Anti-CEA ch2 domain deleted antibody

Abstract

An antibody or antibody fragment, comprising a light chain comprising a VL; a heavy chain, wherein the CH2 domain is removed and comprising a VH domain; wherein the VL domain and VH domain bind Carcinoembryonic antigen (CEA). Methods for detecting, detecting the progression of, treating, or determining the efficacy of treatment for a CEA-positive cancer in a subject comprising administering the antibody or antibody fragment.

Description

ANTI-CEA CH2 DOMAIN DELETED ANTIBODYPRIORITY CLAIM

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 734,399, filed December 16, 2024, which is incorporated by reference herein in its entirety, including drawings.SEQUENCE LISTING

[0002] This application contains a ST.26 compliant Sequence Listing, which was submitted in XML format via Patent Center, and is hereby incorporated by reference in its entirety. The XML copy, created on December 5, 2025, is named 0544358232WO00.xml and is 31 ,000 bytes in size.BACKGROUND

[0003] Molecular imaging has emerged as a non-invasive and quantitative technique for detecting cancer, monitoring its progression, and evaluating therapeutic responses by specific molecular targeting. Immuno-PET imaging leverages the unique properties of antibodies to selectively target antigens on cancer cells, providing detailed insights such as tumor localization, extent of disease, tumor heterogeneity, therapeutic response that aid in the accurate diagnosis and effective treatment management. One such tool is the radiolabeled humanized hT84.66-M5A (M5A) monoclonal antibody (mAb) with high specificity and affinity towards carcinoembryonic antigen (CEA), a tumor-associated antigen

[0033] , CEA is prominently overexpressed in various gastrointestinal (Gl) cancer, including colorectal cancer, making it an ideal target for both diagnostic and therapeutic interventions [12, 25], The radiolabeled M5A mAb is currently being evaluated in the clinic as a [64Cu]PET imaging agent

[0029] and for [90Y] and [225Ac] radioimmunotherapy [1], There is need in the art for novel antibodies that improve imaging efficiency.BRIEF DESCRIPTION OF THE DRAWINGS

[0004] Figure 1A-1 C shows the design of anti-CEA M5AACH2 antibody constructs. (Figure 1 A) Sketch of full-length antibody and smaller recombinant antibody fragments-1 -184600812.1developed to target CEA positive tumors [20, 28, 33], (Figure 1 B) M5AACH2 constructs were designed to include 2, 3 and 5 cysteine bridges in the hinge region. (Figure 1 C) Amino acid sequences of M5AACH2 hinge domain series. Cysteine involved in disulfide bond formation are represented in bold red. (VL: variable light domain, CL: constant light domain, VH: variable heavy domain, CH: constant heavy domain, UH: upper hinge, MH: middle hinge, LH: lower hinge).

[0005] Figure 2A-2D shows HPLC-SEC Superdex 200 analysis after FcXP affinity purification of M5AACH2 antibody fragments, including M5A-DeltaCH2-C2_Tag (Figure 2A), M5A-DeltaCH2-C2_Flex_(Figure 2B), M5A-DeltaCH2-C3 (Figure 2C), and M5A-DeltaCH2- C5 (Figure 2D) percentage value next to the peak on chromatogram representing the percent of monomer present in the sample.

[0006] Figure 3 shows biochemical and stability analysis of the M5AACH2 antibody constructs. (Figure 3A) The purification of the M5AACH2 antibody constructs resulted in a single peak of the expected molecular size by HPLC-SEC analysis and (Figure 3B) were stable up to 9 months. (Figure 3C) Analysis of purified M5AACH2 antibodies on SDS-PAGE under reducing (left panel) and non-reducing conditions (right panel). The M5AACH2 series showed varying expression of two different isoform forms on the non-reduced gel (Form A and Form B). (Figure 3D) For immunoPET imaging, the M5AACH2-C5 was mildly reduced, conjugated with DO3A-VS metal chelator, labeled with [64Cu] and shown to be immunoreactivity to soluble human CEA by a shift in molecular mass on HPLC-SEC.

[0007] Figure 4 shows the M5AACH2 constructs were subjected to reduction by TCEP at varying TCEP:protein ratios. All the constructs were reduced completely at a TCEP:protein ratio of 15:1 or higher (left gel). At lower ratios partially reduced antibody can be seen with various intermediate fragments (right gel).

[0008] Figure 5A-5B shows [64Cu]Cu-DO3A-VS M5AACH2-C5 serial PET imaging and terminal biodistribution studies in 2 colorectal cancer mouse models. (Figure 5A) serial PET imaging of [64Cu]Cu-DO3A-VS M5AACH2-C5 in athymic mice bearing subcutaneous human colorectal cancer LS-174T tumors. (Figure 5B) serial PET imaging of [64Cu]Cu--2-184600812.1D03A-VS M5AACH2-C5 in immunocompetent CEA-transgenic mice bearing MC-38-CEA transfected tumors.

[0009] Figure 6A-6C shows [64Cu]Cu-DO3A-VS M5AACH2-C5 terminal biodistribution and blood clearance curve. (Figure 6A) Biodistribution of [64Cu]Cu-DO3A-VS M5AACH2- C5 in athymic mouse-LS174T model at 48 hrs (Figure 6B) Biodistribution of [64Cu]Cu- DO3A-VS M5AACH2-C5 in TgCEA-MC38-CEA+ mouse model at 48 hr. (Figure 6C) Blood clearance curves of [64Cu]Cu-DO3A-VS M5AACH2-C5 in athymic mice (n=4). The dashed line represents two-phase decay fitting curve.DETAILED DESCRIPTION

[0010] The present technology includes recombinant antibodies and antibody fragments that bind to CEA where the CH2 domain has been removed. The present technology also includes methods of using the antibodies and antibody fragments to image a tumor, to detect cancer, to treat cancer, and / or to determine the efficacy of a cancer treatment.Overview

[0011] Recombinant antibody fragments have demonstrated rapid tumor targeting with a short blood circulation half-life resulting in better tumor to blood ratios compared to a full length antibody for enhanced imaging and reduced bone marrow toxicity

[0030] , The utility of tumor targeting with radiolabeled antibodies can be augmented through antibody engineering approaches to optimize affinity and pharmacokinetics properties. Engineered antibody fragments, such as single domain antibody (sdAb or VHH or nanobody), singlechain variable domain (scFv), Fab, F(ab’)2 diabody, minibody, and scFv-Fc have emerged as powerful tools in tumor imaging [2, 27], These fragments, derived from conventional monoclonal antibodies, retain high specificity and affinity for their target antigens while offering advantages over full-length antibodies such as enhances tumor penetration and accelerated clearance from non-target tissues

[0021] , The rapid pharmacokinetics of engineered antibody fragments allow for earlier imaging, and superior contrast

[0030] , In addition, the engineering flexibility of these fragments also facilitates their conjugation with various imaging agents, such as radionuclides, fluorescent dyes and chemotherapy drugs.-3-184600812.1However, these benefits come with certain trade-offs. Small antibody fragments typically have shorter circulation times, which may limit their uptake in tumors and reduce overall accumulation. They also may lack the effector functions present in full-length antibodies, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), potentially diminishing their immunological therapeutic efficacy.

[0012] Several anti-CEA T84.66 engineered antibody fragments have been evaluated in biodistribution studies targeting colorectal cancer in xenografted mice models. For instance, [1111n and 1311] labeled anti-CEA T84.66 diabody demonstrated rapid clearance from blood and normal tissue while effectively targeting tumors

[0034] , Similarly, the CT84.66 minibody with an intermediate molecular weight (MW 80 kDa) labeled with

[1231] showed excellent tumor targeting and imaging properties in athymic mouse tumor xenograft model

[0028] ,

[0013] Non-specific conjugation procedures, which involve use the stochastic ligation of amine-reactive prosthetic groups to surface lysines, produces heterogeneous conjugates with poor reproducibility and compromise binding capabilities

[0021] , In contrast, site-specific bioconjugation methods yields homogeneous conjugates with superior binding properties

[0022] , Site-specific conjugation of antibodies can be achieved via enzymatic (transglutaminase, glycan-mediated) or chemical (selective reduction of thiol bridges, N- terminal modification) methods. Among these, alkylation of cysteine is a widely used and conventional method for site-specific conjugation. The interchain cysteine in the hinge residues can be selectively reduced using dithiothreitol (DTT) or (tris (2-carboxyethyl) phosphine) (TCEP) under controlled conditions to achieve optimal payload to antibody ratio for enhanced potency [11 , 19],

[0014] The present technology includes a series of CH2 domain deleted antibodies (ACH2) based on the humanized anti-CEA M5A monoclonal antibody. The M5A-ACH2 antibodies feature modified hinge domains for conjugation of additional site-specific payloads and enhanced structural stability (Figure 1 ). This work details the production, and biochemical characterization of the anti-CEA ACH2 antibodies. Notably, the M5AACH2-C5 was labeled with [64Cu] (half-life 12.7 hr, 0.653 MeV positron) and evaluated for PET imaging in 2 human colorectal cancer mouse models. The results showed rapid high tumor-4-184600812.1uptake and fast blood clearance making it an excellent immunoPET imaging agent for advancement toward clinical evaluation.Antibodies and antibody fragments

[0015] The term "antibody" as used herein refers to a protein that specifically binds to an antigen. Antibodies are generally composed of two heavy chains and two light chains, each containing a variable region and a constant region. The variable regions of the heavy and light chains form the antigen-binding site, which is responsible for the specificity of the antibody.

[0016] The term "antibody fragment" as used herein refers to a portion of an antibody that retains the ability to specifically bind to an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-chain variable fragments (scFv), and domain antibodies (dAbs). These fragments can be produced by enzymatic digestion of full-length antibodies or by recombinant DNA technology.

[0017] Antibodies and antibody fragments can be derived from any species, including but not limited to, human, mouse, rat, rabbit, and camelid. They can be produced by hybridoma technology, phage display, or other recombinant methods. Additionally, antibodies and antibody fragments can be modified to improve their stability, affinity, or other properties, such as by humanization, chimerization, or pegylation.

[0018] In the context of this application, the term "antibodies or antibody fragments" encompasses a variety of molecules, including but not limited to, monoclonal antibodies (mAbs), polyclonal antibodies, Fab fragments, Fab' fragments, F(ab')2fragments, singlechain variable fragments (scFv), domain antibodies (dAbs), minibodies, diabodies, nanobodies, or bispecific antibodies. The antibody or antibody fragment may be derived from Immunoglobulin G (IgG), includng subtypes lgG1 , lgG2, lgG3, and lgG4; Immunoglobulin A (IgA), including subtypes lgA1 and lgA2; Immunoglobulin M (IgM); Immunoglobulin E (IgE); or Immunoglobulin D (IgD).

[0019] In some embodiments, the present technology includes a light chain. In some aspects, the light chain comprises a VL domain. In some aspects, the VL domain comprises CDRs having amino acid sequences comprising SEQ ID NOs: 12-14. In some aspects, the-5-184600812.1light chain comprises an amino acid sequence of SEQ ID NO: 15. In some aspects, the light chain is encoded by a nucleotide sequence of SEQ ID NO: 23.

[0020] In some embodiments, the present technology includes a heavy chain, wherein the CH2 domain is removed. In some aspects, the heavy chain. In some aspects, the heavy chain is derived from an lgG1 , lgG2, lgG3, lgG-4, IgA, 1 , lgA2, IgM, IgE, or IgD heavy chain. In some aspects the heavy chain is derived from an IgG 1 heavy chain. In some aspects, the heavy chain comprises a CH3 domain. In some aspects, the CH3 domain comprises an amino acid sequence of SEQ ID NO: 18. In some aspects, the heavy chain comprises a CH1 domain. In some aspects, CH1 domain comprises an amino acid sequence of SEQ ID NO: 17.

[0021] In some aspects the heavy chain comprises a VH domain. In some aspects, the VH domain comprises CDRs having amino acid sequences comprising SEQ ID NOs: 9-11 . In some aspects, the VH domain comprises an amino acid sequence of SEQ ID NO: 16.

[0022] In some aspects, the VL domain and the VH domein bind Carcinoembryonic antigen (CEA). In some aspects, the VL domain comprises CDRs having amino acid sequences comprising SEQ ID NOs: 12-14 and the VH domain comprises CDRs having amino acid sequences comprising SEQ ID NOs: 9-11. In some aspects, the VH domain comprises SEQ ID NO: 16 and the light chain comprises SEQ ID NO: 15.

[0023] In some embodiments, the antibody or antibody fragment comprises a hinge sequence that is joined to the CH3 domain without a CH2 domain. In some aspects, the hinge sequence is joined to the CH3 domain by a linker comprising. In some aspects, the linker comprises serine and glycine amino acids. In some aspects, the linker is a Flex linker. In some aspects, the linker is a TAG linker. In some aspects, the linker comprises an amino acid sequence of SEQ ID NO: 1. In some aspects, the linker comprises an amino acid sequence of SEQ ID NO: 2.

[0024] In some embodiments, the hinge sequence comprises an amino acid sequence configured to form 2, 3, or 5 disulfide bonds. In some aspects, the hinge sequence comprises an amino acid sequence configured to form 2 disulfide bonds. In some aspects, the hinge sequence comprises an amino acid sequence configured to form 3 disulfide bonds. In some-6-184600812.1aspects, the hinge sequence comprises an amino acid sequence configured to form 5 disulfide bonds. In some aspects, the hinge sequence comprises an amino acid sequence of SEQ ID NO: 3. In some aspects, the hinge sequence comprises an amino acid sequence of SEQ ID NO: 4. In some aspects, the hinge sequence comprises an amino acid sequence of SEQ ID NO: 5. In some aspects, the hinge sequence comprises an amino acid sequence of SEQ ID NO: 6. In some aspects, the hinge sequence comprises an amino acid sequence of SEQ ID NO: 7. In some aspects, the hinge sequence comprises an amino acid sequence of SEQ ID NO: 8.

[0025] In some embodiments, the antibody or antibody fragment comprises or consists of a middle hinge (MH) sequence having an amino acid sequence of SEQ ID NO: 3 and a linker sequence having an amino acid sequence of SEQ ID NO: 2. In some aspects, the antibody or antibody fragment comprises an amino acid sequence of SEQ ID NO: 19. In some aspects, the antibody or antibody fragment comprises a heavy chain encoded by SEQ ID NO: 24.

[0026] In some embodiments, the antibody or antibody fragment comprises or consists of a middle hinge (MH) sequence having an amino acid sequence of SEQ ID NO: 3 and a linker sequence having an amino acid sequence of SEQ ID NO: 1. In some aspects, the antibody or antibody fragment comprises an amino acid sequence of SEQ ID NO: 20. In some aspects, the antibody or antibody fragment comprises a heavy chain encoded by SEQ ID NO: 25.

[0027] In some embodiments, the antibody or antibody fragment comprises or consists of a middle hinge (MH) sequence having an amino acid sequence of SEQ ID NO: 4 and a linker sequence having an amino acid sequence of SEQ ID NO: 2. In some aspects, the antibody or antibody fragment comprises an amino acid sequence of SEQ ID NO: 21. In some aspects, the antibody or antibody fragment comprises a heavy chain encoded by SEQ ID NO: 26.

[0028] In some embodiments, the antibody or antibody fragment comprises or consists of a middle hinge (MH) sequence having an amino acid sequence of SEQ ID NO: 5 and a linker sequence having an amino acid sequence of SEQ ID NO: 1. In some aspects, the antibody or antibody fragment comprises an amino acid sequence of SEQ ID NO: 22. In-7-184600812.1some aspects, the antibody or antibody fragment comprises a heavy chain encoded by SEQ ID NO: 27.

[0029] In some embodiments, the antibody or antibody fragment is conjugated to an imaging agent. In some aspects, the imaging agent is a radiolabel. In some aspects, the antibody or antibody fragment is conjugated to an imaging agent by site-specific conjugation. In some aspects, the antibody or antibody fragment is conjugated to an imaging agent by enzymatic (e.g. transglutaminase, glycan-mediated) or chemical (e.g. selective reduction of thiol bridges, N-terminal modification) methods. In some aspects, the antibody or antibody fragment is conjugated to an imaging agent by alkylation of cysteine. In some aspects, the antibody or antibody fragment is conjugated to an imaging agent by selective reduction of the interchain cysteine residues in the hinge sequence using, for example, dithiothreitol (DTT) or (tris (2-carboxyethyl) phosphineaO (TCEP). In some aspects, the imaging agent is a fluorescent dye. The fluorescent dye may be a near infrared (NIR) fluorescent dye, for example IRDye800CW or IRDye700DX.

[0030] In some embodiments, the imaging agent may be conjugated to the antibody or antibody fragment using, for example, DO3A-VS (1 ,4,7,10 tetraazacyclododecane-1 ,4,7- triacetic acid trisodium salt-vinyl sulfone), DOTA (1 ,4,7,10-tetraazacyclododecane-1 ,4,7,10- tetraacetic acid), NOTA (1 ,4,7-triazacyclononane-1 ,4,7-triacetic acid), NHS-Esters, Maleimide Conjugation, Click Chemistry (e.g., azide-alkyne cycloaddition), or DFO (Desferrioxamine). In some aspects, the imaging agent may be conjugated to the antibody or antibody fragment using DO3A-VS.

[0031] In some embodiments, the imaging agent is a radioimaging agent. In some aspects, the radioimaging agent is detectable by a clinical imaging technique such as PET imaging. In some aspects the radioimaging agent is, for example, Fluorine-18 (F-18), Gallium-68 (Ga-68), Technetium-99m (Tc-99m), lodine-123 (1-123), lndium-111 (In-111 ), Carbon-11 (C-11 ), Zirconium-89 (Zr-89), Copper-64 (Cu-64), Yttrium-90 (Y-90), lodine-124 (1-124), Rubidium-82 (Rb-82), Thallium-201 (TI-201 ), Samarium-153 (Sm-153), Lutetium- 177 (Lu-177), or Rhenium-186 (Re-186). In some aspects, the radioimaging agent is Cu-64. In some aspects, the radioimaging agent is Y-90.-8-184600812.1

[0032] In some embodiments, the antibody or antibody fragment may be conjugated to a therapeutic. In some aspects, the therapeutic is a cancer therapeutic. In some aspects, the therapeutic is a cancer therapeutic known in the art for treating a CEA-positive tumor. In some aspects, the therapeutic is cleaved in the tumor microenvironment. Non-limiting examples of therapeutics include Monomethyl Auristatin E (MMAE), Monomethyl Auristatin F (MMAF), Maytansinoids (DM1 , DM4), Calicheamicin, Duocarmycin,Pyrrolobenzodiazepine (PBD) dimers, Doxorubicin, SN-38, Auristatins, Camptothecin derivatives, Tubulysins, Amanitin, or Thailanstatin A.Methods for detecting, treating, and determining the efficacy of treating a CEA-positive tumor

[0033] The present technology also includes methods of detecting, treating, and determining the efficacy of treating a CEA-positive tumor in a subject comprising administering to the subject any of the antibodies or antibody fragments of the present technology.

[0034] In some embodiments, the CEA-positive tumor is a tumor of colorectal cancer, pancreatic cancer, gastric cancer, lung cancer, breast cancer, medullary thyroid cancer, or ovarian cancer. In some aspects, the CEA-positive tumor is a colorectal cancer tumor.

[0035] In some embodiments, the method of detecting a CEA-positive cancer in a subject comprises administering any antibody or antibody fragment of the present technology to the subject and measuring the antibody fragment in the subject.

[0036] In some aspects, the method comprises administering an effective amount of the antibody or antibody fragment to the subject such that the antibody or antibody fragment can be detected in the tumor using an imaging modality. The imaging modality may include PET imaging, Magnetic Resonance Imaging (MRI), Computed Tomography (CT), Single Photon Emission Computed Tomography (SPECT), ultrasound Imaging, optical Imaging, X- ray Imaging, Near-Infrared Spectroscopy (NIRS), Photoacoustic Imaging, or Thermography. In some aspects, the imaging modality is PET imaging.

[0037] In some aspects, the antibody or antibody fragment may be administered by, for example, single dose administration, multiple dose administration, continuous administration, bolus plus infusion, or a dose escalation. As used herein, an “effective-9-184600812.1amount” of an antibody or antibody fragment refers to an approximate concentration of the antibody or antibody fragment that allows for the detection of the CEA-positive tumor in the subject.

[0038] In some embodiments, the antibody or antibody fragment may be administered at about a human equivalent 100 pCi / 10 pg of the antibody or antibody fragment in a single dose in a mouse. As used herein, a “human equivalent” refers to an approximate dose of the antibody or antibody administered to a human subject to elicit a similar effect of a known dose in a mouse. The human equivalent may be estimated using calculations known to one of skill in the art. In some aspects, the antibody or antibody fragment may be administered to the subject in a single dose of the imaging antibody ranging from 0.1 mg / kg to 10 mg / kg of body weight, for example 1 mg / kg.

[0039] The antibody or antibody fragment may be measured during or after the administration of the antibody or antibody fragment. For example, the antibody or antibody fragment may be measured 0, 2, 4, 24, 48, 72 h after the antibody or antibody fragment is administered to the subject.

[0040] In some embodiments, detecting a CEA-positive tumor comprises measuring elevated levels of the antibody or antibody fragment in the subject compared to a healthy subject.

[0041] The present technology also includes a method of treating a CEA-positive cancer is a subject comprising detecting the cancer by the methods described in the present technology and administering a cancer therapeutic to the subject. In some embodiments, the method of treating a CEA-positive cancer comprises administering an antibody or antibody fragment of the present technology conjugated to a cancer therapeutic to a subject in need thereof.

[0042] The present technology also includes a method of determining efficacy of a treatment for a CEA-positive cancer comprising treating the subject according to any of the methods described in the present technology and subsequently administering the subject with a second dose of an antibody or antibody fragment to the subject and measuring the antibody or antibody fragment from the second dose. In some aspects, measuring reduced-10-184600812.1levels of the second dose of the antibody or antibody fragment compared to the levels of the antibody or antibody fragment before the administration of the cancer therapeutic indicates that the treatment is effective.

[0043] The present technology also includes a method of measuring the progression of a CEA-positive cancer comprising detecting a CEA-positive cancer by administering a first dose of the antibody or antibody fragment in a subject and measuring the antibody or antibody fragment from the first dose; and subsequently administering the subject with a second dose of an antibody or antibody fragment to the subject and measuring the antibody or antibody fragment from the second dose. In some aspects, measuring increased levels of the second dose of the antibody or antibody fragment compared to the levels of the first dose of the antibody or antibody fragment before the administration of the cancer therapeutic indicates that the treatment is effective.Example 1 - Design, expression and purification of M5AACH2Methods

[0044] anti-CEA Delta CH2 antibody design and production

[0045] The humanized anti-CEA hT84.66-M5A (M5A) mAb was engineered to a CH2 domain deleted (ACH2) mAb fragment format based on the anti-TAG-72 ACH2 antibody [23, 33], The lgG1 heavy chain CH2 domain was replaced with a linker consisting of serine(S) and glycine(G) amino acids to join the lgG1 lower hinge to the CH3 domain. Two different linkers named Flex (APGGGSSGGGSG), and TAG (GGGSSGGGSG) were incorporated as previously described [10, 13], Modifying the IgG hinge design of Glaser et al, 4 hinge and linker variants were developed having 2, 3 and 5 cysteine disulfide bridges (M5AACH2- C2_Tag, M5AACH2-C2_Flex, M5AACH2-C3_TAG, and M5AACH2-C5_Flex, respectively) as shown in Figure 1

[0010] ,

[0046] cDNA encoding the gene constructs were synthesized by GeneArt (ThermoFisher Scientific, MA). The individual pairs of light and heavy chain genes were subcloned into the pEE12 / 6 dual vector GS expression system (Lonza Biologies, Switzerland). Plasmids encoding the individual delta CH2 antibody constructs were transiently expressed using the ExpiFectamine™ 293 Transfection Kit (Gibco) as per-11-184600812.1manufacturer instructions. The cells were grown in Expi293 expression media at 125 rpm, 37 °C and 8 % CO2 in a humidified incubator and harvested on day 6 post-transfection.

[0047] Purification of M5AACH2 Antibodies

[0048] The cell harvests were clarified by centrifuged (3000 g for 10 min) and filtered to remove particulates. The cell-free harvest was treated with the strong anion AG1 -X8 resin (Bio-Rad, Hercules, CA) (5 % w / v) by incubation on a roller at 4 °C overnight and sterile filtered to provide a clarified feed stream. Chromatography was performed using NGC chromatography system (BioRad) controlled and recorded using ChromLab software (BioRad). For affinity purification an AP-1 column (Waters, MA) was packed with 7 ml of Capture Selecto FcXP affinity matrix (ThermoFisher Scientific). The column was pre-equilibrated with 20 column volume (CV) of phosphate buffered saline (PBS) pH 7.4 at 2 ml / min. The individual harvests were loaded on columns at a flow rate of 1 ml / min. The column was washed with 5 CV PBS, 10 CV of a high salt buffer (0.02M sodium phosphate, 0.02 M sodium citrate, 0.5M NaCI, pH 7.5) and 5 CV of PBS. The M5AACH2 constructs were eluted with 0.1 M glycine pH 3.0 buffer. Eluted protein was neutralized with 0.5M [2-(N- morpholino)ethanesulfonic acid] (MES) pH 8.0 buffer to pH 6.5. The column was cleaned and sanitized with 1 % phosphoric acid and 2 M guanidine hydrochloride and stored at 4 °C in 20 % ethanol.

[0049] Ceramic Hydroxyapatite Type 1 , 20 pm (CHT) (Bio-Rad) chromatography was used as polishing step to remove aggregates at a flow rate of 5 ml / min (except stated otherwise). A 7 ml CHT column was pre-equilibrated with 10 CV of 0.05M MES, 0.01 M potassium phosphate, pH 6.5 buffer. The purified antibody was loaded at 2.5 ml / min followed by 2 CV of wash buffer (0.05M MES, 0.05M potassium phosphate, pH 6.5). A 30 CV linear gradient of 100 % wash buffer to 100 % elution buffer (0.05M MES, 0.2M potassium phosphate, pH 6.5) was used to elute the protein. Monitoring elution by A280, peaks were collected and analyzed for the presence of aggregates by high performance liquid chromatography-size exclusion chromatography (HPLC-SEC) using a Superdex 200 10 / 300 column (Cytiva, Wilmington, DE). The peaks containing the monomeric form were combined, buffer exchanged with PBS using 10K MWCO protein concentrators (ThermoFisher-12-184600812.1Scientific) and sterile filtered. M5AACH2 antibody fragments were stored at a concentration range of 6.5-7.5 mg / ml in PBS at 4 °C.

[0050] Biochemical analysis

[0051] Antibody samples were analyzed by SDS-PAGE under non-reducing and reducing conditions on 10 % Mini Precast Protein Gels (Bio-Rad). Three micrograms of antibody samples were mixed with loading buffer with or without DTT reducing agent. The sample were heated for 5 minutes at 95 °C before loading and electrophoresed at 200 V x 30 min. The gels were imaged on a ChemDoc imaging system (Bio-Rad) and analyzed using the Image-Lab software (Bio-Rad). To compare the thiol stability of M5AACH2 constructs, the samples were incubated with TCEP for 1 hr at room temperature. TCEP: protein molar ratios of 30:1 , 15:1 , 7.5:1 , and 3.5:1 were used. The samples were run as per non-reducing condition as explained earlier in this section. Stability was assessed at serial time points by HPLC-SEC analysis. Anti-CEA immunoreactivity was confirmed by incubating 10 pg of antibody with 50 pg of soluble CEA, (37°C for 30 min) and analyzing for the formation of a 300 kilodaltons (kDa) antibody-antigen complex by HPLC-SEC

[0017] ,

[0052] Surface plasmon resonance (SPR) assays were performed on Biacore X100 (Cytiva) by using recombinant human CEA biotinylated (RayBiotech, Comers, GA) immobilized on sensor chip SA (Cytiva) at a concentration of 5 pg / ml. M5A and M5AACH2 mAb constructs were titrated at 8 concentrations (1000, 500, 250, 125, 62.5, 31.25, 15.62, and 7.8 nM). Each run had 300 s contact time with the analyte, 900 s dissociation time with a flow rate of 30 pl / min followed by 2 regeneration steps of 6 M guanidine hydrochloride with a contact time of 60 s each. The sensograms were analyzed for calculating equilibrium dissociation constant (KD) using biacore evaluation software incorporating the 1 :1 binding model.Results

[0053] A series of recombinant anti-CEA humanized M5A antibody fragments were designed for rapid high-level targeting of gastrointestinal cancers by PET imaging based on the CH2 domain deleted format (named “delta CH2”) previously described [3, 5, 7, 23], For site-specific conjugation of multiple payloads and ensure molecular stability, additional-13-184600812.1disulfide bridges were engineered into the IgG hinge domain adopted from the design of Glaser et al.

[0010] , Based on human lgG1 and lgG3 hinge sequences, 4 different variants were designed with 2, 3 and 5 cysteine (C) disulfide bridges along with the C2 having two different linkers, Flex and TAG to span the lower hinge to the CH3 domain: M5AACH2- C2_Tag, M5AACH2-C2_Flex, M5AACH2-C3_Tag and M5AACH2-C5_Flex as shown in Figure 1 . Transient mammalian expression of the 4 M5AACH2 constructs showed high level expression (ranging from 65-90 pg / ml). Purification employed an affinity capture resin that binds to the human CH3 antibody domain, resulting in the rapid, high-level purification of the M5AACH2 series and removal of aggregates[4, 8, 26], The purified M5AACH2 antibodies were analyzed for presence of aggregates by HPLC-SEC, and the results showed the M5AACH2-C2-Tag, M5AACH2-C2-Flex, M5AACH2-C3, and M5AACH2-C5 contained 10 %, 8 %, 25 %, and 10 % aggregates respectively (Figure 2). A ceramic hydroxyapatite chromatography “polish” step was incorporated, and HPLC-SEC analysis showed that the two-step purification scheme yielded 100 % M5AACH2 monomers with the expected molecular mass and the monomers were stable for 1 year (Figure 3A and 3B).

[0054] The purified M5AACH2 constructs were analyzed by SDS-PAGE gel analysis to determine purity and antibody assembly into covalent disulfide dimers (Figure 3C). Under reducing condition all 4 constructs migrated as two bands, corresponding to the expected light chain and heavy chains molecular weight. However, under non-reducing conditions, heterodimer isoforms were observed that indicated interchain disulfide bonded isoform A and noncovalently assembled isoform B as previously described [10, 15], The M5AACH2- C2-Tag, M5AACH2-C2-Flex, M5AACH2-C3, and M5AACH2-C5 have 68 %, 82 %, 96 %, and 100 % of the disulfide linked isoform A, respectively. M5AACH2-C5 was determined to be the best candidate in terms of hinge stability based on its 100 % isoform A and was selected for in vivo PET imaging. The M5AACH2 constructs were reduced with a varying TCEP:protein molar ratio to test the stability. All the samples were completely reduced with TCEP:protein molar ratio of 15:1 and above. A partial reduction was seen when the samples were reduced using molar ration of 7.5:1 and below (Figure 4).Example 2: Radiolabeling and immunoreactivity of [64CulCu-DO3A-VS-M5AACH2-C5Methods-14-184600812.1

[0055] The M5AACH2-C5 antibody was conjugated with 1 ,4,7,10 tetraazacyclododecane-1 ,4,7-triacetic acid trisodium salt-vinyl sulfone (DO3A-VS) as previously described

[0017] , Briefly, 2 mg of M5AACH2-C5 was added to 376 pl PBS and 24 pl of 10 mM TCEP in a microcentrifuge tube under argon and incubated at room temperature, rocking for 2 hr. The reduced antibody was reacted with 13 pl of DO3A-VS (10 mg / ml stock) and incubated for 2 hours rocking at room temperature. Unconjugated D03A- VS and TCEP were removed by diafiltration with 0.25M ammonium acetate, pH 7 (25 DV) using 10 kDa MWCO ultrafiltration membrane in an Amicon stirred cell (Millipore, MA). The DO3A-VS-M5AACH2-C5 was radiolabeled with [64CuCI] (3D Imaging, Little Rock, AR, specific activity 14.1 pCi / pg, in 1 M HEPES for 1 hr. at 43°C) The radiolabeling efficiency was 98 % by instant thin-layer chromatography. The [64Cu]Cu-DO3A-VS-M5AACH2-C5 was purified by HPLC-SEC. Incubation with soluble CEA (20 molar excess) showed >95 % immunoreactivity by an in vitro molecular weight shift assay and stability study showed the product was stable at least to 72 h by HPLC-SEC as shown in Figure 3D

[0017] ,Results

[0056] All of the M5AACH2 constructs bind to immobilized CEA with similar affinities as of parent M5A mAb when analyzed by surface plasmon resonance on a Biacore X100 instrument (Table 1 ). For animal PET imaging studies, the M5AACH2-C5 was conjugated in the hinge with the thiol-reactive metal chelate, DO3A-VS and radiolabeled with the [64Cu] as previously described

[0017] , The [64Cu]Cu-DO3A-VS-M5AACH2-C5 was purified by SEC- HPLC and shown to be immunoreactive to soluble CEA in preparation for animal studies (Figure 3D).

[0057] Table 1. Kinetic affinity analysis by SPR was performed on the M5A mAb and M5AACH2 antibody binding to CEA using Langmuir 1 :1 binding model. Kd, apparent dissociation constant; Kais the association constant; and KD is equilibrium dissociation constant.-15-184600812.1Example 3: Tumor targeting, biodistribution, and pharmacokineticsMethods

[0058] Animal model and study design

[0059] All applicable institutional and / or national guidelines for the care and use of animals were followed. All mice were handled in the City of Hope (COH) animal care facility as per COH Institutional Animal Care and Use Committee, in accordance with the National Institute of Health Office of Laboratory Animal Welfare guidelines. Two animal models were employed bearing subcutaneous colorectal cancer tumors. Six 6-week old female athymic mice were injected with human colorectal cancer LS174T tumors (106 cells in 100 pl per mice) and six 6-week old female immunocompetent transgenic-CEA (Tg-CEA) mice were injected with MC-38 tumors transfected with the human CEA gene in the flank as previously described [6, 18], After 12 days, four mice from each group were selected based on the tumor size and injected via the tail vein with 100 pCi / 10 pg of [64Cu] Cu-D03A-VS- M5AACH2-C5.

[0060] Imaging and Biodistribution

[0061] Serial imaging studies were conducted using [3-cube and X-cube (MoleCubes, Ghent, Belgium) for PET and computer tomography (CT) scans, respectively. Two mice were selected from each group and PET imaging performed at 0, 3, 24, and 48 hr. post injection. Mice were kept sedate under isoflurane anesthesia during each imaging session. PET and CT scan images were co-registered using manufacture software.

[0062] Blood clearance was measured by microcapillary sampling of 5 pl of blood from the tail vein at 0, 2, 4, 24, 48, 72 h post-injection and counted using a calibrated PerkinElmer gamma counter. After the last blood sample or image was acquired, all animals were euthanized, necropsy performed and organs weighed (tumor, blood, heart, lung, liver, stomach, small and large intestine, spleen, kidneys, right quadricep muscle, carcass) and counted for radioactivity. All data are mean values and have been corrected for radioactive-16-184600812.1decay back to the time of injection, allowing organ uptake to be reported as percent of the injected dose per gram (% IDg-1 ) with standard errors. All statistical analyses were conducted using Prism version 9 (GraphPad Software, San Diego, CA). A two-phase decay non-linear curve fit with constrain (plateau = 0) was used to calculate the half-life.Results

[0063] The [64Cu]Cu-DO3A-VS-M5AACH2-C5 was evaluated for its ability to target CEA-positive tumors in two colorectal cancer mouse models: 1 ) Athymic mice bearing human colorectal cancer LS174T xenografts and 2) Immunocompetent CEA-transgenic mice bearing murine colorectal cancer MC-38 cells transfected with human CEA (CEA is only expressed in higher primates, requiring CEA gene transfection.) [6, 14]. [64Cu]Cu- DO3A-VS-M5AACH2-C5 exhibited excellent tumor targeting in both models as shown in Figure 5A & 5B. Initially, most of the activity can be seen around the thoracic area due to blood pool activity but within the next 3 hours, the blood activity decreased, and tumor uptake was visible. Terminal biodistribution was done immediately after the last PET / CT scan at 48 hrs. In the LS174T model, the tumor tissue reached 34.6 percent injected dose per gram (% ID / g), the highest of all tissues, followed by kidney (16.6 % ID / g) and liver (13.8 % ID / g) shown in Figure 6A. Similarly, in the Tg-CEA MC-38-CEA+ model, the tumor showed the highest accumulation, reaching 22.6 % ID / g, followed by liver (18.4 % ID / g), spleen (16.5 % ID / g), and kidney (13.7 % ID / g) shown in Figure 6B. Blood samples from LS174T mice (n=4) were collected at 0, 2, 4, 24 and 48 hr. and radioactivity counted to determine blood clearance rates. The pharmacokinetics profile of [64Cu]Cu-DO3A-VS-M5AACH2-C5 showed a two-phase clearance with an average 2nd phase half-life (T1 / 2|3) of 8.62 hr. (Figure 6C).

[0064] The present technology demonstrates successful design and production of a series of recombinant humanized anti-CEA M5AACH2 antibody fragments for clinical imaging, for example PET imaging of colorectal cancer. The engineered M5AACH2 constructs, incorporating additional disulfide bridges in the hinge domain, provided multiple sites for site-specific conjugation and demonstrated enhanced stability. The [64Cu]Cu- DO3A-VS-M5AACH2-C5 PET imaging demonstrated high tumor targeting and favorable pharmacokinetics in two CEA-positive colorectal cancer mouse.-17-184600812.1

[0065] From the foregoing, it will be appreciated that specific embodiments of the present technology have been described herein for purposes of illustration, but that various modifications may be made without deviating from the scope of the present technology. Accordingly, the present technology is not limited except as by the appended claims.References1. Akhavan, D., et al., Phase I Study of Yttrium-90 Radiolabeled M5A Anti- Carcinoembryonic Antigen Humanized Antibody in Patients with Advanced Carcinoembryonic Antigen Producing Malignancies. Cancer Biother Radiopharm, 2020. 35(1 ): p. 10-15.2. Bao, G., et al., Nanobody: a promising toolkit for molecular imaging and disease therapy. EJNMMI Research, 2021. 11 (1 ): p. 6.3. Calvo, B., et al., Construction and purification of domain-deleted immunoglobulin variants of the recombinant / chimeric B72.3 (y1 ) monoclonal antibody. Cancer Biother, 1993. 8(1 ): p. 95-109.4. Chen, W., et al., Assessing four subdomain-specific affinity resins' capability to separate half-antibody from intact bispecific antibody. Protein Expr Purif, 2022. 198: p. 106124.5. Chinn, P.C., et al., Pharmacokinetics and tumor localization of (111 )in-labeled HuCC49DeltaC(H)2 in BALB / c mice and athymic murine colon carcinoma xenograft. Cancer Biother Radiopharm, 2006. 21 (2): p. 106-16.6. Clarke, P., et al., Mice transgenic for human carcinoembryonic antigen as a model for immunotherapy. Cancer Res, 1998. 58(7): p. 1469-77.7. De Pascalis, R., et al., Grafting of "abbreviated" complementarity-determining regions containing specificity-determining residues essential for ligand contact to engineer a less immunogenic humanized monoclonal antibody. J Immunol, 2002. 169(6): p. 3076-84.8. Dong, W., D. Zhang, and Y. Li, CaptureSelect FcXP affinity medium exhibits strong aggregate separation capability. Protein Expr Purif, 2024. 220: p. 106503.-18-184600812.19. Ewert, S., et al., Biophysical properties of human antibody variable domains. J Mol Biol, 2003. 325(3): p. 531-53.10. Glaser, S.M., et al., Novel antibody hinge regions for efficient production of CH2 domain-deleted antibodies. J Biol Chem, 2005. 280(50): p. 41494-503.11. Godwin, A., Bridging the Conjugation Gap. Genetic Engineering & Biotechnology News, 2013. 33(3): p. 20-21.12. Hammarstrom, S., The carcinoembryonic antigen (CEA) family: structures, suggested functions and expression in normal and malignant tissues. Semin Cancer Biol, 1999. 9(2): p. 67-81.13. Hu, S., et al., Minibody: A novel engineered anti-carcinoembryonic antigen antibody fragment (single-chain Fv-CH3) which exhibits rapid, high-level targeting of xenografts. Cancer Res, 1996. 56(13): p. 3055-61.14. Kujawski, M., et al., Potent immunomodulatory effects of an anti-CEA-IL-2 immunocytokine on tumor therapy and effects of stereotactic radiation. Oncoimmunology, 2020. 9(1 ): p. 1724052.15. Larson, S.B., et al., The Structure of an Antitumor CH2-domain-deleted Humanized Antibody. Journal of Molecular Biology, 2005. 348(5): p. 1177-1190.16. Lee, N.J., et al., A single-domain antibody library based on a stability-engineered human VH3 scaffold. Sci Rep, 2024. 14(1 ): p. 17747.17. Li, L., et al., A Versatile Bifunctional Chelate for Radiolabeling Humanized Anti-CEA Antibody with In-111 and Cu-64 at Either Thiol or Amino Groups: PET Imaging Of CEA- Positive Tumors with Whole Antibodies. Bioconjug Chem, 2008. 19(1 ): p. 89-96.18. Lwin, T.M., et al., Multimodality PET and Near-Infrared Fluorescence Intraoperative Imaging of CEA-Positive Colorectal Cancer. Mol Imaging Biol, 2023. 25(4): p. 727-734.19. Lyon, R.P., et al., Reducing hydrophobicity of homogeneous antibody-drug conjugates improves pharmacokinetics and therapeutic index. Nature Biotechnology, 2015. 33(7): p. 733-735.-19-184600812.120. Olafsen, T., et al., Covalent disulfide-linked anti-CEA diabody allows site-specific conjugation and radiolabeling for tumor targeting applications. Protein Eng Des Sei, 2004. 17(1 ): p. 21 -7.21. Rodriguez, C., et al., Antibody Engineering for Nuclear Imaging and Radioimmunotherapy. J Nucl Med, 2022. 63(9): p. 1316-1322.22. Sadiki, A., et al., Site-specific conjugation of native antibody. Antib Ther, 2020. 3(4): p. 271 -284.23. Slavin-Chiorini, D.C., et al., Biologic properties of a CH2 domain-deleted recombinant immunoglobulin. Int J Cancer, 1993. 53(1 ): p. 97-103.24. Sundaresan, G., et al., 1241-labeled engineered anti-CEA minibodies and diabodies allow high-contrast, antigen-specific small-animal PET imaging of xenografts in athymic mice. J Nucl Med, 2003. 44(12): p. 1962-9.25. Tiernan, J.P., et al., Carcinoembryonic antigen is the preferred biomarker for colorectal cancer targeting. British Journal of Cancer, 2013. 108(3): p. 662-667.26. Wang, J., et al., Protein A and CaptureSelect FcXP Affinity Resins Possess the Capability of Differentiating Hole-hole Homodimer Isoforms. Protein Pept Lett, 2023. 30(6): p. 498-505.27. Wei, Z., et al., Engineered Antibodies as Cancer Radiotheranostics. Advanced Science, 2024. n / a(n / a): p. 2402361 .28. Wong, J.Y.C., et al., Pilot Trial Evaluating an 1231-Labeled 80-Kilodalton Engineered Anticarcinoembryonic Antigen Antibody Fragment (CT84.66 Minibody) in Patients with Colorectal Cancer. Clinical Cancer Research, 2004. 10(15): p. 5014-5021.29. Wong, J.Y.C., et al., First-In-Human Pilot PET Immunoimaging Study of (64)Cu-Anti- Carcinoembryonic Antigen Monoclonal Antibody (hT84.66-M5A) in Patients with Carcinoembryonic Antigen-Producing Cancers. Cancer Biother Radiopharm, 2022.30. Wu, A.M., Engineered antibodies for molecular imaging of cancer. Methods, 2014. 65(1 ): p. 139-47.-20-184600812.131 . Wu, A.M., et al. , Tumor localization of anti-CEA single-chain Fvs: improved targeting by non-covalent dimers. Immunotechnology, 1996. 2(1 ): p. 21-36.32. Xu, T., et al., Interrogating heterogeneity of cysteine-engineered antibody-drug conjugates and antibody-oligonucleotide conjugates by capillary zone electrophoresis-mass spectrometry. MAbs, 2023. 15(1 ): p. 2229102.33. Yazaki, P.J., et al., Humanization of the anti-CEA T84.66 antibody based on crystal structure data. Protein Eng Des Sei, 2004. 17(5): p. 481-9.34. Yazaki, P.J., et al., Tumor Targeting of Radiometal Labeled Anti-CEA Recombinant T84.66 Diabody and T84.66 Minibody: Comparison to Radioiodinated Fragments. Bioconjugate Chemistry, 2001. 12(2): p. 220-228.Sequences

[0066] SEQ ID NO: 1 - Flex linker - APGGGSSGGGSG

[0067] SEQ ID NO: 2 - TAG linker - GGGSSGGGSG

[0068] SEQ ID NO: 3 - C2 MH - CPPCP

[0069] SEQ ID NO: 4 - C3 MH - CPPCPEPKSCP

[0070] SEQ ID NO: 5 - C5 MH - CPPCPEPKSCDTPPPCPRCP

[0071] SEQ ID NO: 6 - C5 UH and MH - EPKSCDKTHTCPPCPEPKSCDTPPPCPRCP

[0072] SEQ ID NO: 7 - C3 UH and MH - EPKSCDKTHTCPPCPEPKSCP

[0073] SEQ ID NO: 8 - C2 UH and MH - EPKSCDKTHTCPPCP

[0074] SEQ ID NO: 9 - VH CDR1 - DTYMH

[0075] SEQ ID NO: 10 - VH CDR2 - RIDPANGNSKYADSVKG

[0076] SEQ ID NO: 11 - VH CDR3 - FGYYVSDYAMAY

[0077] SEQ ID NO: 12 - VL CDR1 - RAGESVDIFGVGFLH

[0078] SEQ ID NO: 13 - VL CDR2 - RASNLES

[0079] SEQ ID NO: 14 - VL CDR3 - QQTNEDPYT-21-184600812.1

[0080] SEQ ID NO: 15 - Light chainDIQLTQSPSSLSASVGDRVTITCRAGESVDIFGVGFLHWYQQKPGKAPKLLIYRASNLESGVPSRFSGSGSRTDFTLTISSLQPEDFATYYCQQTNEDPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGAASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0081] SEQ ID NO: 16 - VH domainPGGSLRLSCAASGFNIKDTYMHWVRQAPGKGLEWVARIDPANGNSKYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAPFGYYVSDYAMAYWGQGTLVTVSS

[0082] SEQ ID NO: 17 - CH1 domainASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV

[0083] SEQ ID NO: 18 - CH3 domainGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0084] SEQ ID NO: 19 - M5A delCH2 C2_Flex heavy chainPGGSLRLSCAASGFNIKDTYMHWVRQAPGKGLEWVARIDPANGNSKYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAPFGYYVSDYAMAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPGGGSSGGGSGGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0085] SEQ ID NO: 20 - M5A delCH2 C5_Flex heavy chainEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYMHWVRQAPGKGLEWVARIDPANGNSKYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAPFGYYVSDYAMAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPEPKSCDTPPPCPRCPAPGGGSSGGGSGGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP-22-184600812.1SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0086] SEQ ID NO: 21 - M5A delCH2 3C heavy chainEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYMHWVRQAPGKGLEWVARIDPANGNSKYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAPFGYYVSDYAMAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPEPKSCPGGGSSGGGSGGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK

[0087] SEQ ID NO: 22 - M5A delCH2 C2_Tag heavy chainEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYMHWVRQAPGKGLEWVARIDPANGNSKYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAPFGYYVSDYAMAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPGGGSSGGGSGGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0088] SEQ ID NO: 23 - nucleotide sequence encoding light chainGACATCCAGCTGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAGAGTGACCATCACCTGCAGAGCCGGCGAGAGCGTGGACATCTTCGGCGTGGGCTTTCTGCACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACAGAGCCAGCAACCTGGAGAGCGGCGTGCCCAGCAGATTCAGCGGCAGCGGCAGCAGAACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGACCAACGAGGACCCCTACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGAGAACCGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGTCCGGCGCTGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAA-23-184600812.1GGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACAGAGGCGAGTGCTGATAA

[0089] SEQ ID NO: 24 - nucleotide sequence encoding M5A delCH2 Flex heavy chainGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGACTGAGCTGCGCCGCCAGCGGCTTCAACATCAAGGACACCTACATGCACTGGGTGAGACAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCAGAATCGACCCCGCCAACGGCAACAGCAAGTACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCGCCGACACCAGCAAGAACACCGCCTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGCGCCCCCTTCGGCTACTACGTGAGCGACTACGCCATGGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGAGCTGGAACAGCGGCGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCCGCCCCCGGCGGCGGCAGCAGCGGCGGCGGCAGCGGCGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCCAGCAGAGACGAGCTGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGCAA GTGATAA

[0090] SEQ ID NO: 25 - nucleotide sequence encoding M5A delCH2 C5_Flex heavy chainGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGACTGAGCTGCGCCGCCAGCGGCTTCAACATCAAGGACACCTACATGCACTGGGTGAGACAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCAGAATCGACCCCGCCAACGGCAACAGCAAGTACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCGCCGACACCAGCAAGAACACCGCCTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGT-24-184600812.1GTACTACTGCGCCCCCTTCGGCTACTACGTGAGCGACTACGCCATGGCCTACTGGG GCCAGGGCACCCTGGTGACCGTGAGCAGCGCCAGCACCAAGGGCCCCAGCGTGTT CCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACCGCCGCCCTGGGCTGC CTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGAGCTGGAACAGCGGCGCCCT GACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCC TGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGC AACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAG CTGCGACAAGACCCACACCTGCCCCCCCTGCCCCGAGCCCAAGAGCTGCGACACCC CCCCCCCCTGCCCCAGATGCCCCGCCCCCGGCGGCGGCAGCAGCGGCGGCGGCA GCGGCGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCCAGCAGAGACGA GCTGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCG ACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACC CCCCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGA CAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCC TGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGCAAGTGATAA

[0091] SEQ ID NO: 26 - nucleotide sequence encoding M5A delCH2 3C heavy chainGAGGTTCAACTGGTGGAATCTGGCGGCGGACTTGTTCAACCTGGCGGCTCTCTGAGA CTGTCTTGTGCCGCCAGCGGCTTCAACATCAAGGACACCTACATGCACTGGGTCCGA CAGGCCCCTGGAAAAGGACTGGAATGGGTCGCCAGAATCGACCCCGCCAACGGCAA TTCTAAGTACGCCGATAGCGTGAAGGGCAGATTCACCATCAGCGCCGACACCAGCAA GAACACCGCCTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACT ATTGTGCCCCTTTCGGCTACTACGTGTCCGACTACGCCATGGCCTATTGGGGCCAGG GCACACTGGTTACAGTGTCCTCTGCCTCTACAAAGGGCCCCTCCGTTTTTCCACTGG CTCCTAGCAGCAAGAGCACATCTGGTGGAACAGCCGCTCTGGGCTGCCTGGTCAAG GATTACTTTCCTGAGCCTGTGACCGTGTCCTGGAATAGCGGAGCACTGACAAGCGGC GTGCACACATTTCCAGCTGTGCTGCAGAGCAGCGGCCTGTACTCTCTGTCTAGCGTG GTCACAGTGCCTAGCTCTAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCAC AAGCCTAGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGAGCTGCGACAAGAC CCACACCTGTCCTCCATGTCCTGAGCCAAAGAGCTGTCCTGGCGGAGGATCTTCAGG CGGAGGAAGCGGAGGACAGCCTAGAGAACCTCAGGTGTACACACTGCCTCCAAGCA GGGACGAGCTGACCAAGAATCAGGTGTCCCTGACCTGCCTCGTGAAGGGATTCTACC-25-184600812.1CTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCAGAGAACAACTACAAGA CAACCCCTCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAAGCTGACCG TGGACAAGTCCAGATGGCAGCAGGGCAATGTGTTCAGCTGCTCTGTGATGCACGAG GCCCTGCACAACCACTACACCCAGAAGTCTCTGTCTCTGAGCCCCGGCAAGTGATAA

[0092] SEQ ID NO: 27 - nucleotide sequence encoding M5A delCH2 C2_Tag heavy chainGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGACTGAGCTGCGCCGCCAGCGGCTTCAACATCAAGGACACCTACATGCACTGGGTGAGACAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCAGAATCGACCCCGCCAACGGCAACAGCAAGTACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCGCCGACACCAGCAAGAACACCGCCTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGT GTACTACTGCGCCCCCTTCGGCTACTACGTGAGCGACTACGCCATGGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGAGCTGGAACAGCGGCGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCCGGCGGCGGCAGCAGCGGCGGCGGCAGCGGCGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCCAGCAGAGACGAGCTGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGCAAGTGATA A-26-184600812.1

Claims

CLAIMS l / We claim:1 . An antibody or antibody fragment, comprising a light chain comprising a VL; a heavy chain, wherein the CH2 domain is removed and comprising a VH domain; wherein the VL domain and VH domain bind Carcinoembryonic antigen (CEA).

2. The antibody or antibody fragment of claim 1 , wherein the heavy chain domain is derived from an lgG1 heavy chain.

3. The antibody or antibody fragment of claim 1 , wherein the heavy chain comprises a hinge sequence and a CH3 domain.

4. The antibody or antibody fragment of claim 3, wherein the hinge sequence is joined to the CH3 domain of the heavy chain by a linker comprising serine and glycine amino acids.

5. The antibody or antibody fragment of claim 4, wherein the linker comprises a SEQ ID NO: 1 or 2.

6. The antibody or antibody fragment of claim 3, wherein the hinge sequence comprises any of SEQ ID NO: 3-8.

7. The antibody or antibody fragment of claim 6, comprising a first heavy chain and a second heavy chain connected by 2, 3, or 5 disulfide bonds.

8. The antibody or antibody fragment of claim 1 , wherein the VH domain comprises CDR sequences comprising SEQ ID NO: 9-11 .

9. The antibody or antibody fragment of claim 1 , wherein the VL domain comprises CDR sequences comprising SEQ ID NO: 12-14.-27-184600812.

110. The antibody or antibody fragment of claim 1 , wherein the light chain comprises SEQ ID NO: 15.11 . The antibody or antibody fragment of claim 1 , wherein the VH domain comprises SEQ ID NO: 16.

12. The antibody or antibody fragment of claim 1 , wherein the heavy chain comprises a CH1 domain comprising SEQ ID NO: 17.

13. The antibody or antibody fragment of claim 1 , wherein the heavy chain comprises a CH3 domain comprising SEQ ID NO: 18.1 . The antibody or antibody fragment of claim 1 , wherein the heavy chain comprises any of SEQ ID NOs: 19-22.

15. The antibody or antibody fragment of claim 1 , wherein the light chain is transcribed from a nucleotide comprising SEQ ID NO: 23.

16. The antibody or antibody fragment of claim 1 , wherein the heavy chain is transcribed from a nucleotide comprising any of SEQ ID NOs: 24-27.

17. The antibody or antibody fragment of claim 1 conjugated to 1 ,4,7,10 tetraazacyclododecane-1 ,4,7-triacetic acid trisodium salt-vinyl sulfone (DO3A-VS).

18. The antibody or antibody fragment of claim 1 conjugated to a fluorescent dye.

19. A method of detecting a CEA-positive cancer in a subject comprising administering the antibody or antibody fragment of claim 1 to the subject; and measuring the antibody or antibody fragment in the subject; wherein elevated levels of the antibody or antibody fragment compared to a healthy subject indicates the presence of cancer.

20. A method of treating a CEA-positive cancer in a subject comprising: detecting the cancer by administering the antibody or antibody fragment of claim 1 to the subject; and-28-184600812.1measuring the antibody or antibody fragment in the subject; wherein elevated levels of the antibody or antibody fragment compared to a healthy subject indicates the presence of cancer; and administering a cancer therapy.21 . A method of determining efficacy of a treatment for a CEA-positive cancer comprising: treating a subject by detecting the cancer by administering the antibody or antibody fragment of claim 1 to the subject; and measuring the antibody or antibody fragment in the subject; wherein elevated levels of the antibody or antibody fragment compared to a healthy subject indicates the presence of cancer; and administering a cancer therapy; administering a second dose of the antibody or antibody fragment of claim 1 to the subject; and measuring the second dose of the antibody or antibody fragment in the subject; wherein reduced levels of the antibody or antibody fragment after treatment compared to before treatment indicates that the treatment was effective.

22. The method of claim 21 , wherein the antibody or antibody fragment is measured using PET imaging.

23. The method of any one of claims 19-22, wherein the cancer is colorectal cancer.-29-184600812.1

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