Compositions and methods for targeting cell surface markers induced by radiation therapy

Abstract

Described herein are antibodies including a first antigen binding domain which specifically binds a radiation-induced cell-surface marker. The radiolabeled version of these antibodies can be single domain antibodies such as camelid antibodies or shark New Antigen Receptor antibodies, and can also include multi-specific antibodies including one or more additional antigen binding domains. Also described are methods of determining the relative biological effect of a radiotherapy or radiopharmaceutical therapy on the tissues of a patient and methods of determining exposure of a patient to radioactivity using the radiolabeled antibodies.

Description

COMPOSITIONS AND METHODS FOR TARGETING CELL SURFACE MARKERS INDUCED BY RADIATION THERAPYCROSS-REFERENCE TO RELATED APPLICATIONSThis application claims priority to U.S. Provisional Application 63 / 626,612 filed on January 30, 2024, which is incorporated herein by reference in its entirety.FIELD OF THE DISCLOSURE

[0001] The present disclosure is related to compositions and methods for targeting of cell surface markers induced by radiation therapy for the purpose of quantitative molecular imaging, which can be used for dosimetry, or for therapeutic applications in the setting of cancer.SEQUENCE LISTING

[0002] The Instant Application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on January 17, 2025, is named “SEQ LIST— 107668309- P230293W001.xmf’ and is 17,212 bytes in size. The Sequence Listing does not go beyond the disclosure in the application as filed.BACKGROUND

[0003] Radiation therapy (RT) represents a pillar of cancer therapy, used for both curative and palliative treatments for more than half of all cancer patients. In most circumstances, RT is delivered using a linear accelerator, thus is referred to as external beam radiation therapy (EBRT). In patients with multiple sites of metastatic disease, the use of EBRT to target all sites of disease is usually not achievable due to increased side effects as a consequence of radiation dose to normal adjacent organs. Solutions to this limitation of EBRT is the use of molecularly targeted radionuclide therapy (TRT), enabling the delivery RT to multiple sites of disease, as well as the use on non-molecular agents such as Xofigo® and sodium iodide. The TRT agent selectively irradiates and damages cancer cells while limiting radiation exposure of healthy tissue. TRT agents include a radioactive atom (also known as a radionuclide) combined with a targeting molecule that specifically seeks andbinds to, in, or near cancer cells. TRT agents enable the delivery of systemic RT by targeting the delivery’ of radioisotope using a tumor-selective ligand.

[0004] The determination of radiation dose by measurement, calculation, or a combination of measurement and calculation is referred to as dosimetry'. Typically, the radiation dose is calculated as an “absorbed dose’", that is, the amount of radiation energy' that is deposited in tissue divided by the mass of the tissue. The absorbed dose, however, can be different for different patients and even for different lesions in the same patient. In order to deliver an effective dose to a tumor while minimizing potential toxicity7, personalized dosimetry' typically involves imaging the patient and calculating the predicted absorbed dose. While personalized dosimetry is an improvement over one size fits all therapy based on patient weight, for example, the predicted absorbed dose may not be the same as the delivered dose. Moreover, with TRT agents, the dose-rate of RT delivery and the intensity or density7of the deposited dose (linear energy transfer) vary7with the different emission products of different isotopes (e.g., beta, alpha, or electrons) leading to challenges in determining or comparing the biological effect of a dose from one agent with that of another agent or with that from external beam radiation. What is needed are compositions and methods for more accurately determining biological effective dose in vivo using non-invasive imaging methods that provide such determination across all tissues. The compositions can also be used in therapeutic methods.BRIEF SUMMARY

[0005] In one aspect, an antibody comprises a first antigen binding domain which specifically binds a radiation-induced cell-surface marker.

[0006] In another aspect, the antibody comprises a radiolabel, a conjugated chemotherapy agent, a conjugated immunotherapy agent, one or more additional antigen binding domains, or a combination thereof.

[0007] Also included are pharmaceutical compositions comprising the antibodies and methods of treating a cancer in a subject comprising administering to the subject a dose of the pharmaceutical composition.

[0008] In another aspect, a method of determining the relative biological effect of a radiotherapy or radiopharmaceutical therapy on the tissues of a patient comprises administering an administered dose of the radiotherapy or radiopharmaceutical therapy to the patient, followed by administering an imaging dose of an imaging radiolabeled antibody as described herein to the patient, performing quantitative serial imaging of the uptake anddistribution of the imaging radiolabeled antibody, and analyzing the quantitative serial imaging using image-based dosimetry software to provide a three-dimensional spatially resolved measure of the biological effect of the radiotherapy or radiopharmaceutical therapy on the tissues of the patient, wherein the biological effect provides clinical information about the distribution of the radiotherapy or radiopharmaceutical therapy, the absolute level of the radiotherapy or radiopharmaceutical therapy, or the relative degree of the biological effect of the radiotherapy or radiopharmaceutical therapy.

[0009] In a further aspect, a method of determining exposure of a patient to radioactivity comprises administering an imaging dose of an imaging radiolabeled antibody to the patient, wherein the patient has known or suspected exposure to radioactivity; performing quantitative serial imaging of the uptake and distribution of the imaging radiolabeled antibody; and pairing the quantitative serial imaging with image-based dosimetry to provide a three-dimensional spatially resolved measure of the biological effect of the radioactivity on the tissues of the patient, wherein the biological effect is the distribution of the radioactivity’, the absolute level of the radioactivity, or the relative degree of the biological effect of the radioactivity.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Fig. 1 shows a methodology to derive the uniform biologically effective dose (EUBED) using dosimetry and response biomarker imaging.

[0011] Fig. 2 illustrates the Radiopharmaceutical Assessment Platform for Internal Dosimetry (RAPID) mouse dosimetry’ workflow. Following acquisition of p positron emission tomography (PET) / computerized tomography (CT) or p single photon emission computed tomography (SPECT) / CT scans, imaging data at each time point is co-registered and then resampled to match the CT voxel resolution. Radiation transport simulations in RAPID are performed using the Monte Carlo (MC) code Geant4. The nuclear medicine volumes are used in the MC simulation to define the source distribution while the CT images are used to define the material composition and mass density of the simulation geometry. The decay point is sampled uniformly throughout each voxel. Absorbed dose rates in each voxel are then calculated for each time point. All simulations are performed using computational resources in the UW Center for High Throughput Computing (CHTC). Following the simulations, each region of interest (ROI) segmented on the CT w ill be co-registered to a corresponding ROI at a reference time point using normalized mutual information based affine co-registration using the Amira software. Each respective CT transformation matrixwill then be applied to each corresponding dose-rate volume. RAPID also includes a pharmacokinetic (PK) fitting module with six built-in options for fitting and integrating the absorbed dose rate over time to calculate the total absorbed dose (AD).

[0012] Fig. 3 shows cell surface translocation of calreticulin antibodies in CHLA-20 and M21 cells, as measured by flow cytometry'. **** pO.OOOl, ns: not significant (n=3).

[0013] Fig. 4 shows89Zr-labelled calreticulin antibodies being used to perform quantitative molecular imaging of a radiation-induced cell surface marker on tumor that was radiated (right flank) compared to tumor the same tumor line that was not radiated (left flank).

[0014] Fig. 5 is a schematic representation of antibody 2D9-Fc, anti-CALR VNAR- Fc (SEQ ID NO: 16).

[0015] Figs. 6A and B show a comparison of the in vitro binding of a developed shark calreticulin antibody (Shark-CALR) compared to a commercially available calreticulin antibody (Abcam-CALR) in neuroblastoma (CHLA-20) and melanoma (M21) cells. Detection was done by flow cytometry’ and various concentrations of antibody were evaluated. 6A shows mean fluorescence intensity (MFI) by flow cytometry. 6B shows the percent cell positive by flow cytometry.

[0016] The above-described and other features will be appreciated and understood by those skilled in the art from the following detailed description, drawings, and appended claims.DETAILED DESCRIPTION

[0017] Described herein are antibodies and methods of using the antibodies, wherein the antibodies comprise a first antigen binding domain which specifically binds a radiation- induced cell-surface marker. In an aspect, the antibody is a type of radiopharmaceutical, specifically a radiolabeled antibody that comprises a first antigen binding domain which specifically binds a radiation-induced cell-surface marker. The radiation-induced cell surface marker can be expressed on tumor cells or on normal cells. Upon irradiation of a cell, in addition to DNA damage, phenotypic changes are stimulated include cell-surface expression or translocation of damage-associated molecular patterns (e.g., calreticulin), death receptors (e.g., DR5, FAS), and other intracellular peptides. Such radiation-induced cell-surface markers provide novel targets for the antibodies described herein.

[0018] Advantageously, the radiolabeled antibodies described herein can be used to generate radiographic images that enable quantitative measurement of the biologicaleffectiveness of RT dose. Advantageously, the radiolabeled antibodies provide an indirect measure of radiation damage. A downside to traditional dosimetry is that that the predicted absorbed dose, actual absorbed dose, and the biological effect of the absorbed dose of RT may not be the same. For example, different radionuclides exert different biological effectiveness with the distinct forms of radiation they emit. This effect is further compounded by different tissues having different radiation sensitivities. In addition, dose heterogeneity is known to exist with any radiopharmaceutical therapy, however this is not readily measured with current methodologies and this limits radiobiological understanding of the effects of these agents while also posing a challenge for patient selection and dosing clinically. The compositions and methods described herein allow one to image the damage caused by radiation rather than simply predict the radiation dose. As described herein, the determination of the biologically effective dose will allow improved prediction of the efficacy and toxicity of radiation in tumor cells and normal tissues, respectively.

[0019] In an aspect, described herein are antibodies, e.g., radiolabeled antibodies, comprising a first antigen binding domain which specifically binds one or more radiation- induced cell-surface markers.Radiation-induced cell surface markers and antigen binding domains

[0020] Cell-surface markers include, for example, intracellular peptides that are translocated to the cell surface as an immunostimulatory response to radiation therapy. Calreticulin, for example, is a peptide that resides in the endoplasmic reticulum (ER) under normal physiological conditions. However, in response to oxidative stress including RT, calreticulin is translocated to the cell surface, making this peptide a de novo cell surface epitope after radiation therapy and an ideal prototype of a radiation-induced CAR neoantigen. Exemplary radiation-induced cell surface markers comprise calreticulin, TATA-Box Binding Protein Associated Factor 15 (TAF15), Intercellular adhesion molecule-1 (ICAM-1), E- selectin, P-selectin, Glucose-related protein 78 (GRP78), and combinations thereof.

[0021] In an aspect, the antibody can comprise, in addition to an antigen domain binding the targeted cell-surface marker, one or more additional antigen binding domains.

[0022] In an aspect, the antibody is a multi-specific antibody comprising, in addition the cell-surface marker, a tumor-specific antigen.

[0023] Non-limiting examples of tumor-specific antigens include carbonic anhydrase IX (CAIX), carcinoembryomc antigen (CEA), CD8, CD7, CD10, CDI9, CD20, CD22, CD30, CD33, CLL1 , CD34, CD38, CD41 , CD44, CD49f, CD56, CD74, CD133, CD138,CD123, CD44V6, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine-protein kinases erb-B2,3,4 (erb-B2,3,4), folate-binding protein (FBP), fetal acetylcholine receptor (AChR), adult AChR subunits, folate receptor-a, Ganglioside G2 (GD2), Ganglioside G3 (GD3), human Epidermal Growth Factor Receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), Interleukin- 13 receptor subunit alpha-2 (IL-13Ra2), K-light chain, kinase insert domain receptor (KDR), Lewis Y (LeY), LI cell adhesion molecule (LI CAM), melanoma antigen family A, 1 (MAGE-A1), Mucin 16 (MUC16), Mucin 1 (MUC1), Mesothelin (MSLN), ERBB2, MAGEA3, p53, MARTI, GP100, Proteinase3 (PR1), Tyrosinase, Survivin, hTERT, EphA2, NK.G2D ligands, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), R0R1, tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), BCMA, NKCS1, EGF1R, EGFR-vIII, CD99, CD70, ADGRE2, CCR1, LILRB2, PRAME CCR4, CD5, CD3, TRBC1, TRBC2, TIM-3, Integrin B7, ICAM-1, CD70, Tim3, CLEC 12A, ERBB, and combinations thereof.

[0024] More specifically, exemplary tumor-specific antigens include GD2, HER2, EGFR, mesothelin, Claudin-18.2, PSMA, B7-H3, IL-13Ra2, FAP, CA19, CD19, CD5, MUC1, or a combination thereof.

[0025] In another aspect, the one or more additional antigen binding domains of the multi-specific antibody includes antigen binding domains that engage T cells or antigen binding domains that engage other immune effectors.

[0026] Exemplary antigen binding domains that engage T cells include CD3, CD28, CD38, 4-1BB (CD137), and CD122, CD8, and CD4 antigen binding domains.

[0027] Exemplary antigen binding domains that engage immune effectors include CD19, CD20, CD123, CD38, BCMA, CD33, GPRC5D, WT1, FcRH5, CD14, CDl lb, CD56, CD16, CD40, CD27, CD122, NKG2D, NK1.1, CD62L, Ly6G, Ly49, NKG2A / C, CD244, CD226, CD163, CD2, CD1C, CD1A, ITGAE, ITGAM, CXCR1, CD103, BTLA, CADM1, CD8A, CLEC9A, ITGAX, Ly75, THBD, XCR1, MRC1, CD207, SIRPA.Antibodies

[0028] The term “antibody” or “antibody moiety” is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multi specific antibodies (e.g., bispecific antibodies), full-lengthantibodies and antigen-binding fragments thereof, so long as they exhibit the desired antigenbinding activity.

[0029] The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. An IgM antibody includes 5 of the basic heterotetramer units along with an additional polypeptide called a J chain, and contains 10 antigen-binding sites, while IgA antibodies comprise from 2-5 of the basic 4-chain units which can polymerize to form polyvalent assemblages in combination with the J chain. In the case of IgGs, the 4-chain unit is generally about 150,000 Daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the a and y chains and four CH domains for p and 8 isotypes. Each L chain has at the N- terminus, a variable domain (VL) followed by a constant domain at its other end. The VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (CHI). In a 4-chain antibody, the pairing of a VH and VL together forms a single antigenbinding site.

[0030] An “isolated’' antibody (or construct) is one that has been identified, separated and / or recovered from a component of its production environment (e.g., natural or recombinant). Preferably, the isolated polypeptide is free of association with all other components from its production environment. In aspects, the polypeptide will be purified to greater than 95% or even greater than 99 wt% by weight of antibody.

[0031] The term “single-domain antibody'’ or “sdAb’" refers to a single antigenbinding polypeptide having three complementary determining regions (CDRs). The sdAb alone is capable of binding to the antigen without pairing with a corresponding CDR- containing polypeptide. A typical sdAb is a VH domain of a heavy chain-only antibody. In an aspect, the radiolabeled antibody described herein is a single-domain antibody (sdAb).

[0032] The term “heavy chain-only antibody” or “HCAb” refers to a functional antibody, which comprises heavy chains, but lacks the light chains usually found in 4-chain antibodies. Camelid animals (such as camels, llamas, or alpacas) produce HCAbs. Heavychain only antibodies from the Camelid species have a single heavy chain variable region, which is referred to as “VHH”. The variability is concentrated in three segments called complementary determining regions (CDRs) or hypervariable regions (HVRs) both in thelight-chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FR).

[0033] The term '‘hypervariable region,’’ “HVR,” or “HV,” refers to the regions of an antibody variable domain which are hypervariable in sequence and / or form structurally defined loops. Generally , single-domain antibodies comprise three HVRs (or CDRs): HVR1 (or CDR1), HVR2 (or CDR2), and HVR3 (or CDR3).

[0034] The term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and / or post-translation modifications (e.g., isomerizations, amidations) that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins.

[0035] The terms “full-length antibody”, “intact antibody”, or “whole antibody” are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antibody fragment. An “antibody fragment” comprises a portion of an intact antibody, preferably the antigen binding and / or the variable region of the intact antibody. “Functional fragments” of the antibodies described herein comprise a portion of an intact antibody, generally including the antigen binding or variable region of the intact antibody or the Fc region of an antibody which retains or has modified FcR binding capability. Examples of antibody fragments include, but are not limited to Fab, Fab', F(ab')2, Fc and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; single-domain antibodies (such as VHH), and multi-specific antibodies formed from antibody fragments. The Fc fragment comprises the carboxy -terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region, the region which is also recognized by Fc receptors (FcR) found on certain types of cells. “Fv” is the minimum antibody fragment which contains a complete antigen-recognition and -binding site.

[0036] “Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain.Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding.

[0037] The term “diabodies” refers to small antibody fragments prepared by constructing sFv fragments (see preceding paragraph) with short linkers (about 5-10 residues) between the VH and VL domains such that inter-chain but not intra-chain pairing of the V domains is achieved, thereby resulting in a bivalent fragment, i.e., a fragment having two antigen-binding sites. Bispecific diabodies are heterodimers of two “crossover’ sFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains.

[0038] Antibodies include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is(are) identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity. “Humanized antibody” is used as a subset of “chimeric antibodies”

[0039] “Humanized” forms of non-human (e.g., llama or camelid) antibodies are antibodies that contain minimal sequence derived from non-human immunoglobulin. In some embodiments, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from an CDR (hereinafter defined) of the recipient are replaced by residues from an CDR of a non-human species (donor antibody) such as mouse, rat, rabbit, camel, llama, alpaca, or non-human primate having the desired specificity, affinity , and / or capacity. In some instances, framework (“FR”) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody.

[0040] A “human antibody” is an antibody that possesses an amino-acid sequence corresponding to that of an antibody produced by a human and / or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically7excludes a humanized antibody comprising non-human antigen-binding residues.

[0041] A “human consensus framework” or “acceptor human framew ork” is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framew ork sequences.

[0042] An "affinity-matured" antibody is one with one or more alterations in one or more CDRs thereof that result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody that does not possess those alteration(s). In some embodiments, an affinity -matured antibody has nanomolar or even picomolar affinities for the target antigen.

[0043] As used herein, the term "‘specifically binds,” “specifically recognizes,” or is “specific for” refers to measurable and reproducible interactions such as binding between a target and an antigen binding protein (such as a sdAb), which is determinative of the presence of the target in the presence of a heterogeneous population of molecules including biological molecules. For example, an antigen binding protein (such as a sdAb) that specifically binds a target (which can be an epitope) is an antigen binding protein (such as a sdAb) that binds this target with greater affinity, avidity, more readily, and / or with greater duration than it binds other targets. In some embodiments, the extent of binding of an antigen binding protein (such as a sdAb) to an unrelated target is less than about 10% of the binding of the antigen binding protein (such as sdAb) to the target as measured, e.g., by a radioimmunoassay (RIA). In some embodiments, an antigen binding protein (such as a sdAb) that specifically binds a target has a dissociation constant (Ka) of <105M, <10-6M, <10-7M, <10"8M, <109M, <1(T10M, <101 1M, or <10"12M. The dissociation constant can be determined by the Scatchard method using antibodies marked with a variety of marker agents, as well as by using Biacore®X (made by Amersham Biosciences), which is an over-the-counter, measuring kit, or similar kit, according to the user’s manual and experiment operation method attached with the kit. The KD value that can be derived using these methods is expressed in units of M (Mols). In some embodiments, an antigen binding protein specifically binds an epitope on a protein that is conserved among the protein from different species. In some embodiments, specific binding can include, but does not require exclusive binding.

[0044] The term “specificity” refers to selective recognition of an antigen binding protein (such as a sdAb or antigen binding domain) for a particular epitope of an antigen. Natural antibodies, for example, are monospecific, that is, having one or more binding sites each of which bind the same epitope of the same antigen. The term “multispecific” as used herein denotes that an antigen binding protein has poly epitopic specificity7(i.e., is capable of specifically binding to two, three, or more, different epitopes on one biological molecule or is capable of specifically binding to epitopes on two, three, or more, different biological molecules).

[0045] Half maximal inhibitory' concentration (IC50) is a measure of the effectiveness of a substance (such as an antibody) in inhibiting a specific biological or biochemical function. It indicates how much of a particular drug or other substance (inhibitor, such as an antibody) is needed to inhibit a given biological by half. The values are typically expressed as molar concentration. IC50 is comparable to an EC50, the plasma concentration required for obtaining 50% of a maximum effect in vivo. As used herein, an “IC50" is used to indicate the effective concentration of an antibody (such as an anti-MET sdAb) needed to neutralize 50% of the antigen bioactivity' in vitro. IC50 or EC 50 can be measured by bioassays such as inhibition of ligand binding by FACS analysis (competition binding assay), cell based cytokine release assay, or amplified luminescent proximity homogeneous assay (AlphaLISA®).

[0046] In an aspect, the antibodies described herein are single-domain antibodies. Exemplary sdAbs include, but are not limited to, heavy chain variable domains from heavy- chain only antibodies (e.g., VHH (variable domain of the heavy chain of the heavy chain antibody) in Camelidae or VNAR (Variable domain of the shark New Antigen Receptor) in cartilaginous fish), binding molecules naturally devoid of light chains, single domains (such as VH or VL) derived from conventional 4-chain antibodies, humanized heavy-chain only- antibodies, human single-domain antibodies produced by transgenic mice or rats expressing human heavy chain segments, and engineered domains and single domain scaffolds other than those derived from antibodies. The sdAbs may be derived from any species including, but not limited to mouse, rat, human, camel, llama, lamprey, fish, shark, goat, rabbit, and bovine. Single-domain antibodies contemplated herein also include naturally occurring single-domain antibody- molecules from species other than Camelidae and sharks.

[0047] In some embodiments, the sdAb is derived from a naturally occurring singledomain antigen binding molecule known as heavy chain antibody devoid of light chains (also referred herein as “heavy chain-only antibodies”, or “HCAb”). For clarity reasons, the variable domain derived from a heavy chain molecule naturally devoid of light chain is known herein as a VHH to distinguish it from the conventional VH of four chain immunoglobulins. Such a VHH molecule can be derived from antibodies raised in Camelidae species, for example, camel, llama, vicuna, dromedary, alpaca and guanaco. Other species besides Camelidae may produce heavy chain molecules naturally devoid of light chain, and such VHHs are within the scope of the present application.

[0048] In some embodiments, the sdAb is derived from a variable region of the immunoglobulin found in cartilaginous fish. For example, the sdAb can be derived from theimmunoglobulin isotype known as Novel Antigen Receptor (NAR) found in the serum of shark. In some embodiments, the sdAb is recombinant, CDR-grafted, humanized, camelized, de-immunized and / or in vitro generated (e.g., selected by phage display). In some embodiments, the amino acid sequence of the framework regions may be altered by “camelization’" of specific amino acid residues in the framework regions. Camelization refers to the replacement or substitution of one or more amino acid residues in the amino acid sequence of a (naturally occurring) VH domain from a conventional 4-chain antibody by one or more of the amino acid residues that occur at the corresponding position(s) in a VHH domain of a heavy chain antibody. This can be performed in a manner known in the art.

[0049] In some embodiments, the sdAb is a human sdAb produced by transgenic mice or rats expressing human heavy chain segments.

[0050] In some embodiments, naturally occurring VHH domains against a particular antigen or target, can be obtained from (naive or immune) libraries of Camelid VHH sequences. Such methods may or may not involve screening such a library using said antigen or target, or at least one part, fragment, antigenic determinant or epitope thereof using one or more screening techniques. In some embodiments, the sdAbs are generated from conventional four-chain antibodies.

[0051] In some embodiments, the antibody provided herein is a human antibody. Human antibodies can be produced using various techniques known in the art. Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Human antibodies can also be made by hybridoma-based methods. Human antibodies may also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries.

[0052] Antibodies may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities.

[0053] In some embodiments, the radiolabeled antibody described herein may comprise at least a first and a second antigen binding domain. The first and second antigen binding domain can be of any antibody or antibody fragment format, such as a multi-specific sdAb, a full-length antibody, a Fab, a Fab', a (Fab')2, an Fv, a single chain Fv (scFv), an scFv-scFv, a minibody, a diabody, or a sdAb. Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g. E. coli or phage), as described herein. In some embodiments, the first and second antigen binding domain are antibody mimetics, which aresmall engineered proteins comprising antigen-binding domains reminiscent of antibodies. These molecules are derived from existing human scaffold proteins and comprise a single polypeptide. Exemplary antibody mimetics that can be comprised within antibodies described herein can be, but are not limited to, a designed ankyrin repeat protein (DARPin; comprising 3-5 fully synthetic ankyrin repeats flanked by N- and C-terminal Cap domains), an avidi ty multimer (avimer; a high-affinity protein comprising multiple A domains, each domain with low affinity for a target), or an Anticalin (based on the scaffold of lipocalins, with four accessible loops, the sequence of each can be randomized).

[0054] Techniques for making multi-specific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities, and “knob-in-hole” engineering. Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules; cross-linking two or more antibodies or fragments; using leucine zippers to produce bi-specific antibodies; using “diabody” technology for making bispecific antibody fragments; and using single-chain Fv (sFv) dimers; and creating polypeptides comprising tandem single-domain antibodies. Engineered antibodies with three or more functional antigen binding sites, including “Octopus antibodies,” are also included herein.

[0055] In some embodiments, the first and second antigen binding domain of a multispecific antibody can be optionally connected by a peptide linker. The length, the degree of flexibility and / or other properties of the peptide linker(s) may have some influence on properties, including but not limited to the affinity, specificity’ or avidity for one or more particular antigens or epitopes. For example, longer peptide linkers may be selected to ensure that two adjacent domains do not sterically interfere with one another. In some embodiments, a peptide linker comprises flexible residues (such as glycine and serine) so that the adjacent domains are free to move relative to each other. For example, a glycine-serine doublet can be a suitable peptide linker.

[0056] The peptide linker can be of any suitable length. In some embodiments, the peptide linker is at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100 or more amino acids long. In some embodiments, the peptide linker is no more than about any of 100, 75, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or fewer amino acids long.

[0057] The peptide linker may have a naturally occurring sequence, or a non-naturally occurring sequence. Exemplary linkers include a sequence derived from the hinge region of heavy chain only antibodies, a mutated human IgGl hinge, or a flexible linker. Exemplaryflexible linkers include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS (SEQ ID NO: l))n, (GGGS (SEQ ID NO: 2))n, and (GGGGS (SEQ ID NO: 3))n, where n is an integer of at least one, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more, and / or, optionally, up to 10, 15, 20, 25, 30, 35, or more), glycine-alanine polymers, alanine-serine poly mers, and other flexible linkers known in the art. Exemplar}' peptide linkers can include peptide sequences such as GGGGSGGGS (SEQ ID NO: 4), GGGGSGGGGSGGGGS (SEQ ID NO: 5), GPGGP (SEQ ID NO: 6), AALVGPGGQGGGGSGGGGSGGGGSGGGGSGGGGSMA (SEQ ID NO: 7), EPKSSDKTHTSPPSP (SEQ ID NO: 8), and GPGGQGTGPGGS (SEQ ID NO: 9).

[0058] In some embodiments, the antibodies described herein may be further modified to contain additional non-proteinaceous moieties that are known in the art and readily available. The moieties suitable for derivatization of the antibody include but are not limited to water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol / propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3- dioxolane, poly-1, 3, 6-trioxane, ethylene / maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)poly ethylene glycol, propropylene glycol homopolymers, prolypropylene oxide / ethylene oxide copolymers, polyoxy ethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer are attached, they can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.Cell surface marker binding peptides

[0059] As an alternative to the antibodies described herein, a peptide that binds a radiation-induced cell surface marker can be used in the methods described herein. An example is the CALR peptide ofNCBI Reference Sequence: NP_004334.1 (SEQ ID NO: 10; mllsvplllg llglavaepa vyfkeqfldg dgwtsrwies khksdfgkfv Issgkfygde ekdkglqtsq darfyalsas fepfsnkgqt Ivvqftvkhe qnidcgggyv klfpnsldqt dmhgdseyni mfgpdicgpg tkkvhvifnykgknvlinkd irckddefth lytlivrpdn tyevkidnsq vesgsleddw dflppkkikd pdaskpedwd erakiddptd skpedwdkpe hipdpdakkp edwdeemdge weppviqnpe ykgewkprqi dnpdykgtwi hpeidnpeys pdpsiyaydn fgvlgldlwq vksgtifdnf litndeayae efgnetwgvt kaaekqmkdk qdeeqrlkee eedkkrkeee eaedkedded kdedeedeed keedeeedvp gqakdel.

[0060] In a specific aspect, a shark anti-calreticulin antibody designated 2D9-Fc, anti- CALR VNAR-Fc compnses VNAR, CDR1 (DSNCALSS; SEQ ID NO: 11), HVR2 (KSGSTNEERISKG; SEQ ID NO: 12), HVR4 (NSGSKS; SEQ ID NO: 13); CDR3 (WLVSCYGAGSWLDV; SEQ ID NO: 14), and human IgGl CH2+CH3 (GPGGPEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK; SEQ ID NO: 15). In an aspect, the shark anti-calreticulin antibody comprises METDTLLLWVLLLXAAQPAMAARVDQTPQTITKETGESLTINCVLRDSNCALSSTY WYRKKSGSTNEERISKGGRYVETVNSGSKSFSLRINDLTVEDSGTYRCKVWLVSCYG AGSWLDVYGDGTAVTVNGPGGPEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 16, with signal peptide) or ARVDQTPQTITKETGESLTINCVLRDSNCALSSTYWYRKKSGSTNEERISKGGRYVET VNSGSKSFSLRINDLTVEDSGTYRCKVWLVSCYGAGSWLDVYGDGTAVTVNGPGGP EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK (SEQ ID NO: 17, without signal peptide).Radionuclides and conjugation to antibody or peptide

[0061] In an aspect the antibody described herein is a radiolabeled antibody including a radionuclide. Exemplary radionuclides include89Zr,86 / 90Y,niIn,177Lu,225Ac,211At,203 / 212Pb, 124 / 131J, 153Sm134^ 64 / 67^ 66'68^ 18 / 19^ 52^ 44^gadolmlum? or anOther isotope that can be used for imaging or therapy. The radionuclides can emit beta, alpha orAuger electrons. Many of the foregoing radionuclides can be produced using a cyclotron or a linear accelerator.68Ga for example can be produced in a68Ge —68Ga generator.

[0062] In cyclotron production, briefly, a target is irradiated to produce the desired radionuclide which is then purified from the target.

[0063] The radionuclide is then chelated to the antibody using a chelator such as diethylenetriaminepentaacetic acid (DTP A), trans-(S,S)-cyclohexane-DTPA (CHX-A"- DTPA), desferri oxamine (DFO), l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid (DOTA), l,4,7-triazacyclononane-N,N',N"-triacetic acid (NOTA), and 1,4,8,11- tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA). Antibodies can be conjugated to the chelator using N-hydroxysuccinimide (NHS) or isothiocyanate (SCN) chemistry, which react with the e-amino group of lysines on the antibody. Thiol -labeling strategies such as malemide chemistry can also be used to conjugate chelators to solvent-exposed cysteines. Click chemistry can also be employed by engineering strategies such as introducing cysteine residues or unnatural amino acids.Additional conjugated agents

[0064] In addition to, or as an alternative to a radionuclide, the antibodies described herein can be conjugated to a chemotherapy agent or can be genetically fused or otherwise linked to an immunotherapy agent.

[0065] Exemplary chemotherapy agents include alkylating agents such as chlorambucil, cyclophosphamide (Cytoxan®), ifosfamide, and melphalan; Nitrosoureas such as streptozocin, carmustine (BCNU), and lomustine; alkyl sulfonates such as busulfan; triazinessuch as dacarbazine (DTIC) and temozolomide (Temodar®); and ethylenimines: thiotepa and altretamine (hexamethylmelamine); antimetabolites such as -fluorouracil (5-FU), 6-mercaptopurine (6-MP), Capecitabine (Xeloda®), Cytarabine (Ara-C®), Floxuridine, Fludarabine, Gemcitabine (Gemzar®), Hydroxyurea, Methotrexate and Pemetrexed (Alimta®); anthracy clines such as Daunorubicin, Doxorubicin (Adriamycin®), Epirubicin and Idarubicin; anti-tumor antibiotics such as Actinomycin-D, Bleomycin, Mitomycin-C, and Mitoxantrone; topoisomerase inhibitors such as Topotecan and Irinotecan (CPT-11). Topoisomerase II inhibitors include Etoposide (VP-16), Temposide and Mitoxantrone; Mitotic inhibitors such as taxanes: paclitaxel (Taxol®) and docetaxel (Taxotere®); Epothilones: ixabepilone (Ixempra®); Vinca alkaloids: vinblastine (Velban®), vincristine (Oncovin®), and vinorelbine (Navel bine®); and Estramustine (Emcyt®); and the like.

[0066] Exemplary immunotherapy agents include antibodies targeting immune checkpoint ligands or receptors such as PD-1 , PD-L1, CTLA-4, LAG-3, TIGIT, TIM-3.Other agents may include antibodies binding to specific immune cell lineages by lineagespecific markers such asCD19, CD3, NK1.1, CD56. Additional immunotherapy agents include cytokines, chemokines, or receptor traps that bind to these. These include IL2, IL12, IL15, IL21, CXCR3-10, TNF-alpha, TGF-beta, or receptors or binding traps for these. Additional immunotherapy agents include toll-like receptor agonists such as CpG, monophosphoryl lipid A or synthetic derivatives of this like PHAD, 3D-PHAD, 3D-6Acyl- PHAD.Methods of treatment

[0067] In an aspect, a method of treating a cancer in a subject comprises administering to the subject a dose of an antibody as described herein.

[0068] In an aspect, the subject is a mammalian subject, specifically a human or canine subject.

[0069] In an aspect, the compositions and methods described herein are particularly useful to treat cancers such as breast cancer, neuroblastoma, melanoma, sarcoma, neuroendocrine cancer, colorectal cancer, lung cancer, head and neck cancer, prostate cancer, pancreatic cancer, ovarian cancer, glioblastoma, lymphoma, diffuse midline glioma, or a combination thereof.Pharmaceutical Compositions

[0070] Also included herein are pharmaceutical compositions comprising the radiolabeled antibodies and a pharmaceutically acceptable carrier.

[0071] The term, “pharmaceutically -acceptable carrier’" includes any and all drypowder, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, absorption delaying agents, and the like. Pharmaceutically -acceptable carriers are materials, useful for the purpose of administering the radiolabeled antibodies, which are preferably non-toxic, and may be solid, liquid, or gaseous materials, which are otherwise inert and pharmaceutically acceptable, and are compatible with the compounds described herein. Examples of such carriers include, without limitation, various lactose, mannitol, oils such as com oil, buffers such as PBS, saline, polyethylene glycol, glycerin, polypropylene glycol, dimethylsulfoxide, an amide such as dimethylacetamide, a protein such as albumin, and a detergent such as Tween 80, mono- and oligopolysaccharides such as glucose, lactose, cyclodextrins and starch.

[0072] The term “administering"’ or “administration,’" as used herein, refers to providing the compound or pharmaceutical composition of the invention to a subject suffering from or at risk of the diseases or conditions to be treated or prevented.

[0073] A route of administration in pharmacology is the path by which a drug is taken into the body. Routes of administration may be generally classified by the location at which the substance is applied. Common examples may include oral and intravenous administration. Routes can also be classified based on where the target of action is. Action may be topical (local), enteral (system-wide effect, but delivered through the gastrointestinal tract), or parenteral (systemic action, but delivered by routes other than the GI tract), via lung by inhalation. One form of local administration is intratumoral (IT), whereby an agent is injected directly into, or adjacent to, a known tumor site.

[0074] A topical administration emphasizes local effect, and substance is applied directly where its action is desired. Sometimes, however, the term topical may be defined as applied to a localized area of the body or to the surface of a body part, without necessarily involving target effect of the substance, making the classification rather a variant of the classification based on application location. In an enteral administration, the desired effect is systemic (non-local), substance is given via the digestive tract. In a parenteral administration, the desired effect is systemic, and substance is given by routes other than the digestive tract.

[0075] Examples of parenteral administrations may include intravenous (into a vein), e.g. many drugs, total parenteral nutrition intra-arterial (into an artery), e.g., vasodilator drugs in the treatment of vasospasm and thrombolytic drugs for treatment of embolism, intraosseous infusion (into the bone marrow), intra-muscular, intracerebral (into the brain parenchyma), intracerebroventricular (into cerebral ventricular system), intrathecal (an injection into the spinal canal), and subcutaneous (under the skin). Among them, intraosseous infusion is, in effect, an indirect intravenous access because the bone marrow drains directly into the venous system. Intraosseous infusion may be occasionally used for drugs and fluids in emergency medicine and pediatrics when intravenous access is difficult.Methods of use of the radiolabeled antibodies

[0076] The radiolabeled antibodies described herein are particularly useful in theranostic approaches, wherein the radiolabeled antibody is administered to a patient, followed by imaging the expression of the radiolabeled antibody in the tissues of the patient. Visualization of the radiolabeled antibody in the tumor and normal cells of the patient can be used to determine the efficacy of treatment, such as radiotherapy or radiopharmaceuticaltherapy. Based on the results of the imaging, the biological effective dose or the equivalent uniform biological dose of the radiolabeled antibody can be determined and used as a guide to determine a second dose of the radiotherapy or radiopharmaceutical therapy, which may be higher or lower than the first dose depending upon the expression of the radiolabeled antibody in the patient’s tissues. Visualization of the radiolabeled antibody in the tumor and normal cells of the patient can also be used to determine the extent of radiation damage in a patient suspected of exposure to radioactivity.

[0077] In an aspect, a method of determining the relative biological effect of a radiotherapy or radiopharmaceutical therapy on the tissues of a patient comprises administering an administered dose of the radiotherapy or radiopharmaceutical therapy to the patient, followed by administering an imaging dose of an imaging radiolabeled antibody as described herein to the patient, performing quantitative serial imaging of the uptake and distribution of the imaging radiolabeled antibody, and analyzing the quantitative serial imaging using image-based dosimetry software to provide a three-dimensional spatially resolved measure of the biological effect of the radiotherapy or radiopharmaceutical therapy on the tissues of the patient, wherein the biological effect provides clinical information about the distribution of the radiotherapy or radiopharmaceutical therapy, the absolute level of the radiotherapy or radiopharmaceutical therapy, or the relative degree of the biological effect of the radiotherapy or radiopharmaceutical therapy.

[0078] As used herein, radiotherapy refers to radiation therapy in which high doses of radiation are used to kill cancer cells, typically by external beam radiation or internal radiation therapy. Radiopharmaceutical therapy, in contrast, is targeted delivery of radiation, such as radionuclides conjugated to tumor-targeting agents (e.g., nanoparticles, antibodies, peptides, and small molecules), or radionuclides that concentrate in tumors by natural physiological mechanisms such as the use of radioiodine to treat thyroid tumors. In an aspect, the radiopharmaceutical therapy comprises the radiolabeled antibody as described herein, a TRT agent such as NM600, Pluvicto® (lutetium Lu 177 vipovitide tetraxetan), Lutathera® (lutetium Lu 177 dotatate), Dotatate (gallium Ga 68 dotate), Xofigo® (radium Ra 223 dichloride), or others.

[0079] In an aspect, the quantitative serial imaging comprises positron emission tomography (PET), single photon emission computed tomography (SPECT), computerized tomography (CT), or magnetic resonance imaging (MRI).

[0080] In an aspect, the image-based dosimetry comprises the 510 k approved voxelbased dosimetry7software Torch® Dose Assessment for Radiopharmaceutical Therapy (RPT) marketed by Voximetry, or the like.

[0081] The resulting relative biological effect, for example, is similar to biologically effective dose or equivalent uniform biological dose calculations but with the use of actual spatially -resolved measurement of biological effects of radiation. Advantageously, this metric will account for differences in the degree of damage caused by a given dose or type of radiation. A unique feature is that this approach can be used when treatment involves a mixture of high and low linear-energy transfer forms of radiation (e.g., alpha particles), where conventional approaches to dosimetry do not account for biological effect or the variation of this across tissues.

[0082] In an aspect, the method further comprises administering an adjusted dose of the administered radiotherapy or radiopharmaceutical therapy, wherein the adjusted dose is based on the biological effect of the administered dose of the radiotherapy or radiopharmaceutical therapy on the tissues of the patient. Thus, the method may be used to determine the dosing of the administered radiotherapy or radiopharmaceutical therapy.

[0083] In another aspect, the method further comprises administering a third dose of a third radiotherapy or radiopharmaceutical therapy, wherein the third radiotherapy or radiopharmaceutical therapy and dose are selected based on the biological effect of the administered dose of the radiotherapy or radiopharmaceutical therapy on the tissues of the patient, and wherein the third radiotherapy or radiopharmaceutical therapy is different from the administered radiotherapy or radiopharmaceutical therapy. The method may be used to determine that the administered radiotherapy or radiopharmaceutical therapy should be changed to an alternative, arbitrarily referred to as a third, administered radiotherapy or radiopharmaceutical therapy.

[0084] Advantageously, the biological effects of radiotherapy or radiopharmaceutical therapy may be used to guide and personalize the prescription of subsequent doses or cycles of that radiotherapy or radiopharmaceutical therapy or an alternative therapy.

[0085] In an aspect, the radiotherapy or radiopharmaceutical therapy comprises an administered dose of an administered radiolabeled antibody as described herein, wherein the administered radiolabeled antibody and the imaging radiolabeled antibody are the same ordifferent. The quantitative serial imaging of the imaging radiolabeled antibody combined with the image-based dosimetry and biological image-based dosimetry provides the distribution and absolute level or relative degree of the biological effect of the administered radiolabeled antibody in the tissues of the patient. In an aspect, the method can then further comprise administering an adjusted dose of the administered radiolabeled antibodyduring the next cycle of therapy, wherein the adjusted dose is based on the biological effect of the administered dose of the radiotherapy or radiopharmaceutical therapy on the tissues of the patient. For example, if the biological effect is excessive tissue damage, the adjusted dose may be smaller than the administered dose. If the biological effect is insufficient tissue damage, the adjusted dose may be larger than the administered dose. In addition, the method may be used to determine a different radiolabeled antibodyto be administered to the patient.

[0086] Advantageously, the biological effects of radiation therapy may be used to guide and personalize the prescription of subsequent doses or cycles of that radiation therapy or an alternative therapy.

[0087] In the foregoing methods, the administered dose of an administered radiotherapy or radiopharmaceutical therapy (e.g., radiolabeled antibody), the imaging dose and the adjusted dose are determined as activities. In an aspect, the activity of the radiolabeled antibody is 1 pCi to 500 mCi, specifically 100 pCi to 200 mCi. The activity to be administered is dependent on the agent to be used, and can be optimized by the administering physician.

[0088] In another aspect, a method of determining the biological effect of an administered radiotherapy or radiopharmaceutical therapy on the tissues of a patient comprises administering a first imaging dose of the administered radiotherapy or radiopharmaceutical therapy to the patient, performing imaging-based dosimetry and determining a first administered dose of the radiotherapy or radiopharmaceutical therapy, administering the first administered dose of the same or different administered radiolabeled antibody, administering an imaging dose of an imaging radiolabeled antibody as described herein to the patient, performing quantitative serial imaging of the uptake and distribution of the imaging radiolabeled antibody.pairing the quantitative serial imaging with image-based dosimetry to provide a three- dimensional spatially resolved measure of the biological effect of the first administered dose of the radiotherapy or radiopharmaceutical therapy on the tissues of the patient, wherein the biological effect is the distribution of the administered radiolabeled antibody, the absolute level of the administered radiolabeled antibody, or the relative degree of the biological effect of the administered radiolabeled antibody, and based on the biological effect of the first administered dose of the radiotherapy or radiopharmaceutical therapy, administering a second administered dose of the radiotherapy or radiopharmaceutical therapy.

[0089] The method can thus be used to determine subsequent doses of a radiotherapy or radiopharmaceutical therapy for subsequent dosing cycles. The same or a different imaging radiolabeled antibody can be subsequently administered, and the quantitative serial imaging and image-based dosimetry repeated to determine ding for subsequent treatment cycles.

[0090] In another aspect, the radiolabeled antibodies described herein can be used to determine the exposure of a patient to radioactivity.

[0091] In an aspect, a method of determining exposure of a patient to radioactivity, comprises administering an imaging dose of an imaging radiolabeled antibody as described herein to the patient, wherein the patient has known or suspected exposure to radioactivity, performing quantitative serial imaging of the uptake and distribution of the imaging radiolabeled antibody, and pairing the quantitative serial imaging with image-based dosimetry to provide a three- dimensional spatially resolved measure of the biological effect of the radioactivity on the tissues of the patient, wherein the biological effect is the distribution of the radioactivity, the absolute level of the radioactivity, or the relative degree of the biological effect of the radioactivity.

[0092] In this method, the patient has known or suspected exposure to radioactivity’, such as from warfare or an industrial accident. The method does not require identification of the type of radioactivity the patient was exposed to or suspected of exposure to.

[0093] Advantageously, this method can account for differences in the degree of damage caused by a given dose or radiation. A unique feature is that this approach can be used when radiation exposure involves a mixture of high and low linear-energy’ transferforms of radiation (e g. alpha particles), where conventional approaches to dosimetry do not account for biological effect or the variation of this across tissues.

[0094] In an aspect, upon determining the patient has been exposed to potentially lethal levels of radiation to a given tissue, that patient can be afforded early intervention with potentially life-saving or function preserving treatments for the specific tissue that has been exposed (e.g., administering filgrastim, sargramostim, pegfilfrastim, cytokine therapy, or allogeneic hematopoietic cell transplantation for excessive bone marrow dose).

[0095] The invention is further illustrated by the following non-limiting examples.Example: Dosimetry approach using radiolabeled antibody which binds a radiation-induced cell-surface marker

[0096] The initial approach will rely on two image datasets that measure dose rate and treatment response (see Fig. 1). Dose rate distributions (Df(t)) can be predicted as outlined in Fig. 2. Tumor response distributions ( / r(t)) can be measured using imaging biomarkers (PET, immunohistochemistry, immunofluorescence).

[0097] 89Zr-labeled calreticulin antibodies have been produced and dose response has been measured in in irradiated mouse tumors (Figs. 3 and 4). In addition to calreticulin, other response radiation-induced cell-surface markers may be used. By measuringand 7. (t) a modified Lea-Catcheside parameter can be derived and the biological effective dose (BED) at the voxel level can be calculated. The normalized differential BEDVH (p(< ))canbe derived in the tumor (or normal tissues) and the equivalent uniform biological effective dose (EUBED) can be derived by taking the Laplacian ofWithout being held to theory, it is believed that D,(f) and / (canbe fitted with exponentials, but if not the case then other numerical methods will be employed. The EUBED is a useful dosimetry metric to account for variable dose rates and dose heterogeneity in tumors and can be used to compare outcomes.

[0098] A shark calreticulin antibody 2D9-Fc, anti-CALR VNAR-Fc was developed. The sequence is illustrated in Fig. 5. Size-exclusion chromatography shows a single peak at 76.8 kDa (data not shown). The dissociation constants for calreticulin binding are shown in Table 1:Table 1: Calreticulin dissociation constants for 2D9-Fc, anti-CALR VNAR-Fc

[0099] Figs. 6A and B show a comparison of the in vitro binding of 2D9-Fc, anti- CALR VNAR-Fc compared to a commercially available calreticulin antibody (Abcam- CALR) in neuroblastoma (CHLA-20) and melanoma (M21) cells. Detection was done by flow cytometry and various concentrations of antibody were evaluated. 6A shows mean fluorescence intensity (MFI) by flow cytometry. 6B shows the percent cell positive by flow cytometry.

[0100] The use of the terms "‘a” and “an” and “the” and similar referents (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms first, second etc. as used herein are not meant to denote any particular ordering, but simply for convenience to denote a plurality of, for example, layers. The terms ‘"comprising”, “having”, “including”, and “containing” are to be construed as open-ended terms (i.e. , meaning “including, but not limited to”) unless otherwise noted. Recitation of ranges of values are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. The endpoints of all ranges are included within the range and independently combinable. All methods described herein can be performed in a suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”), is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention as used herein.

[0101] While the invention has been described with reference to an exemplary embodiment, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from thescope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims. Any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

Claims1. A method of determining the relative biological effect of a radiotherapy or radiopharmaceutical therapy on the tissues of a patient, comprising administering an administered dose of the radiotherapy or radiopharmaceutical therapy to the patient, followed by administering an imaging dose of an imaging radiolabeled antibody to the patient, wherein the imaging radiolabeled antibody comprises a first antigen binding domain which specifically binds a radiation-induced cell-surface marker and a radiolabel, performing quantitative serial imaging of the uptake and distribution of the imaging radiolabeled antibody, and analyzing the quantitative serial imaging using image-based dosimetry software to provide a three-dimensional spatially resolved measure of the biological effect of the radiotherapy or radiopharmaceutical therapy on the tissues of the patient, wherein the biological effect provides clinical information about the distribution of the radiotherapy or radiopharmaceutical therapy, the absolute level of the radiotherapy or radiopharmaceutical therapy, or the relative degree of the biological effect of the radiotherapy or radiopharmaceutical therapy.

2. The method of claim 1, wherein the radiation-induced cell-surface marker is calreticulin, TATA-Box Binding Protein Associated Factor 15 (TAF15), Intercellular adhesion molecule-1 (ICAM-1), E-selectin, P-selectin, Glucose-related protein 78 (GRP78), or a combinations thereof.

3. The method of claim 1, wherein the antibody is a single-domain antibody (sdAb).

4. The method of claim 3, wherein the antibody is a camelid antibody, a shark New Antigen Receptor antibody, a chimeric antibody, a partially humanized antibody, or a fully humanized antibody.

5. The method of claim 1, wherein the antibody is a heavy-chain only antibody, such as the variable domain of the heavy chain of the heavy chain antibody in Camelidae or the variable domain of the shark New Antigen Receptor in cartilaginous fish.

6. The method of claim 1, wherein the radiolabel is89Zr,86 90Y,niIn,177Lu,225AC,211At, 203 / 212pb;124 / 131J, 153Sm134^ 64 / 67^ 66 / 68^ 18 / 19^ 52^ 44^Qr gadoJinium7. The method of claim 1, wherein the antibody is a multispecific antibody further comprising one or more additional antigen binding domains which specifically bind a tumor-specific antigen, antigen binding domains which specifically engage T cells, antigen binding domains which specifically other immune effectors, and combinations thereof.

8. The method of claim 7, wherein the tumor-specific antigen is carbonic anhydrase IX (CAIX), carcinoembiyonic antigen (CEA), CD8, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CLL1, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, CD123, CD44V6, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine-protein kinases erb-B2,3,4 (erb-B2,3,4), folate-binding protein (FBP), fetal acetylcholine receptor (AChR), adult AChR subunits, folate receptor-a, Ganglioside G2 (GD2), Ganglioside G3 (GD3), human Epidermal Grow th Factor Receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), Interleukin- 13 receptor subunit alpha-2 (IL-13Ra2), K-light chain, kinase insert domain receptor (KDR), Lewis Y (LeY), LI cell adhesion molecule (LI CAM), melanoma antigen family A, 1 (MAGE-A1), Mucin 16 (MUC16), Mucin 1 (MUC1), Mesothelin (MSLN), ERBB2, MAGEA3, p53, MARTI, GP100, Proteinase3 (PR1), Tyrosinase, Survivin, hTERT, EphA2, NKG2D ligands, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), R0R1, tumor- associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), BCMA, NKCS1, EGF1R, EGFR-vIII, CD99, CD70, ADGRE2, CCR1, LILRB2, PRAME CCR4, CD5, CD3, TRBC1, TRBC2, TIM-3, Integrin B7, ICAM-1, CD70, Tim3, CLEC12A, ERBB, or a combination thereof.

9. The method of claim 8, wherein the tumor-specific antigen is GD2, HER2, EGFR, mesothelin, Claudin-18.2, PSMA, B7-H3, IL-13Ra2, FAP, CA19, CD19, CD5, MUC1, or a combination thereof.

10. The method of claim 1, wherein the antibody further comprises a conjugated chemotherapy agent, or a fused immunotherapy agent.

11. The method of claim 1, wherein the antibody comprises VNAR, CDR1 (SEQ ID NO: 11), HVR2 (SEQ ID NO: 12), HVR4 (SEQ ID NO: 13); CDR3 (SEQ ID NO: 14), and human IgGl CH2+CH3 (SEQ ID NO: 15), specifically SEQ ID NO: 17.

12. The method of claim 1, further comprising administering an adjusted dose of the administered radiotherapy or radiopharmaceutical therapy, wherein the adjusted dose is based on the biological effect of the administered dose of the radiotherapy or radiopharmaceutical therapy on the tissues of the patient.

13. The method of claim 11, further comprising administering a third dose of a third radiotherapy or radiopharmaceutical therapy, wherein the third radiolabeled antibody and dose are selected based on the biological effect of the administered dose of the radiotherapy or radiopharmaceutical therapy on the tissues of the patient, and wherein the third radiotherapy or radiopharmaceutical therapy is different from the administered radiotherapy or radiopharmaceutical therapy.

14. The method of claim 1, wherein the radiotherapy or radiopharmaceutical therapy comprises an administered dose of an administered radiolabeled antibody, wherein the administered radiolabeled antibody comprises a first antigen binding domain which specifically binds a radiation-induced cell-surface marker and a radiolabel, wherein the administered radiolabeled antibody and the imaging radiolabeled antibody are the same or different.

15. The method of claim 14, further comprising administering an adjusted dose of the administered radiolabeled antibody, wherein the adjusted dose is based on the biological effect of the administered dose of the radiotherapy or radiopharmaceutical therapy on the tissues of the patient.

16. The method of claim 14, further comprising administering a third dose of a third radiolabeled antibody, wherein the third radiolabeled antibody is selected based on the biological effect of the administered dose of the radiotherapy or radiopharmaceutical therapy on the tissues of the patient.

17. The method of claim 1, wherein the quantitative serial imaging comprises positron emission tomography (PET), single photon emission computed tomography (SPECT), computerized tomography (CT), or magnetic resonance imaging (MRI).

18. The method of claim 1, wherein the administered dose and the imaging dose are determined as activities and are independently 1 pCi to 500 mCi, specifically 100 pCi to 200 mCi.

19. A method of determining exposure of a patient to radioactivity, comprising administering an imaging dose of an imaging radiolabeled antibody, wherein the imaging radiolabeled antibody comprises a first antigen binding domain which specifically binds a radiation-induced cell-surface marker and a radiolabel, and wherein the patient has known or suspected exposure to radioactivity, performing quantitative serial imaging of the uptake and distribution of the imaging radiolabeled antibody, and pairing the quantitative serial imaging with image-based dosimetry to provide a three- dimensional spatially resolved measure of the biological effect of the radioactivity on the tissues of the patient, wherein the biological effect is the distribution of the radioactivity, the absolute level of the radioactivity7, or the relative degree of the biological effect of the radioactivity.

20. The method of claim 19, wherein imaging is positron emission tomography (PET), single photon emission computed tomography (SPECT), computerized tomography (CT), or magnetic resonance imaging (MRI).

21. The method of claim 19, wherein the imaging dose is determined as activity and is 1 pCi to 500 mCi, specifically 100 pCi to 200 mCi.

22. An antibody comprising a first antigen binding domain which specifically binds a radiation-induced cell-surface marker.

23. The antibody of claim 22, wherein the antibody is a single-domain antibody (sdAb).

24. The antibody of claim 23, wherein the antibody is a camelid antibody, a shark New Antigen Receptor antibody, a chimeric antibody, a partially humanized antibody, or a fully humanized antibody.

25. The antibody of claim 22, wherein the antibody is a heavy-chain only- antibody, such as the variable domain of the heavy chain of the heavy chain antibody in Camelidae or the variable domain of the shark New Antigen Receptor in cartilaginous fish.

26. The antibody of claim 22, further comprising a radiolabel.

27. The antibody of claim 26, wherein the radiolabel is89Zr,86 / 90Y,U1ln,177Lu,225AC,211At,203 / 212Pb, 124 / 131J, 153Sm134Ce, 64 / 67^ 66 / 68^ 18 / 19^ 52^ 44^Qr gadollmum28. The antibody of claim 22, wherein the radiation-induced cell-surface marker is calreticulin, TATA-Box Binding Protein Associated Factor 15 (TAF15), Intercellular adhesion molecule-1 (ICAM-1), E-selectin, P-selectin, Glucose-related protein 78 (GRP78), or a combination thereof.

29. The antibody of claim 22, wherein the antibody is a multispecific antibody further comprising one or more additional antigen binding domains which specifically bind a tumor-specific antigen, antigen binding domains which specifically engage T cells, antigen binding domains which specifically other immune effectors, and combinations thereof.

30. The antibody of claim 29, wherein the tumor-specific antigen is carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD8, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CLL1, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, CD123, CD44V6, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine-protein kinases erb-B2,3,4 (erb-B2,3,4), folate-binding protein (FBP), fetal acetylcholine receptor (AChR), adult AChR subunits, folate receptor-a. Ganglioside G2 (GD2), Ganglioside G3 (GD3), human Epidermal Grow th Factor Receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), Interleukin- 13 receptor subunit alpha-2 (IL-13Ra2), K-light chain, kinase insert domain receptor (KDR), Lewis Y (LeY), LI cell adhesion molecule (LI CAM), melanoma antigenfamily A, 1 (MAGE-A1), Mucin 16 (MUC16), Mucin 1 (MUC1), Mesothelin (MSLN), ERBB2, MAGEA3, p53, MARTI, GP100, Proteinase3 (PR1), Tyrosinase, Survivin, hTERT, EphA2, NKG2D ligands, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), R0R1, tumor- associated glycoprotein 72 (TAG-72), vascular endothelial grow th factor R2 (VEGF-R2), Wilms tumor protein (WT-1), BCMA, NKCS1, EGF1R, EGFR-vIII, CD99, CD70, ADGRE2, CCR1, LILRB2, PRAME CCR4, CD5, CD3, TRBC1, TRBC2, TIM-3, Integnn B7, ICAM-1, CD70, Tim3, CLEC12A, ERBB, or a combination thereof.

31. The antibody of claim 30, wherein the tumor-specific antigen is GD2, HER2, EGFR, mesothelin, Claudin-18.2, PSMA, B7-H3, IL-13Ra2, FAP, CA19, CD19, CD5, MUC1, or a combination thereof.

32. The antibody of claim 22, further comprising a conjugated chemotherapy agent, or a fused immunotherapy agent.

33. The antibody of claim 22, wherein the antibody comprises VNAR, CDR1 (SEQ ID NO: 11), HVR2 (SEQ ID NO: 12), HVR4 (SEQ ID NO: 13); CDR3 (SEQ ID NO: 14), and human IgGl CH2+CH3 (SEQ ID NO: 15), specifically SEQ ID NO: 17.

34. A pharmaceutical composition comprising the antibody of claim 22 and a pharmaceutically acceptable carrier.

35. A method of treating a cancer in a subject comprises administering to the subject a dose of the pharmaceutical composition of claim 22.

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