognizes and binds to the radiolabeled DOTA hapten and the A33-target antigen, where the specified complex is designed to be localized in a tumor expressing the A33-target antigen, recognized by the bispecific antibody of the complex.

According to any of the above embodiments of the methods described in the present invention, the complex is administered intravenously, intramuscularly, intraarterially, intrathecally, intracaspularly, intraorbitally, intracutaneously, intraperitoneally, transtracheally, subcutaneously, intracerebroventricularly, orally or intranasally. In some embodiments of the methods described in the present invention, the subject is a human. Additionally or alternatively, according to any of the above embodiments of the methods described in the present invention, the radiolabeled DOTA hapten includes 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, 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 contains an alpha-emitting isotope, a beta-emitting isotope or a radionuclide emitting Auger electrons.

In one aspect, the present invention provides a method of sensitizing a tumor to radiation therapy in a subject diagnosed with A33-positive cancer, comprising (a) administering to said subject an effective amount of an anti-DOTA hapten bispecific antibody according to the technology of the present invention, wherein said bispecific antibody anti-DOTA hapten is designed with the possibility of localization in a tumor expressing the A33 target antigen; and (b) administering to said subject an effective amount of a radiolabeled DOTA hapten, wherein said radiolabeled DOTA hapten is designed to bind to a bispecific anti-DOTA hapten antibody. According to another aspect, the present invention provides a method of treating cancer in a subject in need thereof, comprising (a) administering to said subject an effective amount of an anti-DOTA hapten bispecific antibody according to the technology of the present invention, wherein said anti-DOTA hapten bispecific antibody is designed to localize to a tumor, expressing target antigen A33; and (b) administering to said subject an effective amount of a radiolabeled DOTA hapten, wherein said radiolabeled DOTA hapten is designed to bind to a bispecific anti-DOTA hapten antibody. In some embodiments, the methods of the present invention further comprise administering to the subject an effective amount of a clearance agent prior to administration of the radiolabeled DOTA hapten.

Additionally or alternatively, according to any of the above embodiments of the methods described in the present invention, the radiolabeled DOTA hapten contains 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 contains an alpha-emitting isotope, a beta-emitting isotope or a radionuclide, emitting Auger electrons. In any of the above embodiments of the methods described herein, the subject is a human.

Brief description of graphic materials

In fig. 1(A) shows the design and construction of a humanized anti-A33 bispecific antibody (huA33-BsAb). In fig. 1(B) shows the stability study of purified huA33-BsAb by accelerated aging at 37°C for 4 weeks. In fig. Figure 1(C) shows surface plasmon resonance (SPR) analysis of huA33BsAb at 25°C and 37°C. The data obtained resulted in a 1:1 binding model. In fig. 1(D) shows fluorescence-activated cell sorting (FACS) staining of various tumor cell lines and activated T cells. Mean fluorescence intensity (MFI) values are the geometric mean.

In fig. Figure 2(A) shows the activation of CD25 and CD69 markers on T cells 24 hours after incubation with Colo205 cells and various antibodies. In fig. 2(B) shows quantification of dividing cells based on carboxyfluorescein succinimidyl ester (CFSE) dye dilutions 96 hours after incubation with Colo205 cells and various antibodies.

In fig. 2(C) shows representative views of FIG. 2(B). In fig. 2(D) shows staining of CD45RO cells 96 hours after incubation with Colo205 and huA33-BsAb cells in an independent experiment. In fig. 2(E) shows in vivo huA33-BsAb-stimulated T cell activation and proliferation. Briefly, CFSE-labeled peripheral blood mononuclear cells (PBMCs) were mixed with Colo205 cells and the resulting mixture was implanted subcutaneously into DKO mice. The next day

- 5 046795 intravenous injection of huA33-BsAb was performed and tumors were isolated after 4 additional days and analyzed by FACS analysis.

In fig. Figure 3 shows the profile of secreted cytokines and cytotoxic components of activated huA33-BsAb T cells in the presence of a target tumor. The kinetics of cytokine and cytolytic molecule production by T cells in the presence of huA33-BsAb and Colo205 target cells or negative control SKMEL5 cells were determined over 4 days. Because SKMEL5 secreted large amounts of IL-6, the supernatant from T cells incubated with Colo205 cells in the absence of antibody was used as a negative control in the IL-6 kinetics experiment.

In fig. 4(A) shows huA33-BsAb-induced cytotoxicity against various target tumor cell lines and control cells. Activated T cells were used as effector cells with an effector:target (E:T) ratio of 10:1. Cells were incubated for 16 hours before data reading. In fig. 4(B) shows cytotoxicity to Colo205 cells caused by huA33-BsAb. Sorted fresh T cell subsets were used as effector cells (E:T=5:1). Cells were incubated for 48 hours before reading. The EC50 for memory CD4 T cells was 25 pM.

In fig. 5(A) shows a summary of affinity maturation of huA33 (H2L2) using a yeast screen. In fig. Figure 5 (B) shows a summary of the KD of parent and affinity matured huA33-BsAb (top) and an association/dissociation rate map for different huA33-BsAbs obtained from SPR analysis (bottom). In fig. 5(C) shows the results of a T-cell dependent cellular cytotoxicity (TDCC) assay for parental and affinity matured huA33-BsAb.

In fig. 6(A) shows the growth of LS174T tumor upon subcutaneous injection of cells in the treatment group and control groups. Tumor sizes were assessed by volume (p=0.0133 for the tumor+sc PBMC group compared with the tumor+sc PBMC+N33^Al group; p=0.006 for the tumor only group compared with the tumor+sc PBMC+huBsAb-BsAb group) . In fig. Figure 6(B) shows the growth of the LS174T tumor upon intraperitoneal injection of cells in the treatment group and control groups (top) (p = 0.0125 for the tumor + ATC group compared with the tumor + ATC + NAZ3^AL group; p = 0.0026 for tumor only group compared to tumor+ATC+huBsAb-B3Ab group) and survival of mice in the treatment group and control groups (bottom). In fig. 6(C) shows fluorescent images of LS174T tumor upon abdominal injection of cells in different groups. The tumor only group included one mouse (#4) that did not exhibit tumor engraftment after 21 days and was excluded from the analysis.

In fig. 7(A) shows fluorescent images showing the growth of Colo205 tumor upon subcutaneous injection of cells in different groups. In fig. 7(B) shows the quantification of the signals from FIG. 7(A) (top) and the survival rates of mice from different groups from FIG. 7(A) (bottom). In fig. 7(C) shows fluorescent images of SW1222 tumor upon intraperitoneal injection of cells; mouse #1 from the tumor-only group and mouse #3 from the tumor+ATS group, which did not exhibit tumor engraftment after 21 days, were later excluded from the imaging analyzes and were not included in the survival analysis in Fig. 7(D). In fig. Figure 7(D) shows the survival of mice after intraperitoneal injection of SW1222 tumor cells.

In fig. 8(A) shows the growth of SNU16 tumor upon subcutaneous injection of cells in different groups. In fig. 8(B) shows engraftment of human cells from the blood of mice from FIG. 8(A).

In fig. Figure 9 shows the analysis using R&D of 4 variants of humanized antibodies against A33. All antibodies contained the IgG1 constant domain. 3A3-H1L1, 3A3-H1L2, 3A3-H2L1 and 3A3-H2L2 were 4 variants of humanized 3A3. 3A3-chA33 was a chimeric 3A3.

In fig. 10 shows the results of FACS analysis of various cell lines stained with huA33-BsAb.

In fig. 11(A) shows PD-1 stimulation on T cells activated with huA33-BsAb in the presence of Colo205 cells at 24 hours (left) and 96 hours (right). In fig. 11 (B) shows the absence of T cell division after incubation with SKMEL5 cells in the presence of huA33-BsAb after 96 hours. In fig. 11(C) shows activation of T cell division by huA33-BsAb in the presence of LS174T cells. In fig. 11(B) and 11(C) used the same T-cell preparations.

In fig. 12(A) and 12(B) show CD45RO and CD25 staining on T cells 48 hours after incubation with Colo205 cells in the presence of various antibodies. Cells were obtained from the TDCC assay after using the supernatant for lactate dehydrogenase (LDH) measurement. Fig. 12(A): selection of CD4(+) T cells; Fig. 12(B): selection of CD8(+) T cells.

In fig. 13 shows a strategy for quickly converting scFv to huA33-BsAb format. Expression vector (1) was linearized using HindIII/ApaI restriction enzymes. The promoter fragment (3) was prepared from the SapI digest of the promoter-containing vector. Both the vector and the promoter fragment can be produced in large quantities for higher throughput cloning. VH (4) and V _L (2) were amplified directly from yeast using two 5′ primers to add leader sequences and digested with HindIII/SapI (VL) or ApaI/SapI (VH). The 4 fragments were ligated in a one-step reaction.

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In fig. 14 shows the amino acid sequences of the VH and VL domains of the mouse anti-A33 antibody and their corresponding human homologous sequences (SEQ ID NOs: 1-4). The CDR1, CDR2 and CDR3 regions of the VH and VL domains of the mouse anti-A33 antibody are indicated in bold underline.

In fig. 15 shows the amino acid sequences of the humanized heavy chains huA33-H1 (3A3-H1) (SEQ ID NO: 5) and huA33-H2 (3A3-H2) (SEQ ID NO: 6). The CDR1, CDR2 and CDR3 regions of 3A3-H1 and 3A3-H2 are indicated in bold underline.

In fig. 16 shows the cDNA sequences of the humanized heavy chains huA33-H1 (3A3-H1) (SEQ ID NO: 7) and huA33-H2 (3A3-H2) (SEQ ID NO: 8).

In fig. 17 shows the amino acid sequences of the humanized light chains 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 indicated in bold underline.

In fig. 18 shows the cDNA sequences of the humanized light chains huA33-L1 (3A3-L2) (SEQ ID NO: 11) (SEQ ID NO: 11) and huA33-L2 (SEQ ID NO: 12).

In fig. 19 shows an alignment of the original humanized hA33 sequences according to King et al. (1995), higher than the newly re-humanized huA33 (3A3) sequences.

In fig. 20 shows the binding kinetics of humanized huA33 IgG variants that were assayed for binding to recombinant GPA33 protein using SPR (Biacore T100). All four variants retained high binding affinity for chimeric A33 (chA33).

In fig. 21 shows the binding kinetics of parent humanized hA33 (in the hA33-mC825 bispecific format as described in Cheal et al, Eur. J. Nucl. Med. Mol. Imaging, 43:925-937 (2016)) compared to huA33 (3A3 -H2L2) based on the results of binding to the recombinant GPA33 protein using SPR (Biacore T100). The original humanized hA33 lost a significant degree of affinity compared to huA33.

In fig. 22 shows the binding kinetics of parent humanized hA33 (in the bispecific hA33-mC825 format as described in Cheal et al, Eur. J. Nucl. Med. Mol. Imaging, 43:925-937 (2016) compared to huA33 (3A3- H2L2) according to the results of binding to the recombinant GPA33 protein using SPR (Biacore T100) the original humanized hA33 lost a significant degree of affinity compared to huA33.

In fig. 23 shows humanization analysis of huA33 heavy and light chain sequences (3A3-H1, 3A3-H2, 3A3-L1, 3A3-L2) and hA33 parent sequences. Because all four rehumanized variants retained the high binding affinity of the original chA33, the H2L2 variant was selected for further development based on its higher T20 humanization score.

In fig. 24 shows the amino acid sequences of the light chain and heavy chain of chimeric chA33-IgG1, which correspond to SEQ ID NO: 13 and SEQ ID NO: 14, respectively.

In fig. 25 shows the amino acid sequence and heavy chain cDNA sequence of huA33-IgG1 (H2L2), which correspond to SEQ ID NO: 15 and SEQ ID NO: 16, respectively.

In fig. 26 shows the amino acid sequence and light chain cDNA sequence of huA33-IgG1 (H2L2), which correspond to SEQ ID NO: 17 and SEQ ID NO: 18, respectively.

In fig. 27 shows the amino acid sequence and heavy chain cDNA sequence of the huA33-BsAb T cell recruiter bispecific antibodies, which correspond to SEQ ID NO: 19 and SEQ ID NO: 20, respectively.

In fig. 28 shows the amino acid sequence and light chain cDNA sequence of the huA33-BsAb T cell recruiter bispecific antibodies corresponding to SEQ ID NO: 21 and SEQ ID NO: 22, respectively. The underlined sequences correspond to GS linker sequences.

In fig. 29 shows a summary of potential modifications of the huA33-BsAb T cell recruiter bispecific antibodies described in the present invention.

In fig. 30 shows the heavy chain and light chain amino acid sequences of affinity matured clone 31 in huA33-BsAb format corresponding to SEQ ID NO: 23 and SEQ ID NO: 24, respectively. The underlined sequences correspond to GS linker sequences.

In fig. 31 shows the heavy chain and light chain amino acid sequences of affinity matured clone 32 in huA33-BsAb format corresponding to SEQ ID NO: 25 and SEQ ID NO: 26, respectively. The underlined sequences correspond to GS linker sequences.

In fig. 32 shows the heavy chain and light chain amino acid sequences of affinity matured clone 48 in huA33-BsAb format corresponding to SEQ ID NO: 27 and SEQ ID NO: 28, respectively. The underlined sequences correspond to GS linker sequences.

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In fig. 33 shows the heavy chain and light chain amino acid sequences of affinity matured clone 49 in huA33-BsAb format corresponding to SEQ ID NO: 29 and SEQ ID NO: 30, respectively. The underlined sequences correspond to GS linker sequences.

In fig. 34 shows the heavy chain and light chain amino acid sequences of affinity matured clone 53 in huA33-BsAb format corresponding to SEQ ID NO: 31 and SEQ ID NO: 32, respectively. The underlined sequences correspond to GS linker sequences.

In fig. 35 shows the heavy and light chain amino acid sequences of affinity matured clone 56 in huA33-BsAb format corresponding to SEQ ID NO: 33 and SEQ ID NO: 34, respectively. The underlined sequences correspond to GS linker sequences.

In fig. 36 shows the heavy chain and light chain amino acid sequences of affinity matured clone 57 in huA33-BsAb format corresponding to SEQ ID NO: 35 and SEQ ID NO: 36, respectively. The underlined sequences correspond to GS linker sequences.

In fig. 37 shows the amino acid sequence and heavy chain cDNA sequence of the bispecific antibodies huA33-huC825 (H2L2), which correspond to the sequences of SEQ ID NO: 58 and SEQ ID NO: 59, respectively.

In fig. 38 shows the amino acid sequence and light chain cDNA sequence of the bispecific antibodies huA33-huC825 (H2L2), which correspond to the sequences of SEQ ID NO: 60 and SEQ ID NO: 61, respectively. The underlined sequences correspond to GS linker sequences.

In fig. 39 shows the amino acid sequence of the heavy chain and light chain of the bispecific antibodies huA33-mC825 (H2L2), which correspond to the sequences of SEQ ID NO: 62 and SEQ ID NO: 63, respectively. The underlined sequences correspond to GS linker sequences.

In fig. 40 shows ex vivo biodistribution results for a GPA33-positive (GPA33(+)) human colorectal cancer xenograft targeting assay in mice containing subcutaneously administered GPA33(+) SW1222 human colorectal cancer xenografts treated with the rehumanized bispecific antibody huA33-DOTA , described in the present invention, and indicator doses of ¹⁷⁷ Lu-OOTA-biotin.

Detailed Description of the Invention

It should be understood that the specific aspects, types, embodiments, variations and features of the methods of the present invention described below in varying degrees of detail are provided to provide a practical understanding of the technology of the present invention.

The present invention generally provides compositions based on immunoglobulins (eg, antibodies or antigen-binding fragments thereof) that can specifically bind to and neutralize the biological activity of A33 polypeptides. The immunoglobulin compositions of the technology of the present invention are useful in methods of detecting or treating A33-associated cancers in a subject in need thereof. Accordingly, various aspects of the methods of the present invention relate to the production, characterization and manipulation of anti-A33 antibodies. Compositions based on immunoglobulins according to the technology of the present invention can be used alone or in combination with additional therapeutic agents for the treatment of cancer. In some embodiments, the immunoglobulin composition is a humanized antibody, a chimeric antibody, or a bispecific antibody.

The practice of the methods of the present invention utilizes many standard techniques from molecular biology, protein biochemistry, cell biology, immunology, microbiology, and recombinant DNA. See, for example, Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology., the series Methods in Enzymology (Academic Press, Inc., N.Y.); 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 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 the level of polypeptide gene expression products (i.e., the level

- 8 046795 gene translation) are well known in the art and include the use of polypeptide detection methods such as antibody detection and quantitation methods (see also Strachan & Read, Human Molecular Genetics, Second Edition., John Wiley and Sons, Inc. , NY, 1999).

Definitions.

Unless otherwise specified, all technical and scientific terms used in the present invention generally have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. The use of subjects in the singular in the present invention and the attached claims includes the plural of the specified subjects, unless otherwise clearly appears from the context. For example, a cell reference includes a combination of two or more cells, etc. In general, the nomenclature used in the present invention and the laboratory procedures for cell culture, molecular genetics, organic chemistry, analytical chemistry, nucleic acid chemistry and nucleic acid hybridization described below are well known and commonly used in the art.

When used in the present invention, the term approximately with reference to a number generally includes numbers that are within 1%, 5% or 10% range of the specified number (either side, greater or less), unless otherwise specified or follows from the context (except when such a number is less than 0% or greater than 100% of the possible value).

As used herein, administering an agent or drug to a subject includes any method of administering or delivering a compound to a subject to perform its intended function. Administration can be accomplished by any suitable route, including, but not limited to, oral, intranasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), rectal, intrathecal, intratumoral, or topical administration). Administration includes self-administration and administration with the assistance of another subject.

The term adjuvant refers to one or more substances that cause stimulation of the immune system. In this context, an adjuvant is used to enhance the immune response to one or more vaccine antigens or antibodies. The adjuvant can be administered to the subject before, in combination with, or after administration of the vaccine. Examples of chemical compounds used as adjuvants include aluminum compounds, oils, block polymers, immune system-stimulating complexes, vitamins and minerals (for example, vitamin E, vitamin A, selenium and vitamin B12), Quil A (saponins), coating components bacterial and fungal cells (eg, lipopolysaccharides, lipoproteins and glycoproteins), hormones, cytokines and costimulatory factors.

As used herein, the term antibody generally refers to immunoglobulins or immunoglobulin-like molecules, including, for example, but not limited to, IgA, IgD, IgE, IgG and IgM, combinations thereof and similar molecules produced during an immune response in any vertebrate, for example in mammals such as humans, goats, rabbits and mice, as well as in non-mammalian species such as shark immunoglobulins. When used in the present invention, antibodies (including intact immunoglobulins) and antigen-binding fragments specifically bind to a molecule of interest (or a group of molecules of interest with a high degree of similarity) with substantially less binding to other molecules (e.g., antibodies and antibody fragments that have a binding constant for molecule of interest that is at least 10 ³ M ^-1 , at least 10 ⁴ M ^-1 , or at least 10 ⁵ M ^-1 greater than the binding constant for other molecules in the biological sample). The term antibody also includes genetically engineered forms such as chimeric antibodies (such as humanized murine antibodies), heteroconjugate antibodies (such as bispecific antibodies). See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, ^3rd Ed., W. H. Freeman & Co., New York, 1997.

More specifically, an antibody refers to a polypeptide ligand comprising at least an immunoglobulin light chain variable region or an immunoglobulin heavy chain variable region that specifically recognizes and binds to an epitope of an antigen. Antibodies are composed of a heavy and a light chain, each of which contains a variable region called a heavy chain variable region ( _VH ) and a light chain variable region (VL). Together, the VH region and the VL region are responsible for binding to the antigen recognized by the antibody. Typically, immunoglobulin contains heavy (H) chains and light (L) chains linked by disulfide bonds. There are two types of light chains, lambda (λ) and kappa (k). There are five main classes of heavy chains (or isotypes) that determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each heavy and light chain contains a constant region and a variable region (regions are also known as domains). In combination, the variable regions of the heavy and light chains specifically bind to the antigen. The light and heavy chain variable regions contain a framework region that is interrupted by three hypervariable regions, also called complementarity determining regions, or CDRs. The dimensions of the framework region and CDR have been determined (see Kabat et al., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services,

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1991, which is incorporated herein by reference). The Kabat database is now maintained online. The framework sequences of the various light or heavy chains are relatively conserved within species. The antibody framework regions, which include the framework regions constituting the light and heavy chains, largely adopt a β-sheet conformation, and the CDR regions form loops that connect to, and in some cases form part of, the β-sheet structure. Thus, the framework regions function as a scaffold that positions the CDR regions in the correct orientation through intrastrand noncovalent interactions.

CDR regions are primarily responsible for binding to an antigen epitope. The CDR regions of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting at the N terminus, and also typically include identification by the chain on which the particular CDR is located. Thus, CDR3 VH is located in the variable domain of the antibody heavy chain in which it is found, while CDR1 VL is CDR1 from the variable domain of the light chain of the antibody in which it is found. An antibody that binds to the A33 protein has a specific VH and VL region sequence and thus specific CDR sequences. Antibodies with different specificities (ie, different binding sites for different antigens) have different CDRs. Although the CDR regions vary between antibodies, only a limited number of amino acid positions within the CDR regions are directly involved in antigen binding. These positions within the CDR regions are called specificity determining residues (SDRs). The term immunoglobulin compositions as used herein refers to antibodies (including monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, multispecific antibodies, bispecific antibodies, etc.) as well as antibody fragments. An antibody or antigen-binding fragment specifically binds to an antigen.

As used herein, the term antibody polypeptide means antigen-binding fragments of an antibody, including single chain antibodies, which may contain variable region(s) alone or in combination with all or part of the following polypeptide elements: hinge region, CH1, CH2 and _CH2 domains. CH ₃ antibody molecules. The technology also includes any combination of variable region(s) and hinge region, CH ₁ , CH ₂ and CH ₃ domains. Antibody-related molecules useful in the methods of the present invention include, but are not limited to, Fab, Fab' and F(ab') ₂ , Fd, single chain Fv (scFv), single chain disulfide linked Fv (sdFv), and fragments containing a VL or _VH domain. Examples include: (i) Fab fragment, a monovalent fragment consisting of VL, _VH , CL and CH1 domains; (ii) F(ab') ₂ fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment, consisting of the VL and VH domains of one arm of the antibody, (v) a dAb fragment (Ward et al., Nature 341: 544-546, 1989), which consists of a _VH domain; and (vi) a dedicated complementarity determining region (CDR). In this regard, antibody fragments or antigen-binding fragments may comprise a portion of a full-length antibody, its antigen-binding region, or its variable region. 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.

The term bispecific antibody or BsAb as used in the present invention refers to an antibody that can bind simultaneously to two targets that have different structures, for example, two different target antigens, two different epitopes on the same target antigen, or a hapten and a target antigen, or epitope on the target antigen. Many different structures of bispecific antibodies are known in the art. In some embodiments, each antigen-binding fragment of the bispecific antibody comprises VH and/or VL regions; in some such embodiments, the VH and/or VL regions are regions found in a particular monoclonal antibody. In some embodiments, the bispecific antibody contains two antigen-binding fragments, each of which contains V _H and/or V _L regions from different monoclonal antibodies. In some embodiments, the bispecific antibody comprises two antigen binding fragments, wherein one of the two antigen binding fragments comprises an immunoglobulin molecule having V _H and/or V _L regions containing CDR regions from the first monoclonal antibody, and the other antigen binding fragment contains an antibody fragment (for example, Fab, F(ab'), F(ab') ₂ , Fd, Fv, dAB, scFv, etc.) containing VH and/or VL regions that contain CDR regions from a second monoclonal antibodies.

When used in the present invention, the clearance agent is an agent that binds to an excess amount of the bispecific antibody that, when

- 10 046795 is present in the subject's blood to ensure rapid renal clearance. The use of a clearance agent prior to hapten administration (eg, DOTA) helps achieve a better tumor level:background level ratio in pretargeted radioimmunotherapy (PRIT) systems. Examples of clearance agents include 500 kDa dextran-DOTABn(Y) conjugate (Orcutt et al, Mol Cancer Ther. 11(6): 1365-1372 (2012)), 500 kDa aminodextran-DOTA, anti-pretargeting antibody, etc. .

As used herein, the term conjugated refers to the association of two molecules by any method known to one skilled in the art. Suitable types of association include chemical bonds and physical bonds. Chemical bonds include, for example, covalent bonds and coordination bonds. Physical bonds include, for example, hydrogen bonds, bipolar interactions, van der Waals forces, electrostatic interactions, hydrophobic interactions, and aromatic stacking.

As used herein, the term diabodies refers to small antibody fragments with two antigen-binding sites that contain a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain (VH VL). The use of a linker that is too short to allow pairing between two domains on the same chain forces the domains to pair with complementary domains on the other chain and form two antigen-binding sites. Diabodies are described in more detail, for example, in European patent EP 404 097; publication of international application WO 93/11161; and 30 Hollinger et al, Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).

As used herein, the terms single chain antibody or single chain Fv (scFv) refer to the fusion molecule of the two Fv domains of an antibody fragment, VL and VH. Single chain antibody molecules may contain a polymer of several individual molecules, such as a dimer, trimer, or other polymers. Moreover, despite the fact that the two domains of the Fv fragment - VL and VH - are encoded by separate genes, they can be joined using recombinant methods using a synthetic linker, which ensures their production in the form of a single protein chain in which the V _L and V regions _H combine to form a monovalent molecule (known as 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 produced by recombinant methods or by enzymatic or chemical digestion of intact antibodies.

Any of the above antibody fragments are prepared using standard methods known to one of ordinary skill in the art, and said fragments are screened in the same manner as intact antibodies for binding specificity and neutralization activity.

As used herein, the term antigen refers to a molecule to which an antibody (its antigen-binding fragment) can selectively bind. The target antigen may be a protein, carbohydrate, nucleic acid, lipid, hapten, or other natural or synthetic compound. In some embodiments, the target antigen may be a polypeptide (eg, an A33 polypeptide). The antigen can also be administered to an animal to induce an immune response.

The term antigen-binding fragment refers to a fragment of the full-length immunoglobulin structure that contains the portion of the polypeptide responsible for binding to the antigen. Examples of antigen binding fragments useful in the technology of the present invention include, but are not limited to, scFv, (scFv)2, scFvFc, Fab, Fab' and F(ab')2.

Binding affinity refers to the overall strength of non-covalent interactions between one binding site of a molecule (such as an antibody) and its binding partner (such as an antigen or antigenic peptide). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured using standard methods known in the art, including the methods described in the present invention. A low affinity complex contains an antibody that generally tends to dissociate easily from the antigen, whereas a high affinity complex contains an antibody that generally tends to remain associated with the antigen for a longer time.

As used in the present invention, the term biological sample means sample material derived from living cells. Biological samples may include tissues, cells, protein or membrane cell extracts, and biological fluids (eg, ascites fluid or cerebrospinal fluid (CSF)) isolated from the subject, as well as tissues, cells, and fluids present in the subject. Biological samples according to the technology of the present invention include, but are not limited to, samples taken from breast tissue, kidney tissue, cervix, endometrium, head or neck, gallbladder, parotid tissue, prostate gland, 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, mouth, skin, serum, plasma, CSF, sperm

- 11 046795 we, prostate secretion, seminal fluid, urine, feces, sweat, saliva, sputum, mucus, bone marrow, lymph and tear fluid. Biological samples can also be obtained from biopsies of internal organs or tumors. Biological samples may be obtained from subjects for diagnosis or research, or may be obtained from non-diseased subjects for use as controls or for basic research. Samples can be obtained using standard techniques including, for example, venipuncture and surgical biopsy. In specific embodiments, the biological sample is a sample of breast, lung, colon, or prostate tissue obtained by needle biopsy.

As used herein, the term CDR-grafted antibody means an antibody in which at least one CDR region of an acceptor antibody is replaced by a CDR graft from a donor antibody having the desired antigen specificity.

As used herein, the term chimeric antibody means an antibody in which the Fc constant region of a monoclonal antibody from one species (e.g., a mouse Fc constant region) is replaced with an Fc constant region from an antibody of another species (e.g., a human Fc constant region) using recombinant technology. DNA. See generally Robinson et al, international patent application 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, international patent application publication WO 86/01533; Cabilly et al. US Patent No. 4,816,567; Cabilly et al, European patent application 0125,023; Better et al, Science 240: 1041-1043, 1988; Liu et al, Proc. Natl. Acad. Sci. USA 84: 3439-3443, 1987; Liu et al., J. Immunol 139: 3521-3526, 1987; Sun et al, Proc. Natl. Acad. Sci. USA 84: 214-218, 1987; Nishimura et al., Cancer Res 47: 999-1005, 1987; Wood et al., Nature 314: 446-449, 1885; and Shaw et al., J. Natl. Cancer Inst. 80: 1553-1559, 1988.

As used herein, the term consensus FR refers to the framework region (FR) of an antibody in a consensus immunoglobulin sequence. The FR regions of the antibody do not contact the antigen.

When used in the present invention, a control is an alternative sample used in an experiment for comparison. Control can be positive or negative. For example, when the purpose of an experiment is to determine the correlation of the effectiveness of a therapeutic agent in treating a particular type of disease, typically a positive control (a compound or composition that is known to exhibit the desired therapeutic effect) and a negative control (a subject or sample that is not receiving the therapy) are used. or receives a placebo).

As used herein, the term effective amount refers to an amount sufficient to achieve the desired therapeutic and/or prophylactic effect, for example, an amount that results in the prevention or amelioration of a disease or condition described herein, or one or more signs or symptoms, associated with the disease or condition described in this application. In the context of therapeutic or prophylactic use, the amount of the composition administered to a subject will vary depending on the composition, extent, type and severity of the disease and on characteristics of the subject, such as general health, age, sex, body weight and drug tolerance. One skilled in the art will be able to determine appropriate dosages depending on these and other factors. The compositions may also be administered in combination with one or more additional therapeutic compounds. According to the methods described in the present invention, a therapeutic composition can 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 the level of the composition at which the physiological effects of the disease or condition are attenuated or eliminated. A therapeutically effective amount may be administered in one or more administrations.

As used herein, the term effector cell means a cell of the immune system that is involved in the effector phase of the immune response, as opposed to the cognitive and activation phases of the immune response. Examples of cells of the immune system include cells of myeloid or lymphoid origin, for example, lymphocytes (for example, B cells and T cells, including cytolytic 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. An effector cell can induce Antibody dependent cellular cytotoxicity (ADCC), such as a neutrophil that can induce ADCC. For example, monocytes, macrophages, neutrophils, eosinophils and lymphocytes that express FcaR are involved in the specific killing of target cells and the presentation of antigens to other components of the immune system or binding to antigen-presenting cells.

As used in the present invention, the term epitope means a protein determinant

- 12 046795 capable of specifically binding to the antibody. Epitopes typically consist of chemically active surface groups of molecules, such as amino acids or sugar side chains, and usually have specific three-dimensional structure characteristics as well as specific charge characteristics. Conformational and non-conformational epitopes differ in that binding to conformational epitopes, unlike non-conformational epitopes, is eliminated in the presence of denaturing solvents. In some embodiments, an A33 protein epitope is a region of the protein to which anti-A33 antibodies specifically bind according to the technology of the present invention. In some embodiments, the epitope is a conformational epitope. To screen for anti-A33 antibodies that bind to an epitope, a standard cross-blocking assay, such as the one described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed. This assay can be used to determine whether an anti-A33 antibody binds to the same site or epitope as an anti-A33 antibody according to the technology of the present invention. Alternatively or additionally, epitope mapping can be performed using methods known in the art. For example, the antibody sequence may be subject to mutagenesis, such as alanine scanning, to identify contact residues. In various methods, peptides corresponding to different regions of the A33 protein can be used in competition assays with test antibodies or a test antibody and an antibody with a characterized or known epitope.

As used herein, the term expression includes one or more of the following processes: transcription of a gene into precursor mRNA; splicing and other processing of precursor mRNA to produce mature mRNA; mRNA stabilization; translation of mature mRNA into protein (including codon usage and tRNA availability); and, if necessary, glycosylation and/or other modifications of the translation product for proper expression and function.

As used herein, the term gene means a segment of DNA that contains all the information for the regulated biosynthesis of an RNA product, including promoters, exons, introns and other untranslated regions that control expression.

Homology or identity or similarity refers to the similarity in sequence between two peptides or between two nucleic acid molecules. Homology can be determined by comparing positions within each sequence, which can be aligned for comparison. When a position in the sequence being compared is occupied by the same base or amino acid, then 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. That a polynucleotide or region of a polynucleotide (or polypeptide or region of a polypeptide) has a certain percentage sequence identity with another sequence (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90% , 95%, 98%, or 99%) means that in an alignment, the specified percentage of bases (or amino acids) is the same when comparing two sequences. Said alignment and percent homology or sequence identity can be determined using programs known in the art. In some embodiments, default parameters are used for alignment. One alignment program is BLAST, which uses default parameters. Specifically, the programs are BLASTN and BLASTP using the following default parameters:

Genetic code=standard;

filter=none;

strand=both;

cutoff=60;

expect=10;

Matrix=BLOSUM62;

Descriptions=50 sequences;

sort by=HIGH SCORE;

Data bases=non-redundant,

GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swiss Protein+SPupdate+PIR.

Details of these programs can be found at the National Center for Biotechnology Information. Biologically equivalent polynucleotides are polynucleotides that have a certain percentage homology and encode polypeptides that have the same or similar biological activity. Two sequences are considered unrelated or non-homologous if they are less than 40% or less than 25% identical to each other.

When used in the present invention, humanized forms of non-human (eg, murine) antibodies are chimeric antibodies that contain minimal sequence derived from a non-human immunoglobulin. For the most part

- 13 046795 parts humanized antibodies are human immunoglobulins in which the hypervariable region residues of an acceptor antibody are replaced by the hypervariable region residues of an antibody from a non-human species (such as a mouse, rat, rabbit or non-human primate) having the desired specificity, affinity and capacity (of the antibody donor). In some embodiments, the human immunoglobulin Fv framework region (FR) residues are replaced with corresponding non-human residues. Moreover, humanized antibodies may contain residues that are not found in the acceptor or donor antibody. Such modifications are made to further improve antibody characteristics such as binding affinity. In general, a humanized antibody contains substantially all of at least one, typically two, variable domains (e.g., Fab, Fab', F(ab')2, or Fv) in which all or substantially all of the hypervariable loops correspond to hypervariable loops immunoglobulin of non-human origin, and all or substantially all of the FR regions are human immunoglobulin FR consensus sequence regions, although the FR regions may contain one or more amino acid substitutions that increase binding affinity. The number of specified amino acid substitutions in the FR region, as a rule, is no more than 6 for the H chain and no more than 3 for the L chain. The humanized antibody may also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically human immunoglobulin. For more information, see Jones et al., Nature 321:522-525 (1986); Reichmann et al., Nature 332:323-329 (1988); Presta, Curr. Op.Struct. Biol. 2:593-596 (1992). See, for example, Ahmed & Cheung, FEBS Letters 588(2):288-297 (2014).

As used herein, the term hypervariable region refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region generally contains amino acid residues from the complementarity determining region, or CDR, (for example, approximately the region of residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in V _L and approximately the region of residues 31-35B (H1), 50-65 (H2) and 95-102 (H3) in VH (Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and/or specified residues from the hypervariable loop (for example, residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in V _l u 26-32 (H1), 52A-55 (H2) and 96-101 (H3) in V _H (Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987)).

As used herein, the terms identical or percentage identity, when used in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or partial sequences that are the same or have a certain percentage of identical amino acid residues or nucleotides, i.e. have approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 identity in the indicated region %, 99% or more (e.g., the nucleotide sequence encoding an antibody described herein or the amino acid sequence of an antibody described herein), when compared and aligned for maximum agreement within the comparison window or designated region as measured using BLAST or BLAST 2.0 sequence comparison algorithms with the default parameters described below, or using manual alignment and visual scoring, such as using the NCBI website). Such sequences are then said to be substantially identical. The term also refers or may be applied to a sequence complementary to the sequence of interest. The term also includes sequences that have deletions and/or additions, as well as sequences that have substitutions. In some embodiments, identical regions are regions of at least about 25 amino acids or nucleotides or 50-100 amino acids or nucleotides in length.

As used herein, the term intact antibody or intact immunoglobulin means an antibody that contains at least two heavy (H) chain polypeptides and two light chain (L) polypeptides linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as HCVR or V _H ) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, _CH2 and _CH3 . Each light chain consists of a light chain variable region (abbreviated herein as LCVR or V _L ) and a light chain constant region. The light chain constant region consists of a single domain, C _L . The V _H and V _L regions can be further divided into regions of hypervariability called complementarity determining regions (CDRs) interspersed with regions that are more conserved called framework regions (FR). Each VH and VL consists of three CDRs and four FRs, located from the amino terminus to the carboxy terminus in the following order: FR _b CDR,. FR2, CDR2, FR ₃ , CDR ₃ , FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with the antigen. The constant regions of antibodies can mediate the binding of immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement pathway.

When used in the present invention, the terms individual, patient or subject may

- 14 046795 be a separate organism, vertebrate, mammal or human. In some embodiments, the individual, patient, or subject is a human.

The term monoclonal antibody as used in the present invention refers to an antibody obtained from a population of essentially homogeneous antibodies, i.e. The individual antibodies that make up the population are identical, with the exception of possible natural mutations that may be present in small quantities. For example, a monoclonal antibody may be an antibody derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, but not the method by which the antibody is produced. A monoclonal antibody composition exhibits binding specificity and affinity only for a specific epitope. Monoclonal antibodies are highly specific and directed against a single antigenic site. Moreover, unlike standard (polyclonal) antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single antigen determinant. The definition of monoclonal refers to the characteristic of an antibody that it is obtained from a substantially homogeneous population of antibodies and is not limited to the production of the antibody by any particular method. Monoclonal antibodies can be produced using a wide variety of methods known in the art, including, for example, but not limited to, hybridoma techniques, recombinant technologies and phage display technologies. For example, monoclonal antibodies for use in accordance with the methods of the present invention can be created using the hybridoma method first described by Kohler et al. (Kohler et al., Nature 256:495 (1975)), or can be created using recombinant DNA methods (See, for example, US Patent No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using the methods described in Clackson et al, Nature 352:624-628 (1991), Marks et al, J. Mol. Biol. 222:581-597 (1991), for example.

As used herein, the term pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal compounds, isotonic and absorption delaying compounds, etc., compatible with pharmaceutical administration. Pharmaceutically acceptable carriers and dosage forms thereof are known to one skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences ( ^20th edition, ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, Pa.).

As used herein, the term polyclonal antibody means a preparation of antibodies derived from at least two (2) different antibody-producing cell lines. The term includes preparations of at least two (2) antibodies that contain antibodies that specifically bind to different epitopes or regions of the antigen.

As used herein, the term polynucleotide or nucleic acid means any RNA or DNA, which may be unmodified or modified RNA or DNA. Polynucleotides include, but are not limited to, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, RNA that is a mixture of single- and double-stranded regions, and hybrid molecules containing DNA and RNA, which can be single-stranded or, more often, double-stranded, or a mixture of single- and double-stranded regions. In addition, polynucleotide refers to three-stranded regions containing RNA or DNA or RNA and DNA. The term polynucleotide also includes DNA or RNA molecules containing one or more modified bases, and DNA or RNA molecules having scaffolds modified to enhance stability or for other purposes.

As used in the present invention, the terms polypeptide, peptide and protein are used interchangeably and mean a polymer containing two or more amino acids linked to each other by peptide bonds or modified peptide bonds, i.e. peptide isosteres. A polypeptide refers to both short chains, generally called peptides, glycopeptides or oligomers, and long chains, generally called proteins. Polypeptides may contain amino acids other than the 20 amino acids encoded by the genes. Polypeptides include amino acid sequences modified by natural processes such as post-translational processing or by chemical modification techniques that are well known in the art. Such modifications are well described in basic documents and more detailed monographs, as well as in multi-volume scientific literature.

As used herein, PRRT or pretargeted radioimmunotherapy refers to a multistep process that addresses the problem of slow renal clearance of tumor-targeting antibodies contributing to unwanted toxicity to normal tissues such as bone marrow. In pretargeted therapy, a radionuclide or other diagnostic or therapeutic agent is attached to a small molecule hapten. First, a bispecific antibody for pretargeted therapy is administered, which has binding sites for the hapten as well as the target antigens. The unbound antibody is then allowed to clear from the bloodstream and the hapten is then administered.

- 15 046795

When used in the present invention, the term recombinant in relation to, for example, a cell or nucleic acid, protein or vector indicates that the specified cell, nucleic acid, protein or vector has been modified by introducing a heterologous nucleic acid or protein or by altering a native nucleic acid or protein or that the material comes from a cell so modified. Thus, for example, recombinant cells express genes that are not found in the native (non-recombinant) form of the cells, or express native genes that would otherwise be abnormally expressed, underexpressed, or completely absent.

As used herein, the term separate therapeutic administration 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 administration refers to the administration of at least two active ingredients at different times, where the modes of administration are the same or different. More specifically, sequential administration refers generally to the administration of one of the active ingredients before the administration of the other or other elements. Thus, it is possible to administer one of the active ingredients several minutes, hours or days before the administration of the other active ingredient or ingredients. In this case, simultaneous treatment does not occur.

As used herein, the term specifically binds refers to a molecule (eg, an antibody or an antigen-binding fragment thereof) that recognizes and binds to another molecule (eg, an antigen), but which does not substantially recognize or bind to other molecules. The terms specific binding, specifically binds to, or specific to a particular molecule (e.g., a polypeptide or a polypeptide epitope) as used herein can refer, for example, to a molecule having a KD for the molecule to which it binds of up to about 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 M. The term specific binding can also refer to binding wherein a molecule (eg, an antibody or an antigen-binding fragment thereof) binds to a specific polypeptide (eg, an A33 polypeptide) or an epitope on a specific polypeptide without substantially binding to any other polypeptide or epitope of the polypeptide.

As used herein, the term simultaneous therapeutic use refers to the administration of at least two active ingredients in the same manner 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.

Treating or treatment as used in the present invention includes treating a disease or disorder described in the present invention in a subject, such as a human, and includes: (i) suppressing said disease or disorder, i.e. restraining its development; (ii) alleviation of said disease or disorder, i.e. stimulation of regression of this disorder; (iii) slowing the progression of said disorder and/or (iv) suppressing, alleviating or slowing the progression of one or more symptoms of said disease or disorder. In some embodiments, the term treatment means that symptoms associated with a disease are alleviated, reduced, cured, or put into remission, for example.

It should also be understood that the various methods of treating disorders described in the present invention relate to essentially treatment, including complete treatment, as well as incomplete treatment, in which some biologically or medically relevant result is achieved. Treatment may be continuous, prolonged treatment for a chronic condition, or single or multiple administrations for the treatment of an acute condition.

Modification(s) of the amino acid sequence of the anti-A33 antibodies described in the present invention are contemplated. For example, the modification may be desired to improve binding affinity and/or other biological properties of the antibody. Anti-A33 antibody amino acid sequence variants are generated by introducing suitable nucleotide changes into the antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletions, insertions and substitutions can be made to produce the antibody of interest, as long as the resulting antibody has the desired properties. The modification also involves changing the protein glycosylation pattern. Sites of greatest interest for substitution mutagenesis include hypervariable regions, but changes in FR are also being considered. Conservative substitutions are shown in the table below.

- 16 046795

Amino acid substitutions

Original balance Examples of substitutions Conservative Substitutions Ala (A) val; leu; ile val Arg(R) lys; gin; asn lys Asn(N) gin; his; asp, lys; arg gin Asp (D) glu; asn glu Cys(C) ser; ala ser Gln(Q) asn; glu asn Glu(E) asp; gin asp Gly(G) ala ala His(H) asn; gin; lys; arg arg He(I) leu; val; met; ala; phe; norleucine leu Leu (L) norleucine; ile; val; met; ala; phe ile Lys (K) arg; gin; asn arg Met(M) leu; phe; ile leu Phe (F) leu; val; ile; ala; tyr tyr Pro (P) ala ala Ser(S) thr thr Thr (T) ser ser Trp(W) tyr; phe tyr Tyr(Y) trp; phe; thr; ser phe Vai (V) ile; leu; met; phe; ala; norleucine leu

One type of substitution variant involves replacing one or more hypervariable region residues of the parent antibody. A convenient method for generating such substitution variants involves affinity maturation using phage display. Specifically, mutations are introduced at several hypervariable region sites (eg, sites 6-7) to create all possible amino acid substitutions at each site. Antibody variants generated in this manner are displayed in monovalent form on filamentous phage particles as fusion molecules with the M13 gene III product packaged in each particle. The phage displayed variants are then screened for their biological activity (eg, binding affinity) as described in the present invention. To identify hypervariable region sites that are candidates for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively or additionally, analysis of the crystal structure of the antigen-antibody complex may be preferred to identify contact points between the antibody and the antigen. Such contact residues and adjacent residues are candidates for replacement in accordance with the methods developed in the present invention. After creating these options, the options panel is subjected to

- 17 046795 is screened as described in the present invention, and antibodies with similar or superior properties based on the results of one or more relevant assays can be selected for further development.

CRC and A33.

CRC is a heterogeneous disease and can be divided into 4 consensus molecular subtypes (CMS) based on gene expression analysis, i.e. CMS1-4. MSI tumors primarily belong to CMS1 (14% of all CRC patients) and are characterized by genomic hypermutation and microsatellite instability due to deficiencies in DNA repair pathways. Presumably, hypermutation creates a variety of neoantigens that are presented on the cell surface and attract T cells to tumors. Indeed, CMS 1 has strong molecular signatures of activation and evasion of immune recognition. ICIs act by activating repressed tumor-infiltrating lymphocytes (TILs) to restore their tumoricidal properties and thus MSI tumors are the most susceptible to ICI CRC. However, MSI tumors account for <5% of mCRC; for most mCRCs, the effectiveness of ICI has so far been disappointing. Peritoneal carcinomatosis usually represents the final phase of incurable CRC. Unlike liver and lung metastases, it is usually inoperable and does not respond to chemotherapy and radiation, resulting in significant mortality. Current treatment is primarily palliative, consisting of cytoreductive surgery (CRS) and hyperthermic chemotherapy (HIPEC), which are effective only in a small percentage of patients with low-volume disease.

Human glycoprotein A33 (GPA33 or A33) is a type I membrane protein with a single transmembrane domain that belongs to the CTX family of cell adhesion molecules within the immunoglobulin family. A33 is expressed in 95% of CRC tissues with very limited expression in normal tissues. A33 contains one C2 type Ig-like domain and one V-type Ig-like domain. The predicted mature protein includes a single transmembrane domain, an extracellular region, and an intracellular tail. A33 plays a role in intracellular trafficking, intercellular recognition/signaling, and recycling to the cell surface. The amino acid sequence of the A33 ectodomain (Ile22-Val235) is presented below:

ISVETPQDVLRASQGKSVTLPCTYHTSTSSREGLIQWDKLLLTHTERVVIWPFSNKNYIH

GEL YKNRVSISNNAEQ SD A S IT IDQLTM A DNGT YEC S VSLMSDLEGNTKSRVRLLVLVPP SKPECGIEGETIIGNNIQLTCQSKEGSPTPQYSWKRYNILNQEQPLAQPASGQPVSLKNIST DTSGYYICTSSNEEGTQFCNITVAVRSPSMNV (SEQ Ш NO: 57).

Compositions based on immunoglobulins according to the technology of the present invention.

Existing humanized anti-A33 IgG1 antibodies have been found to be immunogenic in patients suffering from colon cancer. See Ritter G et al., Cancer Res 61:6851-9 (2001). The technology of the present invention describes methods and compositions for creating and using compositions based on anti-A33 immunoglobulin (eg, anti-A33 antibodies or antigen-binding fragments thereof). The anti-A33 immunoglobulin compositions of the present invention may be useful in the diagnosis or treatment of A33-positive cancers. Anti-A33 immunoglobulin compositions within the scope of the technology of the present invention include, for example, but are not limited to, monoclonal, chimeric, humanized antibodies and diabodies that specifically bind to a target polypeptide, homologue, derivative or fragment thereof. The present invention also provides antigen binding fragments of any of the anti-A33 antibodies described herein, wherein said antigen binding fragment is selected from the group consisting of Fab, F(ab)'2, Fab', scF _v and F _v .

In one aspect, the technology of the present invention provides an antibody or antigen-binding fragment thereof comprising an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), wherein (a) the VH comprises the sequence

VH-CDR1 FTFSTYDMS (SEQ ID NO: 37) sequence

VH-CDR2 TISSGGSYTYYLDSVKG (SEQ ID NO: 38) and sequence

VH-CDR3 TTVVPFAY (SEQ ID NO: 39) and/or (b) V _L contains a VL-CDR1 sequence, a VL-CDR2 sequence and a VL-CDR3 sequence 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). In some embodiments, the antibody also comprises an immunoglobulin Fc domain of any isotype, including, but not limited to, IgG (including IgG1, IgG2, IgG3, and IgG4), IgA (including IgA ₁ and IgA ₂ ), IgD, IgE, or IgM, and IgY. Non-limiting examples of constant sequences

- 18 046795 areas include the following sequences:

Human IgD constant region, Uniprot database: P01880 (SEQ ID NO: 46)

APTKAPDVFPIISGCRHPKDNSPVVLACLITGYHPTSVTVTWYMGTQSQPQRTFPEIQRR DSYYMTSSQLSTPLQQWRQGEYKCVVQHTASKSKKEIFRWPESPKAQASSVPTAQPQA EGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAV QDLWLRDKATFTCF VVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRL TLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLL CEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATY TCVVSHEDSRTLLNASRSLEVSYVTDHGPMK

Human IgG1 constant region, Uniprot database: P01857 (SEQ ID NO:47)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVP S S SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Human IgG2 constant region, Uniprot database: P01859 (SEQ ID NO:48)

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNST FRVVSVLTVVHQDW LNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEM TKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Human IgG3 constant region, Uniprot database: P01860 (SEQ ID NO:49)

ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSC DTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVQ FKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMH EALHNRFTQKSLSLSPGK

Human IgM Constant Region, Uniprot Database: P01871 (SEQ ID NO: 50)

GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITLSWKYKNNSDISSTRGFPSVLR GGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPP RDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKV TSTLTIKESDWLGQSM FTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKS TKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGE RFCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPAD VFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEE WNTGETYTCVAHEA LPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY

Human IgG4 constant region, Uniprot database: P01861 (SEQ ID NO:51)

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNST YRVVSVLTVL HQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSLSLSLGK

- 19 046795

Human IgA1 constant region, Uniprot database: P01876 (SEQ ID NO:52) ASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQDAS GDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPS CCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVT FTWTPSSGKSAVQGPPERDLC GCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEEL ALNELVVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILR VAAEDWI<I<GDTFSCMVGHEALPLAFTQI<TIDRLAGI<PTHVNVSVVMAEVDGTCY

Human IgA2 constant region, Uniprot database: P01877 (SEQ ID NO:53) ASPTSPKVFPLSLDSTPQDGNWVACLVQGFFPQEPLSVTWSESGQNVTARNFPPSQDAS

GDLYTTSSQLTLPATQCPDGKSVTCHVKHYTNPSQDVTVPCPVPPPPPCCHPRLSLHRPA LEDLLLGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGC AQPWNHGETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLPPPSEELALNELVTLTCLA RGFSPKDVLVRWLQGSQELPREKYLTWASRQ EPSQGTTTFAVTSILRVAAEDWKKGDT FSCMVGHEALPLAFTQKTIDRMAGKPTHVNVSVVMAEVDGTCY

Human Ig-kappa constant region, Uniprot database: P01834 (SEQ ID NO: 54) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD

SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

In some embodiments, the immunoglobulin compositions of the technology of the present invention contain a heavy chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, is at least 99% or 100% identical to the sequences of SEQ ID NO: 46-53. Additionally or alternatively, according to some embodiments of the invention, immunoglobulin compositions according to the technology of the present invention contain a light chain constant region that is at least 80%, at least 85%, at least 90%, is at least 95%, at least 99%, or equal to 100% identical to the sequence ID NO: 54. In some embodiments, immunoglobulin compositions according to the technology of the present invention bind to an epitope of the A33 polypeptide containing at least five up to eight consecutive amino acid residues of the sequence SEQ ID NO: 57. In some embodiments, the epitope is a conformational epitope.

According to another aspect, the present invention provides an isolated immunoglobulin composition (eg, an antibody or an antigen-binding fragment thereof) 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.

Additionally or alternatively, in some embodiments, the immunoglobulin compositions of the technology of the present invention comprise a light chain (LC) amino acid sequence comprising the sequence 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 containing one or more conservative amino acid substitutions.

In some embodiments, the immunoglobulin compositions of the technology of the present invention comprise an HC amino acid sequence and an LC amino acid sequence 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);

- 20 046795

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), respectively.

In any of the above embodiments of immunoglobulin-based compositions, the sequences of the variable domains HC and LC of the immunoglobulin form an antigen binding site that binds to an epitope of the A33 polypeptide containing at least five to eight 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 variable domain sequences of an immunoglobulin are components of the same polypeptide chain. According to other embodiments of the invention, the sequences of the variable domains HC and LC of the immunoglobulin are components of different polypeptide chains. In certain embodiments, the antibody is a full-length antibody.

In some embodiments, the immunoglobulin compositions of the present invention specifically bind to at least one A33 polypeptide. In some embodiments, immunoglobulin compositions according to the technology of the present invention bind to at least one A33 polypeptide with a dissociation constant (KD) of about 10 ^-3 M, 10 ^-4 M, 10 ^-5 M, 10 ^-6 M, 10 ^-7 M, 10 ^-8 M, 10 ^-9 M, 10 ^-10 M, 10 ^-11 M, or 10 ^-12 M. In specific embodiments of the invention, the immunoglobulin compositions are monoclonal antibodies, chimeric antibodies, humanized antibodies or bispecific antibodies. In some embodiments, the antibodies comprise a framework region of a human antibody.

According to specific embodiments of the invention, the immunoglobulin composition has one or more of the following characteristics: (a) the sequence of the immunoglobulin light chain variable domain is at least 80%, at least 85%, at least 90%, at least is at least 95% or at least 99% identical to the immunoglobulin light chain variable domain sequence present in any of SEQ ID NO: 9. 10, 17, 21, 24, 26, 28, 30, 32, 34, 36, 60 or 63; and/or (b) the immunoglobulin heavy chain variable domain sequence is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the variable domain sequence immunoglobulin heavy chain present in any 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 in the compositions based on immunoglobulins proposed in the present invention, are replaced by another amino acid. The replacement may be a conservative replacement as defined herein.

In some embodiments, the immunoglobulin composition comprises (a) an LC sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the LC sequence present in any of SEQ ID NO: 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 the HC sequence present in any of SEQ ID NO: 5, 6, 15, 19, 23, 25, 27, 29, 31, 33, 35, 58 or 62.

In specific embodiments, the immunoglobulin compositions comprise an IgG1 constant region containing one or more amino acid substitutions selected from the group consisting of N297A and K322A. Additionally or alternatively, according to some embodiments of the invention, the immunoglobulin compositions contain an IgG4 constant region containing the S228P mutation.

In some aspects, the anti-A33 immunoglobulin compositions described herein have structural modifications to facilitate rapid binding and uptake by cells and/or provide slow release. In some aspects, an anti-A33 immunoglobulin composition according to the technology of the present invention (eg, an antibody) may contain a deletion in the heavy chain CH2 constant region to facilitate rapid binding and uptake into the cell and/or provide slow release. In some aspects, the Fab fragment is used to facilitate rapid binding and uptake by the cell and/or to provide slow release. In some aspects, the F(ab)' ₂ moiety is used to facilitate rapid binding and uptake by the cell and/or provide slow release.

In one aspect, the technology of the present invention provides a nucleic acid sequence encoding the heavy chain or light chain of the immunoglobulin composition described herein. Also disclosed herein are recombinant nucleic acid sequences encoding any of the antibodies described herein. According to some embodiments of the invention, the nucleino sequence

- 21046795 voic acid 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 of the present invention provides a host cell expressing any a nucleic acid sequence encoding the heavy chain or light chain of the immunoglobulin composition described herein.

Compositions based on immunoglobulins according to the technology of the present invention (eg, anti-A33 antibody) may be monospecific, bispecific, trispecific, or have a higher degree of multispecificity. Multispecific antibodies may be specific for different epitopes of one or more A33 polypeptides or for A33 polypeptide(s), as well as heterologous compositions such as a heterologous polypeptide or a solid support material (support material. See, for example, International Patent Publication Application WO 93/17715; International Patent Application Publication WO 92/08802; International Patent Application Publication WO 92/05793; ; US patents No. 5,573,920, 4,474,893, 5,601,819, 4,714,681, 6,106,835; Kostelny et al., J. Immunol. 148: 1547-1553 (1992). bulins are chimeric antibodies. In specific embodiments of the invention, the immunoglobulin compositions are humanized antibodies.

Immunoglobulin compositions according to the technology of the present invention may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalent and non-covalent conjugation) to polypeptides or other compositions. For example, immunoglobulin compositions according to the technology of the present invention can be recombinantly fused or conjugated to molecules useful as tags in detection assays and effector molecules, such as heterologous polypeptides, drugs or toxins. See, for example, international patent application publication WO 92/08495; publication of international patent application WO 91/14438; publication of international patent application WO 89/12624; US patent No. 5,314,995 and EP 0 396 387.

According to any of the above embodiments of immunoglobulin compositions according to the technology of the present invention, the antibody or antigen-binding fragment of an antibody may optionally be conjugated to an agent selected from the group consisting of isotopes, dyes, chromogens, contrast agents, drugs, toxins , cytokines, enzymes, enzyme inhibitors, hormones, hormone antagonists, growth factors, radionuclides, metals, liposomes, nanoparticles, RNA, DNA, or any combination thereof. For chemical bonding or physical bonding, the functional group of the immunoglobulin composition is typically bonded to the functional group of the agent. Alternatively, the functional group of the agent is coupled to the functional group of the immunoglobulin composition.

The functional groups of the agent and immunoglobulin compositions can bind directly. For example, a functional group (eg, a sulfhydryl group) of the agent can bind to a functional group (eg, a sulfhydryl group) of an immunoglobulin composition to form a disulfide. Alternatively, functional groups can be connected through a cross-linking agent (ie, a linker). Some examples of cross-linking agents are described below. The cross-linker can be attached to either the agent or the immunoglobulin composition. The amount of immunoglobulin agent or composition in the conjugate is also limited by the number of functional groups present on the binding partner. For example, the maximum amount of agent bound to the conjugate depends on the number of functional groups present on the immunoglobulin composition. Alternatively, the maximum amount of immunoglobulin composition bound to the agent depends on the number of functional groups present on the agent.

According to another embodiment of the invention, the conjugate contains one immunoglobulin composition associated with one agent. According to one embodiment of the invention, said conjugate comprises at least one agent chemically bound (eg, conjugated) to at least one immunoglobulin-based composition. The agent can be chemically coupled to the immunoglobulin composition using any method known to one skilled in the art. For example, a functional group on the agent can be directly attached to a functional group on the immunoglobulin composition. Some examples of suitable functional groups include, for example, an amino group, a carboxy group, a sulfhydryl group, a maleimide group, an isocyanate group, an isothiocyanate group, and a hydroxyl group.

The agent may also be chemically linked to the immunoglobulin composition through cross-linking agents such as dialdehydes, carbodiimides, dimaleimides, etc. Cross-linking agents can, for example, be obtained from Pierce Biotechnology, Inc., Rockford, IL (for assistance, see the Pierce Biotechnology, Inc. website). Additional cross-linkers include platinum-based cross-linkers from Kreatech

- 22 046795

Biotechnology, B.V., Amsterdam, Netherlands, described in US Patent No. 5,580,990; 5,985,566; and 6,133,038.

Alternatively, the functional group on the agent and immunoglobulin compositions may be the same. Homobifunctional cross-linkers are typically used to cross-link identical functional groups. Examples of homobifunctional cross-linkers include EGS (i.e. ethylene glycol bis[succinimidyl succinate]), DSS (i.e. disuccinimidyl suberate), DMA (i.e. dimethyl adipimidate, 2HCl), DTSSP (i.e. 3,3' -dithiobis[sulfosuccinimidylpropionate])), DPDPB (i.e. 1,4-di-[3'-(2'-pyridyldithio)-propionamido]butane) and BMH (i.e. bismaleimidohexane). Such homobifunctional cross-linkers are also available from Pierce Biotechnology, Inc.

In other examples, it may be preferable to cleave the agent from the immunoglobulin composition. The Pierce Biotechnology, Inc. website described above may also assist one of ordinary skill in the art in selecting suitable cross-linkers that can be cleaved, for example, by enzymes in the cell. Thus, the agent can be separated from the immunoglobulin composition. Examples of cleavable linkers include SMPT (i.e., 4-succinimidyloxycarbonylmethyl-a-[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).

According to another embodiment of the invention, the conjugate contains at least one agent physically associated with at least one immunoglobulin-based composition. Any method known to those skilled in the art can be used to physically bind agents to immunoglobulin compositions. For example, immunoglobulin compositions and agents can be mixed together using any method known to those skilled in the art. The order of mixing is not important. For example, agents can be physically mixed with immunoglobulin compositions using any method known to those skilled in the art. For example, the immunoglobulin compositions and agents can be placed in a container and agitated, for example by shaking said container, to mix the immunoglobulin compositions and agents.

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

A. Methods for producing antibodies against A33 according to the technology of the present invention.

General review. First, a target polypeptide is selected to which an antibody can be generated according to the technology of the present invention. For example, an antibody may be generated against the full length A33 protein or a portion of the extracellular domain of the A33 protein. Methods for generating antibodies directed to such target polypeptides are well known to one skilled in the art. Examples of such methods include, for example, but are not limited to, methods involving display libraries, xenohybridomas or human-mouse hybridomas, etc. Target polypeptides within the scope of the technology of the present invention include any polypeptide derived from the A33 protein containing an extracellular domain capable of inducing an immune response. The production of antibodies specific for the A33 protein is illustrated in Examples 1, 2, 3 and 5.

It should be understood that recombinantly engineered antibodies and antibody fragments, such as antibody-related polypeptides that target the A33 protein and fragments thereof, are suitable for use in accordance with the present invention.

Antibodies against A33 that can be used in the methods set forth herein include monoclonal and polyclonal antibodies and antibody fragments such as Fab, Fasb', F(ab') ₂ , Fd, cFv, diabodies, antibody light chains, heavy chains antibodies and/or antibody fragments. Methods have been described that are useful for the highly efficient production of polypeptides containing Fv antibody fragments, for example, Fab' and F(ab') ₂ antibody fragments. See US Patent No. 5,648,237.

In general, the antibody is obtained from the original species. More specifically, a nucleic acid sequence or amino acid sequence of a variable portion of a light chain, a heavy chain, or both chains of an antibody of the parent species having specificity for a target polypeptide antigen is obtained. A parent species is any species useful for generating an antibody according to the technology of the present invention or an antibody library, for example, rat, mouse, rabbit, chicken, monkey, human, etc.

Phage or phagemid display technologies are methods useful for producing antibodies according to the technology of the present invention. Methods for generating and cloning monoclonal antibodies are well known to one skilled in the art. Expression of sequences encoding antibodies according to the technology of the present invention can be carried out

- 23 046795 infuse into E. coli.

Due to the degeneracy of nucleic acid encoding sequences, other sequences that encode substantially the same amino acid sequences as naturally occurring protein sequences may be used in the practice of the technology of the present invention. These sequences include, but are not limited to, nucleic acid sequences, including whole or portions of nucleic acid sequences encoding the above polypeptides, which are altered by substitution of various codons encoding a functionally equivalent amino acid residue within the sequence, thereby resulting in silent change. It should be understood that the nucleotide sequence of an immunoglobulin in accordance with the technology of the present invention can tolerate homologous variation of up to 25% of the sequence, as calculated using 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 variant results in a functioning antibody that recognizes the A33 proteins. For example, one or more amino acid residues within a polypeptide sequence may be replaced by another amino acid of similar polarity, acting as a functional equivalent, resulting in a silent change. Amino acid substituents within a sequence may be selected from other members of the class to which the amino acid belongs. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. Positively charged (basic) amino acids include arginine, lysine and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Also included within the scope of the technology of the present invention are proteins or fragments or derivatives thereof that are modified in various ways during or after translation, for example, by glycosylation, proteolytic cleavage, binding to an antibody molecule or other cellular ligands, etc. Additionally, the nucleic acid sequence encoding the immunoglobulin may be mutated in vitro or in vivo to create and/or destroy translation, initiation and/or termination sequences or create variations in the coding regions and/or create new endonuclease restriction sites or destroy pre-existing ones specified sites to facilitate additional in vitro modification. Any mutagenesis method known in the art can be used, including, but not limited to, in vitro site-directed mutagenesis, J. Biol. Chem. 253:6551, use of Tab linkers (Pharmacia), etc.

Preparation of polyclonal antiserum and immunogens. Methods for generating antibodies or antibody fragments according to the technology of the present invention generally involve immunizing a subject (generally a non-human subject such as a mouse or rabbit) with purified A33 protein or a fragment thereof or a cell expressing the A33 protein or fragment thereof . A suitable immunogenic preparation may comprise, for example, a recombinantly expressed A33 protein or a chemically synthesized A33 peptide. The A33 protein ECM, or a portion or fragment thereof, can be used as an immunogen to generate an anti-A33 antibody that binds to the A33 protein, or a portion or fragment thereof, using standard polyclonal and monoclonal antibody production methods.

The full-length A33 protein or fragments thereof are useful in fragment form as immunogens. In some embodiments, the A33 fragment contains at least five to eight contiguous amino acid residues of the amino acid sequence SEQ ID NO: 57 and contains an epitope of the A33 protein such that an antibody raised against the peptide forms a specific immune complex with the A33 protein.

In some embodiments, the A33 antigenic peptide overlapping the A33 ectodomain (Ile22-Val235) contains 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 in accordance with methods well known to one skilled in the art. Multimers of a given epitope are sometimes more effective than monomers.

If necessary, the immunogenicity of the A33 protein (or a fragment thereof) can be increased by fusion or conjugation with a hapten such as keyhole limpet hemocyanin (KLH) or ovalbumin (OVA). Many such haptens are known in the art. It is also possible to combine the A33 protein with a standard adjuvant, such as Freund's complete or incomplete adjuvant, to enhance the immune response to the polypeptide in a subject. Various adjuvants useful to enhance the immunological response include, but are not limited to, Freund's adjuvant (complete or incomplete), mineral gels (e.g., aluminum hydroxide), surfactants (e.g., lysolecithin, Pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), human adjuvants such as Bacillus Calmette-Guerin and Corynebacterium parvum or

- 24 046795 similar immunostimulating compounds. These methods are standard in the art.

In describing the technology of the present invention, immune responses may be described as primary or secondary immune responses. The primary immune response, which is also described as a protective immune response, refers to the immune response that occurs in an individual as a result of some initial exposure (eg, initial immunization) to a specific antigen, such as the A33 protein. In some embodiments, immunization may result from vaccinating a subject with a vaccine containing an antigen. For example, the vaccine may be an A33 vaccine containing one or more antigens derived from the A33 protein. The primary immune response may decrease or weaken over time and may even disappear or at least become so weakened that it cannot be detected. Accordingly, the technology of the present invention also relates to the secondary immune response, which is also described herein as an immunological memory response.

The term secondary immune response refers to an immune response that occurs in a subject after a primary immune response has already occurred.

Thus, a secondary immune response may occur, for example, to enhance an existing immune response that has become diminished or weakened, or to recreate a previous immune response that has disappeared or is no longer detectable. The secondary or immunological memory response can be either a humoral response (antibody based) or a cellular response. The secondary or humoral immunological memory response occurs by stimulating the memory B cells that were formed when the antigen was first presented. Delayed-type hypersensitivity reactions (DTH) are a type of cellular secondary or immune memory response that is mediated by CD4+ T cells. The first exposure to an antigen primes the immune system, and additional exposure(s) leads to HRT.

After proper immunization, anti-A33 antibody can be obtained from the subject's serum. If desired, antibody molecules directed against the A33 protein can be isolated from the mammalian body (eg, blood) and further purified using well known methods such as polypeptide A chromatography to obtain an IgG fraction.

Monoclonal antibody. In one embodiment of the technology of the present invention, the antibody is an anti-A33 monoclonal antibody. For example, in some embodiments, the anti-A33 monoclonal antibody may be a human or murine anti-A33 monoclonal antibody. To obtain monoclonal antibodies directed to the A33 protein, or its derivatives, fragments, analogues or homologs, any method that provides the production of antibody molecules by continuous cultivation of cell lines can be used. Such methods include, but are not limited to, the hybridoma method (see, for example, Kohler & Milstein, 1975. Nature 256: 495-497); trioma method; the human B cell hybridoma method (see, e.g., Kozbor, et al., 1983. Immunol. Today 4: 72) and the EBV-hybridoma method 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 of the present invention and can be produced using human hybridomas (see, for example, Cote, et al., 1983. Proc. Natl. Acad. Sci. USA 80: 2026-2030) or by transforming human B cells with Epstein-Barr virus in vitro (see, eg, Cole, et al., 1985. In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). For example, a population of nucleic acids that encode antibody regions can be isolated. PNRs using primers derived from sequences encoding conserved regions of antibodies are used to amplify sequences encoding portions of antibodies from a population, and then DNA encoding the antibodies or fragments thereof, such as variable domains, are reconstructed from the amplified sequences. Such amplified sequences can also be fused to DNA encoding other proteins, such as bacteriophage coat or bacterial cell surface protein, for expression and display of the fusion polypeptides on the phage or bacteria. The amplified sequences can then be expressed and further selected or isolated based on, for example, the affinity of the expressed antibody or fragment thereof for an antigen or epitope present on the A33 protein. Alternatively, hybridomas expressing monoclonal antibodies against A33 can be obtained by immunizing a subject and then isolating the hybridomas from the subject's spleen using standard methods. See, for example, Milstein et al. (Galfre and Milstein, Methods Enzymol (1981) 73: 3-46). Screening for hybridomas using standard methods produces monoclonal antibodies of varying specificity (i.e., for different epitopes) and affinities. A selected monoclonal antibody with the desired properties, for example binding to A33, can be used. Since the antibody is expressed in the hybridoma, it can be linked to a molecule such as polyethylene glycol (PEG) to alter its properties, or the cDNA encoding it can be isolated, sequenced, and manipulated in various ways. Synthetic dendrimers can be added to reactive amino acid side chains, such as lysine, to enhance the immunogenic properties of the A33 protein. Additionally, CPG dinucleotide techniques can be used to enhance the immunogenic properties of the A33 protein. Other manipulations include replacement or removal of certain amino acid residues that contribute to the instability of the antibody during storage or after administration to a subject, and methods for maturing the affinity of the antibody for the A33 protein.

Hybridoma method. In some embodiments, an antibody according to the technology of the present invention is an anti-A33 monoclonal antibody produced by a hybridoma that includes a B cell derived from a transgenic non-human animal, such as a transgenic mouse, having a genome comprising a heavy chain transgene and a light chain transgene human circuits fused with an immortalized cell. Hybridoma methods include those known in the art and described 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 for producing hybridomas and monoclonal antibodies are well known to those skilled in the art.

Phage display method. As noted above, antibodies according to the technology of the present invention can be obtained using recombinant DNA and phage display methods. For example, antibodies against A33 can be obtained using various phage display methods known in the art. In phage display methods, the functional domains of antibodies are displayed on the surface of a phage particle, which contains the polynucleotide sequences encoding them. Phages with the desired binding properties are selected from a combinatorial antibody repertoire or library (eg, human or mouse) by selection directly using an antigen, typically an antigen bound or captured on a solid surface or bead. Phages used in these methods are typically filamentous phages, including fd and M13, in which Fv, Fv, or disulfide-stabilized Fv domains of antibodies are recombinantly fused to a phage protein III or protein VIII gene. In addition, the methods can be adapted to generate Fab expression libraries (see, for example, Huse, et al., Science 246: 1275-1281, 1989) to provide rapid and efficient identification of monoclonal Fab fragments with the desired polypeptide specificity A33, for example, a polypeptide or derivative thereof, fragments, analogues or homologues. Other examples of phage display methods that can be used to produce antibodies according to the technology of the present invention include those described 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; publication of international applications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; WO 96/06213; WO 92/01047 (Medical Research Council et al.); WO 97/08320 (Morphosys); WO 92/01047 (CAT/MRC); WO 91/17271 (Affymax); and US Patents No. 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, 25, 5,658,727 and 5,733,743. Methods useful for displaying polypeptides on the surface of bacteriophage particles by attaching polypeptides via disulfide bonds have been described by Lohning (US Pat. No. 6,753,366). As described in the above references, once a phage is selected, antibody coding regions from the phage can be isolated and used to generate full length antibodies, including human antibodies, or any other desired antigen binding moiety, and expressed in any desired host, including mammalian cells, insect cells, cells of plants, yeast and bacteria. For example, methods for recombinantly producing Fab, Fab' and F(ab') ₂ fragments using methods known in the art, such as those described in international patent application publication WO 92/22324; Mullinax et al., BioTechniques 12: 864-869, 1992; and Sawai et al., AJRI 34: 26-34, 1995; Better et al., Science 240: 1041-1043, 1988.

In general, fusion antibodies or fusion antibody fragments that are cloned into a display vector can be selected using the appropriate antigen to identify variants that support good binding activity as the antibody or antibody fragment will be present on the surface of the phage or phagemid particle. See, for example, Barbas III et al., Phage Display, A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001). However, other vector formats can be used for this process, such as cloning a library of antibody fragments into a lytic phage vector (modified T7 or lambda-Zap systems) for selection and/or screening.

Expression of recombinant antibodies against A33. As noted above, antibodies according to the technology of the present invention can be obtained using the recombinant DNA method. Recombinant polynucleotide constructs encoding an anti-A33 antibody according to the technology of the present invention typically include an expression control sequence operably linked to the coding sequences of the anti-A33 antibody chains, including naturally associated or heterologous promoter regions. In this regard, another aspect of techno

- 26 046795 logi includes vectors containing one or more nucleic acid sequences encoding an anti-A33 antibody according to the technology of the present invention. For recombinant expression of one or more polypeptides according to the technology of the present invention, a nucleic acid containing all or part of the nucleotide sequence encoding an anti-A33 antibody is inserted into a suitable cloning vector or expression vector (i.e., a vector that contains the necessary elements for transcription and translation of the inserted polypeptide coding sequence) using recombinant DNA techniques well known in the art and described in detail below. Methods for producing diverse vector populations have been described by Lerner et al., US Patent Nos. 6,291,160 and 6,680,192.

In general, expression vectors useful in recombinant DNA techniques are often presented in the form of plasmids. According to the present invention, the terms plasmid and vector can be used interchangeably, since plasmid is the most commonly used form of vector. However, it is contemplated that the technology of the present invention includes such other forms of expression vectors that are not technically plasmids, such as viral vectors (eg, retroviruses, adenoviruses, and replication-deficient adeno-associated viruses) that perform equivalent functions. Such viral vectors enable infection of a subject and expression of the construct in said subject. In some embodiments, the expression control sequences are eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. After insertion of the vector into a suitable host, the host is maintained under conditions suitable for high-level expression of nucleotide sequences encoding the anti-A33 antibody and collection and purification of the anti-A33 antibody, eg, cross-reacting anti-A33 antibodies. See generally US Patent Application 2002/0199213. These expression vectors are typically capable of replicating in host organisms either as episomes or as an integral part of the host's chromosomal DNA. Typically, expression vectors contain selectable markers, such as an ampicillin resistance or hygromycin resistance gene, to enable detection of said cells transformed with the desired DNA sequences. Vectors may also encode a signal peptide, such as pectate lyase, useful for targeted secretion of extracellular antibody fragments (see US Pat. No. 5,576,195).

Recombinant expression vectors according to the technology of the present invention contain a nucleic acid encoding a protein having the properties of binding to A33 in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors contain one or more regulatory sequences selected taking into account a host cell to be used for expression, wherein said sequences are operably linked to a nucleic acid sequence to be expressed. In the context of a recombinant expression vector, operably linked means that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell where the vector is introduced into the specified host cell). The term regulatory sequence is intended to include promoters, enhancers, and other expression control elements (eg, 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 sequences that direct constitutive expression of a nucleotide sequence in many types of host cells and that direct expression of a nucleotide sequence only in certain host cells (eg, 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 host cell to be transformed, the desired level of expression of the polypeptide, etc. Typical regulatory sequences useful as promoters of expression of a recombinant polypeptide (eg, anti-A33 antibodies) include, for example, but are not limited to, promoters of 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, among others, promoters from alcohol dehydrogenase, isocytochrome C, and maltose and galactose utilization enzymes. In one embodiment, a polynucleotide encoding an anti-A33 antibody according to the technology of the present invention is operably linked to the ara B promoter and is expressible in a host cell. See US Patent 5,028,530. Expression vectors according to the technology of the present invention can be introduced into host cells to thereby produce polypeptides or peptides, including fusion polypeptides encoded by nucleic acids as described herein (eg, anti-A33 antibody, etc.).

Another aspect of the technology of the present invention relates to anti-A33 antibody-expressing host cells that contain a nucleic acid encoding one or more anti-A33 antibodies. Recombinant expression vectors according to the present technology

- 27 046795 of the invention can be engineered to express anti-A33 antibodies in prokaryotic or eukaryotic cells. For example, an anti-A33 antibody can be expressed in bacterial cells such as Escherichia coli cells, insect cells (using baculovirus expression vectors), fungal cells such as yeast, yeast cells or mammalian cells. Suitable host cells are further discussed in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example, using the T7 regulatory sequence promoter and T7 polymerase. Methods useful for producing and screening polypeptides having a predetermined property, for example, anti-A33 antibodies, through the expression of stochastically generated polynucleotide sequences have been described previously. See US Patent Nos. 5,763,192; 5,723,323; 5,814,476; 5,817,483; 5,824,514; 5,976,862; 6,492,107; 6,569,641.

Expression of polypeptides in prokaryotes is most often accomplished in E. coli with vectors containing constitutive or inducible promoters that direct the expression of fusion polypeptides or non-fusion polypeptides. Fusion vectors add a series of amino acids to the polypeptide it encodes, usually at the amino terminus of the recombinant polypeptide. Such fusion vectors typically serve three purposes: (i) to enhance expression of the recombinant polypeptide; (ii) to increase the solubility of the recombinant polypeptide; and (iii) to aid in the purification of the recombinant polypeptide by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion fragment and the recombinant polypeptide to allow separation of the recombinant polypeptide from the fusion fragment after purification of the fusion polypeptide. Such enzymes and their cognate recognition sequences include factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, MA), and PRIT5 (Pharmacia, Piscataway, NJ), which mediate fusion glutathione-transferase (GST), which binds maltose E polypeptide or polypeptide A, respectively, to the recombinant target polypeptide.

Examples of suitable inducible E. coli expression vectors that are not fusion vectors include pTrc (Ammann 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 the targeted assembly of individual active domains of a peptide or protein to produce multifunctional polypeptides by polypeptide fusion have been described by Pack et al., US Patent Nos. 6,294,353; 6,692,935. One strategy for maximizing the expression of a recombinant polypeptide, for example, an anti-A33 antibody, in E. coli is to express the polypeptide in a host bacterium with an impaired ability to proteolytically cleave the recombinant polypeptide. See, for example, Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to change the sequence of the nucleic acid to be inserted into the expression vector so that the individual codons for each amino acid are the codons preferentially used by the host cell, eg E. coli, for expression (see, for example, Wada, et al. al., 1992. Nucl. Acids Res. 20: 2111-2118). Such alteration of nucleic acid sequences according to the technology of the present invention can be accomplished using standard DNA synthesis techniques.

According to another embodiment of the invention, the anti-A33 antibody expression vector is a yeast expression vector. Examples of expression vectors from the yeast Saccharomyces cerevisiae include pYepSec1 (Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, Cell 30: 933-943, 1982), pJRY88 (Schultz et al. , Gene 54: 113-123, 1987), pYES2 (Invitrogen Corporation, San Diego, CA) and picZ (Invitrogen Corp, San Diego, CA). Alternatively, anti-A33 antibody can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of polypeptides, such as anti-A33 antibodies, in cultured insect cells (eg, SF9 cells) include the pAc group (Smith, et al., Mol Cell. Biol. 3: 2156-2165, 1983) and the pVL group ( Lucklow and Summers, 1989. Virology 170: 31-39).

According to another embodiment of the invention, a nucleic acid encoding an anti-A33 antibody according to the technology of the present invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include, for example, 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, expression vector control functions are 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 for both prokaryotic and eukaryotic cells useful for expressing anti-A33 antibodies according to the technology of the present invention, see, for example, Chaps. 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd

- 28 046795 ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

According to another embodiment of the invention, the recombinant mammalian expression vector is capable of directing nucleic acid expression to a specific cell type (eg, 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 tissue-specific promoters (Calame and Eaton, Adv. Immunol. 43: 235-275 , 1988), T cell receptor promoters (Winoto and Baltimore, EMBO J. 8: 729-733, 1989) and immunoglobulins (Banerji, et al., 1983. Cell 33: 729-740; Queen and Baltimore, Cell 33: 741-748, 1983.), neuron-specific promoters (eg, neurofilament gene promoter; Byrne and Ruddle, Proc. Natl. Acad. Sci. USA 86: 5473-5477, 1989), pancreas-specific promoters (Edlund, et al. al., 1985. Science 230: 912-916) and mammary gland-specific promoters (eg, whey protein gene promoter; US Patent No. 4,873,316 and European Application Publication No. 264,166). Developmentally regulated promoters are also included, for example, the mouse hox gene promoters (Kessel and Gruss, Science 249: 374-379, 1990) and the α-fetoprotein promoter (Campes and Tilghman, Genes Dev. 3: 537-546, 1989) .

Another aspect of the methods of the present invention relates to host cells into which a recombinant expression vector has been introduced according to the technology of the present invention. The terms host cell and recombinant host cell are used interchangeably in this application. It should be understood that such terms refer not only to a particular cell of a subject, but also to the progeny or potential progeny of such cell. Since specific modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may not actually be identical to the original cell, but are still included within the scope of the term as used herein.

The host cell can be any prokaryotic or eukaryotic cell. For example, an anti-A33 antibody may be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells. Mammalian cells are a suitable host for the expression of nucleic acid segments encoding immunoglobulins or fragments thereof (see Winnacker, From Genes To Clones, (VCH Publishers, NY, 1987). A number of suitable host cell lines have been developed in the art that are capable of secrete intact heterologous proteins and include a Chinese hamster ovary (CHO) cell line, various COS cell lines, HeLa cells, L cells, and myeloma cell lines. In some embodiments, the cells are non-human cells. Expression vectors for the cells may be present. include expression of control sequences such as the origin of replication, promoter, enhancer, and essential information processing sites such as ribosome binding sites, RNA splicing 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 virus, adenovirus, bovine papillomavirus, etc. (Co et al., J Immunol. 148: 1149, 1992). Other suitable host cells are known to one skilled in the art.

The DNA vector can be introduced into prokaryotic or eukaryotic cells using standard transformation or transfection methods. As used herein, the terms transformation and transfection are intended to refer to a variety of methods known in the art for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride coprecipitation, 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. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) and other laboratory instructions. Vectors containing DNA segments of interest can be transferred into a host cell using well known methods depending on the type of host cell.

It is known that in the case of stable transfection of mammalian cells, depending on the expression vector and transfection method used, only a small fraction of cells is capable of incorporating foreign DNA into its genome. To identify and select these cells capable of incorporating foreign DNA, a gene encoding a selectable marker (eg, an antibiotic resistance gene) is generally introduced into host cells along with the gene of interest. Various selectable markers include markers that confer resistance to drugs such as 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 on a separate vector. Cells stably transfected with the introduced nucleic acid can be

- 29 046795 identified by drug-based selection (eg, cells that have inserted a selectable marker gene will survive while other cells die).

A host cell containing an anti-A33 antibody according to the technology of the present invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (ie, express) a recombinant anti-A33 antibody. According to one embodiment of the invention, the method includes culturing a host cell (into which a recombinant expression vector encoding an anti-A33 antibody has been introduced) in a suitable medium such that production of an anti-A33 antibody occurs. According to another embodiment of the invention, the method also includes the step of isolating the anti-A33 antibody from the environment or said host cell. Once expressed, the collected anti-A33 antibodies, such as anti-A33 antibodies or anti-A33-related polypeptides, are purified from the culture medium and host cells. The anti-A33 antibody can be purified according to standard procedures in the art, including purification by HPLC, column chromatography, gel electrophoresis, etc. In one embodiment, the anti-A33 antibody is produced in the host using a method according to Boss et al., US Pat. No. 4,816,397. Typically, anti-A33 antibody chains are expressed with signal sequences and are thus released into the culture medium. However, if anti-A33 antibody chains are not naturally secreted by host cells, said anti-A33 antibody chains can be released by treatment with a weak detergent. Purification of recombinant polypeptides is well known in the art and includes ammonium sulfate precipitation, affinity chromatography purification method, column chromatography, ion exchange purification method, gel electrophoresis, etc. (See generally Scopes, Protein Purification (Springer-Verlag, N.Y., 1982).

Polynucleotides encoding anti-A33 antibodies, for example, sequences encoding an anti-A33 antibody, can be inserted into transgenes for introduction into the genome of a transgenic animal and subsequent expression in the milk of said transgenic animal. See, for example, US Pat. Nos. 5,741,957, 5,304,489, and 5,849,992. Suitable transgenes include transgenes encoding light and/or heavy chain sequences operably linked to a promoter and enhancer from a mammary gland-specific gene, such as casein or P-lactoglobulin. To obtain transgenic animals, these transgenes can be introduced by microinjection into fertilized eggs or can be inserted into the genome of embryonic stem cells and the nuclei of such cells transferred into nucleated eggs.

Single chain antibodies. In one embodiment, the anti-A33 antibody of the technology of the present invention is a single-chain anti-A33 antibody. In accordance with the technology of the present invention, methods can be adapted to produce single chain antibodies specific for the A33 protein (see, for example, US Pat. No. 4,946,778). Examples of methods that can be used to produce single-chain Fv fragments and antibodies according to the technology of the present invention include methods described in US patent No. 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 Skerrae et al., Science 240: 1038-1040, 1988.

Chimeric and humanized antibodies. In one embodiment, the anti-A33 antibody of the technology of the present invention is a chimeric anti-A33 antibody. In one embodiment, the anti-A33 antibody of the technology of the present invention is a humanized anti-A33 antibody. According to one embodiment of the technology of the present invention, the donor antibody and the acceptor antibody are monoclonal antibodies from different species. For example, the acceptor antibody is a human antibody (to minimize its antigenicity in humans), in which case the resulting CDR-grafted antibody is called a humanized antibody.

Recombinant anti-A33 antibodies, such as chimeric and humanized monoclonal antibodies containing both human and non-human antibody portions, can be generated using standard recombinant DNA techniques and are within the scope of the technology of the present invention. For certain applications, including in vivo use in humans of anti-A33 antibodies according to the technology of the present invention, as well as the use of these agents in in vitro detection assays, it is possible to use chimeric or humanized anti-A33 antibodies. Such chimeric and humanized monoclonal antibodies can be produced using recombinant DNA techniques known in the art. Such applicable methods include, for example, but are not limited to, those described in International Patent Application No. PCT/US86/02269; US Patent No. 5 225539; European patent No. 184187; European patent No. 171496; European patent No. 173494; publication of international application WO 86/01533; US Patent No. 4,816,567; 5,225,539; European patent No. 125023; Better, et al., 1988. Science 240: 1041-1043; Liu, et al., 1987. Proc. Natl. Acad. Sci. USA 84: 3439-3443; Liu, et al., 1987. J. Immunol. 139: 3521-3526; Sun, et al., 1987. Proc. Natl. Acad. Sci. USA 84: 214-218; Nishimura, et al., 1987.

- 30 046795

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; US Patent No. 5,807,715; and Beidler, et al., 1988. J. Immunol. 141:4053-4060. For example, antibodies can be humanized using various methods, including the CDR vaccination (European Patent EP 0 239 400; Publication of the International Patent Application Wo 91/09967; US patents No. 5.530.101; 5.585.089; 5,859,205; 6,248,516; European patent ER460167), viniding and rem Deling surfaces (European patent EP 0 592 106; European patent EP 0 519 596; Padlan E. A., Molecular Immunology, 28: 489-498, 1991; Studnicka et al., Protein Engineering 7: 805-814, 1994; Roguska et al. ., PNAS91: 969-973, 1994) and circuit shuffling (US Patent No. 5,565,332). In one embodiment, the cDNA encoding the mouse anti-A33 monoclonal antibody is digested with a restriction enzyme selected to specifically remove the sequence encoding the Fc constant region, which is replaced by an equivalent portion of the cDNA encoding the human Fc constant region (see Robinson et al. , PCT/US86/02269; Akira et al., European patent application 184,187; Taniguchi, European patent application 171,496; Morrison et al., European patent application 173,494; ; Cabilly et al. US Patent No. 4,816,567; European Patent Application 125,023; Science 240: 1041-1043; USA 84: 3439-3443; JImmunol 139: 3521-3526; Proc. Acad. USA 84: 214-218; Cancer Res 47: 999-1005; Wood et al. (1985) Nature 314: 446-449; (1988)J. Natl. Cancer Inst. 80: 1553-1559; US Patent No. 6,180,370; US Patents No. 6,300,064; 6,696,248; 6,706,484; 6,828,422).

In one embodiment, the technology of the present invention enables the construction of humanized anti-A33 antibodies that are low likely to elicit an anti-mouse antibody (hereinafter referred to as HAMA) response in humans while maintaining the effective effector function of the antibody. As used herein, the terms human and humanized in relation to antibodies refer to any antibody that is expected to elicit a therapeutically tolerable mild immunogenic response in a human subject. In one embodiment, the technology of the present invention provides humanized anti-A33 antibodies, immunoglobulin heavy chains and light chains.

CDR antibodies. In some embodiments, the anti-A33 antibody of the technology of the present invention is an anti-A33 CDR antibody. In general, the donor and acceptor antibodies used to create anti-A33 CDR antibodies are monoclonal antibodies from various species, typically the antihelo-acceptor is a human antibody (to minimize its antigenicity in humans), in this case obtained as a result, the CDR-grafted antibody is called a humanized antibody. A single CDR region (or even portions of a single CDR) may be grafted within a single VH or VL region of an acceptor antibody, or multiple CDRs (or portions thereof) may be grafted within one or both of the VH and _VL . Often, all three CDR regions from all variable domains of an acceptor antibody are replaced with the corresponding donor CDRs, although only enough CDRs need to be replaced to ensure proper binding to the A33 protein of the resulting CDR-grafted antibody. Methods for generating CDR-grafted and humanized antibodies are described in Queen et al. US Patents No. 5,585,089; No. 5,693,761; US No. 5,693,762; and Winter, US Patent No. 5,225,539 and European Patent EP 0682040. Methods useful for preparing VH and VL polypeptides are described in Winter et al., US Patent No. 4,816,397; 6,291,158; 6,291,159; 6,291,161; 6 545 142; European patent EP 0368684; EP0451216; and EP0120694.

After selecting suitable framework candidates from the same family and/or the same family member, either or both of the heavy and light chain variable regions are produced by grafting CDRs from the parent species into the hybrid frameworks. The assembly of hybrid antibodies or hybrid antibody fragments having hybrid variable chain regions in relation to any of the above aspects can be accomplished using standard methods known to one skilled in the art. For example, DNA sequences encoding the hybrid variable domains described herein (ie, framework regions based on the target species and CDR regions from the parent species) can be obtained by oligonucleotide synthesis and/or PNR. The nucleic acid encoding the CDR regions can also be isolated from antibodies of the parent species using suitable restriction enzymes and ligated into the backbone of the target species by ligation using suitable ligation enzymes. Alternatively, the framework regions of the parent antibody variable chains can be altered by site-directed mutagenesis.

Since hybrids are constructed from a selection of a plurality of candidates corresponding to each framework region, there are many combinations of sequences that are amenable to design in accordance with the principles described herein. Accordingly, they can

- 31 046795 libraries of hybrids containing members with different combinations of individual frame regions can be assembled. Such libraries may be electronic collections in the form of sequence databases or physical collections of hybrids.

This process generally does not change the FR of the acceptor antibody flanking the grafted CDRs. However, one of ordinary skill in the art can sometimes improve the antigen binding affinity of the resulting CDR-grafted anti-A33 antibody by replacing certain residues of a given FR to make the FR more similar to the corresponding FR of the donor antibody. Suitable substitution positions include amino acid residues adjacent to the CDR or residues capable of interacting with the CDR (see, for example, US Pat. No. 5,585,089, particularly columns 12-16). One skilled in the art may also start with the donor FR and modify it so that the region is more similar to the acceptor FR or the human consensus FR region. Methods for creating these modifications are known in the art. In particular, if the resulting FR matches the human consensus FR at that position or is at least 90% or more identical to the specified consensus FR, such modifications may not significantly increase the antigenicity of the resulting modified CDR-grafted anti-A33 antibody compared to the same an antibody containing a fully human FR region.

Bispecific antibodies (BsAb). A bispecific antibody is an antibody that binds simultaneously to two targets that have different structures, such as two different target antigens, two different epitopes on the same target antigen, or a hapten and a target antigen or an epitope on a target antigen. BsAbs can be produced, for example, by combining heavy chains and/or light chains that recognize different epitopes of the same or different antigens. In some embodiments, in terms of molecular function, the bispecific binding agent binds one antigen (or epitope) on one of two binding arms (one VH/VL pair) and binds another antigen (or epitope) on its second arm (the other VH pair). /VL). By this definition, a bispecific binding agent has two different antigen-binding arms (both specificity and CDR) and is monovalent for each antigen to which it binds.

Bispecific antibodies (BsAb) and bispecific antibody fragments (BsFab) according to the technology of the present invention contain at least one arm that specifically binds, for example, to A33, and at least one other arm that specifically binds to a second target antigen. In some embodiments, the second target antigen is an antigen or epitope of a B cell, T cell, myeloid cell, plasma cell, or mast cell. Additionally or alternatively, according to specific embodiments of the invention, 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, gamma/delta TCR, NKp46 and KIR. In specific embodiments, the BsAbs are capable of binding to tumor cells that express the A33 antigen on the cell surface. In some embodiments, BsAbs have been engineered to promote tumor cell killing by targeting (or recruiting) cytotoxic T cells to the tumor site. Other examples of BsAbs include antibodies with a first antigen-binding site specific for A33 and a second antigen-binding site specific for a low molecular weight hapten (e.g., DTP A, IMP288, DOTA, DOTA-Bn, DOTAdesferrioxamine, other DOTA chelates, described herein, biotin, fluorescein or haptens described in Goodwin, D A. et al., 1994, Cancer Res 54(22):5937-5946).

Various bispecific fusion proteins can be produced using molecular engineering techniques. For example, BsAbs have been designed that utilize the entire immunoglobulin framework (eg, IgG), a single chain variable fragment (scFv), or a combination thereof. In some embodiments, the bispecific fusion protein is divalent, comprising, for example, a scFv with a single binding site for one antigen and a Fab fragment with a single binding site for a second antigen. In other embodiments, the bispecific fusion protein is tetravalent and comprises, for example, an immunoglobulin (eg, IgG) with two binding sites for one antigen and two identical scFvs for a second antigen. BsAbs consisting of two scFv units in tandem have been shown to be a clinically successful bispecific antibody format. In some embodiments, BsAbs have been designed to contain two single chain variable fragments (scFvs) in tandem such that a scFv that binds a tumor antigen (eg, A33) is linked to a scFv that attracts T cells (eg, by CD3 binding). As a consequence, T cells are recruited to the tumor site in such a way that they can mediate cytotoxic killing of tumor cells. See, for example, Dreier et al., J. Immunol. 170:4397-4402 (2003); Bargou et al., Science 321:974-977 (2008)).

Recently developed methods for producing BsAbs involve engineered recombinant monoclonal antibodies that contain additional cysteine residues such that the

- 32 046795 antibodies have stronger cross-linking compared to more common immunoglobulin isotypes. See, for example, FitzGerald et al, Protein Eng. 10(10): 1221-1225 (1997). Another approach is the engineering of recombinant fusion proteins that bind two or more different single-chain antibodies or antibody fragment segments with the requirement of dual specificity. See, for example, Coloma et al., Nature Biotech. 15:159-163 (1997). A variety of bispecific fusion proteins can be produced using molecular engineering.

Bispecific fusion proteins linking two or more different single chain antibodies or antibody fragments are prepared in a similar manner. Recombinant methods can be used to produce a variety of fusion proteins. In some specific embodiments, a BsAb according to the technology of the present invention comprises an immunoglobulin that contains a heavy chain and a light chain and a scFv. In some specific embodiments, the scFv is linked to the C-terminus of the heavy chain of any A33 immunoglobulin described herein. In some specific embodiments, the scFvs are linked to the C-terminus of the light chain of any A33 immunoglobulin described herein. According to various embodiments of the invention, scFvs are linked to heavy or light chains via a linker sequence. Suitable linker sequences for in-frame joining of the Fd and scFv heavy chains are introduced into the _kappa VL and V domains via PCR reactions. The DNA fragment encoding the scFv is then ligated into a Staging vector containing the DNA sequence encoding the CH1 domain. The resulting scFv-CH1 construct is excised and ligated into a vector containing the DNA sequence encoding the VH region of antibody A33. The resulting vector can be used to transfect a suitable host cell, such as 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 amino acids in length. In some embodiments, the linker is characterized in that it tends not to adopt a rigid three-dimensional structure, but rather provides flexibility to the polypeptide (eg, the first and/or second antigen binding sites). In some embodiments, the linker used in the BsAb described herein is selected based on specific properties inherent in the BsAb, such as, for example, increasing stability. In some embodiments, the BsAb of the present invention comprises a G4S linker. In some specific embodiments, the BsAb of the present invention comprises a (G ₄ S) _n linker, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 1, 12, 13, 14 15 or more.

Fc modifications. In some embodiments, anti-A33 antibodies of the technology of the present invention comprise a variant Fc region, wherein said variant Fc region contains at least one amino acid modification compared to a wild-type Fc region (or parent Fc region) such that said molecule has altered affinity for an Fc receptor (e.g., FcyR), provided that said variant Fc region does not contain a substitution at positions directly in contact with the Fc receptor as determined by crystallographic and structural analysis of the Fc/Fc receptor interaction, such as analyzes described in Sondermann et al., Nature, 406: 267-273 (2000). Examples of positions in the Fc region that make direct contact with an Fc receptor such as FcyR include amino acids 234-239 (hinge region), amino acids 265-269 (B/C-loop), amino acids 297-299 (C7-loop) and amino acids 327 -332 (F/G loop).

In some embodiments, an anti-A33 antibody according to the technology of the present invention has altered affinity for activating and/or inhibitory receptors having a variant Fc region with one or more amino acid modifications, wherein said one or more amino acid modifications are a substitution of N297 for alanine or replacing K322 with alanine.

Modifications of glycosylation. In some embodiments, anti-A33 antibodies according to the technology of the present invention contain a variant glycosylation Fc region compared to the parent Fc region. In some embodiments, the variant glycosylation includes the absence of fucose; in some embodiments, the variant glycosylation results from expression in GnT1-deficient CHO cells.

In some embodiments, antibodies according to the technology of the present invention may contain a modified glycosylation site compared to a suitable reference antibody that binds to an antigen of interest (eg, A33) without changing the functionality of the antibody, such as antigen binding activity. When used in the present invention, glycosylation sites include any specific amino acid sequence of the antibody to which it is specifically and covalently attached.

- 33 046795 oligosaccharide (i.e. a carbohydrate containing two or more simple sugars linked together).

Oligosaccharide side chains are typically linked to the antibody backbone via N- or O-links. N-linked glycosylation refers to the addition of an oligosaccharide moiety to the side chain of an aspartic residue. O-linked glycosylation refers to the addition of an oligosaccharide moiety to a hydroxyamino acid, such as serine, threonine. For example, an Fc-glycoform (huA33-IgG1n), which lacks certain oligosaccharides, including fucose and terminal N-acetylglucosamine, can be produced in dedicated CHO cells and exhibit enhanced ADCC effector function.

In some embodiments, the carbohydrate content of the immunoglobulin composition described herein is modified by the addition or removal of a glycosylation site. Methods for modifying the carbohydrate content of antibodies are well known in the art and are included in the technology of the present invention (see, for example, US Patent No. 6218149; European Patent EP 0359096B1; US Patent Application Publication No. 2002/0028486; International Patent Application Publication WO 03 /035835; publication of US patent application No. 2003/0115614; US patent No. 6472511; all of which are incorporated herein by reference in their entirety. In some embodiments, the carbohydrate content of the antibody (or a corresponding portion thereof) component) is modified by removing one or more endogenous carbohydrate moieties of the antibody. In some specific embodiments, the technology of the present invention includes removing a glycosylation site of the Fc region of the antibody by modifying position 297 from asparagine to alanine.

The engineered glycoforms may be useful for a variety of purposes, including, but not limited to, enhancing or decreasing effector function. Engineered glycoforms can be produced by any method known to one skilled in the art, for example, using engineered or variant expression strains, by coexpression with one or more enzymes, for example DI N-acetylglucosaminyltransferase III (GnTIII), by expressing a molecule containing the Fc region in various organisms or cell lines from different organisms, or by modifying the carbohydrate(s) after the molecule containing the Fc region has been expressed. Methods for preparing 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; US Patent No. 6,602,684; US Patent Applications No. 10/277,370; 10/113.929; publications of international applications WO 00/61739A1; WO 01/292246A1; WO 02/311140A1; WO 02/30954A1; POTILLEGENT™ technologies (Biowa, Inc. Princeton, N.J.); GLYCOMAB™ glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland), each of which is incorporated herein by reference in its entirety. See, for example, international patent application publication WO 00/061739; publication of US patent application No. 2003/0115614; Okazaki et al., 2004, JMB, 336: 1239-49.

Fusion proteins. In one embodiment, the anti-A33 antibody of the technology of the present invention is a fusion protein. Antibodies against A33 according to the technology of the present invention, when fused with a second protein, can be used as an antigenic tag. Examples of domains that can be fused to polypeptides include not only heterologous signal sequences, but also other heterologous functional regions. The fusion does not have to be direct, but can occur through linker sequences. Moreover, fusion proteins according to the technology of the present invention can also be engineered to improve the performance of anti-A33 antibodies. For example, a region of additional amino acids, particularly charged amino acids, can be added to the N-terminus of an anti-A33 antibody to improve stability and stability during purification from the host cell or subsequent processing and storage. In addition, peptide fragments can be added to the anti-A33 antibody to facilitate purification. Such regions can be removed before final production of the anti-A33 antibody. The addition of peptide fragments to facilitate processing of polypeptides is an established and routine technique in the art. The anti-A33 antibody according to the technology of the present invention can be fused to marker sequences, such as a peptide, which facilitates the purification of the fusion polypeptide. In selected embodiments, the marker amino acid sequence is a hexahistidine peptide, such as the tag provided in the pQE vector (QIAGEN, Inc., Chatsworth, Calif.), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86: 821-824, 1989, for example, hexa-histidine provides convenient purification of fusion protein. Another peptide tag useful for purification, the HA- tag, corresponds to an epitope derived from the influenza virus hemagglutinin protein. Wilson et al., Cell 37: 767, 1984.

Thus, any of the above fusion proteins can be constructed using

- 34 046795 polynucleotides or polypeptides according to the technology of the present invention. In addition, according to some embodiments of the invention, the fusion proteins described herein have an increased half-life in vivo.

Fusion proteins having disulfide-linked dimeric structures (via IgG) may be more effective in binding and neutralizing other molecules compared to a monomeric secreted protein or a protein fragment alone (Fountoulakis et al., J. Biochem. 270: 3958-3964, 1995).

Similarly, European patent EP-A-0 464 533 (Canadian equivalent 2045869) describes fusion proteins containing various parts of the constant region of immunoglobulin molecules together with another human protein or fragment thereof. In many cases, the Fc portion of the fusion protein is advantageous for therapy and diagnosis and can thus lead, for example, to improved pharmacokinetic properties (see EP-A 0232 262). Alternatively, it may be desirable to remove or modify the Fc portion after the fusion protein has been expressed, detected and purified. For example, the Fc portion may interfere with therapy and diagnosis if the fusion protein is used as an antigen for immunization. For drug discovery, human proteins, such as hIL-5, are fused to Fc moieties for high-throughput screening assays to identify hIL-5 antagonists. Bennett et al., J. Molecular Recognition 8: 52-58, 1995; Johanson et al., J. Biol. Chem., 270: 9459-9471, 1995.

Labeled anti-ASS antibodies. In one embodiment, an anti-A33 antibody according to the technology of the present invention is coupled to a labeling moiety, i.e. detectable group. The particular label or detectable group conjugated to the anti-A33 antibody is not a critical aspect of the technology, provided that it does not significantly interfere with the specific binding of the anti-A33 antibody of the present invention to the A33 protein. A detectable group can be any material having a detectable physical or chemical property. Such detectable tags are well developed in the field of immunoassays and imaging. In general, almost any label useful in such methods can be used according to the technology of the present invention. Thus, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical methods. Tags useful in the practice of the technology of the present invention include magnetic beads (eg, Dynabeads™), fluorescent dyes (eg, fluorescein isothiocyanate, Texas Red, rhodamine, etc.), radiolabels (eg, 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 TC, ¹¹¹ In (for single photon emission tomography), enzymes (for example, horseradish peroxidase, alkaline phosphatase and other tags commonly used in ELISA) and calorimetric tags such as colloidal gold or colored glass or plastic beads (for example, polystyrene, polypropylene, latex and etc.). All patents that describe the use of such tags include US Patent No. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241, each of which is incorporated herein by reference in its entirety and for all purposes. See also Handbook of Fluorescent Probes and Research Chemicals ( ^6th Ed., Molecular Probes, Inc., Eugene OR).

The label may be coupled directly or indirectly to the desired assay component in accordance with methods well known in the art. As stated above, a wide variety of labels can be used, with the choice of label depending on factors such as the required sensitivity, ease of conjugation to the compound, required stability, available equipment and disposal capabilities.

Non-radioactive tags are often attached by indirect methods. In general, ligand molecules (eg biotin) are covalently attached to the molecule. The ligand then binds to a molecule directed against the ligand (eg, streptavidin), which is either itself detectable or covalently linked to a signaling system, such as a detectable enzyme, fluorescent compound, or chemiluminescent compound. A number of ligands and anti-ligand compounds can be used. When a natural compound directed against a ligand exists (eg, as in the case of biotin, thyroxine and cortisol), the ligand can be used in conjunction with the labeled compound. Alternatively, any hapten or antigenic compound can be used in combination with an antibody, for example an anti-A33 antibody.

The molecules can also be conjugated directly to signal-generating compounds, for example by conjugation with an enzyme or fluorochrome. Enzymes of interest useful as tags are primarily hydrolases, in particular phosphatases, esterases and glycosidases or oxidoreductases, in particular peroxidases. Fluorescent compounds useful as labeling moieties include, but are not limited to, fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc. Chemiluminescent compounds useful as labeling moieties include, but are not limited to,

- 35 046795 e.g. luciferin and 2,3-dihydrophthalazinediones, e.g. luminol. For a review of various labeling or signal-producing systems that may be used, see US Patent No. 4,391,904.

Methods for detecting labels are well known to those skilled in the art. Thus, for example, when the label is a radioactive label, detection methods include a scintillation counter or photographic film, as in the case of autoradiography. When the label is a fluorescent label, it can be detected by exciting the fluorochrome with a suitable wavelength of light and detecting the resulting fluorescence. Fluorescence can be detected visually using photographic film, by using electron detectors such as charge coupled devices (CCDs) or photomultiplier tubes, etc. Similarly, enzyme tags can be detected by providing suitable substrates for the enzyme and detecting the resulting reaction product. Finally, simple colorimetric tags can be detected simply by observing the color associated with the tag. Thus, in various test strip assays, the conjugated gold often appears pink, whereas when using various conjugated beads, the beads appear colored.

Some assay formats do not require the use of labeled components. For example, agglutination assays can be used to detect the presence of target antibodies, such as anti-A33 antibodies. In this case, antigen-coated particles are agglutinated by samples containing target antibodies. With this format there is no need to label any of the components; the presence of the target antibody is detected by simple visual assessment.

B. Identification and characterization of anti-A33 antibodies according to the technology of the present invention.

Methods for identifying and/or screening anti-A33 antibodies according to the technology of the present invention. Methods useful for identifying and screening antibodies against A33 polypeptides for antibodies having the desired specificity for the A33 protein include any immunologically mediated methods known in the art. Components of the immune response can be detected in vitro using various methods that are well known to those skilled in the art. For example, (1) cytotoxic T lymphocytes can be incubated with radioactively labeled target cells, and lysis of said target cells is detected by the release of radioactivity; (2) helper T lymphocytes can be incubated with antigens and antigen-presenting cells and the synthesis and secretion of cytokines is measured using standard methods (Windhagen A et al., Immunity, 2: 373-80, 1995); (3) antigen-presenting cells can be incubated with whole protein antigen, and antigen presentation on the MHC is detected either by T-lymphocyte activation assays or by biophysical methods (Harding et al., Proc. Natl. Acad. Sci., 86: 4230-4, 1989); (4) mast cells can be incubated with reagents that cross-link their Fc epsilon receptors, and histamine release is measured by enzyme immunoassay (Siraganian et al., TIPS, 4: 432-437, 1983); and (5) enzyme-linked immunosorbent assay (ELISA).

Likewise, products of the immune response in any model organism (eg, mouse) or human subject can also be detected using various methods that are well known to those skilled in the art. For example, (1) antibody production in response to vaccination can be easily detected using standard methods currently used in clinical laboratories, such as ELISA; (2) migration of immune cells to sites of inflammation can be detected by scratching the surface of the skin and placing a sterile container to capture the migrating cells at the site of scratch (Peters et al., Blood, 72: 1310-5, 1988); (3) proliferation of peripheral blood mononuclear cells (PBMCs) in response to mitogens or a mixed lymphocyte response can be measured using ³ H-thymidine; (4) the phagocytic ability of granulocytes, macrophages and other phagocytes in PBMCs can be measured by placing PBMCs in wells along with labeled particles (Peters et al., Blood, 72: 1310-5, 1988); and (5) immune cell differentiation can be measured by labeling PBMCs with antibodies to CD molecules such as CD4 and CD8 and measuring the fraction of PBMCs expressing these markers.

According to one embodiment of the invention, anti-A33 antibodies according to the technology of the present invention are selected using the display of A33 peptides on the surface of replicated units of genetic information. See, for example, US Patent Nos. 5,514,548; 5,837,500; 5,871,907; 5,885,793; 5,969,108; 6,225,447; 6,291,650; 6,492,160; European patents EP 585 287; EP 605522; EP 616640; EP 1024191; EP 589 877; EP 774 511; EP 844 306. Methods useful for the production/selection of a filamentous bacteriphage particle containing a phagemid genome encoding a binding molecule having the desired specificity have been described. See, for example, European patent EP 774 511; US patents US 5871907; US 5969108; US 6225447; US 6291650; US 6492160.

In some embodiments, anti-A33 antibodies according to the technology of the present invention are selected using the display of A33 peptides on the surface of a host yeast cell. Methods useful for isolating scFv polypeptides using yeast surface display have been described in Kieke et al., Protein Eng. 1997 Nov; 10(11): 1303-10.

According to some embodiments of the invention, anti-A33 antibodies according to the technology

- 36 046795 according to the present invention is selected using ribosomal display. Methods useful for identifying ligands in peptide libraries using ribosomal display have been described in Mattheakis et al., Proc. Natl. Acad. Sci. USA 91: 9022-26, 1994; Hanes et al., Proc. Natl. Acad Sci. USA 94: 4937-42, 1997.

In specific embodiments, anti-A33 antibodies according to the technology of the present invention are selected using tRNA display of A33 peptides. Methods useful for in vitro selection of ligands using tRNA display have been described by Merryman et al. (Chem. Biol, 9: 741-46, 2002).

According to one embodiment of the invention, anti-A33 antibodies according to the technology of the present invention are selected using RNA display. Methods useful for selecting peptides and proteins using RNA display libraries have been described by Roberts et al. and Nemoto et al. (Roberts et al. Proc. Natl. Acad. Sci. USA, 94: 12297-302, 1997; and Nemoto et al. et al., FEBS Lett., 414: 405-8, 1997). Methods useful for selecting peptides and proteins using non-natural RNA display libraries have been described by Frankel et al. (Curr. Opin. Struct. Biol., 13: 506-12, 2003).

In some embodiments, anti-A33 antibodies according to the technology of the present invention are expressed in the periplasm of gram-negative bacteria and mixed with labeled A33 protein. See international patent application publication WO 02/34886. In clones expressing recombinant polypeptides with affinity for A33 protein, the concentration of labeled A33 protein bound to anti-A33 antibody is increased and allows cells to be isolated from the remainder of the library, as described in Harvey et al., Proc. Natl. Acad. Sci. 22: 9193-98 2004 and U.S. Pat. Publication no. 2004/0058403.

It is contemplated that once the desired anti-A33 antibodies have been selected, said antibodies can be produced in large quantities using any method known to one skilled in the art, for example, expression in a prokaryotic or eukaryotic cell, etc. Anti-A33 antibodies, which are, for example, but not limited to, anti-A33 fusion antibodies or fragments thereof, can be produced using standard methods for constructing an expression vector that encodes an antibody heavy chain in which the CDR regions and, if necessary, the minimal a portion of the variable region framework that is required to maintain the binding specificity of an antibody from the parent species (designed in accordance with the procedures described herein) is derived from the antibody from the parent species, and the remainder of the antibody is derived from immunoglobulin of the target species that can be manipulated as described herein, thereby obtaining a vector for expressing a hybrid antibody heavy chain.

A33 binding measurement. In some embodiments, an A33 binding assay refers to an assay format in which an A33 protein and an anti-A33 antibody are mixed under conditions suitable for binding between the A33 protein and the anti-A33 antibody and quantifying the binding between them. Quantitative binding is compared to a suitable control, which may be quantitative binding in the absence of A33 protein, quantitative binding in the presence of a non-specific immunoglobulin composition, or in the absence of A33 protein and in the presence of a non-specific immunoglobulin composition.

Quantitative binding can be assessed by any suitable method. Binding assay methods include, for example, ELISA, radioimmunoassay, scintillation proximity assay, fluorescence energy transfer assay, liquid chromatography, membrane filtration assay, etc. Biophysical assays for directly measuring the binding of A33 protein to anti-A33 antibody are, for example, nuclear magnetic resonance, fluorescence, fluorescence polarization, surface plasmon resonance (BIACORE chips), etc. Specific binding is determined using standard assays known in the art, for example, radioligand binding assays, ELISA, FRET, immunoprecipitation, SPR, NMR (2O-NMR). mass spectrometry, etc. If the specific binding of a candidate anti-A33 antibody is at least 1 percent greater than the binding observed in the absence of the candidate anti-A33 antibody, the antibody is useful as an anti-A33 antibody according to the technology of the present invention.

Measurement of neutralization A33. As used herein, A33 neutralization refers to the reduction in activity and/or expression of A33 protein as a result of binding to an anti-A33 antibody. The ability of anti-A33 antibodies according to the technology of the present invention to neutralize A33 activity/expression can be assessed in vitro or in vivo using methods known in the art.

Methods of using antibodies against A33 according to the technology of the present invention.

General provisions. Anti-A33 antibodies according to the technology of the present invention are useful in methods known in the art related to the localization and/or quantification of A33 protein (for example, for use in measuring the level of A33 protein in suitable physiological samples, for use in diagnostic methods, for use in polypeptide visualization, etc.). Antibodies according to the technology of the present invention are applicable

- 37 046795 for the isolation of protein A33 using standard methods such as affinity chromatography or immunoprecipitation. The anti-A33 antibody of the technology of the present invention can facilitate the purification of naturally occurring immunoreactive A33 proteins from biological samples, such as serum or mammalian cells, as well as recombinantly produced immunoreactive A33 proteins expressed in a host system. Moreover, anti-A33 antibodies can be used to detect immunoreactive A33 protein (eg, in plasma, cell lysate or cell supernatant) to assess the amount and expression pattern of the immunoreactive polypeptide. Anti-A33 antibodies according to the technology of the present invention can be used for diagnostic purposes to monitor the level of immunoreactive A33 protein in tissue as part of a clinical trial procedure, for example, to determine the effectiveness of a given treatment regimen. As noted above, detection can be facilitated by coupling (ie, physical binding) of anti-A33 antibodies according to the technology of the present invention with a detectable substance.

Detection of protein A33. An example of a method for determining the presence or absence of A33 immunoreactive protein in a biological sample involves obtaining a biological sample from a test subject and subjecting said biological sample to an anti-A33 antibody according to the technology of the present invention capable of detecting A33 immunoreactive protein such that the presence of said A33 immunoreactive protein is determined in a biological sample. Detection can be accomplished using a detectable label attached to the antibody.

The term labeled with respect to an anti-A33 antibody is intended to include direct labeling of the antibody by coupling (i.e., physically binding) a detectable substance to the antibody, as well as indirect labeling of the antibody by reacting with another compound that is directly labeled, such as secondary antibody. Examples of indirect labeling include detection of a primary antibody using fluorescently labeled secondary antibodies and end-labeling of a DNA probe with biotin so that it can be detected using fluorescently labeled streptavidin.

In some embodiments, the anti-A33 antibodies described herein are conjugated to one or more detectable tags. For such applications, anti-A33 antibodies can be labeled with a detectable label by covalently or non-covalently attaching a chromogenic, enzymatic, radioisotope, isotopic, fluorescent, toxic, chemiluminescent agent, nuclear magnetic resonance contrast agent, or other label.

Examples of suitable chromogenic labels include diaminobenzidine and 4-hydroxyazobenzene-2carboxylic acid. Examples of suitable enzymatic tags include malate dehydrogenase, staphylococcal nuclease, Δ-5-steroid isomerase, yeast alcohol dehydrogenase, α-glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, goat 6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.

Examples of suitable radioisotope labels include ³ H, 'In. ¹²⁵ I, ¹³¹ I, ³² P, ³⁵ S, ¹⁴ C, ⁵¹ Cr, ⁵⁷ To, ⁵⁸ Co, ⁵⁹ Fe, ⁷⁵ Se, ¹⁵² Eu, ⁹⁰ Y, ⁶⁷ Cu, ²¹⁷ Ci, ²¹¹ At, ²¹² Pb, ⁴⁷ Sc , ¹⁰⁹ Pd, etc. ¹¹¹ In is an example of an isotope for which in vivo imaging is useful because it avoids the problems associated with liver dehalogenation of ¹²⁵ I- or ¹³¹ Io-labeled A33-binding antibodies. In addition, this isotope emits gamma radiation energy that is more preferable 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, ¹¹¹ In conjugated to monoclonal antibodies with 1-(P-isothiocyanatebenzyl)DPTA shows weak uptake into non-tumor tissues, particularly the liver, and increased tumor site specificity (Esteban et al., J. Nucl. Med. 28 :861-870 (1987)). Examples of suitable non-radioactive isotopic tracers include ¹⁵⁷ Gd, ⁵⁵ Mn, ¹⁶² Dy, ⁵² Tr and ⁵⁶ Fe.

Examples of suitable fluorescent labels include ¹⁵² Eu label, fluorescein label, isothiocyanate label, rhodamine label, phycoerythrin label, phycocyanin label, allophycocyanin label, green fluorescent protein, o-phthaldehyde label and fluorescemicamine label. Examples of suitable toxin-based tags include diphtheria toxin, ricin, and cholera toxin.

Examples of chemiluminescent labels include luminol, isoluminol, aromatic acridine ester, imidazole label, acridine salt, axalate ester, luciferin, luciferase and aequorin. Examples of nuclear magnetic resonance contrast agents include heavy metal nuclei such as Gd, Mn and iron.

The detection method according to the technology of the present invention can be used to detect the presence of immunoreactive A33 protein in a biological sample in vitro as well as in vivo. In vitro methods for detecting immunoreactive protein A33 include enzyme-linked immunosorbent assays (ELISA), Western blotting, immunoprecipitation, radioimmunoassay and immunofluo

- 38 046795 review. Moreover, in vivo methods for detecting immunoreactive A33 protein involve administering a labeled anti-A33 antibody to the subject. For example, an anti-A33 antibody may be labeled with a radioactive marker, the presence and location of which in a subject can be detected using standard imaging techniques. According to one embodiment of the invention, the biological sample contains A33 protein molecules from a test subject.

Immunoassay and imaging. The anti-A33 antibody of the technology of the present invention can be used to analyze the level of immunoreactive A33 protein in a biological sample (eg, human plasma) using antibody-based methods. For example, protein expression in tissues can be studied using classical methods of immunohistology (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 assay (ELISA) and radioimmunoassay (RIA). Suitable labels for antibody-based assays are known in the art and include enzymatic labels such as glucose oxidase, and radioisotopes or other radioactive agents such as iodine ( ¹²⁵ I, ¹²¹ I, ¹³¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S), tritium ( ³ H), indium ( ¹¹² In) and technetium ( ⁹⁹ mTc), and fluorescent tags such as fluorescein, rhodamine and green fluorescent protein (GFP), as well as biotin.

In addition to analyzing the level of immunoreactive A33 protein in a biological sample, anti-A33 antibodies according to the technology of the present invention can be used for in vivo imaging of A33. Antibodies useful for this method include those detectable by radiography, nuclear magnetic resonance (NMR) or electron paramagnetic resonance (EPR). For radiography, suitable tracers include radioisotopes such as barium or cesium, which emit detectable radiation but do not cause obvious harm to the subject. Suitable markers for NMR and EPR include markers with a detectable characteristic spin, such as deuterium, which can be incorporated into anti-A33 antibodies by labeling nutrients for the corresponding scFv clone.

The subject is administered (eg, parenterally, subcutaneously, or intraperitoneally) an anti-A33 antibody that has been labeled with a suitable detectable moiety for imaging, such as a radioisotope (eg, ¹³¹ I, ¹¹² In, ⁹⁹ mTc), a radiocontrast agent, or a material detectable with nuclear magnetic resonance. One skilled in the art will appreciate that the size of the subject and the imaging system used will determine the amount of imaging fragment required to obtain diagnostic images. In the case of a radioactive isotopic fragment for a human subject, the administered radioactive dose is typically from 5 to 20 millicuries ^{of 99} mTc. The labeled anti-A33 antibody then accumulates at the site of cells that contain the specific target polypeptide. For example, labeled anti-A33 antibodies according to the technology of the present invention will accumulate in a subject in cells and tissues in which the A33 protein is localized.

Thus, the technology of the present invention provides a method for diagnosing a medical condition that includes: (a) analyzing the expression of immunoreactive A33 protein by measuring the binding of an anti-A33 antibody according to the technology of the present invention in cells or body fluid of a subject; (b) comparing the amount of immunoreactive A33 protein present in the sample with a standard standard/reference sample, wherein an increase or decrease in the level of immunoreactive A33 protein compared to the standard is an indicator of a medical condition.

Affinity purification. Antibodies against A33 according to the technology of the present invention can be used to purify immunoreactive A33 protein from a sample. In some embodiments, the antibodies are immobilized on a solid support. Examples of such solid carriers include plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, acrylic resins and such as polyacrylamide and latex beads. Methods 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, N.Y. (1974)).

The simplest way to bind an antigen to an antibody carrier matrix is to collect the beads in a column and pass the antigen solution through said column. The effectiveness of this method depends on the interaction time between the immobilized antibody and the antigen, which can be increased by reducing the flow rate. The immobilized antibody captures the antigen as it passes. Alternatively, the antigen can interact with the antibody carrier matrix by mixing a solution of the antigen with the carrier (eg, beads) and rotating or rocking the resulting suspension to ensure maximum contact between the antigen and the immobilized antibody. After completion of the binding reaction, the suspension is applied to a bead collection column. The beads are washed using a suitable wash buffer and then pure or substantially pure antigen is eluted.

The antibody or polypeptide of interest can be conjugated to a solid support such

- 39 046795 like a granule. In addition, the first solid support, such as a bead, may also optionally be conjugated to a second solid support, which may be a second bead or other support, by any suitable methods, including methods described herein for conjugating a polypeptide to a carrier . Thus, any of the conjugation methods and means described herein with reference to conjugating a polypeptide to a solid support can also be used to conjugate a first support to a second support, wherein said first and second supports may be the same or different.

Suitable linkers, which may be cross-linking agents, for use in conjugating a polypeptide to a solid support include a variety of agents that can react with a functional group present on the surface of the support or with the polypeptide, or with the specified functional group and the polypeptide. Reagents useful as cross-linking agents include homobifunctional and, in particular, heterobifunctional reagents. Useful bifunctional cross-linking agents include, but are not limited to, N-SIAB, dimaleimide, DTNB, N-SATA, N-SPDP, SMCC and 6-HYNIC. The cross-linking agent may be selected to provide a selectively cleavable linkage between the polypeptide and the solid support. For example, a photolabile cross-linker such as 3-amino(2-nitrophenyl)propionic acid can be used as a means to cleave the polypeptide from the solid support (Brown et al., Mol. Divers, pp, 4-12 (1995) ; Rothschild et al., Nucl. Acids Res., 24:351-66 (1969); US Pat. No. 5,643,722. Other cross-linking reagents are well 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, such as a bead, through a covalent amide bond formed between the bead containing the carboxyl functional group and the amino terminus of the polypeptide, or, conversely, through a covalent amide bond formed between the bead containing the amino functional group and carboxy terminus of the polypeptide. In addition, the bifunctional trityl linker can be attached to a support, for example, to a 4-nitrophenyl active ester resin such as Wang's resin via an amino group or a carboxy group on the resin via an amino resin. When using a bifunctional trityl linker approach, it may be necessary to treat the solid support with a volatile acid such as formic acid or trifluoroacetic acid to allow cleavage of the polypeptide and allow its removal. In such a case, the polypeptide can be applied as an application without the use of beads to the bottom of a well of a solid support or to the flat surface of a solid support. After adding the matrix solution, the polypeptide can be desorbed for MS.

Hydrophobic trityl linkers can also be used as acid-labile linkers using a volatile acid or a suitable matrix solution, for example a matrix solution containing 3-HPA, to cleave the amino-linked trityl group from the polypeptide. Acid lability may also be altered. For example, trityl, monomethoxytrityl, dimethoxytrityl or trimethoxytrityl of a polypeptide can be replaced by corresponding p-substituted or more acid-labile tritylamine derivatives, that is, trityl ether and tritylamine linkages can be created in the polypeptide. Accordingly, the polypeptide can be removed from the hydrophobic linker, for example, by breaking the hydrophobic attraction or by cleaving tritylether or tritylamine bonds under acidic conditions, including, if desired, typical MS conditions, where a matrix such as 3-HPA acts in as an acid.

Orthogonally cleavable linkers may also be useful for linking a first solid support, such as a bead, to a second solid support or for linking a polypeptide of interest to a solid support. By using such linkers, a first solid support, such as a bead, can be selectively cleaved from a second solid support without cleaving the polypeptide from the support; thereafter, said polypeptide can be later cleaved from the bead. For example, a disulfide linker, which can be cleaved using a reducing agent such as DTT, can be used to link the bead to a second solid support, and an acid-cleavable bifunctional trityl group can be used to immobilize the polypeptide on the support. If desired, the bond of the polypeptide to the solid support may be cleaved first, for example, while maintaining the bond between the first and second support intact. Tretyl linkers can provide covalent or hydrophobic conjugation and, regardless of the nature of the conjugation, the trityl group is readily cleaved under acidic conditions.

For example, the bead may be associated with a second support through a linking group, which may be selected such that the group has a length and chemical nature that provides high-density binding of the beads to a solid support or high-density binding of polypeptides to the beads. Such a linking group may have, for example, a tree structure providing multiple functional groups at the site of attachment to the solid support. Examples of such a linking group include polylysine, polyglutamic acid, penta

- 40 046795 erythrol and tris-hydroxy-aminomethane.

Connection by non-covalent binding. The antibody or polypeptide may be conjugated to a solid support, or the first solid support may also be conjugated to a second solid support, through a non-covalent interaction. For example, a magnetic bead made of a ferromagnetic material capable of magnetization may be attracted to a magnetic solid carrier and may be released from the carrier when the magnetic field is removed. Alternatively, the solid support may be provided with an ionic or hydrophobic moiety allowing the interaction of said ionic or hydrophobic moiety, respectively, with a polypeptide, for example a polypeptide containing an attached trityl group, or with a second solid support having hydrophobic characteristics.

The solid support may also be provided with a specific binding partner and therefore may be conjugated to a polypeptide or a second solid support containing a complementary binding moiety. For example, an avidin- or streptavidin-coated bead may be associated with a polypeptide containing a biotin moiety incorporated therein, or with a second solid support coated with biotin or a biotin derivative, such as iminobiotin.

It should be understood that any of the binding partners described in the present invention or otherwise known in the art can be interchanged. Thus, biotin, for example, may be incorporated into a polypeptide or solid carrier, and conversely, avidin or other biotin-binding moiety may be incorporated into the carrier or polypeptide, respectively. Other specific binding pairs contemplated for use in the present invention include, but are not limited to, hormones and their receptors, enzymes and their substrates, a nucleotide sequence and its complementary sequence, an antibody and the antigen with which it specifically interacts, and other such pairs , known to those skilled in the art.

A. Methods for the diagnostic use of anti-A33 antibodies according to the technology of the present invention.

General provisions. Antibodies against A33 according to the technology according to the present invention are useful in diagnostic methods. Thus, the technology of the present invention provides methods for using antibodies in diagnosing A33 activity in a subject. Anti-A33 antibodies according to the technology of the present invention can be selected to have any level of epitope binding specificity and very high binding affinity to the A33 protein. In general, the higher the binding affinity of an antibody, the more stringent washing conditions can be implemented in an immunoassay to remove nonspecifically bound material without removing the target polypeptide. Accordingly, anti-A33 antibodies according to the technology of the present invention useful in diagnostic assays typically have a binding affinity of about 10 ⁸ M ^-1 , 10 ⁹ M ^-1 , 10 ¹⁰ M ^-1 , 10 ¹¹ M ^-1 or 10 ¹² M ^-1 . In addition, it is desirable that anti-A33 antibodies used as diagnostic reagents have sufficient association rate kinetics to reach equilibrium under standard conditions within at least 12 hours, at least five (5) hours, or at least one (1) ) hours.

Anti-A33 antibodies can be used to detect immunoreactive A33 protein in a variety of standard assay formats. Such formats include immunoprecipitation, Western blotting, ELISA, radioimmunoassay, and immunometric assays. See Harlow & Lane, Antibodies, A Laboratory Manual (Cold Spring Harbor Publications, New York, 1988); US Patents No. 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's body. In certain embodiments, the subject has early stage cancer. In one embodiment, early stage cancer is determined by the level or pattern of A33 protein expression in a sample obtained from the subject. In specific embodiments of the invention, the sample is selected from the group consisting of urine, blood, serum, plasma, saliva, amniotic fluid, cerebrospinal fluid (CSF), and body tissue biopsy.

Immunometric or sandwich assays are one format of diagnostic methods according to the technology of the present invention (see US patents 4,376,110, 4,486,530, 5,914,241 and 5,965,375). Such assays use one antibody, for example, an anti-A33 antibody, or a population of anti-A33 antibodies, immobilized on a solid support, and another anti-A33 antibody, or a population of anti-A33 antibodies, in solution. Typically, an anti-A33 antibody in solution or a population of anti-A33 antibodies in a solution is labeled. When using a population of antibodies, the population may contain antibodies that bind to different antigenic determinants on the target polypeptide. Accordingly, the same population can be used both as a solid phase and as antibodies in solution. When using anti-A33 monoclonal antibodies, first and second anti-A33 monoclonal antibodies having different binding specificities are used for the solid phase and the solution phase. Solid phase antibodies (also called capture) and solution antibodies (also called detector) can bind to anti

- 41 046795 target genome in any order or simultaneously. If reaction with the solid phase antibody occurs first, the assay is called a direct assay. Conversely, if the reaction occurs first with the antibody in solution, the assay is called a reverse assay. If the target is exposed to both antibodies at the same time, the assay is called a simultaneous assay. After reacting the A33 protein with the anti-A33 antibody, the sample is incubated for a time, which typically ranges from about 10 minutes to about 24 hours and is typically about 1 hour. A washing step is then performed to remove sample components that do not bind specifically to the anti-A33 antibody used as the diagnostic reagent. When solid phase antibodies and solution antibodies are bound in separate steps, washing can be performed after one or both of the binding steps. After washing, binding is quantified, typically by detecting label bound to the solid phase by binding of the labeled antibody from solution. Typically, for a given pair of antibodies or populations of antibodies and given reaction conditions, a calibration curve is constructed from samples containing known concentrations of the target antigen. Concentrations of immunoreactive A33 protein in test samples are then determined by interpolating data from a calibration curve (ie, standard curve). The amount of analyte can be measured either by the amount of labeled antibody from solution bound at equilibrium or by kinetic measurements of bound labeled antibody from solution at several time points until equilibrium is reached. The slope of such a curve is an indicator of the concentration of A33 protein in the sample.

Suitable carriers for use in the above methods include, for example, nitrocellulose membranes, nylon membranes and derivatized nylon membranes, and also particles such as agarose, dextran gel, test strips, microparticles, microspheres, magnetic particles, test tubes, wells microtiter plate, SEPHADEX™ (Amersham Pharmacia Biotech, Piscataway, NJ), etc. Immobilization can be accomplished by absorption or covalent attachment. Optionally, anti-A33 antibodies can be coupled to a linker molecule, such as biotin, to attach to a surface-bound linker, such as avidin.

In some embodiments, the present invention provides an anti-A33 antibody according to the technology of the present invention conjugated to a diagnostic agent. The diagnostic agent may contain a radioactive or non-radioactive tracer, a contrast agent (such as an agent for magnetic resonance imaging, computed tomography or ultrasound analysis), and the radioactive tracer may be an isotope emitting gamma, beta, alpha particles, Auger electrons or positrons. The diagnostic agent is an administered molecule conjugated to an antibody fragment, i.e. an antibody or a fragment or subfragment thereof, and useful for diagnosing or detecting a disease by determining the location of cells containing the antigen.

Useful diagnostic agents include, but are not limited to, radioisotopes, dyes (such as biotin-streptavidin complex dyes), contrast agents, fluorescent compounds or molecules and enhancers (eg, paramagnetic ions) for magnetic resonance imaging (MRI). US Patent No. 6,331,175, which is incorporated herein by reference in its entirety, describes an MRI method and the production of antibodies conjugated to an MRI enhancer. In some embodiments, the diagnostic agents are selected from the group consisting of radioisotopes, enhancers for use in magnetic resonance imaging, and fluorescent compounds. Loading an antibody component with radioactive metals or paramagnetic ions may require it to react with a reagent having a long tail to which a plurality of chelating groups are attached to bind the ions. Such a tail may be a polymer such as a polylysine, polysaccharide or other derivatized or derivatizable chain having side groups to which chelating groups can bind, such as, for example, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrins, polyamines, crown ethers, bis-thiosemicarbazones, polyoximes and the like groups which are known to be useful for this purpose. Chelates can be combined with antibodies according to the technology of the present invention using standard chemistry techniques. The chelate is normally coupled to the antibody by a moiety that allows for formation of a bond to the molecule with minimal loss of immunoreactivity and minimal aggregation and/or internal cross-linking. Other methods and reagents for conjugating chelates with antibodies are described in US Pat. No. 4,824,659. In particular, useful metal-chelate combinations include 2-6οη3κλ^ΤΡΑ and its monomethyl and cyclohexyl analogues used with diagnostic isotopes for radioimaging. These same chelates, when combined with non-radioactive metals such as manganese, iron and gadolinium, are useful for MRI when used along with anti-A33 antibodies according to the technology of the present invention.

Macrocyclic chelates such as NOTA (1,4,7-triazacyclononane-H,Ni,Ni-triacetic acid), DOTA and TETA (p-bromoacetamidobenzyltetraethylaminetetraacetic acid) are used with a variety of metals and radioactive metals such as gallium, yttrium and copper radionuclides

- 42 046795 respectively. Such metal-chelate complexes can be stabilized by adjusting the ring size to the metal of interest. Examples of other DOTA chelates include (i) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH ₂ ;

(ii) Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH ₂ ;

(iii) DOTA-D-Asp-D-Lys(HSG)-D-Asp-D-Lys(HSG)-NH ₂ ;

(iv) DOTA-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH ₂ ;

(v) DOTA-D-Tyr-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH ₂ ;

(vi) DOTA-D-Ala-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH ₂ ;

(vii) DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH2;

(viii) Ac-D-Phe-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-NH2;

(ix) Ac-D-Phe-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH ₂ ;

(x) Ac-D-Phe-D-Lys(Bz-DTPA)-D-Tyr-D-Lys(Bz-DTPA)-NH ₂ ;

(xi) Ac-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH2;

(xii) DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH2;

(xiii) (Tscg-Cys)-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(DOTA)-NH2;

(xiv) Tscg-D-Cys-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2;

(xv) (Tscg-Cys)-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2;

(xvi) Ac-D-Cys-D-Lys(DOTA)-D-Tyr-D-Ala-D-Lys(DOTA)-D-Cys-NH2;

(xvii) Ac-D-Cys-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH2;

(xviii) Ac-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-D-Lys(Tscg-Cys)-NH2 and (xix) Ac-D-Lys(DOTA)-D-Tyr- D-Lys(DOTA)-D-Lys(Tscg-Cys)-NH2.

Other ring-type chelates such as macrocyclic polyethers are also being considered and are of interest when stably binding nuclides such as 223Ra are used for RAIT.

B. Therapeutic use of anti-A33 antibodies according to the technology of the present invention.

Compositions based on immunoglobulins (eg, antibodies or antigen-binding fragments thereof) according to the technology of the present invention are useful for the treatment of A33-associated types of cancer. Such treatment can be used in patients who have been diagnosed with abnormally high levels of A33 (for example, patients who have been diagnosed using the methods described herein) or in patients who have been diagnosed with a disease known to be associated with such abnormal level. In one aspect, the present invention provides a method of treating A33-associated cancer in a subject in need thereof, comprising administering to said subject an effective amount of an antibody (or antigen-binding fragment thereof) according to the technology of the present invention. Examples of cancer that can be treated with an antibody according to the technology of the present invention include, but are not limited to: colorectal cancer, pseudomyxoma peritonei, appendix cancer, pancreatic cancer and gastric cancer. A33-associated cancer may be colorectal cancer with the MSI genotype or the MSS genotype. Additionally or alternatively, in some embodiments, colorectal cancer is associated with a KRAS G12D mutation or a p53 mutation.

Compositions according to the technology of the present invention can be used in conjunction with other therapeutic agents useful in the treatment of A33-associated cancer types. For example, antibodies according to the technology of the present invention can be administered separately, sequentially or simultaneously with at least one additional therapeutic agent selected from the group consisting of alkylating agents, platinum-based agents, taxanes, vinca-based agents, anti-estrogen drugs, inhibitors aromatase, ovarian suppressive agents, VEGF/VEGFR inhibitors, EGF/EGFR inhibitors, PARP inhibitors, cytostatic alkaloids, cytotoxic antibiotics, antimetabolites, endocrine/hormonal agents, bisphosphonate therapy agents and targeted biological therapy agents (eg, therapeutic peptides described in the publication US Patent Application 6306832, International Patent Application Publication WO 2012007137, International Patent Application Publication WO 2005000889, International Patent Application Publication WO 2010096603, etc.). In some embodiments, the at least one additional therapeutic agent is a chemotherapeutic agent. Specific chemotherapeutic agents include, but are not limited to, cyclophosphamide, fluorouracil (or 5-fluorouracil or 5-FU), methotrexate, edatrexate (10-ethyl-10-deazaminopterin), thiotepa, carboplatin, cisplatin, taxanes, paclitaxel, protein paclitaxel, docetaxel, vinorelbine, tamoxifen, raloxifene, toremifene, fulvestrant, gemcitabine, irinotecan, ixabepilone, temozolmide, topotecan, vincristine, vinblastine, eribulin, mutamicin, capecitabine, anastrozole, exemestane, letrozole, leuprolide, abarelix, buserlin, goserelin, strol acetate, risedronate, pamidronate, ibandronate, alendronate, denosumab, zoledronate, trastuzumab, ticurb, anthracyclines (eg, daunorubicin and doxorubicin), bevacizumab, oxaliplatin, melphalan, etoposide, mechlorethamine, bleomycin, microtubule poisons, anone acetogenins, or

- 43 046795 their combinations.

Compositions according to the technology of the present invention may optionally be administered as a single bolus dose to a subject in need thereof. Alternatively, the dosage regimen may include multiple administrations administered at different times after tumor onset.

Administration can be accomplished by any suitable route, including oral, intranasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), rectal, intracranial, intrathecal, or topical administration. Administration includes self-administration and administration with the assistance of another subject. It should also be understood that the various methods of treating the described medical conditions are intended to mean treatment per se, including complete as well as incomplete treatment, in which some biologically or medically relevant result is achieved.

In some embodiments, the antibodies of the technology of the present invention comprise pharmaceutical dosage forms that can be administered to subjects in need thereof in one or more doses. Dosage regimens can be adjusted to obtain the desired response (eg, therapeutic effect).

Typically, an effective amount of an antibody composition according to the technology of the present invention sufficient to achieve a therapeutic effect ranges from about 0.000001 mg per kilogram of body weight per day to about 10,000 mg/kg body weight per day. Typically, dosage ranges are from about 0.0001 mg/kg body weight per day to about 100 mg/kg body weight per day. For administration of anti-A33 antibodies, the dose ranges from about 0.0001 to 100 mg/kg, and typically ranges from 0.01 mg/kg body weight to 5 mg/kg body weight of the subject per week, every two weeks, or every three weeks. For example, doses may be 1 mg/kg body weight or 10 mg/kg body weight per week, every two weeks or every three weeks, or within the range of 1-10 mg/kg per week, every two weeks or every three weeks. According to one embodiment of the invention, one dose of antibody ranges from 0.1-10,000 micrograms per kg of body weight. According to one embodiment of the invention, the concentration of antibody in the carrier varies from 0.2 to 2000 micrograms per milliliter delivered. Examples of treatment regimens include once every two weeks or once every month or once every 3-6 months. Antibodies against A33 can be administered periodically. The intervals between single doses may be an hour, a day, a week, a month or a year. The intervals may also vary and be determined based on measuring the level of antibody in the subject's blood. In some methods, the dose is adjusted to achieve a serum antibody concentration in the subject of from about 75 μg/ml to about 125 μg/ml, from about 100 μg/ml to about 150 μg/ml, from about 125 μg/ml to about 175 µg/ml or from about 150 µg/ml to about 200 µg/ml. Alternatively, anti-A33 antibodies can be administered in a sustained release dosage form, which requires less frequent administration. The dose and frequency vary depending on the half-life of the antibody in the subject's body. The dosage and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. When used prophylactically, a relatively low dose is administered at relatively long intervals over a long period of time. When used therapeutically, it is sometimes necessary to administer a relatively high dose at relatively short intervals until disease progression is reduced or terminated or until the subject experiences partial or complete relief of disease symptoms. The patient can then be administered according to the prophylactic regimen.

According to another aspect, the present invention provides a method of detecting a tumor in a subject in vivo, comprising (a) administering to said subject an effective amount of an antibody (or an antigen-binding fragment thereof) according to the technology of the present invention, wherein said antibody is engineered to localize to a tumor expressing A33 is a labeled radioisotope; and (b) detecting the presence of a tumor in the subject by detecting a level of antibody radioactivity greater than a control value. In some embodiments, the control value is expressed in units of injectable dose per gram (%I/g). The reference value can be calculated by measuring the level of radioactivity in non-tumor (normal) tissues and calculating the mean level of radioactivity in non-neoplastic (normal) tissues ± standard deviation. In some embodiments, the radioactivity ratio between tumor and normal tissue is approximately 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10: 1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1 or 100:1.

In some embodiments, the subject is diagnosed or suspected of having cancer. The level of radioactivity of an antibody can be determined using positron emission tomography or single photon emission computed tomography.

Additionally or alternatively, according to some embodiments of the invention, the method also includes administering to the subject an effective amount of an immunoconjugate comprising an antibody according to the technology of the present invention conjugated to a radionuclide. Co

- 44 046795 According to some embodiments of the invention, the radionuclide is an alpha emitting isotope, a beta emitting isotope, an Auger electron emitting radionuclide, or any combination thereof. Examples of beta-emitting isotopes include ⁸⁶ Y, ⁹⁰ Y, ⁸⁹ Sr, ¹⁶⁵ Dy, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁷⁷ Lu, and ⁶⁷ Cu. Examples of alpha-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 electron-emitting radionuclides include ¹¹¹ In, ⁶⁷ Ga, ⁵¹ Cr, ⁵⁸ Co, ^99m Tc, ^103m Rh, ^195m Pt, ¹¹⁹ Sb, ¹⁶¹ Ho, ^189m Os, ¹⁹² Ir, ²⁰¹ T1 and ²⁰³ Pb. In some embodiments of the method, non-specific FcR-dependent binding in normal tissues is eliminated or reduced (eg, by using the N297A mutation in the Fc region, resulting in aglycosylation). The therapeutic efficacy of such an immunoconjugate can be determined by calculating the area under the curve (AUC) ratio of the tumor! P PC normal tissue. In some embodiments, the ratio of tumor:normal tissue AUC for the immunoconjugate is approximately 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10 :1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1 , 75:1, 80:1, 85:1, 90:1, 95:1 or 100:1.

PREV. In one aspect, the present invention provides a method for detecting solid tumors in a subject in need thereof, comprising (a) administering to said subject an effective amount of a complex comprising a radiolabeled DOTA hapten and a bispecific antibody according to the technology of the present invention that binds to said radiolabeled DOTA -hapten and A33 antigen, where the specified complex is designed with the possibility of localization in a solid tumor expressing the A33 antigen, recognized by the bispecific antibody of the complex; and (b) detecting the presence of solid tumors in the subject by detecting a level of radioactivity of the complex in excess of a control value. In some embodiments, the subject is a human.

According to another aspect, the present invention provides a method of selecting a subject for pre-targeted radioimmunotherapy, comprising (a) administering to the subject an effective amount of a complex comprising a radiolabeled DOTA hapten and a bispecific antibody according to the technology of the present invention that binds to the radiolabeled DOTA hapten and an A33 antigen, where the specified complex is designed with the possibility of localization in a solid tumor expressing the A33 antigen, recognized by the bispecific antibody of the complex; (b) detecting the level of radioactivity of the complex; and (c) selecting for pretargeted radioimmunotherapy a subject whose level of complex radioactivity exceeds a control value. In some embodiments, the subject is a human.

Examples of DOTA haptens include (i) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH2;

(ii) Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH2;

(iii) DOTA-D-Asp-D-Lys(HSG)-D-Asp-D-Lys(HSG)-NH2;

(iv) DOTA-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2;

(v) DOTA-D-Tyr-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2;

(vi) DOTA-D-Ala-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2;

(vii) DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH2;

(viii) Ac-D-Phe-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-NH2;

(ix) Ac-D-Phe-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH ₂ ;

(x) Ac-D-Phe-D-Lys(Bz-DTPA)-D-Tyr-D-Lys(Bz-DTPA)-NH ₂ ;

(xi) Ac-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH2;

(xii) DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH2;

(xiii) (Tscg-Cys)-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(DOTA)-NH2;

(xiv) Tscg-D-Cys-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2;

(xv) (Tscg-Cys)-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2;

(xvi) Ac-D-Cys-D-Lys(DOTA)-D-Tyr-D-Ala-D-Lys(DOTA)-D-Cys-NH2;

(xvii) Ac-D-Cys-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH2;

(xviii) Ac-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-D-Lys(Tscg-Cys)-NH2;

(xix) Ac-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-D-Lys(Tscg-Cys)-NH ₂ and (xx) DOTA.

The radioactive tracer may be an alpha-emitting isotope, a beta-emitting isotope, or an Auger electron-emitting radionuclide. Examples of radioactive tracers 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. According to some embodiments of the method described in the present invention, the level of radioactivity of the complex is detected using positron emission tomography or single photon emission computed tomography. Additionally or alternatively, in some embodiments of the methods described herein, the subject is diagnosed or suspected of having A33-positive cancer, such as colorectal cancer, pseudomyxoma peritonei, appendiceal cancer, pancreatic cancer, and gastric cancer.

- 45 046795

Additionally or alternatively, according to some embodiments of the methods described in the present invention, the complex is administered intravenously, intramuscularly, intraarterially, intrathecally, intracaspularly, intraorbitally, intradermally, intraperitoneally, transtracheally, subcutaneously, intracerebroventricularly, orally or intranasally. In specific embodiments, the complex is administered to the cerebrospinal fluid or blood of a subject.

According to some embodiments of the methods described in the present invention, the level of radioactivity of the complex is detected between 2 and 120 hours after administration of the complex. In specific embodiments of the methods described in the present invention, the level of radioactivity of the complex is expressed as a percentage of the injected dose per gram of tissue (%I/g). The reference value can be calculated by measuring the level of radioactivity in non-tumor (normal) tissues and calculating the mean level of radioactivity in non-neoplastic (normal) tissues ± standard deviation. In some embodiments, the reference value is a standardized accumulation value (SUV, see Thie JA, J Nucl Med. 45(9): 1431-4 (2004). In some embodiments, the ratio of radiation levels between tumor and normal cloth is approximately 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1 , 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90 :1, 95:1 or 100:1.

According to another aspect, the present invention provides a method of sensitizing a tumor to radiation therapy in a subject diagnosed with A33-positive cancer, comprising (a) administering to said subject an effective amount of an anti-DOTA hapten bispecific antibody according to the technology of the present invention, wherein said bispecific antibody anti-DOTA hapten is designed to localize to a tumor expressing the A33 target antigen; and (b) administering to said subject an effective amount of a radiolabeled DOTA hapten, wherein said radiolabeled DOTA hapten is designed to bind to a bispecific anti-DOTA hapten antibody. In some embodiments, the subject is a human.

The anti-DOTA hapten bispecific antibody is administered under conditions and for a period of time (eg, according to a dosage regimen) sufficient to saturate the tumor cells. In some embodiments, the unbound anti-DOTA hapten bispecific antibody is removed from the bloodstream following administration of the anti-DOTA hapten bispecific antibody. In some embodiments, the radiolabeled DOTA hapten is administered after a period of time that may be sufficient to allow clearance of the unbound bispecific anti-DOTA hapten antibody.

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

Additionally or alternatively, in some embodiments, the method also includes administering to the subject an effective amount of a clearance agent prior to administration of the radiolabeled DOTA hapten. The clearance agent can be any molecule (dextran or dendrimer or polymer) that can be conjugated to a C825 hapten. In some embodiments, the clearance agent has a molecular weight of no more than 2000 kDa, 1500 kDa, 1000 kDa, 900 kDa, 800 kDa, 700 kDa, 600 kDa, 500 kDa, 400 kDa, 300 kDa, 200 kDa, 100 kDa , 90 kDa, 80 kDa, 70 kDa, 60 kDa, 50 kDa, 40 kDa, 30 kDa, 20 kDa, 10 kDa or 5 kDa. In some embodiments, the clearance agent is a 500 kDa aminodextran-DOTA conjugate (e.g., 500 kDa dextran-DOTA-Bn (Y), 500 kDa dextran-DOTA-Bn (Lu), or 500 kDa dextran-DOTA-Bn ( In) etc.). In some embodiments, the clearance agent and radiolabeled DOTA hapten are administered without additional administration of a bispecific anti-DOTA hapten antibody according to the technology of the present invention. For example, in some embodiments, an anti-DOTA hapten bispecific antibody according to the technology of the present invention is administered in accordance with a regimen that includes at least one cycle of: (i) administration of a bispecific anti-DOTA hapten antibody according to the technology of the present invention (optional so that the corresponding tumor cells are saturated); (ii) administering a radiolabeled DOTA hapten and optionally a clearance agent; (iii) optional additional administration of radiolabeled DOTA hapten and/or

- 46 046795 providing clearance agent without additional administration of a bispecific antibody against DOTA hapten. In some embodiments, the method may include multiple such cycles (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more cycles).

Additionally or alternatively, in some embodiments of the method, a bispecific anti-DOTA hapten antibody and/or a radiolabeled DOTA hapten is administered intravenously, intramuscularly, intraarterially, intrathecally, intracaspularly, intraorbitally, intracutaneously, intraperitoneally, transtracheally, subcutaneously, intracerebroventricularly, orally. or intranasally. In one aspect, the present invention provides a method of sensitizing a tumor to radiation therapy in a subject diagnosed with A33-positive cancer, comprising administering to said subject an effective amount of a complex comprising a radiolabeled DOTA hapten and a bispecific antibody according to the technology of the present invention that recognizes and binds with the specified radioactively labeled DOTA hapten and the A33 target antigen, where the specified complex is designed to be localized in a tumor expressing the A33 target antigen recognized by the bispecific antibody of the complex. The specified complex can be administered intravenously, intramuscularly, intraarterially, intrathecally, intracaspularly, intraorbitally, intracutaneously, intraperitoneally, transtracheally, subcutaneously, intracerebroventricularly, orally or intranasally. In some embodiments, the subject is a human.

In another aspect, the present invention provides a method of treating cancer in a subject in need thereof, comprising (a) administering an effective amount of an anti-DOTA hapten bispecific antibody according to the technology of the present invention to the subject, wherein said anti-DOTA hapten bispecific antibody is designed to localize to a tumor , expressing the A33 target antigen; and (b) administering an effective amount of a radiolabeled DOTA hapten to a subject, wherein said radiolabeled DOTA hapten is designed to bind to a bispecific anti-DOTA hapten antibody. The anti-DOTA hapten bispecific antibody is administered under conditions and for a period of time (eg, according to a dosage regimen) sufficient to saturate the tumor cells. In some embodiments, the unbound anti-DOTA hapten bispecific antibody is removed from the bloodstream following administration of the anti-DOTA hapten bispecific antibody. In some embodiments, the radiolabeled DOTA hapten is administered after a period of time that may be sufficient to allow clearance of the unbound bispecific anti-DOTA hapten antibody. In some embodiments, the subject is a human.

Accordingly, in some embodiments, the method also includes administering to the subject an effective amount of a clearance agent prior to administration of the radiolabeled DOTA hapten. The radiolabeled DOTA hapten can be administered any time between 1 and 4 minutes or more than a day after administration of the anti-DOTA hapten bispecific antibody. For example, in some embodiments, the radiolabeled DOTA hapten is administered at 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes. , 45 minutes, 50 minutes, 55 minutes, 1 hour, 1.25 hours, 1.5 hours, 1.75 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 11 hours, 12 hours , 13 o'clock, 14 o'clock, 15 o'clock, 16 o'clock, 17 o'clock, 18 o'clock, 19 o'clock, 20 o'clock, 21 o'clock, 22 o'clock, 23 o'clock, 24 o'clock, 48 o'clock, 72 o'clock, 96 o'clock or any range within the indicated values after administration of a bispecific antibody against DOTA hapten. Alternatively, radiolabeled DOTA hapten can be administered at any time 4 or more days after administration of the anti-DOTA hapten bispecific antibody.

The clearance agent may be a 500 kDa aminodextranDOTA conjugate (e.g., 500 kDa dextran-DOTA-Bn (Y), 500 kDa dextran-DOTA-Bn (Lu), or 500 kDa dextran-DOTA-Bn (In), etc. ). In some embodiments, the clearance agent and radiolabeled DOTA hapten are administered without additional administration of a bispecific anti-DOTA hapten antibody. For example, in some embodiments, the anti-DOTA hapten bispecific antibody is administered in accordance with a regimen that includes at least one cycle of: (i) administering the anti-DOTA hapten bispecific antibody according to the technology of the present invention (optionally in a manner that occurs saturation of the corresponding tumor cells); (ii) administering a radiolabeled DOTA hapten and, optionally, a clearance agent; (iii) optionally additional administration of a radiolabeled DOTA hapten and/or a clearance agent without additional administration of a bispecific anti-DOTA hapten antibody. In some embodiments, the method may include multiple such cycles (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more cycles).

The present invention also provides methods for treating cancer in a subject in need thereof, comprising administering to said subject an effective amount of a complex containing

- 47 046795 radiolabeled DOTA hapten and bispecific antibody according to the technology of the present invention, which recognizes and binds to said radiolabeled DOTA hapten and A33 target antigen, wherein said complex is designed to localize to a tumor expressing the A33 target antigen, recognized by the bispecific antibody of the complex. The therapeutic efficacy of such a complex can be determined by calculating the ratio of the area under the curve (AUC) of the tumor: AUC of normal tissue. In some embodiments, the PIC tumor VIC normal tissue ratio for the complex is approximately 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10: 1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1 or 100:1.

Toxicity. An optimally effective amount (eg, dose) of an anti-A33 antibody described herein provides a therapeutic benefit and is substantially free of toxicity in a subject. The toxicity of the anti-A33 antibody described in the present invention can be determined using standard pharmaceutical procedures in cell cultures or experimental animals, for example, by determining the LD ₅₀ (dose lethal to 50% of the population) or LD100 (dose lethal to 100% of the population) . The ratio between doses causing toxic and therapeutic effects is the therapeutic index. Data obtained from the three cell culture assays and animal studies can be used to develop dose ranges that are not toxic for use in humans. The dose of the anti-A33 antibody described herein is within the range of circulating antibody concentrations that include an effective dose with little or no toxicity. The dose may vary within the specified range depending on the dosage form used and the route of administration used. The exact dosage form, route of administration and dose may be selected by the attending physician based on the condition of the subject. See, for example, Fingl et al., In: The Pharmacological Basis of Therapeutics, Ch. 1 (1975).

Dosage forms of pharmaceutical compositions. In accordance with the methods of the technology of the present invention, the anti-A33 antibody can be included in pharmaceutical compositions suitable for administration. Pharmaceutical compositions generally contain a recombinant or substantially purified antibody and a pharmaceutically acceptable carrier in a form suitable for administration to a subject. Pharmaceutically acceptable carriers are determined in part by the particular composition administered, as well as the particular route of administration of said composition employed. Accordingly, there is a wide variety of suitable dosage forms of pharmaceutical compositions for administering antibody compositions (see, for example, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA 18 ^th ed., 1990). Pharmaceutical compositions are generally prepared in a sterile, substantially isotonic form in strict accordance with all US Food and Drug Administration (FDA) Good Manufacturing Practice (GMP) regulations.

The terms pharmaceutically acceptable, physiologically acceptable, and grammatical variations thereof, when used in relation to compositions, carriers, diluents, and reagents, are used interchangeably and mean that the materials can be administered to a subject or applied to the body of a subject without causing undesirable physiological effects to a degree that would interfere with introduction of the specified composition. For example, a pharmaceutically acceptable excipient means an excipient that is useful in the preparation of a pharmaceutical composition that is generally safe, non-toxic and desirable and includes excipients that are acceptable for veterinary use as well as for pharmaceutical use in humans. Such excipients may be solid, liquid, semi-solid or gaseous in the case of an aerosol composition. The term pharmaceutically acceptable salts and esters means salts and esters that are pharmaceutically acceptable and have the desired pharmacological properties. Such salts include salts that can be formed by the ability of the acidic protons present in the composition to react with inorganic or organic bases. Suitable inorganic salts include those formed with alkali metals, for example sodium and potassium, magnesium, calcium and aluminum. Suitable organic salts include those formed with organic bases such as amine bases, for example ethanolamine, diethanolamine, triethanolamine, tromethamine, Nmethylglucamine, etc. Such salts also include acid addition salts formed with inorganic acids (eg, hydrochloric and hydrobromic acids) and organic acids (eg, acetic acid, citric acid, maleic acid, and alkane and arenesulfonic acids such as methanesulfonic acid and benzenesulfonic acid). Pharmaceutically acceptable esters include those derived from the carboxy-, sulfonyloxy-phosphonoxy groups present in the anti-A33 antibody, for example, C _1-6 alkyl esters. When two acid groups are present, the pharmaceutically acceptable salt or ester may be a monoacid/monosalt or ester, or a dibasic salt or ester; and likewise, if more than two acid groups are present, some or all such groups

- 48 046795 can be converted into salts or esterified. The anti-A33 antibody referred to in the technology of the present invention may be present in a non-salt or non-esterified form or a salt and/or esterified form, and reference to such anti-A33 antibody is intended to include both the original (non-salt and non-esterified form) of the compound and its pharmaceutically acceptable salts and esters. In addition, certain embodiments of the technology of the present invention may be present in more than one stereoisomeric form. Reference to such anti-A33 antibody is intended to include all individual stereoisomers and all mixtures (racemic or otherwise) of such stereoisomers. One skilled in the art will readily determine the appropriate timing, sequence, and dosage of administration for specific drugs and compositions of the technology of the present invention.

Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin solution. Liposomes and non-aqueous carriers such as fatty oils can also be used. The use of such media and compounds for pharmaceutically active substances is well known in the art. Unless any conventional carrier or compound is incompatible with the anti-A33 antibody, its use in the compositions is contemplated. Additional active compounds may also be included in the compositions.

The pharmaceutical composition according to the technology of the present invention is formulated so that it is compatible with the intended route of administration. Anti-A33 antibody compositions according to the technology of the present invention can be administered parenterally, topically, intravenously, orally, subcutaneously, intraarterially, intradermally, transdermally, rectally, intracranially, intrathecally, intraperitoneally, intranasally or intramuscularly or by inhalation. An anti-A33 antibody may optionally be administered in combination with other agents that are at least partially effective in treating various A33-associated cancers.

Solutions or suspensions used for parenteral, intradermal or subcutaneous use may include the following components: a sterile diluent such as water for injection, saline, fatty oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates; and tonicity control compounds such as sodium chloride or dextrose. The pH can be adjusted using acids or bases such as hydrochloric acid or sodium hydroxide. The drug for parenteral administration may be contained in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injection include sterile aqueous solutions (as long as they are water soluble) or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable vehicles include saline, bacteriostatic water, Cremophor EL™ (BASF, Parsipanny, NJ.) or saline buffered saline (PBS). In all cases, the composition must be sterile and must be liquid to such an extent that it can be easily syringed. Said composition must be stable under production and storage conditions and must be protected from contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg, glycerin, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be achieved using various antibacterial and antifungal compounds, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases it is desirable to include isotonic compounds in the composition, for example sugars, polyalcohols such as mannitol, sorbitol, sodium chloride. Prolonged absorption of injectable compositions can be achieved by including an absorption delaying compound, such as aluminum monostearate and gelatin, in the composition.

Sterile injection solutions can be prepared by incorporating the required amount of anti-A33 antibody according to the technology of the present invention into a suitable diluent with one or a combination of the ingredients listed above, as required, followed by sterilization by filtration. In general, dispersions are prepared by incorporating the anti-A33 antibody into a sterile vehicle that contains the basic dispersion medium and the necessary other ingredients listed above. In the case of sterile powders for the preparation of sterile injection solutions, the preparation methods are vacuum drying and freeze drying, which result in a powder of the active ingredient with any additional

- 49 046795 with the desired ingredient from its pre-sterilized solution by filtration. Antibodies according to the technology of the present invention can be administered as a slow-release injection or an implantable preparation, which can be formulated to provide a sustained or intermittent release of the active ingredient.

Compositions for oral administration generally include an inert diluent or edible carrier. They may be enclosed in gelatin capsules or compressed into tablets. For oral therapeutic administration, the anti-A33 antibody may be presented along with excipients and used in the form of tablets, lozenges or capsules. Compositions for oral administration can also be prepared using a liquid carrier for use as a mouthwash, wherein the compound in the liquid carrier is applied to the mouth, rinsed and spit out or swallowed. Pharmaceutically compatible binding compounds and/or adjuvant materials may be included in the composition. Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients or compounds of a similar nature: a binder such as microcrystalline cellulose, tragacanthum or gelatin; an excipient such as starch or lactose; a disintegrating compound such as alginic acid, Primogel or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as fumed silica; a sweetener such as sucrose or saccharin; or a flavoring such as peppermint, methyl salicylate, or orange flavor.

For administration by inhalation, the anti-A33 antibody is delivered as an aerosol spray from a pressurized container or nebulizer that contains a suitable propellant, for example a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be carried out by the transmucosal or transdermal route. For transmucosal or transdermal administration, the product uses penetrants suitable for penetration through the appropriate barrier. Such penetrants are generally known in the art and include, for example, in the case of mucosal administration, detergents, bile salts and fusidic acid derivatives. Transmucosal administration can be achieved by using nasal sprays or suppositories. For transdermal administration, anti-A33 antibody is formulated as ointments, balms, gels or creams as is generally known in the art.

The anti-A33 antibody can also be formulated as pharmaceutical compositions in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention microenemas for rectal delivery.

In one embodiment, the anti-A33 antibody is formulated with carriers that protect the anti-A33 antibody from rapid elimination from the body, such as controlled release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid can be used. Methods for preparing such compositions will be apparent to those skilled in the art. Materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (including liposomes targeted to infected cells using monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. They can be prepared in accordance with methods known to those skilled in the art, for example, as described in US Pat. No. 4,522,811.

C. Sets.

The technology of the present invention provides kits for the detection and/or treatment of A33-associated cancers containing at least one immunoglobulin composition according to the technology of the present invention (for example, any antibody or antigen-binding fragment described herein) or a functional variant thereof (for example, containing a replacement option). Optionally, the above-described components of kits according to the technology of the present invention are packaged in suitable containers and labeled for the diagnosis and/or treatment of A33-associated cancer. The above components may be stored in single or multi-dose containers, for example, sealed ampoules, vials, bottles, syringes and test tubes, as an aqueous, preferably sterile, solution or as a lyophilized, preferably sterile, reconstitution composition. The kit may further comprise a second container that contains a diluent suitable for diluting the pharmaceutical composition to obtain a larger volume. Suitable diluents include, but are not limited to, a pharmaceutically acceptable pharmaceutical excipient and saline solution. In addition, the kit may contain instructions for diluting the pharmaceutical composition and/or instructions for administering the diluted or undiluted pharmaceutical composition. Containers can be made from a variety of materials, such as glass or plastic, and can have a sterile access port (for example, the container can be a race bag).

- 50 046795 solution for intravenous administration or a bottle with a stopper that can be pierced with a hypodermic needle). The kit may also contain additional containers containing a pharmaceutically acceptable buffer such as phosphate buffered saline, Ringer's solution and dextrose solution. In addition, it may contain other materials as desired from a commercial and user point of view, including other buffers, diluents, filters, needles, syringes, culture media for one or more suitable host cells. The kits may optionally include instructions typically found in commercial packages of therapeutic or diagnostic products that contain information, for example, about the indications, use, dosage, manufacture, administration, contraindications and/or warnings regarding the use of such therapeutic or diagnostic products.

The kits are useful for detecting the presence of immunoreactive protein A33 in a biological sample, for example, any body fluid, including, for example, but not limited to, serum, plasma, lymph, cystic fluid, urine, stool, cerebrospinal fluid, ascitic fluid or blood and including samples biopsy of body tissue. For example, the kit may contain one or more humanized, chimeric or bispecific anti-A33 antibodies according to the technology of the present invention (or antigen binding fragments thereof) capable of binding to the A33 protein in a biological sample; means for determining the amount of A33 protein in a sample; and a means for comparing the amount of immunoreactive A33 protein in the sample with a standard. One or more anti-A33 antibodies may be labeled. Kit components (eg reagents) may be packaged in a suitable container. The kit may further contain instructions for use of the kit for detection of immunoreactive protein A33.

In the case of antibody-based kits, the kit may contain, for example, 1) a first antibody, for example, a humanized, chimeric or bispecific anti-A33 antibody according to the technology of the present invention (or an antigen-binding fragment thereof), which binds to the A33 protein, attached to a solid carrier, and optionally; 2) a second, different antibody that binds to either the A33 protein or the first antibody and is conjugated to a detectable label.

The kit may also contain, for example, a buffering agent, preservative or protein stabilizing agent. The kit may further contain components necessary to detect a detectable label, such as an enzyme or substrate. The kit may also contain a control sample or set of control samples that can be analyzed and compared with the test sample. Each component of the kit may be contained in an individual container, and all of the different containers may be presented in a single package along with instructions for interpreting the results of the assay performed using said kit. Kits according to the technology of the present invention may contain a written application on or within the container of the kit. The written application describes how to use the reagents contained in the kit, for example, to detect the A33 protein in vitro or in vivo or to treat A33-associated cancers in a subject in need thereof. According to specific embodiments of the invention, the reagents can be used in accordance with the methods of the technology according to the present invention.

Examples

The technology of the present invention is further illustrated by the following Examples, which are not intended to limit the present invention in any way. The following Examples demonstrate the production, characterization and use of exemplary anti-A33 antibodies according to the technology of the present invention. The following Examples demonstrate the production of chimeric, humanized and bispecific antibodies according to the technology of the present invention, as well as the characterization of their binding specificity and in vivo biological activity.

Example 1: Materials and methods for creating and characterizing antibodies against A33 according to the technology of the present invention.

Human cell lines and leukocytes. Cell lines LS174T, Colo205, SNU-16, and 293T cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA); SW1222 were obtained from the European Collection of Animal Cell Cultures (ECACS, Salisbury, UK). All cells were authenticated using STR loci typing. Cells were maintained in RPMI medium supplemented with 10% fetal bovine serum (FBS, Sigma, St. Louis, MO), 0.03% L-glutamine (Gibco Laboratories, Gaithersburg, MD), and penicillin/streptomycin solution (Gibco Laboratories, Gaithersburg, MD). Maryland). Buffy coats from healthy donors were obtained from the New York Blood Center, New York, NY, and human PBMCs were isolated from buffy coats using a Ficoll gradient.

Establishment of cell lines expressing luciferase. 293T cells were first transfected with a retroviral construct containing the luciferase and GFP genes using PolyJet transfection reagent (SignaGen, Rockville, MD) according to the manufacturer's instructions. Thirty-six hours later, the virus-containing supernatant was collected and filtered through a filter

- 51 046795 (0.45 microns). Two milliliters of the filtered supernatant was aliquoted into each well of a 12-well plate precoated with retronectin (Clontech Laboratories, Mountain View, CA). The plate was spun at 3500 rpm at 4°C for 45 minutes. The process was repeated 2 to 3 times. After unscrewing, the plate was washed once with PBS, after which tumor cells at a concentration of 0.2x10 ⁶ were seeded and incubated at 37°C. Inoculation by centrifugation was repeated again after 24 hours. Cells were then further incubated for at least 48 hours before sorting to detect GFP-expressing cells. For cell sorting, transduced cells were distributed into at least 4 wells of a 96-well plate at a density of 1 cell/well and incubated for 2 weeks before collecting colonies. Harvested colonies were scored and selected based on luciferase, GFP, and GPA33 expression compared to parental cell lines.

Analysis by size exclusion HPLC. The size and purity of huA33-BsAb were analyzed using an HPLC system (Shimadzu Scientific Instruments Inc., Columbia, Maryland). Monomeric variants were identified using molecular weight standards (Bio-Rad Laboratories, Hercules, CA) and the percentage of monomer was calculated based on the relative area under the curve (AUC) of various non-buffer-bound peaks.

Humanization of mouse A33. Humanization of mouse A33 was carried out to obtain IgG ₁ by grafting with CDR. Two different V _H and V _L sequences were combined to produce 4 different humanized antibodies against A33. The binding kinetics were compared with the binding kinetics of the chimeric antibody chA33 using surface plasmon resonance (SPR) analysis. The heavy chain sequence was SEQ ID NO: 6 (3A3-H2), and the light chain sequence was SEQ ID NO: 10 (3A3-L2).

The heavy chain sequence including the leader sequence was MGWSCIILFLVATATGEVOLVESGGGLVKPGGSLRLSCAASGFAFSTYDMSWVROAPG

KRLEWVATIS SGGS YT YYLD S VKGRFTISRDN A KNS LYLQMNSLR AEDT A V Y YC ARTT VVPFAYWGQGTLVTVSS (SEQ Ш NO: 55), and the light chain sequence including the leader sequence was MGWSCIILFLVATATGDIOMTOSOSSLSTSVGDRVTITCKASONVRTVVAWYOOKPGKS

PKTLIYLASNRHTGVPSRFSGSGSGTEFTLTISNVQPEDFADYFCLQHWSYPLTFGSGTKL

The underlined sequence corresponds to the leader sequence.

SPR analysis. Human GPA33 (Novoprotein, Summit, NJ) was immobilized on CM5 chips. Five concentrations of huA33 IgG1 or huA33-BsAb (starting at 20 nM) in 2-fold serial dilutions were run on the chip using the Biacore™ T100 system (GE Healthcare, Chicago, IL). huA33 binding kinetics were measured at 25°C and huA33-BsAb binding kinetics were measured at 25°C and 37°C. Sensograms were fit to a 1:1 binding model for both antibodies to derive kinetic parameters.

Generation of bispecific antibody huA33-BsAb. HuA33-BsAb was constructed by fusing the humanized OKT3 scFv to the C-terminus of the huA33 antibody light chain via a (G ₄ S) ₃ linker as previously described in: Xu H et al., Cancer Immunology Research 3:266-277 (2015) and Lopez-Albaitero A et al., Oncoimmunology 6:el267891 (2017). Mutations N297A and K322A were introduced into the Fc region of the antibody to eliminate FcR and complement binding activity, 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 construct was then transfected into CHO-S cells and stable clones were selected to produce high levels of antibody. To purify the antibody on a larger scale, the selected stable clone was propagated in mixing flasks. The bispecific antibody was purified from the supernatant using one-step protein A affinity chromatography.

T-cell cytotoxicity (TDCC) assays. Cytotoxicity assays were performed using the ⁵¹ Cr release assay and LDH release assay, Pierce (Thermo Fisher Scientific, Cambridge, MA). For both assays, T cells activated by treatment for 14 days with Dynabeads microparticles (anti-CD3/anti-CD28) were subsequently used as effector cells, with the exception of sorted cells from PBMCs, which were used for the TDCC assay without prestimulation. ^51Cr analysis was performed as previously described in Cheng M et al., International Journal of Cancer 136:476-486 (2015). The LDH assay was performed according to the manufacturer's instructions with the following modifications. Briefly, in each assay well of a 96-well round bottom plate, target cells at a concentration of 1.5 x 10 ⁴ were incubated with varying numbers of effector cells depending on the expected E:T ratios. Antibodies were then added at various dilutions and the plates were incubated at 37°C for 16 hours. Each experimental variant was carried out in triplicate. The supernatant was then transferred to a flat bottom plate containing the reaction substrate and incubated for 30 min before reading the plates

- 52 046795 at 490 nm using 680 nm as a reference wavelength. EC ₅₀ values were calculated by fitting the curves to a 4-parameter nonlinear regression model using GraphPad Prism.

Analysis of the release of cytokines and cytolytic molecules. T cells were purified from PBMCs using a total T cell fraction isolation kit (Miltenyi Biotec, Cambridge, MA). Target and control tumor cells were incubated in triplicate in 24-well plates at a concentration of 2x10 ⁶ cells/well in a total volume of 2 ml per well at an effector:target ratio of 5:1. Culture supernatants were collected at 24 hours, 48 hours, 72 hours, and 96 hours, and cytokine levels were measured by flow cytometry using the LEGENDplex™ human CD8/NK panel (Biolegend, San Diego, CA) 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., Carlsbard, CA) for 5 min at room temperature, followed by neutralization with PBS containing 5% FBS. Target cells were then incubated with labeled PBMCs under various conditions at 37°C before analyzing the expression of surface markers of activation and T cell proliferation at 24 hours and 96 hours.

In vivo tumor therapy. For subcutaneous (SC) tumor injection models, LS174T, Colo205, or SNU16 cells were combined with fresh PBMCs at a 1:1 ratio, mixed with Matrigel (cells:gel=1:2 by volume), and implanted (100 μl/mouse) into the flank of Balb mice /c Rag2 ^-/- IL2RY ^-/- (DKO) (currently available commercially from Taconic as CIEA BRG mice). Treatment was initiated after confirmation of tumor presence with a twice weekly (BIW) regimen of 100 μg/mouse and continued for 3–4 weeks. Tumor growth was monitored by weekly tumor volume measurements using a caliper or digital Peira TM900 Scanner (Peira Scientific Instruments, Turnhout, Belgium).

For intraperitoneal (IP) tumor injection models, luciferase-expressing LS174T-luc or SW1222-luc cells were resuspended in RPMI medium and injected intraperitoneally into DKO mice. Human effector cells were activated T cells that were injected intravenously. The mice underwent treatment cycles 2-3 times a week according to the antibody-T-cell-antibody regimen, where the interval between administrations of each component was 3-4 days. Tumor growth was monitored weekly by measuring luminescent signals on an IVIS Spectrum in vivo imaging system (PerkinElmer Inc., Waltham, MA) after injection of 3.3 mg/mouse luciferin. Luminescence was analyzed and calculated using Living Image Software (PerkinElmer Inc., Waltham, MA).

Antibodies and flow cytometry. Antibodies against hCD25-PE, against CD69-PE, against hCD8-APC, against hCD45-PECy7, against hCD4-PE, against hCD4-BV421, against hCD62L-PercpCy5.5, against hPD1-BV421, against hCD45RO-PECy7, were obtained from Biolegend (San Diego, CA). Goat anti-human IgG (IgG-PE) was obtained from SouthernBiotech (Birmingham, AL). Streptavidin-PE, anti-hCD4-APC, and anti-hCD25-APC antibodies were obtained from BD Biosciences (San Jose, CA). All FACS analyzes were performed using the FACSCalibur or LSRII system (BD Biosciences, San Jose, CA) and analyzed using the FlowJo system (FLOWJO, Ashland, OR).

Affinity ripening using yeast display. The original huA33 was converted into scFv format with a (G4S)4 linker containing 20 amino acids and cloned into a yeast display vector. HuA33 scFv was randomly mutated using the GeneMorph II mutagenesis kit (Agilent Technologies, Santa Clara, CA). The PCR products were electroporated along with the linearized vector into yeast and the library was subjected to 4 rounds of sorting using biotinylated GPA33. Individual clones from the last cycle were amplified by PCR and sequenced to analyze the mutation pattern. Conversion of selected scFv clones to the huA33-BsAb format was accomplished using a one-step 4-fragment ligation method using 50 ng of lyanized vector and insertion vector at a 1:3 molar ratio to the other 3 components. Ligation was performed using a Rapid DNA ligation kit (Thermo Fisher Scientific, Cambridge, MA) at room temperature for 1 hour. The type II restriction enzyme SapI (New England Biolabs, Ipswich, MA) was used to ensure continuous coupling of the various components (Fig. 13). Transient expression of selected clones was performed using the Expi293 expression system (Thermo Fisher Scientific, Cambridge, MA) according to the manufacturer's instructions. Supernatant from Expi293 cells, obtained after 4-5 days of culture in mixing flasks, was used for antibody purification using MabSelect SuRe (GE Healthcare, Chicago, IL) and dialyzed against citrate buffer (pH 8.0) on a dialysis membrane (Spectrum Laboratories, Inc., Rancho Dominguez, California).

Statistical analysis. Significance (p < 0.05) was assessed using Student's t test.

Example 2: Structure and binding affinity of humanized anti-A33 antibodies according to the technology of the present invention.

In fig. 14 shows the amino acid sequences of the VH and _VL domains of a mouse antibody.

- 53 046795 against A33 and corresponding human homologous sequences (SEQ ID NO: 1-4). The amino acid sequences of the VH and VL domains of 3A3-H1/L1, 3A3-H1/L2, 3A3-H2/L1 and 3A3-H2/L2 of humanized anti-A33 antibodies according to the technology of the present invention are shown in FIG. 15 and fig. 17. The cDNA sequences of the VH and VL domains of 3A3-H1/L1, 3A3-H1/L2, 3A3-H2/L1 and 3A3-H2/L2 of the humanized A33 antibodies are shown in FIG. 16 and FIG. 18. In FIG. 19 shows an alignment of the original humanized amino acid sequences of hA33 from King et al. (1995), higher than the newly rehumanized amino acid sequences of huA33 (3A3).

In fig. 24 shows the amino acid sequences of the light chain and heavy chain of the chimeric chA33-IgG1, which correspond to the sequences of SEQ ID NO: 13 and SEQ ID NO: 14, respectively. In fig. 25 shows the amino acid sequence and heavy chain cDNA sequence of huA33-IgG1 (H2L2), which correspond to SEQ ID NO: 15 and SEQ ID NO: 16, respectively. In fig. 26 shows the amino acid sequence and light chain cDNA sequence of huA33-IgG1 (H2L2), which correspond to SEQ ID NO: 17 and SEQ ID NO: 18, respectively.

When compared with the chimeric anti-A33 antibody, all four newly humanized anti-A33 antibodies had slightly improved koff when binding to immobilized GPA33 in the NIR assay (Fig. 9 and Fig. 20). Based on molecular weight (kDa), stability at 37°C and T20 humanization score, the H2L2 huA33 clone was selected for further development (see FIG. 23). Fig. 21 and fig. 22 demonstrate that the original humanized antibody hA33 (hA33-mC825 BsAb) described in Cheal et al. (2016), above, there was a significant decrease in binding affinity compared to huA33-H2L2.

These results demonstrate that antibodies according to the technology of the present invention or antigen-binding fragments thereof specifically bind to the A33 antigen with high binding affinity. Accordingly, the immunoglobulin compositions described herein are useful for detecting A33 protein in a sample.

Example 3: Structure and binding affinity of HuA33-BsAb T cell recruiter antibodies according to the technology of the present invention.

The huA33 antibody was converted into a bispecific format by fusing the humanized OKT3 cFv to the C-terminus of the light chain via a flexible GS linker (Figure 1(A)). The DNA construct was used to establish a stable CHO-S cell line and huA33-BsAb was purified from the supernatant. Protein yields using Protein A chromatography ranged from 50 mg/L to 100 mg/L without intensive optimization. Similar yields were observed using the Expi293 transient expression system (data not shown). One-step purification with Protein A generally resulted in protein with a purity greater than 90% as measured by size exclusion HPLC. Four freeze-thaw cycles did not cause noticeable changes in the size-exclusion HPLC profile (data not shown). After incubating the molecules at 37°C for 4 weeks, only a minimal decrease in the percentage of monomers was observed, as shown in FIG. 1(B). These data suggest that huA33-BsAb had good solubility, purity and thermal stability, all of which are critical characteristics for further downstream development.

In fig. 27 shows the amino acid sequence and heavy chain cDNA sequence of the bispecific T cell recruiter antibodies huA33-BsAb, which correspond to the sequences of SEQ ID NO: 19 and SEQ ID NO: 20, respectively. In fig. 28 shows the amino acid sequence and light chain cDNA sequence of the bispecific T cell recruiter antibodies huA33-BsAb, which correspond to the sequences of SEQ ID NO: 21 and SEQ ID NO: 22, respectively. In fig. 29 shows a summary of potential modifications for the huA33-BsAb bispecific T cell recruiter antibodies described in the present invention.

The avidity of huA33-BsAb for GPA33 at temperatures of 25°C and 37°C was measured using CM5 chips with immobilized GPA33. As shown in FIG. 1(C), huA33-BsAb bound GPA33 with high apparent affinity of about 0.2 nM, slightly lower than the 0.13 nM observed for parent huA33. FACS analysis of a panel of cell lines derived from various cancer types showed that huA33-BsAb stained colon cancer cell lines and one gastric cancer cell line, but did not stain the neuroblastoma cell line GPA33(-) IMR32, the osteosarcoma cell line TC32, or the melanoma line SKMEL5 (Fig. 1(D) and Fig. 10), demonstrating that huA33-BsAb retained the specificity of the parent A33 antibody in binding target antigens on colon cancer cells and a subset of gastric cancer cells. Staining of activated T cells also showed that huA33-BsAb bound to CD3 on the surface of T cells. See fig. 1(D).

These results demonstrate that antibodies according to the technology of the present invention or antigen-binding fragments thereof specifically bind to the A33 antigen with high binding affinity. Accordingly, the immunoglobulin-based compositions described in

- 54 046795 of the present invention, are applicable for detecting protein A33 in a sample.

Example 4: Biological activity of HiA33-BsAb T-cell recruiter antibodies according to the technology of the present invention.

NiA33-BsAb activated fresh T cells and induced their entry into the cell cycle. To test the ability of huA33-BsAb to activate unstimulated 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). huA33-C825, which contained an irrelevant scFv (Cheal SM et al., Eur J Nucl MedMol Imaging 43:925–37 (2016)) instead of an anti-CD3 scFv, as well as an irrelevant BsAb T cell recruiter antibody L1CAM-BsAb against the L1CAM antigen (L1CAMxCD3 ), which does not bind to L1CAM(-) Colo205 by FACS, were used as negative controls.

After 24 and 96 hours, cells were stained with various T-cell activation markers to assess T-cell activation and proliferation status. As early as 24 hours, huA33-BsAb caused activation of both CD4(+) and CD8(+) T cells, as shown by the up-regulation of cell surface markers CD25 and CD69 (Fig. 2(A)). In contrast, huA33-C825 and L1CAM-BsAb caused only minimal up-regulation of CD25. L1CAM-BsAb did not increase CD69 expression, particularly in CD4(+) T cells, possibly due to L1CAM expression on T cells. Similarly, up-regulation of PD-1 expression was observed at 24 hours, which persisted up to 96 hours (Fig. 11(A)). Cell division, measured by CFSE dye dilution, was observed for both CD4(+) and CD8(+) T cells at 96 hours (Fig. 2(B)). CD8(+) T cells showed more cell division cycles, indicating that CD8(+) T cells were dividing faster than CD4(+) T cells (Figure 2(C)). The huA33-C825 control antibody did not stimulate significant amounts of cell division in any of the T cell subsets, confirming that CD3 activation is required for T cell division. L1CAM-BsAb induced a low level of cell division, consistent with the low level of activation observed above. When using GPA33(-) SKMEL5 target cells, huA33-BsAb did not activate T cells (Fig. 11(B)). These results demonstrate that activation of T cells by huA33-BsAb depends on the presence of cognate antigens on tumor cells. It was also observed that cell division was associated with the expansion of CD45RO(+) effector/memory cells (Fig. 2(D)), indicating the importance of this subpopulation in mediating T-BsAb activity, as confirmed below. These assays were repeated using another colon cancer cell line, LS174T, for which similar conclusions were obtained (FIG. 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 the mixture was implanted subcutaneously into DKO mice. The next day, animals were intravenously injected with huA33-BsAb, and after another 4 days, tumors were isolated and analyzed using FACS analysis. As shown in FIG. 2(E), approximately 25% of CD4(+) and CD8(+) T cells increased CD25 expression as they progressed through cell division (progressive halving of CFSE fluorescence), suggesting that huA33-BsAb is also capable of promoting activation and T cell proliferation in vivo.

HuA33-BsAb induced the secretion of inflammatory cytokines and cytolytic molecules. The secreted cytokine profile of T cells activated by huA33-BsAb in the presence of target tumors was studied. The total T cell fraction was purified from PBMCs and cultured in the presence of Colo205 tumor cells at an effector:target ratio of 5:1. To assess nonspecific activation, SKMEL5 cells were used as a negative control. Cell culture supernatants were collected daily for 4 days and levels of cytokines and cytotoxic molecules were measured using a multiplex flow cytometry-based method. As shown in FIG. 3, both Th1 cytokines (IL-2, ZFNy, TNFa) and Th2 cytokines (IL-4, LL-10) were secreted by activated T cells, although IL-4 was secreted at a much lower level than other cytokines. Likewise, significant amounts of IL-6 were secreted by activated T cells. Th17 cell cytokine IL-17a was also secreted at high levels. All cytotoxic components sFasL, sFas, granzyme A, granzyme B, perforin and granulysin were released into the supernatant.

HuA33-BsAb redirected T cells to specifically kill colon and gastric cancer cells. The ability of huA33-BsAb to redirect T cells to kill cancer cells was tested. Based on studies of GPA33 expression in multiple human cancer cell lines (Figure 10), 3 colon cancer cell lines (LS174T, SW1222 and Colo205) and one gastric cancer cell line (SNU16) were selected for cytotoxicity studies. These cell lines were classified into MSI (LS174T) or MSS (SW1222, Colo205, SNU16) subtypes based on their microsatellite instability profile (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)). In addition, LS174T cells had the KRAS G12D mutation, while other cells had 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 J-L & Park J-G: Cancer Research and Treatment: Official Journal of Korean Cancer Association 37:1-19 (2005); al.,Molecular can

- 55 046795 cer therapeutics 5:2606-2612 (2006)).

Cancer cells were incubated with activated T cells at an effector:target ratio of 10:1 in the presence of huA33-BsAb at 10-fold serial dilutions. The GPA33(-) melanoma cell line SKMEL5 and the osteosarcoma cell line TC32 were used as negative controls. As shown in FIG. 4 (A), huA33-BsAb redirected T cells to specifically kill all GPA33-expressing cancer cells, regardless of their genetic background, while T cells were not affected by SKMEL5 and TC32, confirming the antigen specificity of huA33-BsAb-mediated TDCC. It seemed that the maximum level of cytotoxicity correlated with the level of staining in FACS analysis (Fig. 1(D) and Fig. 4(A)). Moreover, TDCC induced by huA33-BsAb was strong with EC50 values in the picomolar range. See fig. 4(A).

To determine which T cell subpopulations were mobilized by the huA33-BsAb antibody, memory cell subpopulations, CD45RA(+)CD62L(+) and CD45RA(-)CD45RO(+)CD62L(-) were sorted from CD4(+) and CD8(+) T cells. The sorted cells were cultured in the presence of Colo205 tumor cells at an E:T ratio of 5:1 for 48 hours before measuring cytotoxicity using a lactate dehydrogenase (LDH) assay. As shown in FIG. 4(B), CD4(+) and CD8(+) memory T cell subsets were able to induce cytotoxicity, with CD8(+) memory T cells providing more efficient killing at higher concentrations. The CD45RA(+)CD62L(+) subpopulations of both the CD4(+) and CD8(+) populations, the majority of which were naïve T cells, were able to induce cytotoxicity at 48 hours, although the activity was less compared to T − memory cells.

When T cells from 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)) , which confirms that huA33-BsAb can activate both naïve and memory T cells. The majority of CD45RA(+)CD62L(+) T cells remained CD45RO(-) after incubation, with CD45RO expression increasing in a small but important population, particularly among CD8(+) cells. See fig. 12(B). However, this population could arise either as a result of the maturation of naïve T cells or as a result of the proliferation of rare CD45RO+ cells present in the original culture. Together, these data demonstrate that huA33-BsAb can induce potent cytotoxicity against colon and gastric cancer cells in a GPA33-dependent manner by mobilizing both CD4 and CD8 T cells, especially those with a memory cell phenotype.

These results demonstrate that the antibodies of the technology of the present invention or antigen binding fragments thereof specifically bind to the A33 antigen with high binding affinity. Accordingly, the immunoglobulin-based compositions described herein are useful for detecting A33 protein in a sample.

Example 5: Affinity maturation of huASS-BsAb using yeast display.

In an attempt to further improve the activity of huA33-BsAb, a yeast display method was used to promote affinity maturation of huA33-derived scFv. Because scFv tends to aggregate easily, a method for rapidly converting scFv into huA33-BsAb T cell recruiters (which is a more suitable format and can be easily produced in high yield and purity) was developed (Figure 13).

The method was based on a one-step 4-fragment ligation method that used the SapI type II enzyme to ensure seamless joining of different fragments. This method could be easily adapted to other vectors or scaled to a high-throughput workflow. In-Fusion® multi-fragment cloning technology (Clontech Laboratories, Mountain View, CA) was also tested successfully but was found to be less reliable (data not shown), possibly due to specific expression vector sequences.

Based on the results of sequence analysis of 60 individual clones, 7 clones were selected (Fig. 5(A)) for further characterization by NIR and TDCC analysis. For all 7 clones, an increase in binding affinity was observed compared to the parent antibody, ranging from a 4.3- to 51-fold increase (Fig. 5(B)). The heavy chain and light chain amino acid sequences of the seven affinity matured clones are shown in Figures 30-36. Most of the improvements were attributed to the slower dissociation rate. For all clones, there was a slight improvement in maximum killing in TDCC assays (Fig. 5(C)).

These results demonstrate that antibodies according to the technology of the present invention or antigen-binding fragments thereof specifically bind to the A33 antigen with high binding affinity. Accordingly, the immunoglobulin compositions described in the present invention are useful for detecting A33 protein in a sample.

Example 6: In vivo therapeutic studies using huASS-BsAb.

The efficacy of huA33-BsAb was studied in vivo in the following xenograft models of humanized DKO mice using two different tumor models: (1) subcutaneous tumor + subcutaneous effectors and (2) intraperitoneal tumor + i.v.

- 56 046795 denition of effectors.

HuA33-BsAb provided treatment for MSI tumor LS174T in a model of subcutaneous xenograft injection and suppressed tumor growth in a model of intraperitoneal injection. LS174T cells were mixed with PBMCs at a 1:1 ratio and implanted subcutaneously into DKO mice. As shown in FIG. 6(A), both groups not treated with the antibody, the tumor only group and the tumor+PBMC group, experienced rapid tumor growth. Mice in the tumor+PBMC group developed tumor ulceration and had to be sacrificed. In contrast, administration of 6 doses of huA33-BsAb over 3 weeks resulted in effective treatment in mice—the animals did not develop tumors for at least 120 days.

To stimulate malignant ascites, a common finding in colon cancer, luciferase-expressing LS174T cells were injected intraperitoneally into DKO mice. Once tumor growth was confirmed by luminescence, mice were randomized into different treatment groups, a group receiving no antibody treatment (tumor only), a tumor + T cells only group (tumor + ATC), or a group receiving intravenous injections of huA33-BsAb (7 doses per for 3 weeks) and weekly T-cell injections for 3 weeks. Treatment began on day 5 after tumor implantation. All T cells were injected in the retroorbital manner.

As shown in FIG. 6(B) and fig. 6(C), all control groups (no antibody group or tumor + T cell only group) showed exponential tumor growth in the peritoneal cavity. By day 28, 3 of 4 mice in the no-antibody group and 3 of 5 mice in the tumor + T-cell group had died of tumors, respectively. In contrast, huA33-BsAb significantly suppressed the metastatic growth of LS174T tumor in the abdominal regions of treated mice. All mice remained alive for at least 60 days without further treatment. These results demonstrate that huA33-BsAb was effective against CRC tumors with the MSI genotype. However, as mentioned earlier, MSI tumors represent only a minority of patients with CRC cancer. Most patients with CRC have the MSS genotype. Therefore, the efficacy of huA33-BsAb was further tested on MSS tumors.

HuA33-BsAb induced treatment of MSS tumor COLO205 in a subcutaneous xenograft model and suppressed the growth of metastatic MSS tumor SW1222. Two colon cancer cell lines Colo205 and SW1222 with the MSS genotype were studied. For Colo205 cells, tumors were implanted subcutaneously using PBMCs as effector cells. In this model, 4 doses of huA33-BsAb were sufficient to completely kill Colo205 tumors, and mice remained tumor-free for at least 4 months after treatment with a total of 6 doses of antibody (Fig. 7(A) and Fig. 7(B) ).

For the SW1222 cell line, tumors were inoculated intraperitoneally and animals were treated with 6 intravenous doses of antibody over 3 weeks and intravenous T cell administration weekly for 2 weeks. As shown in FIG. 7(C) and FIG. 7(D), the two control groups showed tumor growth distributed throughout the abdominal region, while treatment with huA33-BsAb suppressed tumor growth and significantly prolonged the survival of mice (p=0.0125). These results demonstrate that the effectiveness of huA33-BsAb in the treatment of MSS tumors was comparable to that in the treatment of MSI tumors. Additional cycles of treatment are expected to completely eliminate tumor growth, as in subcutaneous models.

HuA33-BsAb inhibited the growth of gastric tumors in a model of subcutaneous (SC) xenograft administration. GPA33 is expressed in a subpopulation of gastric cancer cells. FACS and in vitro TDCC assays showed that SNU16 cells expressed GPA33 and were sensitive to killing by huA33-BsAb-redirected T cells. The SNU16 cell xenograft was administered subcutaneously after mixing with human PBMCs. huA33-BsAb provided effective treatment to mice containing subcutaneously injected tumors (Fig. 8(A)) and significantly prolonged survival. Unlike NSG mice, DKO mice were more resistant to engraftment of human PBMCs. In the SNU16 subcutaneous injection model at day 58 (Fig. 8(B)), most mice exhibited low levels of blood chimerism with large variations.

As shown above, the efficacy of huA33-BsAb was not dependent on the genetic background or mutational status of CRC tumors, a critical biological property with desirable applicability to most CRCs. In contrast to CAR-modified T cells, huA33-BsAb redirected T cells did not undergo cell exhaustion and continuously demonstrated quantitative tumor homing properties for >3 weeks. Treatment of tumors was achieved despite the presence of PD1 and PD-L1, and the addition of checkpoint blockade at subtherapeutic doses of huA33-BsAb further enhanced these antitumor properties. The immunoglobulin-based compositions described herein provide both an FcR-dependent and a T-cell-dependent immunotherapeutic strategy for the diagnosis and therapy of A33-associated cancer.

Together, the results demonstrate that antibodies or antigen-binding fragments thereof according to the technology of the present invention can detect tumors and inhibit tumor progression and/or metastasis. Accordingly, the immunoglobulin compositions described herein are useful for the detection and treatment of A33-positive cancer in a subject in need thereof.

Example 7: Use of huA33-C825 antibodies according to the technology of the present invention in pretargeted radioimmunotherapy.

The major disadvantages in developing antibody-based agents for pretargeted radioimmunotherapy (PRIT) are excessive exposure of normal tissues, immunogenicity, suboptimal tumor dose, and low therapeutic index. This Example will demonstrate that huA33-DOTA bispecific antibodies according to the technology of the present invention are useful in PRT for the treatment of cancers expressing human A33 antigen, such as colorectal cancer.

A33-DOTA bispecific antibodies according to the technology of the present invention contain a first antigen binding site based on the humanized A33 antibodies described in this application, and a second antigen binding site that binds to a low molecular weight hapten (for example, benzyl-1,4, 7, 10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid [DOTA-Bn]). A single chain Fv fragment of an anti-DOTA-Bn antibody (ScFv), derived from an affinity matured 2D 12.5 antibody, is coupled to the carboxyl terminus of the humanized A33 light chain. In fig. 37 shows the amino acid sequence and heavy chain cDNA sequence of the bispecific antibodies huA33-huC825 (H2L2), which correspond to the sequences of SEQ ID NO: 58 and SEQ ID NO: 59, respectively. In fig. 38 shows the amino acid sequence and light chain cDNA sequence of the bispecific antibodies huA33-huC825 (H2L2), which correspond to the sequences of SEQ ID NO: 60 and SEQ ID NO: 61, respectively. In fig. 39 shows the amino acid sequence of the heavy chain and light chain of the bispecific antibodies huA33-mC825 (H2L2), which correspond to the sequences of SEQ ID NO: 62 and SEQ ID NO: 63, respectively.

Tumor cell lines and cell culture reagents. The human colorectal cancer cell line SW1222 was maintained by serial passage. Cells were cultured in minimal growth medium supplemented with 10% heat-inactivated fetal bovine serum, 2.0 mM glutamine, 100 units/ml penicillin and 100 units/ml streptomycin at 37°C with 5% CO2. Cultures were established and cryopreserved in small aliquots to limit passaging to less than three months and periodically tested for mycoplasma according to the manufacturer's specifications using a commercial kit (Lonza, Portsmouth, NH). A solution of 0.25% trypsin/0.53 mM EDTA in Hanks' balanced salt solution without calcium and magnesium was used for trypsinization during cell passaging and collection. huA33-huC825 and huA33-mC825 were produced in CHO cells in a mammalian expression vector and purified by protein A affinity chromatography.

Surface plasmon resonance studies. The Biacore T100 biosensor, CM5 sensor chip, and associated reagents were purchased from GE Healthcare (Chicago, IL). Recombinant human A33 protein was purchased from Novoprotein Scientific, Inc. (Summit, New Jersey). The BSA-(Y)-DOTA-Bn conjugate was prepared as described in: Cheal et al., Mol. Cancer Ther. 13 (7) (2014). A33 and DOTA antigens were immobilized using the Amino Coupling kit (GE Healthcare, Chicago, IL). Purified bispecific antibodies and control antibodies were analyzed and data normalized to a bivalent analyte model using Biacore T100 evaluation software as described in Cheal et al., (2014).

Reagents and protocol for PRIT and xenograft studies. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Memorial Cancer Center. Sloan Kettering (Institutional Animal Care and Use Committee of Memorial Sloan Kettering Cancer Center), and were conducted in accordance with institutional guidelines for the proper and humane use of animals in research. Female nu/nu nude mice (6–8 weeks old; Harlan Sprague Dawley) were allowed to acclimate to the animal facility for at least one week. Groups of animals were injected subcutaneously with A33-positive SW1222 cells in the left flank with a concentration of tumor cells of ^5x106 , which were mixed with Matrigel (BD Biosciences, San Jose, CA) in a 1:1 ratio, and behind established tumors (100-900 mm2) observed for 7-10 days using the ellipsoid volume formula (V=4/3n; (length/2xwidth/2xheight/2). All reagents were administered intravenously (IV) through the posterior tail vein. The PRIT protocol included injections: huA33-huC825 or huA33-mC825 [t=-28 h] followed 24 h later by administration of a clearance agent (500 kDa dextran-(Y)-DOTA-Bn conjugate prepared according to Orcutt et al., Nucl. Med. Biol. 38: 223-233 (2011) and diluted in saline injection). The substitution rate for moles of (Y)-DOTA-Bn per moles of dextran was 61 ((Y)-DOTA-Bn/dextran)) [t=4 h]. ¹⁷⁷ Lu-DOTA-Bn (prepared as previously described by incubating aminobenzyl-DOTA (p-NH2Bn-DOTA) from macrocyclic compounds and ¹⁷⁷ LuCl ₃ (specific activity -30 Ci/mg; Perkin Elmer) and diluted in saline for injections) were administered after 4 hours [t=0 hours]. In addition, huA33

- 58 046795

C825 was labeled with a radioactive ^1,131 tracer to assess tumor uptake during PRIT.

The IODOGEN method (Cheal, S. et al., Mol. Cancer Ther. 13(7): 1-10 (2014)) is used to obtain ¹³¹ I-huA33-mC825 or ¹³¹ I-huA33-huC825 (final specific activity 95. 5 MBq/mg spiked with cold huA33-huC825 or huA33-mC825 to achieve the desired mg dose, radiochemical purity >98% by high-pressure exclusion liquid chromatography) and in vitro cell-binding immunoreactivity were assessed using SW1222 cells essentially as described in Lindmo, T. et al., J. Immunol. Meth. 126(2): 183-189 (1990)). For PRIT using nonspecific IgG-C825, an equivalent dose (in mg) of a bispecific antibody targeting GD2 (hu3F8-C825) was used instead of huA33-mC825 or huA33-huC825.

For ex vivo biodistribution analysis, mice were sacrificed by CO2 (gas) asphyxia, tumors and selected organs were collected, washed with water and allowed to air dry, weighed, and radioanalyzed by gamma scintillation counting (Perkin Elmer Wallac Wizard 3 PerkinElmer Inc. , Waltham, MA). Emission intensities were corrected for background and attenuation, converted to activity values using a system calibration factor, normalized to the injected activity, and expressed as percent injected dose per gram (%I/g). Differences in ¹⁷⁷ Lu activity concentrations between the tumor and different tissues were analyzed using an unpaired Student's t test when appropriate.

Estimation of absorbed doses. Groups of A33-positive mice bearing SW1222 tumors (n=4-5) were treated with 0.25 mg huA33-C825 (either huA33-huC825 or huA33-mC825), a clearance agent (62.5 μg; 25% mass)) and 1.85-2.0 MBq (-10 pmol) ¹⁷⁷ Lu-DOTA-Bn and animals were sacrificed 2, 24 and 120 hours after administration. For each tissue, uncorrected activity concentration-time data were fitted using Excel to a 1-component, 2-component, or more complex exponential function as appropriate, and integrated analytically to obtain the cumulative activity concentration per unit administered activity (MBq-h/ g per MBq). The ¹⁷⁷ Lu dose equilibrium constant for non-penetrating radiation (8.49 g-cGy/MBq-h) was used to estimate the doses absorbed by a given tissue for different tumors and selected organs, based on the total local absorption of ¹⁷⁷ Lu beta rays only, excluding gamma rays and contribution from absorption by other tissues. To determine the effect of ¹⁷⁷ Lu-DOTA-Bn dose on the relative uptake ^{of 177} Lu-DOTA-Bn in the tumor and selected tissues with the highest absorbed doses (i.e. blood, liver, spleen and kidney), groups of female nude mice (n=5 /group) containing SW1222 tumors were administered 0.25 mg (1.19 nmol) huA33-C825 (either huA33-huC825 or huA33-mC825) at t = -28 hours and 62.5 μg of clearance agent per time point t=-4 hours followed by administration of 11.1 MBq (11.14-11.40 MBq), 55.5 MBq (54.61-55.06 MBq) or 111 MBq (109.52-112.5 MBq). Animals of all groups were sacrificed 24 hours after administration of ¹⁷⁷ Lu-DOTA-Bn (i.e., at the time of maximum tumor uptake) to analyze the biodistribution of ¹⁷⁷ Lu activity.

PET imaging PRIT + 86Y-DOTA-Bn. One group of mice containing A33-positive SW1222 tumors in the brachial region (n=5) was administered 0.25 mg huA33-C825 (huA33-huC825 or huA33-mC825), a clearance agent (62.5 μg 25% (w/w) )) and 8.6-8.8 MBq (-50 pmol) ⁸⁶ Y-DOTA-Bn and noninvasively imaged using a microPET Focus 120 system (CTI Molecular Imaging, Inc. Knoxville, TN) approximately 2 and 20 h after introduction. The following image acquisition parameters were used: energy window - 350-750 keV, coincidence time window -6 nsec and exposure time - 20 min. The resulting list-format data were sorted into 2D histograms using Fourier resampling, and cross-sectional images were converted using filtered back projection into a 128x128x95 matrix (the converted spatial resolution was 2.6 mm at half maximum width (FWHM)). Image data were corrected for scanner response heterogeneity, radiation loss during attenuation, physical attenuation (at the time of injection), and the ⁸⁶ Y positron decay factor. No correction for attenuation, scattering, or partial volume averaging was applied. An empirically determined system calibration factor (i.e., μCi/mL/cPs/voxel) for mice was used to convert voxel emission intensities into activity concentration values. The resulting image data were then normalized to the injected activity to determine, by region of interest analysis, the percent injected dose per gram (%W/g) of tissue, corrected for radioactive decay at the time of injection. AsiPRO VM 5.0 software (Concorde Microsystems, Knoxville, TN) was used to perform imaging and region of interest (ROI) analysis (maximum ROI, %W/g). Animals were sacrificed 24 h after administration for ex vivo biodistribution analysis.

Autoradiography and immunohistochemistry. Frozen and OCT-embedded tumors and kidneys from selected mice that received PRIT using huA33-C825 (huA33-huC825 or huA33-mC825) followed by 11.1 (11.14-11.40 MBq), 55.5 (54.61-55.06 MBq) or 111 MBq (109.52-112.5 MBq) ¹⁷⁷ Lu-DOTA-Bn (killing time: 24 hours after administration) were cut into 10 μm sections using a cryostat (Avantik , Springfield, NJ) and immediately transferred to an X-ray plate (Fuji Photo Film, Kanagawa, Japan) for 72 h and then scanned

- 59 046795 were scanned using a Typhoon FLA 7000 scanner (GE, Pittsburgh, PA). The same sections were stained with hematoxylin and eosin and scanned under an Olympus BX60 microscope equipped with a controlled moving stage (Olympus, Central Valley, PA). Both autoradiogram and microscope images were processed and analyzed using ImageJ software (NIH, Bethesda, MD).

Therapy and scintillation studies. Groups of mice containing subcutaneously established A33-positive SW1222 xenografts were treated with huA33-C825 (huA33-huC825 or huA33-mC825) or nonspecific (ns) IgG-C825-based PRIT (i.e., single-cycle treatment, injection ¹⁷⁷ Lu-DOTA-Bn on day 7 after tumor inoculation) or two cycles of PRIT (i.e., single-cycle therapeutic study, injections of ¹⁷⁷ Lu-DOTA-Bn on days 10 and 17 after tumor inoculation). For the two-cycle therapeutic study, tumor volume at day 10 post-inoculation (TV 10) (ie, the day of the first injection of ¹⁷⁷ Lu-DOTA-Bn) was recorded and expressed (if appropriate) as mean ± SD. The following definitions are used to describe response to treatment: complete response (CR) is defined as tumor volume reduction to <100 mm ³ . Durable response (DR) is defined as survival 140 days after treatment. Excess tumor burden is defined as tumor volume >2000 mm ³ . For scintigraphic studies, selected groups of mice containing A33-positive SW1222 tumors treated were placed under gas inhalation anesthesia prior to scanning on a nanoSPECT system (Bioscan, Washington, DC) 20 hours after injection for 30 minutes (~ ¹⁰⁵ pulses). per image) using a low-energy, high-resolution collimator and a window set at 208 keV. Images were converted to a 256x256 matrix using Bioscan HiSPECT software and loaded into ASIPro VM software for analysis.

Results. The dose of huA33-C825 (huA33-huC825 or huA33-mC825) was selected based on pilot biodistribution studies in mice bearing SW1222 tumors 24 hours after injection of ¹⁷⁷ Lu-DOTA-Bn using 0.1-0.6 mg of ImL33-C825 (0.48-2.86 nmol) and fixed ratios of the clearance agent and ¹⁷⁷ Lu-DOTA-Bn (5.6 MBq). Additional biodistribution experiments were then performed to optimize the dose of the clearance agent during ART. Groups of tumor bearing mice (n=3-4 per group) were administered huA33-C825 (huA33-huC825 or huA33-mC825) followed 24 hours later by either: saline (i.e. vehicle), 2.4% (w/w) , relative to the preselected dose of huA33-C825), 5% (by weight), 10% (by weight) or 25% (by weight) of the dose of clearance agent (0-62.5 μg/mouse). After another 4 h, mice were injected with 5.6 MBq ^{of 177} Lu-DOTA-Bn and the animals were sacrificed after 24 h for biodistribution analysis. It is expected that the dose of the clearance agent will make a significant contribution to the activity of ¹⁷⁷ Lu in the circulation (ie, blood) and may reduce the ability of subsequent uptake ^{of 177} Lu-DOTA-Bn into the tumor.

Dose titration studies of ¹⁷⁷ Lu-DOTA-Bn were then performed using optimized PRIT doses for huA33-C825 (huA33-huC825 or huA33-mC825) and clearance agent. For Lu-DOTA-Bn dose titration studies, ¹⁷⁷ Lu activity biodistribution data for the tumor and critical selected tissues (blood, liver, spleen and kidney) were compared between groups receiving different doses of ¹⁷⁷ Lu-DOTA-Bn, as %W/g, and by absolute absorption (kBq/g). Finally, a single-stage biodistribution experiment was performed 24 hours after injection of ^131β -indicator-labeled huA33-C825 (0.39-0.40 MBq with the addition of cold huA33-C825 to a concentration of 1.19 nmol) in mice bearing SW1222 tumors to assess absolute uptake huA33-C825 antibodies (huA33-huC825 or huA33-mC825) in tumor (pmol/g) during PRIT.

The biodistribution of radiolabeled DOTA-Bn was determined and absorbed doses were assessed in mice implanted with SW1222 tumor cells. PRIT was performed on groups of mice bearing A33-positive SW1222 tumors, with the administration of optimal doses of huA33-C825 and a clearance agent followed by 2.0 MBq (-10 pmol) ^{of 177} Lu-DOTA-Bn and a biodistribution study was carried out after 2-120 h after administration of ¹⁷⁷ Lu-DOTA-Bn to determine the retention time of ¹⁷⁷ Lu activity in the tumor and various normal tissues. Briefly, ¹⁷⁷ Lu activity in tumor and various normal tissues was determined using a biodistribution assay and subsequent PRIT using optimal doses of A33-C825 (huA33-huC825 or huA33-mC825) and a dextran-based clearance agent and 2.0 MBq (-10 pmol) ¹⁷⁷ Lu-DOTA-Bn. Groups of mice bearing the SW1222 tumor (n=4-5) received huA33-C825 followed 24 hours later by administration of a dextran-based clearance agent and 2.0 MBq (-10 pmol) ^{of 177} Lu-DOTA-Bn via an additional 4 h. Animals of one group were sacrificed 2, 24 and 120 hours after administration of ¹⁷⁷ Lu-DOTA-Bn for biodistribution analysis. These data were used as described in the present invention to estimate absorbed doses during radioimmunotherapy using ¹⁷⁷ Lu-DOTA-Bn. Tumor uptake ^{of 177} Lu is expected to occur very quickly after administration and will decrease over the next 96 hours through 120 hours after administration. For each target area, the absorbed dose was calculated as the product of ^{the 177} Lu equilibrium dose constant for

- 60 046795 non-penetrating radiation (i.e. beta rays) and accumulated ¹⁷⁷ Lu activity in the target area, based on the total local absorption of ¹⁷⁷ Lu beta rays without taking into account gamma rays and the contribution of dose absorption by other tissues. It is expected that a high therapeutic tumor/blood index will be observed during single-cycle treatment and little or no toxicity will be observed over extended periods of time in subjects who exhibit durable responses. When evaluated using PET imaging, tumor uptake results similar to those obtained after PRET are expected to be obtained.

This Example demonstrates the effectiveness of PRIT using A33-DOTA bispecific antibodies according to the technology of the present invention against A33-positive tumors. It is expected that a progressive increase in the therapeutic index and a decrease in absolute tumor uptake will be observed with increasing doses. Accordingly, huA33-DOTA bispecific antibodies according to the technology of the present invention are useful for treating cancer types expressing human A33 antigen, such as colorectal cancer.

Example 8: In vivo therapeutic effects of huA33-C825 antibodies according to the technology of the present invention.

This Example illustrates the in vivo effectiveness of the bispecific antibody huA33-C825 in PRIT to mediate a reduction in tumor burden in mice harboring A33-positive cancer cells. Specifically, this Example describes the effect of single-cycle and double-cycle therapy on tumor burden in SW1222 tumor-bearing mice.

During a single-cycle therapeutic study, 5 groups of tumor-containing mice (n=6-8 per group) were treated with any of the following: vehicle (i.e. no treatment, n=8, TV ₇ : 76±15 ^mm3 ), only 33.3 MBq ¹⁷⁷ Lu-DOTA-Bn (injected with vehicle instead of injections of bispecific antibody and clearance agent, n=6, TV 7: 116±23 mm ³ ), one cycle of IgG-C825 PRIT+33.3 MBq ¹⁷⁷ Lu-DOTA -Bn (non-specific treatment, administered IgG-C825 instead of huA33-C825, n=8, TV?: 100±10 ^mm3 ) or one cycle of huA33-C825 (huA33-huC825 or huA33-mC825) PRIT+11.1 MBq or 33.3 MBq ¹⁷⁷ Lu-DOTA-Bn (for both groups n=8, TV7: 103±17 mm ³ and TV7: 93±15 mm ³ , respectively). The relative tumor uptake is expected to decrease with increasing dose of ¹⁷⁷ Lu-DOTA-Bn during treatment. Groups of tumor-containing mice either untreated or treated with only 33.3 MBq ¹⁷⁷ Lu-DOTA-Bn or one cycle of IgG^825 PRIT + 33.3 MBq ¹⁷⁷ Lu-DOTA-Bn are expected to be tumor-free. answer. The scintigraphic study for the latter two groups receiving ¹⁷⁷ Lu-DOTA-Bn is expected to show minimal activity at the tumor site. In contrast, groups treated with one cycle of huA33-C825 PRIT (huA33-huC825 or huA33-mC825)+11.1 MBq or 33.3 MBq ¹⁷⁷ Lu-DOTA-Bn are expected to see a slowdown in tumor growth after treatment.

The second therapeutic study evaluated huA33-C825-based PRIT (huA33-huC825 or huA33-mC825) in two cycles. All mice from any untreated groups (n=5/ _TV10 : 314±77 mm ³ ) were sacrificed within 30 days due to excess tumor burden. Treatment with two cycles of PRIT+11.1 MBq ¹⁷⁷ Lu-DOTA-Bn (total dose of ¹⁷⁷ Lu-DOTA-Bn 22.2 MBq) (n=5/TV ₁₀ : 462±179 mm ³ ) is expected to result in complete tumor control response and/or inhibition of tumor growth in treated subjects.

Toxicity studies. Briefly, a total of six mice were treated with two cycles of PRIT+11.1 ¹⁷⁷ MBq ¹⁷⁷ Lu-DOTA-Bn (n=3) or two cycles of PRIT+1.5 mCi ¹⁷⁷ Lu-DOTA-Bn (n=3 ), anatomical pathology of the kidneys, bone marrow, liver and spleen was assessed up to 9 weeks after treatment. It is expected that the kidneys, bone marrow, liver and spleen will be normal in treated animals and that PRRT-based treatment will not be associated with radiation-induced toxicity.

Curative treatment and diagnostic PRIT. Nude mice bearing established SW1222 tumors after subcutaneous injection of xenografts (n=20; tumor volume=102±40 ^mm3 ; mean±standard deviation (SD)) were treated (n=5-10/group) with any of the following: no treatment (n=5), ¹⁷⁷ Lu-DOTA-Bn alone (n=5) or a three-cycle PRRT regimen consisting of PRRT huA33-C825 (huA33-huC825 or huA33-mC825) + 55 MBq of ¹⁷⁷ Lu-DOTA- Bn (n=10; total: 165 MBq). Serial nanoSPECT/CT imaging was performed on five randomly selected mice that were exposed to D-PRIT for up to 160 hours after the first cycle of ¹⁷⁷ Lu-DOTA-Bn injection for dosimetric calculation. D-PRIT is expected to induce a complete tumor response in all treated animals with no apparent toxicity to the kidney, liver, spleen or bone marrow. Imaging of lutetium177 using the nanoSPECT/CT system in animals treated with PRIT with a three-cycle regimen is expected to show strong differences with visible uptake in the tumor and minimal background levels in normal tissues. These results demonstrate that the huA33-DOTA bispecific antibodies according to the technology of the present invention are useful for reducing tumor burden in vivo and that PRIT-based diagnostic and treatment therapy can have a therapeutic effect

- 61 046795 and/or be applicable to detect a tumor in a subject.

Therapeutic-diagnostic simultaneous treatment and real-time image-corrected dosimetry. NanoSPECT/CT was used for high-resolution quantitative imaging in mice treated with ¹⁷⁷ Lu-D-PRIT for real-time dosimetry. A nude mouse bearing a SW1222 tumor (volume: 100 mm ³ as measured by caliper) was treated with huA33-C825-based PRIT with one cycle (huA33-huC825 or huA33-mC825) + 55 MBq ¹⁷⁷ LuDOTA-Bn and imaged using the nanoSPECT system /CT three times after injection of ¹⁷⁷ Lu-DOTA-Bn: 1, 24 and 160 hours after injection. Images were corrected for attenuation at the time of injection and data were calibrated using known activity standards. The concentration of activity in the tumor was determined using region of interest analysis on calibrated images.

Accordingly, the huA33-DOTA bispecific antibodies according to the technology of the present invention are useful for the treatment of A33-positive cancer types and are useful for detecting a tumor in a subject.

Example 9: Ex vivo biodistribution studies using huASS-DOTA bispecific antibodies according to the technology of the present invention.

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 growth medium supplemented with 10% heat-inactivated fetal bovine serum, 2.0 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. All cells were maintained in a 37°C environment with 5% CO ₂ (gas). After cell line acquisition, cultures were established and cryopreserved in small aliquots to limit cell passage time to less than three months and periodically examined for the absence of mycoplasma using a commercial kit (Lonza, Basel, Switzerland). A solution of 0.25% trypsin/0.53 mM EDTA in Hanks' balanced salt solution without calcium and magnesium was used for trypsinization during subculture and collection of cells; to establish the SW1222 tumor, groups of mice were inoculated with cells at a concentration of 5.0 x 10 ⁶ in 200 μl of cell suspension in a 1:1 mixture of medium and reconstituted basement membrane (BD Matrigel, Collaborative Biomedical Products Inc., Bedford, MA) into the lower flank via subcutaneous (SC) injection and tumor establishment (100-300 mm ³ ) occurred within 7- 10 days.

For all intravenous injections, mice were gently warmed using an IR lamp and placed in a restrainer. Mice had their tails sterilized using alcohol pads and injections were made into the posterior tail vein.

For biodistribution analysis following radiolabel administration, mice were sacrificed by CO2(gas)-mediated asphyxia and tumors and selected organs were collected, washed with water and allowed to air dry, weighed, and radioanalyzed by gamma scintillation counting (Wallac Wizard 3, Perkin Elmer, Waltham, Massachusetts). Emission intensities were corrected for background and attenuation, converted to activity values using an isotope-specific system calibration factor, normalized to the injected activity, and expressed as mean ± SD in %W/g unless otherwise noted.

To determine the biodistribution of huA33-DOTA BsAb in mice containing the SW1222 tumor (number of groups: n=2 for groups that included the clearance agent (CA) step, and n=1 for the control that received saline vehicle; tumor weight ex vivo ranged from 225 to 571 mg at the time of biodistribution assessment) three separate reagents were administered intravenously via the posterior tail vein: 0.25 mg (1.19 nmol) huA33-C825 (BC155-3, clone 2G7) at t=-28 h followed by administration of a clearance agent consisting of a 500 kDa dextran-DOTA(Y) complex at t=-4 h and a [ ¹⁷⁷ Lu]Lu-DOTAbiotin complex (50 μCi) at t=0 h. Research biodistribution was carried out 24 hours after injection of the [ ¹⁷⁷ Lu]Lu-DOTA-biotin tag complex. The 500 kDa dextran-DOTA(Y) clearance agent and the [ ¹⁷⁷ Lu]Lu-DOTA-6uotuh complex were prepared using the methods described in: Orcutt, KD et al., Mol Cancer Ther 11:1365-1372 (2012); An identical radiochemical protocol described for the analysis of the [ ¹⁷⁷ Lu]Lu-DOTA-benzene complex was used for the analysis of [ ¹⁷⁷ Lu]Lu-DOTA-biotin. %W/g=percentage of injected dose per gram of tissue.

As shown in FIG. 40, animals subjected to PRIT using huA33-DOTA bispecific antibodies according to the technology of the present invention showed high levels of antibody penetration into the tumor with or without the use of a clearance agent step (approximately 20-25% ID/g localized at GPA33 (+) tumors), thus demonstrating that huA33-DOTA bispecific antibodies can effectively target tumors in vivo.

Accordingly, the huA33-DOTA bispecific antibodies according to the technology of the present invention are useful for the treatment of A33-positive cancers and useful for the detection of tumors.

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Claims (27)

hotley in the subject. Equivalents. The technology of the present invention is not limited to the specific embodiments described in the present invention, which are intended to be provided solely as an illustration of certain aspects of the technology of the present invention. It is within the scope and spirit of the invention that many modifications and variations of the technology of the present invention are possible, as will be apparent to those skilled in the art. Functionally equivalent methods and devices within the scope of the technology of the present invention, other than those listed herein, will be apparent to those skilled in the art from the prior art descriptions. Such modifications and variations are intended to be within the scope of the technology of the present invention. It should be understood that this technology is not limited to specific methods, reagents, compounds, compositions or biological systems, which, of course, may vary. It should also be understood that the terminology used in the present invention is provided solely for the purpose of describing specific embodiments and does not limit the present invention. In addition, when features or aspects of the invention are described in terms of Markush groups, it will be apparent to one skilled in the art that the invention is thus also described in terms of any individual member or subgroup of members of the Markush group. As will be appreciated by one skilled in the art, for any and all purposes, particularly to provide a written description, all ranges described in the present invention also include any and all possible sub-bands and combinations of sub-bands. Any range listed can easily be defined as a similar range that sufficiently describes and provides the same range, broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed in the present invention can be easily broken down into a lower third, a middle third and an upper third, etc. It will also be apparent to one skilled in the art that all statements such as up to at least more than, less than, etc. include the number indicated and refer to ranges that can be further broken down into sub-ranges such as discussed above. Finally, as one skilled in the art will appreciate, the range includes each individual term. Thus, for example, a group containing 1-3 cells refers to groups containing 1, 2 or 3 cells. Similarly, a group containing 1-5 cells refers to groups containing 1, 2, 3, 4 or 5 cells, etc. All patents, patent applications, provisional applications and publications referred to or cited herein are herein incorporated by reference in their entirety, including all figures and tables, provided they do not expressly conflict with the principles of the present invention. CLAIM 1. An anti-A33 antibody or an antigen-binding fragment thereof, containing an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), where: (a) VH contains the VH-CDR1 sequence FTFSTYDMS (SEQ ID NO: 37), the VH-CDR2 sequence TISSGGSYTYYLDSVKG (SEQ ID NO: 38) and the VH-CDR3 sequence TTVVPFAY (SEQ ID NO: 39); and (b) V _L contains a VL-CDR1 sequence, a VL-CDR2 sequence and a VL-CDR3 sequence selected from the group consisting of: KASQNVRTVVA (SEQ ID NO: 40), LASNRHT (SEQ ID NO: 41) and QYWSYPLT (SEQ ID NO: 42); KASQNVRTWA (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). 2. The antibody or antigen-binding fragment of an antibody according to claim 1, comprising an Fc domain of an antibody having an isotype selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD and IgE. 3. The antigen-binding fragment of an antibody according to claim 1, characterized in that said antigen-binding fragment is selected from the group consisting of Fab, F(ab') ₂ , Fab', scF _v and F _v . 4. The antibody or antigen-binding fragment of an antibody according to claim 1, wherein said antibody comprises a heavy chain (HC) amino acid sequence and a light chain (LC) amino acid sequence selected from the group consisting of: SEQ ID NO: 25 and SEQ ID NO: 26; SEQ ID NO: 29 and SEQ ID NO: 30; SEQ ID NO: 31 and SEQ ID NO: 32; And SEQ ID NO: 35 and SEQ ID NO: 36, respectively. 5. An antibody or antigen-binding fragment of an antibody according to any one of claims 1 to 4, wherein said - 63 046795 The antibody is a monoclonal antibody, a chimeric antibody or a humanized antibody. 6. An antibody or antigen-binding fragment of an antibody according to any one of claims 1 to 5, characterized in that said antibody or antigen-binding fragment thereof binds to an epitope of the A33 protein containing at least five to eight consecutive amino acid residues of the sequence SEQ ID NO: 57. 7. An antibody or antigen-binding fragment of an antibody according to any one of claims 1 to 6, wherein said antibody comprises an IgG1 constant region containing one or more amino acid substitutions selected from the group consisting of N297A and K322A or an IgG4 constant region containing the S228P mutation . 8. The antibody or antigen-binding fragment of an antibody according to any one of claims 1 or 2, comprising a VL sequence that is at least 95% identical to the VL sequence that is present in any of SEQ ID NO: 26, 30, 32 or 36; and/or a VH sequence that is at least 95% identical to the VH sequence present in any of SEQ ID NO: 25, 29, 31 or 35. 9. A conjugate of an antibody or antigen-binding fragment of an antibody according to any one of claims 1 to 8, wherein said antibody or antigen-binding fragment is conjugated to an agent selected from the group consisting of isotopes, dyes, chromogens, contrast agents, drugs, toxins , cytokines, enzymes, enzyme inhibitors, hormones, hormone antagonists, growth factors, radionuclides, metals, liposomes, nanoparticles, RNA, DNA, or any combination thereof. 10. A recombinant nucleic acid sequence encoding an antibody or antigen binding fragment of an antibody according to any one of claims 1 to 8, wherein said antibody or antigen binding fragment of an antibody is capable of binding to the A33 protein. 11. An expression vector containing the recombinant nucleic acid sequence according to claim 10. 12. A host cell for expressing an antibody or an antigen-binding fragment of an antibody according to any one of claims 1 to 8, containing a recombinant nucleic acid sequence according to claim 10 or a vector according to claim 11. 13. A composition for the treatment of cA33-associated cancer, containing in an effective amount an antibody or antigen-binding fragment according to any one of paragraphs. 1 to 8 or the antibody or antigen binding fragment conjugate of claim 9 and a pharmaceutically acceptable carrier. 14. A bispecific antibody comprising an antibody or antigen-binding fragment of an anti-A33 antibody according to claim 1 and an antibody or antigen-binding fragment of an antibody that: binds to T cells, B cells, myeloid cells, plasma cells, mast cells, CD3, CD4 , CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, gamma/delta TCR, NKp46, KIR or low molecular weight DOTA hapten. 15. A method of treating A33-associated cancer in a subject in need thereof, comprising administering to said subject an effective amount of an antibody or antigen-binding antibody fragment of any one of claims 1 to 8, or an antibody conjugate or antigen-binding fragment of claim 9, wherein said antibody or antibody fragment specifically binds and neutralizes the activity of the A33 protein. 16. The method of claim 15, wherein the cancer associated with A33 is colorectal cancer, pseudomyxoma peritonei, appendiceal cancer, pancreatic cancer or gastric cancer. 17. The method of claim 15 or 16, wherein said antibody or antigen-binding antibody fragment or conjugate is administered to said subject separately, sequentially or simultaneously with an additional therapeutic agent. 18. A method of detecting a tumor in a subject in vivo, comprising (a) administering to said subject an effective amount of an antibody or antigen-binding fragment of an antibody according to any one of claims 1 to 8 or an antibody conjugate or antigen-binding fragment according to claim 9, wherein said antibody or fragment is constructed with the possibility of localization in a tumor expressing A33 and labeled with a radioisotope; and (b) detecting the presence of a tumor in said subject by detecting levels of radioactivity emitted by said antibody or antibody fragment in excess of a control value. 19. The method of claim 18, wherein said levels of radioactivity emitted by said antibody or antibody fragment are detected using positron emission tomography or single photon emission computed tomography. 20. A kit for detection of A33 associated cancer, containing an antibody or antigen binding fragment for binding A33 according to any one of claims 1 to 8 or a conjugate of an antibody or antigen binding fragment according to claim 9 and instructions for use. 21. A kit for the treatment of A33-associated cancer, containing an antibody or antigen-binding fragment for binding A33 according to any one of claims 1-8 or a conjugate of an antibody or antigen-binding fragment according to claim 9 and instructions for use. 22. Method for detecting a solid tumor expressing A33 in a patient in need - 64 046795 to a subject, comprising (a) administering to said subject an effective amount of a complex comprising a radiolabeled DOTA hapten and an anti-A33 bispecific antibody according to claim 14, wherein said anti-A33 bispecific antibody binds to a radiolabeled DOTA hapten and an A33 antigen, and wherein said complex is designed to localize to an A33-expressing solid tumor; and (b) detecting the presence of a solid tumor in said subject by detecting levels of radioactivity emitted by said complex in excess of a control value. 23. A method of selecting a subject for pretargeted radioimmunotherapy of a tumor expressing A33, comprising (a) administering to said subject an effective amount of a complex containing a radiolabeled DOTA hapten and a bispecific anti-A33 antibody according to claim 14, wherein said bispecific anti-A33 antibody binds to the radiolabeled DOTA hapten and A33 antigen and where the specified complex is designed with the possibility of its localization in a solid tumor expressing A33; (b) detecting levels of radioactivity emitted by said complex; and (c) selecting said subject to undergo pretargeted radioimmunotherapy if said levels of radioactivity emitted by said complex exceed a control value. 24. A method of treating A33-associated cancer in a subject in need thereof, comprising administering to said subject an effective amount of a complex comprising a radiolabeled DOTA hapten and an anti-A33 bispecific antibody according to claim 14, wherein said anti-A33 bispecific antibody binds to a radiolabeled DOTA-hapten. hapten and A33 antigen and where the specified complex is designed to be localized in an A33-expressing tumor. 25. The method of claim 24, wherein said A33-associated cancer is colorectal cancer, pseudomyxoma peritonei, appendiceal cancer, pancreatic cancer or gastric cancer. 26. A method of treating A33-associated cancer in a subject in need thereof, comprising (a) administering an effective amount of an anti-A33 bispecific antibody according to claim 14, wherein said anti-A33 bispecific antibody binds to a radiolabeled DOTA hapten and an A33 antigen, and wherein said anti-A33 bispecific antibody is designed to localize to an A33-expressing tumor; and (b) administering an effective amount of a radiolabeled DOTA hapten to said subject, wherein said radiolabeled DOTA hapten is designed to bind to said anti-A33 bispecific antibody. 27. The method of claim 26, further comprising administering an effective amount of the clearance agent to the subject prior to administering the radiolabeled DOTA hapten. -