Methods for production of radioimmunoconjugates

Abstract

The disclosure provided herein relates to the field of radioimmunoconjugates, specifically the radiolabeling of immunoconjugates with Actinium-225 (225AC) for therapeutic and diagnostic applications. The disclosure encompasses improved methods for producing radioimmunoconjugates, including streamlined processes that eliminate intermediate purification steps, optimize radiochemical yield and stability, and enable scalability for clinical and commercial manufacturing. The disclosure further encompasses compositions of radioimmunoconjugates, optimized reaction conditions, and scalable production methodologies designed to enhance efficiency, reduce production time, and ensure compliance with Good Manufacturing Practice (GMP) standards. Additionally, the disclosure provides pharmaceutical compositions comprising radioimmunoconjugates and methods of use for treating diseases such as cancer.

Description

Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]METHODS FOR PRODUCTION OF RADIOIMMUNOCONJUGATESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 749,281 filed on January 24, 2025, the disclosure of which is hereby incorporated by reference in its entirety.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. The XML copy, created on January 26, 2026, is named 065768-220W01-SequenceListing.xml and is 17,557 bytes in size.TECHNICAL FIELD

[0003] The disclosure provided herein relates to the field of radioimmunoconjugates, specifically the radiolabeling of immunoconjugates with Actinium-225 (223Ac). The disclosure further provides methods for optimizing the radiochemical yield, stability, and scalability of radioimmunoconjugate compositions for clinical and commercial applications.BACKGROUND

[0004] Radiopharmaceuticals represent a powerful class of cancer therapeutics due to their ability to deliver cytotoxic radiation directly to tumor cells while minimizing damage to healthy tissues. Actinium-225 (225Ac), a radionuclide emitting high-energy alpha particles, has gained significant attention for its potential in targeted alpha therapy (TAT). Alpha radiation offers the advantage of high linear energy transfer (LET), resulting in localized cytotoxicity to cancer cells while sparing adjacent healthy tissues.

[0001] Despite the therapeutic promise of225Ac, its clinical utility is limited by several challenges, including:1. Inefficient Labeling Processes: Existing methods for chelating225Ac to immunoconjugates often result in low radiochemical yields and suboptimal efficiency of the radiolabeling reaction.2. Scalability Issues: Radiolabeling processes designed for small-scale laboratory experiments are not easily adaptable to larger-scale production, which is critical for commercial manufacturing.3. Stability Concerns: Radiolabeled drug products tend to degrade during storage due to radiolysis, which can compromise radiochemical purity and therapeutic efficacy.Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]4. Formulation Complexity: The interaction between radionuclides and immunoconjugates requires carefully optimized buffers to maintain stability and prevent aggregation.

[0005] Accordingly, there is a need for robust, scalable, and efficient methods for radiolabeling immunoconjugates with Actinium-225 (225Ac) to overcome the limitations of existing processes.SUMMARY

[0006] Provided herein is a process for producing a radioimmunoconjugate comprising Actinium-225 (225Ac) chelated to an immunoconjugate, wherein the immunoconjugate comprises a chelating agent conjugated to an antibody, or an antigen-binding fragment thereof, the process comprising:(a) mixing the immunoconjugate with225Ac in a reaction buffer, wherein the reaction buffer comprises:(i) a radioprotectant,(ii) a buffering agent, and(iii) a surfactant, to form a reaction mixture, wherein the ratio of immunoconjugate to 225 Ac is at least about 440 to 1;(b) incubating the reaction mixture of (a) under conditions sufficient to form the radioimmunoconjugate; and(c) filtering the reaction mixture through a sterile filter to produce a sterile radioimmunoconjugate; andwherein the specific activity of the radioimmunoconjugate is at or below about 200 pCi / mg, and wherein the process excludes intermediate purification steps between mixing, incubation, and filtration.

[0007] In certain embodiments, the specific activity is adjusted for example, by dilution prior to or after sterile filtration, while maintaining radiochemical purity and excluding intermediate purification steps.

[0008] Also provided here is a process for producing a radioimmunoconjugate comprising Actinium-225 (22?Ac) chelated to an immunoconjugate, wherein the immunoconjugate comprises a chelating agent conjugated to an antibody, or an antigen-binding fragment thereof, the process comprising:(a) mixing the immunoconjugate with225Ac in a reaction buffer to form a reaction mixture, wherein the reaction buffer comprises:(i) a radioprotectant,Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT](ii) a buffering agent, and(iii) a surfactant,(b) incubating the reaction mixture of (a) at a temperature of 33°C to 39°C for up to 4 hours to form the radioimmunoconjugate,optionally, the process further comprising:(c) filtering the reaction mixture through a sterile filter to produce a sterile radioimmunoconjugate, wherein the process excludes intermediate purification steps between mixing, incubation, and filtration.

[0009] In some embodiments, the chelating agent is or comprises 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or a derivative thereof.

[0010] In some embodiments, the radioprotectant is sodium ascorbate.

[0011] In some embodiments, the buffering agent is sodium acetate.

[0012] In some embodiments, the surfactant is Polysorbate 20.

[0013] In some embodiments, the reaction buffer comprises sodium ascorbate at about 0.1% to about 5% (w / v), sodium acetate at about 0.01 M to about 1 M, and Polysorbate 20 at about 0.01% to about 1% (v / v).

[0014] In some embodiments, the reaction buffer comprises sodium ascorbate at about 1% to about 8% (w / v), sodium acetate at about 0.1 M to about 1 M, and Polysorbate 20 at about 0.01% to about 1% (v / v).

[0015] In some embodiments, the pH of the reaction mixture is or is adjusted to about 6.0-6.5 before incubation.

[0016] In some embodiments, the incubation is performed at about 37 °C.

[0017] In some embodiments, the incubation is performed at about 35 °C.

[0018] In some embodiments, the incubation is carried out for about 2 hours.

[0019] In some embodiments, the incubation is carried out for about 3 hours.

[0020] In some embodiments, the sterile filter has a pore size of about 0.22 pm.

[0021] In some embodiments, the radioimmunoconjugate is directly filtered into a sterile container.

[0022] In some embodiments, the radioimmunoconjugate is prepared at a scale of about 0.1 mCi to about 50 mCi.

[0023] In some embodiments, the process is scaled for large-scale production with automated or semi-automated systems.Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]

[0024] In some embodiments, the process further comprises diluting the reaction mixture after step (b) or step (c) with a dilution buffer comprising a second radioprotectant, a second buffering agent and a second surfactant. In some embodiment, the dilution buffer comprises sodium ascorbate, acetic acid, and / or Polysorbate 20. In some embodiment, the dilution buffer comprises about 0.1-5% (w / v) sodium ascorbate, about 0.5 - 10 mM acetic acid, and about 0.01% to about 1% (v / v) Polysorbate 20.

[0025] In some embodiments, the radioimmunoconjugate has a radiochemical purity of at least 95% as determined by instant thin-layer chromatography (iTLC).

[0026] In some embodiments, the process further comprises performing a stability challenge using diethylenetriamine pentaacetate (DTP A) to confirm chelation stability of theradi oi mmunoconj ugate .

[0027] In some embodiments, the DTPA challenge shows at least 95% of the225Ac remains chelated after 30 minutes of incubation with DTPA.

[0028] In some embodiments, the process further comprises analyzing the radioimmunoconjugate using size-exclusion high-performance liquid chromatography (SEC-HPLC) to confirm protein integrity and radiochemical purity.

[0029] In some embodiments, the radiochemical purity remains at least about 90% after 96 hours of storage at 4 °C.

[0030] In some embodiments, the process does not comprise a step of mixing the radioimmunoconjugate recovered at step (c) with unlabeled immunoconjugate.

[0031] In some embodiments, the immunoconjugate comprises an antibody, or an antigen binding fragment thereof, that specifically binds to human kallikrein-related peptidase 2 (hK2).

[0032] In some embodiments, the hK2 antibody, or antigen-binding fragment thereof, comprises:a heavy chain CDR1 (HCDR1) of SEQ ID NO: 5;a heavy chain CDR2 (HCDR2) of SEQ ID NO: 6;a heavy chain CDR3 (HCDR3) of SEQ ID NO: 7;a light chain CDR1 (LCDR1) of SEQ ID NO: 8;a light chain CDR2 (LCDR2) of SEQ ID NO: 9; anda light chain CDR3 (LCDR3) of SEQ ID NO: 10.Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]

[0033] In some embodiments, the hK2 antibody, or antigen-binding fragment thereof, comprises a variable heavy chain (VH) sequence of SEQ ID NO: 1 and a variable light chain (VL) sequence of SEQ ID NO: 2.

[0034] In some embodiments, the hK2 antibody, or antigen-binding fragment thereof, comprises a heavy chain (HC) sequence of SEQ ID NO: 3 and a light chain (LC) sequence of SEQ ID NO: 4.

[0035] In some embodiments, the hK2 antibody is a humanized antibody, e.g., a humanized monoclonal antibody, such as hllB6.

[0036] In some embodiments, the antibody (e.g., the hK2 antibody, such as the antibody of

[0030] -

[0034] ) or antigen-binding fragment thereof is conjugated to the chelator (e.g., to DOTA) via a linker. The linker may e.g., be or comprise 2-isothiocyanatobenzyl.

[0037] In some embodiments, the antibody or antigen-binding fragment thereof is conjugated to 2-(4-isothiocyanatobenzyl)-l ,4,7, 10-tetraazacyclododecane- 1 ,4,7, 10-tetraacetic acid, more particularly to one or more 2-(4-isothiocyanatobenzyl)-l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid groups. The one or more 2-(4-isothiocyanatobenzyl)-l,4,7,10-tetraazacy cl ododecane- 1,4, 7,10-tetraacetic acid group(s) may e.g., be covalently linked to one or more amino acid residues in the Fc region of the antibody or antigen-binding fragment thereof.

[0038] In some embodiments, the radioimmunoconjugate may comprise an average of from about 1 to about 4 chelator molecules conjugated to the antibody or antigen binding fragment thereof.

[0039] In some embodiments, the process is a GMP-compliant process.

[0040] In some embodiments, the process is for producing a radioimmunoconjugate for the treatment of prostate cancer.

[0041] In some embodiments, the radioimmunoconjugate is obtained or obtainable by the process disclosed herein.

[0042] In some embodiments, a composition comprising the radioimmunoconjugate is obtained or obtainable by the process disclosed herein.

[0043] In some embodiments, an intermediate composition is obtained or obtainable by implementing the process described herein, wherein the intermediate composition comprises a radioimmunoconjugate and a buffer solution, and wherein the intermediate composition is obtained or obtainable at step (b) or (c) of the process.Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]

[0044] In another aspect, disclosed herein is a method of treating prostate cancer comprising administering a therapeutically effective amount of a radioimmunoconjugate produced by the process disclosed herein.

[0045] The skilled person will understand that the described process and methods may be specified in medical use format, for example in the form of a radioimmunoconjugate for use in the treatment of prostate cancer. This skilled person will also understand that the methods may be specified in so-called Swiss form, for example in the form of the use of a radioimmunoconjugate for the manufacture of a medicament for the treatment of prostate cancer. This applies throughout the disclosure.BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The foregoing and other objects, aspects, features, and advantages of exemplary embodiments will become more apparent and may be better understood by referring to the following description taken in conjunction with the accompanying drawings.

[0047] FIG. 1 : iTLC chromatogram of the radiolabeling reaction. Region 1 (baseline) corresponds to the radiolabeled conjugate. The solvent front is marked on the x-axis, with radioactivity counts on the y-axis.

[0048] FIG. 2: iTLC chromatogram after DTPA challenge. The absence of radioactivity at the solvent front indicates stable chelation of225Ac.

[0049] FIG. 3 A and FIG. 3B: SEC-HPLC chromatograms of the drug product at t = 0 hours after end of synthesis. (FIG. 3A) UV chromatogram at 280 nm; (FIG. 3B) Reconstructed radiochromatogram.

[0050] FIG. 4A and FIG. 4B: SEC-HPLC chromatograms of the drug product at t = 96 hours after end of synthesis. (FIG. 4A) UV chromatogram at 280 nm; (FIG. 4B) Reconstructed radi ochromatogram .

[0051] FIG. 5: iTLC of the Ac-225 labeling reaction solution. Region 1 (baseline) corresponds to the radiolabeled conjugate. The solvent front is marked on the x-axis, with radioactivity counts on the y-axis.

[0052] FIG. 6: DTPA challenge of the drug product. The absence of radioactivity at the solvent front indicates stable chelation of225Ac.

[0053] FIG 7 A and FIG. 7B: sec-HPLC UV at 280 nm chromatogram (FIG. 7A) and reconstructed radio-chromatogram (FIG. 7B) of the drug product at t = 0 h after end of synthesis.Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]

[0054] FIG. 8 A and FIG. 8B: sec-HPLC UV at 280 nm chromatogram (FIG. 8 A) and reconstructed radio-chromatogram (FIG. 8B) of the drug product at t = 96 h after the end of synthesis. Note: A retention time shift from Rt = 10.8 min at t = 0 h to Rt = 12.3 min at t = 96 h was observed due to a reduction in HPLC flow rate caused by mobile phase leakage.

[0055] FIG. 9: iTLC of the225Ac labeling reaction solution targeting specific activity = 100 pCi / mg. Region 1 (baseline) corresponds to the radiolabeled conjugate. The solvent front is marked on the x-axis, with radioactivity counts on the y-axis.

[0056] FIG. 10: DTPA challenge of the225Ac labeling reaction solution targeting specific activity = 100 pCi / mg. The absence of radioactivity at the solvent front indicates stable chelation of225Ac.

[0057] FIG. 11 : iTLC of the225Ac labeling reaction solution targeting specific activity = 200 pCi / mg. Region 1 (baseline) corresponds to the radiolabeled conjugate. The solvent front is marked on the x-axis, with radioactivity counts on the y-axis.

[0058] FIG. 12: DTPA challenge of the225Ac labeling reaction solution targeting specific activity = 200 pCi / mg. Absence of radioactivity at the solvent front would indicate stable chelation of225Ac.

[0059] FIG. 13: iTLC of the225Ac labeling reaction solution targeting specific activity = 400 pCi / mg. Region 1 (baseline) corresponds to the radiolabeled conjugate. The solvent front is marked on the x-axis, with radioactivity counts on the y-axis.

[0060] FIG. 14: DTPA challenge of the225Ac labeling reaction solution targeting specific activity = 400 pCi / mg. Absence of radioactivity at the solvent front would indicate stable chelation of225Ac.

[0061] FIG. 15: iTLC of the225Ac labeling reaction solution targeting specific activity = 800 pCi / mg. Region 1 (baseline) corresponds to the radiolabeled conjugate. The solvent front is marked on the x-axis, with radioactivity counts on the y-axis.

[0062] FIG. 16: DTPA challenge of the223Ac labeling reaction solution targeting specific activity = 800 pCi / mg. Absence of radioactivity at the solvent front would indicate stable chelation of225Ac.

[0063] FIG. 17: iTLC of the225Ac labeling reaction solution targeting specific activity = 1600 pCi / mg. Region 1 (baseline) corresponds to the radiolabeled conjugate. The solvent front is marked on the x-axis, with radioactivity counts on the y-axis.

[0064] FIG. 18: DTPA challenge of the223Ac labeling reaction solution targeting specific activity = 1600 pCi / mg. Absence of radioactivity at the solvent front would indicate stable chelation of225Ac.Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0065] The present disclosure relates to a process for producing radioimmunoconjugates, wherein actinium-225 (223Ac) is chelated to an immunoconjugate. The disclosed process provides efficient, scalable, and GMP-compliant approaches for producing radioimmunoconjugates for therapeutic or diagnostic purposes. The process also encompasses compositions and intermediate products produced by the described methods.

[0066] The disclosed process and methods can be understood more readily by reference to the following detailed description. It is to be understood that the disclosed methods are not limited to the specific methods described and / or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed methods. All patents, published patent applications and publications cited herein are incorporated by reference as if set fourth fully herein.

[0067] Unless otherwise defined herein, technical and scientific terms used in the present description have the meanings that are commonly understood by those of ordinary skill in the art. For purposes of interpreting this specification, the following description of terms will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any description of a term set forth conflicts with any document incorporated herein by reference, the description of the term set forth below shall control.

[0068] In an attempt to help the reader of the present application, the description has been separated in various paragraphs or sections. These separations are not considered as disconnecting the substance of a paragraph or section from the substance of another paragraph or section. To the contrary, the present description encompasses all the combinations of the various sections, paragraphs and sentences that can be contemplated.DEFINITIONS

[0069] Various terms relating to aspects of the description are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein.

[0070] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “an antibody” includes a combination of two or more antibodies, and the like.Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]

[0071] The term “about” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of up to ±10% from the specified value, as such variations are appropriate to perform the disclosed methods. Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0072] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0073] The term “comprising” is intended to include examples encompassed by the terms “consisting essentially of’ and “consisting of’; similarly, the term “consisting essentially of’ is intended to include examples encompassed by the term “consisting of.” Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

[0074] As used herein, the term "actinium-225" or223Ac refers to a radionuclide that emits alpha particles. Actinium-225 is used in targeted alpha-particle radiotherapy for the treatment of cancer and other diseases.

[0075] As used herein, the term "chelated" means the process or state in which a radionuclide, such as actinium-225, is bound to a chelating agent that forms a stable complex, preventing the radionuclide from dissociating under physiological conditions.

[0076] As used herein, the term "reaction buffer" means a solution used to maintain the stability and pH of a reaction mixture during the production of radioimmunoconjugates. A reaction buffer described herein may comprise buffering agents, radioprotectants, and surfactants.Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]

[0077] As used herein, the term "radioprotectant" means a compound included in the reaction mixture to protect biomolecules, such as antibodies or antigen binding fragments thereof, from degradation caused by radiation emitted from the radionuclide. In some embodiments, the radioprotectant is sodium ascorbate.

[0078] As used herein, the term "buffering agent" means a compound or combination of compounds that maintain the pH of a solution within a specified range during a chemical or biological reaction. In some embodiments, the buffering agent is sodium acetate.

[0079] As used herein, the term "surfactant" means a compound that reduces surface tension in the reaction mixture, improving solubility, dispersion, or stability of components. In some embodiments, the surfactant is Polysorbate 20.

[0080] As used herein, the term "reaction mixture" means the combination of components, including the immunoconjugate, radionuclide, reaction buffer, and other reagents, that participate in the radiolabeling process to produce a radioimmunoconjugate.

[0081] " Radiochemical purity" refers to the percentage of the radiolabeled product in the desired chemical form, as determined by well-known analytical methods. In some embodiments, the radiochemical purity is determined by iTLC or SEC-HPLC.

[0082] As used herein, the term "intermediate purification steps" means any steps performed during the radiolabeling process to isolate or purify the product prior to final filtration. Examples of intermediate purification steps include chromatography or diafiltration.

[0083] As used herein, the term "GMP-compliant process" means a manufacturing process that adheres to Good Manufacturing Practice (GMP) regulations, ensuring that the product is consistently produced and controlled according to quality standards required for pharmaceutical applications.

[0084] The terms “treating” or “treatment” refer to any success or indicia of success in the attenuation or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement, remission, diminishing of symptoms or making the condition more tolerable to the patient, slowing in the rate of degeneration or decline, making the final point of degeneration less debilitating, improving a subject’s physical or mental well-being, or prolonging the length of survival. The treatment may be assessed by objective or subjective parameters; including the results of a physical examination, neurological examination, or psychiatric evaluations.Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]

[0085] An "effective amount" or "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of a radioimmunoconjugate described herein may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the radioimmunoconjugate are outweighed by the therapeutically beneficial effects.

[0086] “Kallikrein related peptidase 2”, “hK2”, or “KLK2” refer to a known protein which is also called kallikrein-2, grandular kallikrein 2, or HK2. hK2 is produced as a preproprotein and cleaved during proteolysis to generate active protease. All hK2 isoforms and variants are encompassed in “hK2”. The amino acid sequences of the various isoforms are retrievable from GenBank® (N1H genetic sequence database) accession numbers NP_005542.1, NP_001002231.1 and NP_001243009. The amino acid sequence of a full length hK2 is shown in SEQ ID NO: 11. The sequence includes the signal peptide (amino acid residues 1-18) and the pro-peptide region (amino acid residues 19-24).SEQ ID NO: 11 MWDLVLSIALSVGCTGAVPLIQSRIVGGWECEKHSQPWQVAVYSHGWAHC GGVLVHPQWVLTAAHCLKKNSQVWLGRHNLFEPEDTGQRVPVSHSFPHPL YNMSLLKHQSLRPDEDSSHDLMLLRLSEPAKITDVVKVLGLPTQEPALGT TCYASGWGSIEPEEFLRPRSLQCVSLHLLSNDMCARAYSEKVTEFMLCAG LWTGGKDTCGGDSGGPLVCNGVLQGITSWGPEPCALPEKPAVYTKVVHYR KWIKDTIAANP

[0087] “Antibody” refers to all isotypes of immunoglobulins (IgG, IgA, IgE, IgM, IgD, and IgY) including various monomeric, polymeric and chimeric forms, unless otherwise specified.Specifically encompassed by the term “antibody” are polyclonal antibodies, monoclonal antibodies (mAbs), and antibody-like polypeptides, such as chimeric antibodies and humanized antibodies.

[0088] The term “antigen-binding fragment” refers to a fragment of the antigen-binding arm containing an antigen-binding domain. Antigen-binding fragments include those provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques. Some antigen-binding fragments are composed of portions of intact antibodies that retain antigenbinding specificity of the parent antibody molecule. For example, antigen-binding fragments may comprise at least one variable region (either a heavy chain or light chain variable region) or one orAttorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]more CDRs of an antibody known to bind a particular antigen. Examples of suitable antigenbinding fragments include, without limitation diabodies and single-chain molecules as well as Fab, F(ab’)2, Fc, Fabc, and Fv molecules, single chain (Sc) antibodies, individual antibody light chains, individual antibody heavy chains, chimeric fusions between antibody chains or CDRs and other proteins, protein scaffolds, heavy chain monomers or dimers, light chain monomers or dimers, dimers consisting of one heavy and one light chain, a monovalent fragment consisting of the VL, VH, CL and CHI domains, or a monovalent antibody as described in W02007059782, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region, a Fd fragment consisting essentially of the VH and CHI domains; a Fv fragment consisting essentially of the VL and VH domains of a single arm of an antibody, a dAb fragment (Ward et al., Nature 341, 544-546 (1989)), which consists essentially of a VH domain and also called domain antibodies (Holt et al; Trends Biotechnol. 2003 Nov.; 21(ll):484-90); camelid or nanobodies (Revets et al; Expert Opin Biol Ther. 2005 Jan.; 5(1): 111-24); an isolated complementarity determining region (CDR), and the like; and antibodies formed from antibody fragments. All antibody isotypes may be used to produce antigen-binding fragments. Additionally, antigen-binding fragments may include non-antibody proteinaceous frameworks that may successfully incorporate polypeptide segments in an orientation that confers affinity for a given antigen of interest, such as protein scaffolds.Antigen-binding fragments may be recombinantly produced or produced by enzymatic or chemical cleavage of intact antibodies. The phrase “an antibody or antigen-binding fragment thereof’ may be used to denote that a given antigen-binding fragment incorporates one or more amino acid segments of the antibody referred to in the phrase.

[0089] The terms “CDR”, and its plural “CDRs”, refer to a complementarity determining region (CDR) of which three make up the binding character of a light chain variable region (CDRL1, CDRL2 and CDRL3) and three make up the binding character of a heavy chain variable region (CDRH1, CDRH2 and CDRH3). CDRs contribute to the functional activity of an antibody molecule and are separated by amino acid sequences that comprise scaffolding or framework regions. The exact definitional CDR boundaries and lengths are subject to different classification and numbering systems. CDRs may therefore be referred to herein by Kabat, Chothia, AbM, contact or any other boundary definitions. Despite differing boundaries, each of these systems has some degree of overlap in what constitutes the so called “hypervariable regions” within the variable sequences. CDR definitions according to these systems may therefore differ in length and boundaryAttorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]areas with respect to the adjacent framework region. See for example Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. NIH Publication No. 91-3242 (1991); Chothia et al., “Canonical Structures For the Hypervariable Regions of Immunoglobulins,” J. Mol. Biol. 196:901 (1987); and MacCallum et al., “Antibody-Antigen Interactions: Contact Analysis and Binding Site Topography,” J. Mol. Biol. 262:732 (1996)), each of which is hereby incorporated by reference in its entirety.

[0090] Typically, CDRs form a loop structure that can be classified as a canonical structure. The term “canonical structure” refers to the main chain conformation that is adopted by the antigen binding (CDR) loops. From comparative structural studies, it has been found that five of the six antigen binding loops have only a limited repertoire of available conformations. Each canonical structure can be characterized by the torsion angles of the polypeptide backbone. Correspondent loops between antibodies may, therefore, have very similar three-dimensional structures, despite high amino acid sequence variability in most parts of the loops (Chothia et al., “Canonical Structures For the Hypervariable Regions of Immunoglobulins,” J. Mol. Biol. 196:901 (1987); Chothia et al., “Conformations of Immunoglobulin Hypervariable Regions,” I 342:877 (1989); Martin and Thornton, “Structural Families in Loops of Homologous Proteins: Automatic Classification, Modelling and Application to Antibodies,” J. Mol. Biol. 263:800 (1996), each of which is incorporated by reference in its entirety). Furthermore, there is a relationship between the adopted loop structure and the amino acid sequences surrounding it. The conformation of a particular canonical class is determined by the length of the loop and the amino acid residues residing at key positions within the loop, as well as within the conserved framework (i.e., outside of the loop). Assignment to a particular canonical class can therefore be made based on the presence of these key amino acid residues.

[0091] “Specifically binds” or “binds specifically” or derivatives thereof when used in the context of antibodies, or antibody fragments, represents binding via domains encoded by immunoglobulin genes or fragments of immunoglobulin genes to one or more epitopes of a protein of interest, without preferentially binding other molecules in a sample containing a mixed population of molecules. Typically, an antibody binds to a cognate antigen with a Ka of less than about 1x10'8M, as measured by a surface plasmon resonance assay or a cell-binding assay. Phrases such as “[antigen]-specific” antibody (e.g., hK2-specific antibody) are meant to convey that the recitedAttorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]antibody specifically binds the recited antigen. Wherever the term “binds” is used herein it is intended that this encompasses “specifically binds” and the terms may be interchanged as desired.

[0092] ‘ ‘Pharmaceutical composition” refers to composition that comprises an active ingredient and a pharmaceutically acceptable carrier.

[0093] “Pharmaceutically acceptable carrier” or “excipient” refers to an ingredient in a pharmaceutical composition, other than the active ingredient, which is nontoxic to a subject.

[0094] The embodiments described herein are not limited to particular methods, reagents, compounds, compositions or biological systems, which can, of course, vary.OVERVIEW OF THE PROCESSGeneral Overview:

[0095] In one aspect, the disclosure provides a process for producing a radioimmunoconjugate, wherein the radioimmunoconjugate comprises225Ac chelated to an immunoconjugate.

[0096] In some embodiments, the immunoconjugate comprises a chelating agent conjugated to an antibody or an antigen-binding fragment thereof.

[0097] In some embodiments, the process for producing the radioimmunoconjugate comprises the steps of(a) mixing the immunoconjugate with22?Ac in a reaction buffer, wherein the reaction buffer comprises:(i) a radioprotectant,(ii) a buffering agent, and(iii) a surfactant, to form a reaction mixture;(b) incubating the reaction mixture of (a) under conditions sufficient to form the radioimmunoconjugate; and(c) filtering the reaction mixture through a sterile filter to produce a sterile radioimmunoconjugate.

[0098] In some embodiments, the reaction mixture formed in (a) comprises a ratio of immunoconjugate to 225Ac of at least about 440 to 1.

[0099] In some embodiments, the specific activity of the radioimmunoconjugate is at or below about 200 pCi / mg.

[0100] In some embodiments, the process for producing the radioimmunoconjugate comprises the steps of:Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT](a) mixing the immunoconjugate with225Ac in a reaction buffer to form a reaction mixture, wherein the reaction buffer comprises:(i) a radioprotectant,(ii) a buffering agent, and(iii) a surfactant, and(b) incubating the reaction mixture of (a) at a temperature of 33°C to 39°C for up to 4 hours to form the radioimmunoconjugate. In some embodiment, the process further comprising: (c) filtering the reaction mixture through a sterile filter to produce a sterile radioimmunoconjugate, wherein the process excludes intermediate purification steps between mixing, incubation, and filtration.

[0101] In some embodiment, the incubating step (b) comprises incubating the reaction mixture of (a) at a temperature of 33°C to 39°C, such as 33°C, 34°C, 35°C, 36°C, 37°C, 38°C or 39°C, for up to 4 hours, such as for 1 hour, 2 hours, 3 hours or 4 hours, to form the radioimmunoconjugate.

[0102] In some embodiment, the process further comprises diluting the reaction mixture after step (b) or step (c) with a dilution buffer. In some embodiment, the dilution buffer comprises a radioprotectant, a buffering agent and a surfactant.

[0103] The process does not require and therefore can exclude an intermediate purification step (e.g., on a purification column) between mixing and incubation and / or between incubation and filtration. Advantageously, the process excludes intermediate purification steps between mixing, incubation, and filtration.EMBODIMENTS OF THE PROCESSStep (a): Mixing

[0104] In some embodiments, the radioimmunoconjugate that is produced by the process described herein comprises the step of mixing an immunoconjugate with225Ac in a reaction buffer.Consideration of Actinium-225 Source Variability:

[0105] In some embodiments, the process described herein accounts for the variability in225Ac sources, which may contain adventitious non-radioactive metal impurities such as Fe, Ca, Mn, or Al, in addition to other metals in lesser quantities. These impurities can significantly impact the efficiency of the chelation process by competing with actinium for binding to the DOTA chelators. The disclosed process is designed to accommodate actinium grades obtained from multiple suppliers, each with varying levels of non-radioactive metal content.Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]

[0106] In some embodiments, the potential variability in225Ac sources is compensated by increasing the ratio of immunoconjugate to225Ac. The increase in ratio of immunoconj ugate to223Ac ensures that sufficient unoccupied DOTA chelators are available to achieve near-quantitative chelation of225Ac, even in the presence of these competing metals.

[0107] This flexibility in handling actinium sources from different suppliers enhances the robustness and scalability of the process. It mitigates the risk of batch failures due to variable metal impurity levels, ensuring consistent radiochemical yields and eliminating the need for postchelation purification to remove unchelated actinium. This adaptability makes the process suitable for both clinical and commercial production, where reliable sourcing of actinium with consistent quality may be challenging.

[0108] In some embodiments, the immunoconjugate that is mixed with225Ac in a reaction buffer comprises a chelating agent conjugated to an antibody or an antigen-binding fragment thereof.

[0109] In some embodiments, the reaction buffer comprises a radioprotectant.

[0110] In some embodiments, the radioprotectant is sodium ascorbate.[OHl] In some embodiments, the amount of sodium ascorbate is about from about 0.1 to about 8% (w / v), or about 0.1 to about 7% (w / v), or about 0.1 to about 6% (w / v), or about 0.1 to about 5 % (w / v), or about from 0.1% to 1.0% (w / v), or about from 0.1% to 0.5% (w / v), or about from 0.2% to 0.7% (w / v), or about from 0.5% to 1.0% (w / v), or about from 1.0% to 2.0% (w / v), or about from 1.0% to 1.5% (w / v), or about from 1.2% to 1.8% (w / v), or about from 1.5% to 2.0% (w / v), or about from 2.0% to 3.0% (w / v), or about from 2.0% to 2.5% (w / v), or about from 2.2% to 2.8% (w / v), or about from 2.5% to 3.0% (w / v), or about from 3.0% to 5.0% (w / v), or about from 3.0% to 4.0% or about 0.1% (w / v), or about 0.2% (w / v), or about 0.3% (w / v), or about 0.4% (w / v), or about 0.5% (w / v), or about 0.6% (w / v), or about 0.7% (w / v), or about 0.8% (w / v), or about 0.9% (w / v), or about 1.0% (w / v), or about 1.1% (w / v), or about 1.2% (w / v), or about 1.3% (w / v), or about 1.4% (w / v), or about 1.5% (w / v), or about 1.6% (w / v), or about 1.7% (w / v), or about 1.8% (w / v), or about 1.9% (w / v), or about 2.0% (w / v), or about 2.1% (w / v), or about 2.2% (w / v), or about 2.3% (w / v), or about 2.4% (w / v), or about 2.5% (w / v), or about 2.6% (w / v), or about 2.7% (w / v), or about 2.8% (w / v), or about 2.9% (w / v), or about 3.0% (w / v), or about 3.1% (w / v), or about 3.2% (w / v), or about 3.3% (w / v), or about 3.4% (w / v), or about 3.5% (w / v), or about 3.6% (w / v), or about 3.7% (w / v), or about 3.8% (w / v), or about 3.9% (w / v), or about 4.0% (w / v), or about 4.1% (w / v), or about 4.2% (w / v), or about 4.3% (w / v), or about 4.4% (w / v), or about 4.5% (w / v), or about 4.6% (w / v), or about 4.7%Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT](w / v), or about 4.8% (w / v), or about 4.9% (w / v), or about 5.0% (w / v), or about 6.0% (w / v), or about 7.0% (w / v), or about 8.0% (w / v).

[0112] In another embodiment, the radioprotectant is gentisic acid.

[0113] In another embodiment, the radioprotectant is L -methionine.

[0114] In another embodiment, the radioprotectant is N-acetyl-cysteine.

[0115] In some embodiments, the reaction buffer comprises a buffering agent.

[0116] In some embodiments, the buffering agent is sodium acetate.

[0117] In some embodiments, the concentration of sodium acetate is about from 0.1 M to 1.0 M, or about 0.1 M to 0.5 M, or about 0.1 M to 0.3 M, or about 0.2 M to 0.4 M, or about 0.3 M to 0.5 M, or about from 0.5 M to 1.0 M, or about 0.5 M to 0.7 M, or about 0.6 M to 0.8 M, or about 0.7 M to 1.0 M, or about 0.1 M, or about 0.2 M, or about 0.3 M, or about 0.4 M, or about 0.5 M, or about 0.6 M, or about 0.7 M, or about 0.8 M or about 0.9 M, or about 1.0 M, or about 0.15 M, or about 0.25 M, or about 0.35 M, or about 0.45 M, or about 0.55 M, or about 0.65 M, or about 0.75 M, or about 0.85 M, or about 0.95 M.

[0118] In another embodiment, the buffering agent is HEPES.

[0119] In another embodiment, the buffering agent is TRIS.

[0120] In another embodiment, the buffering agent does not comprise phosphate.

[0121] In some embodiments, the reaction buffer comprises a surfactant.

[0122] In some embodiments, the surfactant is Polysorbate 20.

[0123] In some embodiments, the concentration of Polysorbate 20 is about 0.01% to about 1% (v / v), or about 0.01% to 0.5% (v / v), or about 0.01% to 0.1% (v / v), or about 0.1% to 0.3% (v / v), or about 0.3% to 0.5% (v / v), or about 0.5% to 1.0% (v / v), or about 0.5% to 0.7% (v / v), or about 0.7% to 0.9% (v / v), or about 0.9% to 1.0% (v / v), or about 0.01% to 0.05% (v / v), or about 0.05% to 0.1% (v / v), or about 0.1% to 0.2% (v / v), or about 0.2% to 0.3% (v / v), or about 0.3% to 0.4% (v / v), or about 0.4% to 0.5% (v / v), or about 0.5% to 0.6% (v / v), or about 0.6% to 0.7% (v / v), or about 0.7% to 0.8% (v / v), or about 0.8% to 0.9% (v / v), or about 0.9% to 1.0% (v / v), or about 0.01% (v / v), or about 0.02% (v / v), or about 0.03% (v / v), or about 0.04% (v / v), or about 0.05% (v / v), or about 0.06% (v / v), or about 0.07% (v / v), or about 0.08% (v / v), or about 0.09% (v / v), or about 0.1% (v / v), or about 0.2% (v / v), or about 0.3% (v / v), or about 0.4% (v / v), or about 0.5% (v / v), or about 0.6% (v / v), or about 0.7% (v / v), or about 0.8% (v / v), or about 0.9% (v / v), or about 1.0% (v / v).

[0124] In another embodiment, the surfactant is Polysorbate 80.Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]

[0125] In another embodiment, the surfactant is Triton™-X(t-octylphenoxypolyethoxy ethanol).pH:

[0126] The pH of the reaction mixture is an important parameter that influences the efficiency of actinium-225 (223Ac) chelation to the immunoconjugate, as well as the stability and overall quality of the resulting radioimmunoconjugate. The present disclosure provides a range of pH values optimized for the process, ensuring reproducibility and high radiochemical yield across various conditions.

[0127] In some embodiments, the pH of the reaction mixture is adjusted to fall within a range of about 4.0 to about 8.0. This range ensures that both the immunoconjugate and the chelating agent maintain structural integrity while enabling efficient chelation with22?Ac.

[0128] In particular embodiments, the pH is adjusted to more specific ranges to optimize chelation and minimize side reactions.

[0129] In some embodiments the pH (before incubation) is adjusted to fall within a range of about 5.0 to about 7.0, or about 5.5 to about 6.5, or about 6.0 to about 6.5 or about 6.0 to about 6.2, or about 6.2 to about 6.5.

[0130] In some embodiments, the pH (before incubation) of the reaction mixture is adjusted to about 5.5, or about 5.6, or about 5.7, or about 5.8, or about 5.9, or about 6.0, or about 6.1 or about 6.2, or about 6.3, or about 6.4 or about 6.5.

[0131] In some embodiments, the pH of the reaction mixture may be adjusted using standard laboratory techniques. In some embodiments, the pH may be adjusted by adding small amounts of diluted hydrochloric acid or acetic acid to lower the pH. In some embodiments, the pH may be adjusted by adding sodium hydroxide or sodium acetate solution to raise the pH. In some embodiments, the pH may be adjusted by using a pH meter or calibrated test strips to monitor and verify the pH during preparation. In some embodiments, the reaction buffer is pre-formulated to achieve the desired pH without requiring additional adjustment.Mixing Conditions:

[0132] In some embodiments, the reaction mixture does not require agitation.

[0133] In some embodiments, the reaction mixture is mixed manually. The skilled artisan will appreciate that manual mixing can be performed using methods such as by pipetting up and down, by gently swirling the container in a slow circular motion or by gently stirring with a sterile rod.Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]

[0134] In some embodiments, the duration of mixing will vary range depending on the scale and method.REACTION VOLUME VARIATION AND EMBODIMENTS:

[0135] The process for producing a radioimmunoconjugate described herein is designed to accommodate a wide range of reaction volumes, enabling its use in small-scale research applications as well as large-scale clinical and commercial manufacturing. This adaptability is achieved by maintaining consistent reaction conditions across varying volumes, ensuring efficiency, product consistency, and reproducibility regardless of scale.Small Reaction Volume Embodiments:

[0136] In some embodiments, the reaction is performed in small volumes suitable for research, development, or preclinical applications.

[0137] In some embodiments, the reaction volume ranges from about 0.1 mL to about 5 mL.

[0138] In some embodiments, the reaction volume is about 0.1 mL, or about 0.5 mL, or about 1.0 mL, or about 2.0 mL, or about 5.0 mL.

[0139] Small-volume reactions are typically performed in microcentrifuge tubes, small reaction vials, or similar laboratory containers. Mixing may be achieved through manual pipetting.Medium Reaction Volume Embodiments:

[0140] In some embodiments, the reaction is performed in medium reaction volumes.

[0141] In some embodiments, the reaction volume ranges from about 5 mL to about 50 mL.

[0142] In some embodiments, the reaction volume is about 10 mL, or about 20 mL, or about 30 mL, or about 50 mL.

[0143] Medium-volume reactions are conducted in sterile reaction vessels, or polymer containers, that provide adequate space for mixing and processing. Mixing is achieved by gentle pipetting up and down or leaving to stand at 37 °C permitting thermal diffusion.Large Reaction Volume Embodiments:

[0144] In some embodiments, the process is scaled to large reaction volumes for clinical or commercial production.

[0145] In some embodiments, the reaction volume ranges from about 50 mL to about 500 mL.

[0146] In some embodiments, the reaction volume is about 100 mL, or about 200 mL, or about 300 mL, or about 400 mL, or about 500 mL.Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]

[0147] Large-volume reactions are conducted in industrial-grade, sterile reaction vessels equipped with mixing systems, such as magnetic stirrers, overhead stirrers, or rocking platforms.Volume Adjustment Considerations:

[0148] In adjusting the reaction volumes, the geometry and material of the reaction vessel are selected to minimize surface adsorption of reactants and ensure uniform mixing at all scales.

[0149] In adjusting the reaction volumes, mixing methods are optimized for each volume range to ensure homogeneity of the reaction mixture, prevent localized concentration gradients, and maximize chelation efficiency.ACTINIUM-225 CONCENTRATION VARIATION

[0150] The present disclosure encompasses embodiments for varying the concentration of actinium-225 (225AC). Such concentration variations allow for flexibility in adapting the process to different production scales, specific activities, or application requirements.

[0151] In some embodiments, the concentration of225Ac solution ranges from about 5 mCi / mL to about 50 mCi / mL, or about 10 mCi / mL to about 40 mCi / mL, or about 15 mCi / mL to about 30 mCi / mL. In other embodiments,22?Ac may be prepared at a target concentration of about 5 mCi / mL, or about 10 mCi / mL, or about 20 mCi / mL, or about 30 mCi / mL, or about 40 mCi / mL, or about 50 mCi / mL. The optimal concentration of225Ac can be selected based on the desired specific activity and the total radioactivity required for the production batch.Step (b): Incubation

[0152] In some embodiments, the reaction mixture is incubated under conditions that promote efficient chelation of225Ac to the immunoconjugate.Temperature:

[0153] In some embodiments, the reaction mixture is incubated at a particular temperature.

[0154] In some embodiments, the incubation temperature is about 20°C to about 50°C, or about 20°C to 30°C, or about 20°C to 25°C, or about 25°C to 30°C, or about 30°C to 40°C, or about 30°C to 35°C, or about 35°C to 40°C, or about 40°C to 50°C, or about 40°C to 45°C, or about 45°C to 50°C, or about 20°C, or about 21°C, or about 22°C, or about 23°C, or about 24°C, or about 25°C or about 26°C, or about 27°C, or about 28°C, or about 29°C, or about 30 °C, or about 31°C, or about 32°C, or about 33°C, or about 34°C, or about 33°C or about 35°C, or about 37°C or about 38°C, or about 39°C, or about 40°C, or about 41 °C, or about 42°C, or about 43 °C, or about 44°C, or about 45°C, or about 46°C, or about 47°C, or about 48°C, or about 49°C, or about 50°C.Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]Time:

[0155] In some embodiments, the reaction mixture is incubated for a particular incubation period.

[0156] In some embodiments, the incubation period is about 5 minutes to about 5 hours, or about 5 minutes to 60 minutes, or about 5 minutes to 30 minutes, or about 30 minutes to 60 minutes, or about 60 minutes to 120 minutes, or about 60 minutes to 90 minutes, or about 90 minutes to 120 minutes, or about 120 minutes to 300 minutes, or about 120 minutes to 180 minutes, or about 180 minutes to 240 minutes, or about 240 minutes to 300 minutes, or about 5 minutes, or about 6 minutes, or about 7 minutes, or about 8 minutes, or about 9 minutes, or about 10 minutes, or about 11 minutes, or about 12 minutes, or about 13 minutes, or about 14 minutes, or about 15 minutes, or about 16 minutes, or about 17 minutes, or about 18 minutes, or about 19 minutes, or about 20 minutes, or about 21 minutes, or about 22 minutes, or about 23 minutes, or about 24 minutes, or about 25 minutes, or about 26 minutes, or about 27 minutes, or about 28 minutes, or about 29 minutes, or about 35 minutes, or about 40 minutes, or about 45 minutes, or about 50 minutes, or about 60 minutes (1 hour), or about 70 minutes, or about 80 minutes, or about 90 minutes (1.5 hours), or about 100 minutes, or about 110 minutes, or about 120 minutes (2 hours), or about 130 minutes, or about 140 minutes, or about 150 minutes (2.5 hours) or about 160 minutes, or about 170 minutes, or about 180 minutes (3 hours) or about 190 minutes, or about 200 minutes, or about 220 minutes, or about 240 minutes (4 hours) or about 260 minutes, or about 280 minutes, or about 300 minutes (5 hours).

[0157] In some embodiments, the incubation period is of about 2 hours.

[0158] In some embodiments, the incubation period is of about 3 hours.

[0159] In some embodiments, the incubation period is not more than about 4 hours.Step (c): Filtration

[0160] The reaction mixture is filtered through a sterile filter to isolate the radioimmunoconjugate without intermediate purification steps. As used herein, “pore size” refers to the size of the pores in a sterile filter used to remove particulate contaminants, microorganisms, and impurities from the reaction mixture during the filtration step. The pore size is a critical parameter that determines the level of sterility and purity achieved in the process.

[0161] In some embodiments, pore size of the sterile filter may range from about 0.1 pm to about 1.0 pm.Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]

[0162] In some embodiments, the sterile filter pore size is about 0.1 pm, or about 0.2 pm or about 0.22 pm, or about 0.3 pm, or about 0.45 pm, or about 0.5 pm, or about 0.6 pm, or about 0.8 pm, or about 1.0 pm.

[0163] In some embodiments, the sterile filter pore size selected is tailored to a specific application.

[0164] In some embodiments, the sterile filter pore size selected is tailored for bacterial sterilization.

[0165] In some embodiments, the sterile filter pore size selected is tailored for viral removal.

[0166] In some embodiments, the sterile filter pore size selected is tailored for large-particle filtration.

[0167] In some embodiments, the selection of the sterile filter pore size may align with regulatory guidelines for sterility. In a particular embodiment, the selection of the sterile filter pore size aligns with Good Manufacturing Practices (GMP). In another embodiment, the selection of the sterile filter pore size aligns with research-grade processes.Container:

[0168] In some embodiments, the radioimmunoconjugate is directly filtered into a sterile container.

[0169] In some embodiments, the sterile container is a polymer container, suitable for pharmaceutical use.Dilution

[0170] As used herein, “dilution” refers to reducing radioactive concentration and / or specific activity of a reaction mixture or radioimmunoconjugate by increasing the effective solution volume, including by addition of a dilution buffer, formulation buffer, vessel rinsing, filter washing, formulation volume adjustment, or combinations thereof.

[0171] In some embodiment, the process further comprises diluting the reaction mixture after step (b) and / or after step (c) with a dilution buffer. In some embodiment, the diluting step is performed after the filtering step (c) with a sterile dilution buffer. In some embodiment, the diluting step is performed after step (b) and before step (c). For example, after step (b), the radiolabeled reaction can be diluted with a dilution buffer and the diluted reaction mixture can be filtered through a sterile filter to produce a sterile radioimmunoconjugate.

[0172] In some embodiment, the dilution buffer comprises a radioprotectant, a buffering agent and a surfactant. In some embodiment, the dilution buffer comprises sodium ascorbate, acetic acid, and / or Polysorbate 20.Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]

[0173] In some embodiment, the dilution buffer comprises about 0.1-5% (w / v) sodium ascorbate, about 0.5 - 10 mM acetic acid, and about 0.01% to about 1% (v / v) Polysorbate 20.

[0174] In some embodiment, the dilution buffer may also function as a formulation buffer for the final drug product.

[0175] In certain embodiments, dilution of the reaction mixture facilitates handling, formulation, sterile filtration, and control of the specific activity of the radioimmunoconjugate. In other embodiments, particularly at lower activity scales, the process may be performed without a discrete dilution step, for example where an equivalent reduction in radioactive concentration or specific activity is achieved through vessel rinsing, filter washing, formulation volume adjustment, or combination thereof, as described herein.MOLAR RATIOS AND SPECIFIC ACTIVITYMolar Ratio Embodiments

[0176] The molar ratio of the immunoconjugate (e.g., DOTA-hl lb6) to the radionuclide (225Ac) may be an important factor in achieving high chelation efficiency, ensuring product stability, and eliminating the need for intermediate purification steps.General Ratios

[0177] In some embodiments, the molar ratio of the immunoconjugate to225Ac is maintained at or above a minimum threshold to ensure sufficient chelators are available for chelating22?Ac and competing non-radioactive metals.

[0178] In some embodiments, the molar ratio of the immunoconjugate to225Ac is at least about 440 to 1.

[0179] In some embodiments, the molar ratio of the immunoconjugate to225Ac is about 500 to 1 to about 1000 to 1.

[0180] In a particular embodiment, the molar ratio of the immunoconjugate to225Ac is approximately 880 to 1.

[0181] High Ratios for Chelation Efficiency: In one embodiment, maintaining a molar ratio of the immunoconjugate to225Ac of at least about 440 to 1 ensures high chelation efficiency, with at least about 95% of the225Ac chelated to the immunoconjugate. This reduces the presence of unchelated225AC and minimizes the risk of requiring additional purification steps.

[0182] Very High Ratios: In another embodiment, a molar ratio of the immunoconjugate to225Ac of about 880:1 is employed to provide near-quantitative chelation, ensuring robust and reliableAttorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]labeling yields across a wide range of225Ac sources with varying levels of non-radioactive contaminants.

[0183] Lower Ratios and Challenges: In contrast, embodiments wherein a molar ratio of the immunoconjugate to225Ac drops below 440:1 may result in decreased chelation efficiency, requiring intermediate purification steps to remove unchelated225Ac or byproducts. In one such embodiment, a molar ratio of the immunoconjugate to225Ac of 220:1 yields lower efficiency and stability, while a molar ratio of the immunoconjugate to225Ac of approximately 110: 1 demonstrates a significant reduction in chelation efficiency.Specific Activity Embodiments

[0184] Specific activity, defined as the radioactivity per unit mass of immunoconjugate (e.g., pCi / mg), may be an important parameter influencing the chelation efficiency and stability of the final product. Embodiments of the process described herein address specific activities optimized for clinical and commercial production.

[0185] The relationship between molar ratio, specific activity, chelation efficiency, and product stability described herein further elucidate experimental observations disclosed herein, including radiochemical purity and stability assessments such as DTPA challenge and chromatographic analyses performed at varying activity levels.Low to Moderate Specific Activities

[0186] In some embodiments, the specific activity of the radioimmunoconjugate is maintained at or below about 200 pCi / mg to achieve high chelation efficiency and robust product stability.

[0187] In some embodiments, specific activities at approximately 100 pCi / mg demonstrate chelation efficiencies of about 99.6% and stability of about 98% under DTPA challenge.Threshold Specific Activities

[0188] In another embodiment, the specific activity threshold of 200 pCi / mg is defined as the upper limit for maintaining high efficiency (e.g., >99.5%) without requiring intermediate purification steps.

[0189] High Specific Activities and Challenges: Embodiments addressing higher specific activities, such as about 400 pCi / mg or above, may demonstrate a decline in chelation efficiency and stability; the examples below show that:• at 400 pCi / mg, chelation efficiency may decrease to about 95.3%, and stability under DTPA challenge is reduced to about 88.1%; and thatAttorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]• at 800 pCi / mg, chelation efficiency may further decline to about 20%, with stability under DTPA challenge dropping to about 13%.

[0190] At 1600 pCi / mg, chelation efficiency and stability are substantially compromised.

[0191] Optimized Specific Activities: Embodiments may target specific activities of about 100 pCi / mg to about 200 pCi / mg as optimal ranges for ensuring reliable and reproducible production processes, while avoiding the need for intermediate purification steps.

[0192] Clinical Applications: In certain embodiments, specific activities of about 100 pCi / mg to about 200 pCi / mg are selected to support therapeutic applications requiring consistent and high-quality radiolabeling. For diagnostic purposes, higher specific activities may be tolerated but may require additional processing steps.

[0193] Scalability with Specific Activity: In one embodiment, the specific activity is carefully controlled during large-scale production to ensure that product consistency and chelation efficiency are maintained at a high level. For example, scaling up to produce radioimmunoconjugates with a total radioactivity of about 50 mCi to about 1000 mCi would still maintain a specific activity of about 100 pCi / mg to about 200 pCi / mg.

[0194] Combined Embodiments: The interplay between molar ratio and specific activity is critical to the process described herein:

[0195] High Molar Ratios at Optimal Specific Activities: In one embodiment, maintaining a molar ratio of at least 440: 1 ensures that specific activities at or below 200 pCi / mg yield high chelation efficiencies (>99%) and robust stability under DTPA challenge.

[0196] Lower Molar Ratios and High Specific Activities: Embodiments where specific activity exceeds 200 pCi / mg or where the molar ratio drops below 440: 1 may necessitate additional purification steps to remove free225Ac and byproducts.

[0197] In some embodiments, the molar ratio of the immunoconjugate to225Ac is of at least about 440: 1 and the specific activity is at or below 200 pCi / mg.

[0198] In some embodiments, the molar ratio of the immunoconjugate to225Ac is of at least about 440: 1 and the specific activity is of about 100 pCi / mg to about 200 pCi / mg.

[0199] In some embodiments, the molar ratio of the immunoconjugate to225Ac is of at least about 440:1 and the specific activity is of about 100 pCi / mg.

[0200] In some embodiments, the molar ratio of the immunoconjugate to225Ac is of at least about 440:1 and the specific activity is of about 200 pCi / mg.Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]

[0201] In some embodiments, the molar ratio of the immunoconjugate to225Ac is about 500: 1 to about 1000:1 and the specific activity is at or below 200 pCi / mg.

[0202] In some embodiments, the molar ratio of the immunoconjugate to225Ac is about 500: 1 to about 1000:1 and the specific activity is of about 100 pCi / mg to about 200 pCi / mg.

[0203] In some embodiments, the molar ratio of the immunoconjugate to225Ac is about 500: 1 to about 1000:1 and the specific activity is of about 100 pCi / mg.

[0204] In some embodiments, the molar ratio of the immunoconjugate to225Ac is about 500: 1 to about 1000:1 and the specific activity is of about 200 pCi / mg.

[0205] In some embodiments, the molar ratio of the immunoconjugate to225Ac is of about 880:1 and the specific activity is at or below 200 pCi / mg.

[0206] In some embodiments, the molar ratio of the immunoconjugate to225Ac is of about 880: 1 and the specific activity is of about 100 pCi / mg to about 200 pCi / mg.

[0207] In some embodiments, the molar ratio of the immunoconjugate to225Ac is of about 880:1 and the specific activity is of about 100 pCi / mg.

[0208] In some embodiments, the molar ratio of the immunoconjugate to225Ac is of about 880:1 and the specific activity is of about 200 pCi / mg.

[0209] In some embodiments, maintaining both a high molar ratio (e.g., 880:1) and a specific activity of <200 pCi / mg enables robust and scalable production without intermediate purification steps, ensuring compliance with GMP requirements.INTERMEDIATE PURIFICATION STEPS

[0210] In traditional processes for producing radioimmunoconjugates, intermediate purification steps are often required to ensure the removal of unreacted reagents, by-products, and impurities before proceeding to subsequent steps in the production workflow. These intermediate purification steps may involve techniques such as:• Size Exclusion Chromatography (SEC): Used to separate free radionuclide or chelator molecules from the immunoconjugate.• Ultrafiltration / Diafiltration: Applied to concentrate the reaction mixture and exchange buffer systems.• Precipitation and Centrifugation: Used to remove unreacted reagents or insoluble impurities.• Vacuum Filtration or Gravity Filtration: To clarify the reaction mixture and removeparticulates.Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]CHALLENGES OF INTERMEDIATE PURIFICATION STEPS IN LARGE-SCALE MANUFACTURING:Time Consumption:

[0211] Intermediate purification processes require extended setup and runtime due to the careful handling of radioactive materials. For example:a. Setup Time: Equipment must be assembled, sterilized, and validated before use. b. Run Time: Chromatographic purification, particularly on a large scale, can take hours to complete as the process depends on the flow rates and capacity of the columns used.c. Post-Processing Time: Cleaning, decontaminating, and validating equipment between batches can further delay production.d. Equipment Complexitye. The need for multiple purification units (e.g., ultrafiltration devices, chromatography columns) complicates the process. On a large scale, this requires specialized infrastructure, such as:• Clean rooms for sterile operations.• Radiation shielding for handling radionuclides.• Dedicated equipment to prevent cross-contamination.Product Yield Loss:

[0212] Each intermediate step introduces opportunities for product loss:• Adsorption of the immunoconjugate to the filtration membrane or column resin.• Dilution during diafiltration or buffer exchange steps.• Physical loss during transfers between vessels or devices.Risk of Contamination:

[0213] Every additional step increases the risk of microbial or particulate contamination, especially in a manufacturing environment. The use of intermediate purification requires heightened monitoring and validation of sterility, adding further costs and regulatory burdens.ELIMINATING INTERMEDIATE PURIFICATION STEPS:

[0214] The process described herein eliminates the need for intermediate purification steps entirely. The described process significantly streamlines production by combining all critical steps into a seamless workflow, from mixing to radiolabeling and final filtration.Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]

[0215] In preferred embodiments, the process excludes intermediate purification steps between mixing, incubation, and sterile filtration. In other embodiments, one or more additional processing or handling steps may be employed, provided that such steps do not materially affect radiochemical purity, product stability, or specific activity.

[0216] Key aspects of the disclosed process that enable this simplification include:Optimized Reaction Conditions:

[0217] The inclusion of a radioprotectant, buffering agent, and surfactant in the reaction buffer minimizes the formation of impurities, enabling the reaction to proceed to completion without additional purification steps.

[0218] Precise control of pH and temperature ensures efficient chelation of actinium-225 to the immunoconjugate, reducing the presence of unreacted radionuclide.Direct Sterile Filtration:

[0219] The reaction mixture is directly filtered through a sterile 0.22 pm filter (or other suitable pore size), removing particulate contaminants and achieving sterility in a single step.

[0220] This eliminates the need for size exclusion chromatography, ultrafiltration, or buffer exchange, which are typically required to achieve similar levels of purity and sterility.BENEFITS OF AVOIDING INTERMEDIATE PURIFICATION STEPS:

[0221] Increased Efficiency: Reducing the process to a single reaction and filtration step saves time and simplifies workflows, enabling faster production cycles.

[0222] Reduced Costs: Eliminating the need for intermediate purification steps reduces the material, labor, and equipment costs associated with large-scale production.

[0223] Improved Yield: The lack of intermediate steps minimizes product loss, ensuring higher recovery of the radioimmunoconjugate.

[0224] Enhanced Sterility Assurance: Direct filtration through a sterile filter minimizes the risk of contamination, ensuring compliance with regulatory standards for sterile pharmaceuticals.

[0225] Simplicity in Scale-Up: The streamlined process reduces the complexity of scaling up production, enabling consistent results from small-batch to large-batch manufacturing. Notably, eliminating all intermediate chromatographic step enables effective scale-up and facilitates process automation. Automation is a critical aspect of scale-up feasibility as it minimizes or entirely eliminates production personnel's exposure to high levels of radiation during manufacturing.Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]

[0226] In some embodiments, sterile filtration is performed on the reaction mixture, on a diluted reaction mixture, and / or on a final formulation to produce a sterile radiolabeled immunoconjugate.IMMUNOCONJUGATES

[0227] The present disclosure provides processes and compositions related to immunoconjugates, which serve as the foundational targeting components for the radiolabeled constructs described herein. Immunoconjugates are broadly defined as conjugates formed by covalently linking an antibody or antigen-binding fragment thereof to a functional moiety, such as a chelating agent, for subsequent radiolabeling. The immunoconjugates described herein are specifically designed to facilitate efficient chelation with radionuclides, such as225Ac, and to maintain their targeting and functional integrity throughout the labeling and purification processes.General Characteristics of Immunoconjugates:

[0228] As used herein, the term "immunoconjugate" refers to any antibody, antibody fragment, or antigen-binding fragment that has been chemically or biologically conjugated to a functional moiety capable of binding a radionuclide. The immunoconjugates of the present disclosure possess several desirable characteristics:• High Binding Specificity: The antibody or antigen-binding fragment retains its specificity for a target antigen, enabling selective delivery of the radionuclide to the desired tissue or cell type.• Stability: The covalent linkage between the antibody and the chelating agent is robust, ensuring that the functional moiety remains conjugated under physiological and manufacturing conditions.• Compatibility with Radiolabeling: The chelating agent is selected and optimized for strong and specific binding to radionuclides, such as225Ac, facilitating efficient radiolabeling. Antibodies and Antigen-binding fragments:

[0229] In some embodiments, the immunoconjugate comprises a full-length antibody. In some embodiments, the full-length antibody is monoclonal. In some embodiments, the full-length antibody is polyclonal. In some embodiments, the full-length antibody is chimeric. In some embodiments, the full-length antibody is humanized. In one embodiment described herein, the immunoconjugate comprises a full-length humanized monoclonal antibody.

[0230] In some embodiments, the immunoconjugates comprises antigen-binding fragments. In some embodiments, the antigen-binding fragment is a Fab. In some embodiments, the antigen-Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]binding fragment is a Fab'. In some embodiments, the antigen-binding fragment is a F(ab')2. In some embodiments, the antigen-binding fragment is a single-chain variable fragment (scFv).Target Specificity of the Immunoconjugates:

[0231] The antibodies or antigen-binding fragments used in the immunoconjugates are designed to target specific antigens associated with particular diseases or conditions.

[0232] In some embodiments, the immunoconjugate targets a specific antigen associated with prostate cancer.

[0233] In some embodiments, the immunoconjugate comprises an antibody or antigen-binding fragment thereof that specifically binds to human kallikrein-related peptidase 2 (hK2).

[0234] In some embodiments, the antibody is or comprises a heavy chain CDR1 (HCDR1) of SEQ ID NO: 5;a heavy chain CDR2 (HCDR2) of SEQ ID NO: 6;a heavy chain CDR3 (HCDR3) of SEQ ID NO: 7;a light chain CDR1 (LCDR1) of SEQ ID NO: 8;a light chain CDR2 (LCDR2) of SEQ ID NO: 9; anda light chain CDR3 (LCDR3) of SEQ ID NO: 10.

[0235] In some embodiments, the antibody is or comprises a variable heavy chain (VH) sequence of SEQ ID NO: 1 and a variable light chain (VL) sequence of SEQ ID NO: 2.

[0236] In some embodiments, the antibody is or comprises a heavy chain (HC) sequence of SEQ ID NO: 3 and a light chain (LC) sequence of SEQ ID NO: 4.

[0237] In some embodiments, the antibody is or comprises a humanized antibody, e g., a humanized monoclonal antibody, such as hl 1B6.Chelating Agents:

[0238] The immunoconjugates described herein incorporate a chelating agent that is covalently attached to the antibody or an antigen-binding fragment thereof. The chelating agent serves as the site for binding the radionuclide.

[0239] In some embodiments, the chelating agent is or comprises a macrocyclic chelating moiety. Examples of macrocyclic chelating moi eties include, without limitation, 1,4,7,10-tetraazacy cl ododecane- 1,4, 7, 10, tetraacetic acid (DOTA), S-2-(4-isothiocyanatobenzyl)-l,4,7-triazacyclononane-l,4,7-triacetic acid (NOTA), 1,4,8, 11-tetraazacyclodocedan-l, 4,8,11-tetraacetic acid (TETA), 3,6,9,I5-tetraazabicyclo[9.3.1]-pentadeca-l(15),l l,13-triene-4-(S)-(4-Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]isothiocyanatobenzyl)-3,6,9-triacetic acid (PCTA), 5-S-(4-aminobenzyl)-l-oxa-4,7,10-triazacyclododecane-4,7,10-tris(acetic acid) (D03A), or a derivative thereof. In some embodiments, the chelating agent is or comprises 1,4,7,10-tetraazacyclododecane-1,4, 7, 10, tetraacetic acid (DOTA). In some embodiments, the chelating agent is or comprises S-2-(4-isothiocyanatobenzyl)-l,4,7-triazacyclononane-l,4,7-triacetic acid (NOTA). In some embodiments, the chelating agent is or comprises 1,4, 8,1 l-tetraazacyclodocedan-1,4,8,11-tetraacetic acid (TETA). In some embodiments, the chelating agent is or comprises 3,6,9,15-tetraazabicyclo[9.3.1] -pentadeca- 1(15), 1 l,13-triene-4-(S)-(4-isothiocyanatobenzyl)-3,6,9-triacetic acid (PCTA). In some embodiments, the chelating agent is or comprises 5-S-(4-aminobenzyl)-l-oxa-4,7,10- triazacyclododecane-4,7,10-tris(acetic acid) (DO3A). In some embodiments, the chelating agent is or comprises DOTA, DFO (desferrioxamine), DTPA (calcium trinatrium diethylenetriaminepentaacetic acid), NOTA, or TETA.

[0240] In some embodiments, the skilled person will select an alternative chelating agent known in the art, depending on the radionuclide and application.

[0241] The chelating agent is typically conjugated to the antibody or antigen-binding fragment thereof through reactive functional groups, such as isothiocyanate or NHS-ester groups, which form covalent bonds with lysine residues on the antibody or an antigen binding fragment thereof. The chelator-to-antibody ratio (CAR) is carefully controlled to balance radiolabeling efficiency and immunoconjugate functionality.

[0242] In some embodiments, the antibody (e.g., the antibody of

[0225] -

[0229] ) or antigen-binding fragment thereof) is conjugated to the chelator (e.g., to DOTA) via a linker. The linker may e g., be or comprise 2-isothiocyanatobenzyL

[0243] In some embodiments, the antibody or antigen-binding fragment thereof is conjugated to 2-(4-isothiocyanatobenzyl)-l,4,7, 10-tetraazacyclododecane-l,4,7, 10-tetraacetic acid, more particularly to one or more 2-(4-isothiocyanatobenzyl)-l, 4,7, 10-tetraazacyclododecane-l, 4, 7, 10-tetraacetic acid groups. The one or more 2-(4-isothiocyanatobenzyl)- 1,4, 7, 10-tetraazacyclododecane-l, 4, 7, 10-tetraacetic acid group(s) may e.g., be covalently linked to one or more amino acid residues in the Fc region of the antibody or antigen-binding fragment thereof.

[0244] In some embodiments, the radioimmunoconjugate may comprise an average of from about 1 to about 4 chelator molecules conjugated to the antibody or antigen binding fragment thereof.

[0245] Methods of Preparing Immunoconjugates:Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]

[0246] In some embodiments, the preparation of immunoconjugates comprises two main steps:• conjugation of the chelating agent to the antibody or an antigen-binding fragment thereof;and• purification of the resulting conjugate.

[0247] In an exemplary embodiment, the method of preparing immunoconjugates comprises:Conjugation:

[0248] The antibody or an antigen-binding fragment is dissolved in a suitable buffer to optimize conjugation conditions.

[0249] A solution of the chelating agent (e.g., p-SCN-Bn-DOTA) is added to the antibody or antigen-binding fragment there at a molar ratio sufficient to achieve the desired CAR, typically ranging from 2:1 to 10:1.

[0250] The reaction mixture is incubated at a temperature of about 25°C to about 37°C for 1 to 24 hours, with gentle agitation to ensure uniform mixing.Purification:

[0251] The reaction mixture is purified to remove excess chelating agent and by-products using techniques known in the art, such as ultrafiltration, diafiltration, or size exclusion chromatography (SEC).

[0252] The purified immunoconjugate is buffer-exchanged into a suitable storage medium.

[0253] The purified immunoconjugate is characterized using analytical techniques such as size exclusion high-performance liquid chromatography (SEC-HPLC), mass spectrometry, or ultraviolet (UV) spectrophotometry. The CAR is determined to ensure consistent product quality.

[0254] Properties of Immunoconjugates: The immunoconjugates prepared according to the methods described herein exhibit the following properties:• High Purity: The immunoconjugates are free from significant levels of unreacted chelator or antibody.• Defined Composition: The CAR is consistent across batches, ensuring reproducibility in radiolabeling.• Biological Activity: The immunoconjugates retain their antigen-binding capability and are suitable for use in therapeutic or diagnostic applications.

[0255] Advantages of the Immunoconjugates in Radiolabeling: The immunoconjugates described herein are specifically optimized for radiolabeling with actinium-225. Advantages include:Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]• High Labeling Efficiency: The chelating agents form stable complexes with225Ac, reducing the presence of free radionuclide.• Reduced Radiolysis: The inclusion of radioprotectants in the reaction buffer minimizes damage to the immunoconjugate during radiolabeling.• Scalability: The methods for preparing immunoconjugates are compatible with large-scale manufacturing processes.

[0256] Applications of Immunoconjugates: The immunoconjugates described herein can be used in a wide range of applications, including:• Therapeutics: Delivery of radionuclides for targeted alpha therapy (TAT) in cancer and other diseases.• Diagnostics: Radioimmunoconjugates can serve as imaging agents for positron emission tomography (PET) or single-photon emission computed tomography (SPECT).• Research Tools: Immunoconjugates can be used to study antigen expression and distribution in preclinical models.RADIOIMMUNOCONJUGATES

[0257] The present disclosure provides compositions and processes for the production and use of radioimmunoconjugates, which represent a class of biologically targeted radiopharmaceuticals. Radioimmunoconjugates are molecular constructs comprising a radionuclide that is chelated to a conjugated targeting moiety. In some embodiments, the targeting moiety is an immunoconjugate comprising an antibody or antigen-binding fragment thereof. Radioimmunoconjugates produced by the process described herein combine the targeting specificity of immunoconjugates with the therapeutic or diagnostic capabilities of radionuclides, enabling highly selective applications in cancer treatment, imaging, and other therapeutic areas.General Characteristics of Radioimmunoconjugates:

[0258] As used herein, the term "radioimmunoconjugate" refers to a complex formed by the radiolabeling of an immunoconjugate with a radionuclide. In some embodiments, the immunoconjugate comprises an antibody or antigen-binding fragment thereof covalently linked to a chelating agent that binds the radionuclide with high specificity and stability. The radioimmunoconjugates described herein exhibit the following characteristics:Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]• High Targeting Specificity: The antibody or antibody fragment binds selectively to its antigen, ensuring precise delivery of the radionuclide to the target tissue or cells.• Radiochemical Stability: The chelating agent forms a stable complex with the radionuclide, minimizing the release of free radionuclide during storage or in vivo application.• Therapeutic Efficacy: The radionuclide delivers localized radiation to the target tissue, resulting in therapeutic effects, such as cell killing in the context of cancer treatment.• Diagnostic Utility: Certain radionuclides can be used for imaging applications, providing real-time visualization of target tissues.Radionuclide Component:

[0259] In some embodiments, the radionuclide used in the radioimmunoconjugates of the present disclosure225Ac, a radionuclide with properties that make it particularly well-suited for targeted alpha therapy (TAT).

[0260] Actinium-225 Properties:• Radiation Type:225Ac emits alpha particles, which have high linear energy transfer (LET) and a short path length, ensuring localized and potent cytotoxicity.• Half-Life:225Ac has a half-life of about 10 days, providing a balance between therapeutic efficacy and practical storage and logistics.• Decay Chain: The decay of223Ac produces daughter isotopes, including bismuth-213 (213Bi), which contribute additional therapeutic effects.

[0261] The radionuclide is introduced into the immunoconjugate through the process described herein.

[0262] Chelation of Actinium-225: Chelation is a critical step in the preparation of radioimmunoconjugates, ensuring the stable incorporation of223Ac into the immunoconjugate. In some embodiments, the chelating agent, (l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid (DOTA) or its derivatives), is covalently attached to the immunoconjugate described herein. Key properties of the chelating agents used in the described methods include:• High Affinity for225Ac: The chelating agent forms a stable coordination complex with the radionuclide.• Resistance to Radiolysis: The chelate is designed to withstand the radiolytic environment created by223Ac and its daughter isotopes.Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]• Biological Compatibility: The chelated radionuclide does not interfere with the biological activity or targeting specificity of the immunoconjugate.

[0263] Quality Control of Radioimmunoconjugates: The radiochemical purity of the radioimmunoconjugate produced by the described process is assessed using techniques such as instant thin-layer chromatography (iTLC) or size-exclusion high-performance liquid chromatography (SEC-HPLC). In some embodiments, the radiochemical purity achieved is at least 95%.STABILITY CHALLENGE TO CONFIRM CHELATION STABILITY OF THE RADIOIMMUNOCONJUGATE

[0264] The stability of the described radioimmunoconjugate, particularly the chelation of225Ac to the immunoconjugate, is critical for ensuring the efficacy and safety of the resulting product for therapeutic or diagnostic applications. As such, performing a stability challenge is an integral part of the process to confirm that the chelation of the radiometal and the conjugate remains intact under various conditions, including those mimicking physiological environments.

[0265] Method for performing a stability assessment includes, e.g., DTPA Stability Challenge.

[0266] The method described herein to confirm chelation stability involves subjecting the radioimmunoconjugate to a chelating agent such as diethylenetriaminepentaacetic acid (DTPA). DTPA is used as a competitive chelator to challenge the strength of the225Ac-chelating agent complex.

[0267] In some embodiments, the radioimmunoconjugate is incubated with an excess of DTPA under controlled conditions and a temperature of about 37°C.

[0268] In some embodiments, the incubation is carried out for a defined period. In some embodiments, the incubation is carried out for 30 minutes. In some embodiments, the incubation is carried out for 1 hour. In some embodiments, the incubation is carried out for or up to 24 hours. In some embodiments, the incubation is carried out depending on the specific experimental design.

[0269] Following incubation with DTPA, the proportion of225Ac that remains bound to the immunoconjugate is assessed.

[0270] In one embodiment, the radioimmunoconjugate is considered stable if at least 95% of the225AC remains chelated to the immunoconjugate after 30 minutes of incubation with DTPA.

[0271] To ensure robustness, in some embodiments, the stability of the radioimmunoconjugate may also be assessed under various conditions to mimic storage, handling, and in vivo environments.Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]

[0272] In some embodiments, periodic assessments by sec-HPLC are conducted to confirm that the radiochemical purity remains above 95%.

[0273] In some embodiments, stability is tested under storage and physiological conditions, such as incubation in formulation buffer, phosphate-buffered saline (PBS), or human serum at 37°C, to simulate various environments. The stability test of225Ac by the immunoconjugate is measured over time (e.g., 1 hour, 24 hours, or 96 hours) to ensure minimal decomposition under the conditions described.

[0274] In some embodiments, the stability assessment is performed to evaluate the stability of the radioimmunoconjugate under non-ideal conditions, such as elevated temperatures (e.g., 50°C) or extreme pH ranges (e.g., pH 3-10).

[0275] In some embodiments, the results of the stability assessment are documented to confirm that the radioimmunoconjugate exhibits sufficient chelation stability and radiochemical purity for clinical or commercial use.

[0276] In a particular embodiment, the radioimmunoconjugate is reported as stable if• At least 95% of the225Ac remains chelated to the immunoconjugate after DTPA challenge.• Radiochemical purity remains above 90% during storage at 4°C for at least 96 hours.• The radioimmunoconjugate retains chelation stability under physiological conditions for clinically relevant timeframes.SCALABILITY EMBODIMENTS

[0277] The process described herein for producing a radioimmunoconjugate of the disclosure is inherently designed to be scalable, enabling efficient and reproducible production across a wide range of radioactivity levels, from research-scale preparations to clinical and commercial-scale manufacturing. The described process eliminates intermediate purification steps and utilizes optimized reaction conditions, facilitating its adaptation to higher production volumes without compromising product quality, radiochemical yield, or sterility.Small-Scale Production

[0278] In some embodiments, the process described herein is performed on a small scale, suitable for research or preclinical applications. In some embodiments, the radioactivity of the radioimmunoconjugate produced by the process described herein ranges from about 0.1 mCi to about 5 mCi, or about 0.5 mCi to about 2 mCi or about 1 mCi to about 5 mCi. This scale isAttorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]particularly useful for optimizing reaction conditions, evaluating chelation efficiency, and conducting stability studies under controlled laboratory conditions.Medium- Scale Production

[0279] In some embodiments, the process described herein is performed on a medium scale, suitable for early-stage clinical trials or limited therapeutic applications. In some embodiments, the process described herein can be scaled to produce the radioimmunoconjugate with a final radioactivity of about 5 mCi to about 20 mCi, or about 10 mCi to about 15 mCi, or about 15 mCi to about 20 mCi. In such embodiments, adjustments may be made to reaction vessel sizes, mixing methods, and sterile filtration systems to accommodate increased reaction volumes.Large-Scale Production

[0280] In some embodiments, the process described herein is performed on a large scale, suitable for commercial production. In some embodiments, the process described herein can be scaled up to produce the radioimmunoconjugate with a final radioactivity of about with a final radioactivity of about 20 mCi to about 1,000 mCi, or about 50 mCi to about 500 mCi, or about 500 mCi to about 1,000 mCi. In such embodiments, the reaction mixture volume may be scaled proportionally while maintaining critical component concentrations (e.g., chelating agent, radioprotectant, buffering agent, and surfactant). In such embodiments, the mechanical mixing methods such as orbital shaking, magnetic stirring, or use of industrial-grade rotary mixers may be employed to ensure uniform distribution of reactants in larger volumes. In such embodiments, sterile filtration may be performed using larger filtration systems, such as high-throughput sterile filtration units.RADIOIMMUNOCONJUGATE COMPOSITIONS AND METHODS OF USE:

[0281] Embodiments of the present disclosure provide methods of treating cancer in a patient, the method comprising administering to the patient a therapeutically effective amount of a radioimmunoconjugate produced by the process provided herein. According to an embodiment, the method comprises administering to the patient a therapeutically effective amount of a composition comprising a radioimmunoconjugate and one or more pharmaceutically acceptable excipients.

[0282] Embodiments of the present disclosure are particularly useful in treating patients that have been diagnosed with prostate cancer; for example, patients that have late-stage prostate cancer. According to an embodiment, the cancer is non-localized prostate cancer. According to another embodiment, the cancer is metastatic prostate cancer. According to another embodiment, the cancer is castration-resistant prostate cancer (CRPC). According to another embodiment, the cancer isAttorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]metastatic castration-resistant prostate cancer (mCRPC). According to another embodiment, the cancer is mCRPC with adenocarcinoma. According to particular embodiments, testosterone castrate levels of the patient are about 50 ng / dL or less. According to additional embodiments, the patient had prior exposure to at least one androgen receptor (AR) targeted therapy; for example, abiraterone acetate, enzalutamide, apalutamide, darolutamide, or combinations of any of the foregoing. According to additional embodiments, the patient had prior chemotherapy; for example, the chemotherapy involved administration of taxane. According to another embodiment, the patient had prior orchiectomy or medical castration. According to another embodiment, the patient is receiving ongoing androgen deprivation therapy with a gonadotropin releasing hormone (GnRH) agonist or antagonist.

[0283] In accordance with embodiments of the treatment methods described herein, the radioimmunoconjugate administered to the patient is produced by the process described herein.

[0284] Radioimmunoconjugate compositions of the present disclosure may be administered via any suitable route known to those skilled in the art. For example, the compositions may be administered parenterally. Non-limiting examples of routes of administration include intravenous (IV), intramuscular or subcutaneous, or they may be administered by infusion techniques. In certain aspects, the methods of treatment herein comprise injecting the composition intravenously.

[0285] It will be appreciated by persons skilled in the art that pharmaceutical compositions of the present disclosure may be administered alone or in combination with one or more additional therapeutic agents or imaging agents or modalities as determined by the attending physician. A composition of the present disclosure may be administered to the patient before or concurrently with other therapeutic modalities for the treatment of prostate cancer.

[0286] According to particular embodiments, radioimmunoconjugates of the present disclosure are administered in admixture with one or more pharmaceutically acceptable excipients. The compositions may be prepared using the process described herein. In particular embodiments, the compositions are sufficiently storage stable and suitable for administration to humans.

[0287] The excipients may be selected by one skilled in the art and may take a wide variety of forms depending upon the desired route of administration. For example, for parenteral administration, the excipients may include sterile water, and other ingredients may be added to increase solubility and preservation of the composition. Injectable suspensions or solutions mayAttorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]also be prepared utilizing excipients that comprise aqueous carriers and / or appropriate additives such as, solubilizers and preservatives.

[0288] In additional embodiments, the excipients comprise a diluent. The diluent may be an aqueous or non-aqueous solution with the purpose of diluting the radioimmunoconjugate composition. For example, a diluent may comprise one or more of saline, water, polyethylene glycol, propylene glycol, ethanol or oils (such as safflower oil, corn oil, peanut oil, cottonseed oil or sesame oil).

[0289] The radioimmunoconjugate compositions produced by the process described herein may be subjected to conventional pharmaceutical operations such as sterilization and / or may contain conventional adjuvants. Compositions may also contain aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats and / or solutes which render the formulation isotonic with the blood of the intended recipient.

[0290] In still other embodiments, the excipients may comprise one or more of a binder, carbohydrate, coating agent, coloring agent, disintegrating agent, dispersing agent, emulsifier, filler, flavoring agent, granulating agent, lipid, lubricant, mineral, polymer, preservative, radioprotectant, solubilizing agent, stabilizer, suspending agent, sweetener, thickening agent, wetting agent, or combinations thereof.

[0291] According to particular embodiments, the excipients comprise at least one radioprotectant. In certain embodiments, the radioimmunoconjugate composition comprises: a radioimmunoconjugate and one or more pharmaceutically acceptable excipients, wherein the radioimmunoconjugate is produced by the process provided herein, and the one or more pharmaceutically acceptable excipients comprise one or more radioprotectants.INTERMEDIATE COMPOSITIONS

[0292] The present disclosure provides intermediate compositions that are formed during the process of producing the radioimmunoconjugates described herein. These intermediate compositions are critical transitional products that maintain the integrity of the immunoconjugate, facilitate the efficient chelation of radionuclides, and enable the seamless progression of the manufacturing process without the need for intermediate purification steps. As used herein, the term "intermediate composition" refers to a reaction product or mixture obtained at a specific stage of the process for producing radioimmunoconjugates prior to the final sterilization and isolation step. Intermediate compositions may contain the partially or fully radiolabeled immunoconjugate inAttorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]combination with components of the reaction mixture, including but not limited to unreacted radionuclides, excess immunoconjugates, reaction buffers, radioprotectants, surfactants, and any byproducts generated during the process.

[0293] Components of the Intermediate Composition:a. Partially or Fully Radiolabeled Immunoconjugates:

[0294] In some embodiments, the intermediate composition may comprise the immunoconjugate partially or fully chelated with225Ac.b. Reaction Buffer Components:

[0295] In some embodiments, the intermediate composition may comprise buffering agents (e.g., sodium acetate) to maintain the optimal pH for chelation.

[0296] In some embodiments, the intermediate composition may comprise radioprotectants (e.g., sodium ascorbate) to protect the immunoconjugate and radionuclide from radiolysis.

[0297] In some embodiments, the intermediate composition may comprise surfactants (e.g., Polysorbate 20) to prevent aggregation of the immunoconjugate and promote uniform mixing.c. Unreacted Materials and By-products:

[0298] In some embodiments, the intermediate composition may comprise minor amounts of unreacted radionuclides.d. Physical and Chemical Properties:

[0299] In some embodiments, the intermediate composition is in an aqueous solution with a pH optimized for chelation, such as about 6.0-6.5. It is designed to remain stable under ambient or controlled conditions (e.g., 4°C) during the transition between steps in the production process.

[0300] In some embodiments, the intermediate composition maintains the biological activity of the immunoconjugate and the stability of the radionuclide chelation throughout the process.Examples of Intermediate Compositions:

[0301] Intermediate Composition After Mixing (Step (a)):

[0302] In some embodiments, the intermediate composition may form following the mixing step. At this stage, the immunoconjugate may be partially chelated, and the reaction mixture is prepared for incubation to complete the chelation process.

[0303] Intermediate Composition After Incubation (Step (b)):

[0304] In some embodiments, the intermediate composition may form after incubation. In this context, the intermediate composition primarily consists of fully chelated radioimmunoconjugatesAttorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]along with reaction buffer components and residual unreacted materials. This intermediate composition is suitable for direct sterile filtration without the need for intermediate purification.

[0305] The intermediate compositions described herein may be analyzed to monitor key parameters such as chelation efficiency, pH, and the stability of the immunoconjugate. Techniques such as instant thin-layer chromatography (iTLC) or size-exclusion high-performance liquid chromatography (sec-HPLC) may be used.

[0306] In some embodiments, the intermediate composition demonstrates minimal aggregation or degradation of the immunoconjugate, with radiochemical purity exceeding 90% at this stage.

[0307] In some embodiments, the Intermediate composition allows for real-time adjustments to reaction conditions, such as pH, temperature, or mixing, to maximize product yield and quality.NUMBERED EMBODIMENTS

[0308] The disclosure provided herein also provides the following non-limiting numbered embodiments.1. A process for producing a radioimmunoconjugate comprising Actinium-225 (225Ac) chelated to an immunoconjugate, wherein the immunoconjugate comprises a chelating agent conjugated to an antibody or an antigen-binding fragment thereof, the process comprising:(a) mixing the immunoconjugate with225Ac in a reaction buffer, wherein the reaction buffer comprises:(i) a radioprotectant,(ii) a buffering agent, and(iii) a surfactant, to form a reaction mixture;(b) incubating the reaction mixture of (a) under conditions sufficient to form the radioimmunoconjugate; and(c) filtering the reaction mixture through a sterile filter to produce a sterile radioimmunoconjugate.2. The process of embodiment 1, wherein the process excludes intermediate purification steps between mixing, incubation, and filtration.3. The process of embodiment 1 or 2, wherein the chelating agent is or comprises DOTA, NOTA, TETA, PCTA, DO3 A, DFO or DTP A, for example DOTA, DFO, DTP A, NOTA or TETA.Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]The process of any one of embodiments 1-3, wherein the chelating agent is or comprises DOTA or a derivative thereof.The process of any one of embodiments 1-4, wherein the antibody or antigen-binding fragment thereof is conjugated to the chelating agent (e.g., to DOTA) via a linker.The process of embodiment 5, wherein the linker is or comprises 2-isothiocyanatobenzyl.The process of any one of embodiments 1-6, wherein the antibody or antigen-binding fragment thereof is conjugated to 2-(4-isothiocyanatobenzyl)-l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid, and wherein the conjugation comprises attachment of one or more molecules of 2-(4-isothiocyanatobenzyl)-l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid.The process of embodiment 7, wherein the one 2-(4-isothiocyanatobenzyl)-l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid group is covalently linked to an amino acid residue in the Fc region of the antibody or antigen-binding fragment thereof, or wherein each of the more than one 2-(4-isothiocyanatobenzyl)-l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid groups are covalently linked to (different) amino acid residues in the Fc region of the antibody or antigen-binding fragment thereof.The process of any one of embodiments 1-8, wherein the radioimmunoconjugate comprises an average of from about 1 to about 4 chelator molecules conjugated to the antibody or antigen binding fragment thereof.The process of any one of embodiments 1- 9, wherein the radioprotectant is sodium ascorbate, gentisic acid, L-methionine or N-acetyl-cysteine.The process of any one of embodiments 1- 10, wherein the radioprotectant is sodium ascorbate. The process of any one of embodiments 1- 11, wherein the buffering agent is sodium acetate, HEPES or TRIS.The process of any one of embodiments 1-12, wherein the buffering agent is sodium acetate. The process of any one of embodiments 1- 13, wherein the surfactant is Polysorbate 20, Polysorbate 80, or Triton™ X.The process of any one of embodiments 1- 14, wherein the surfactant is Polysorbate 20.The process of any one of embodiments 1- 15, wherein the radioprotectant is sodium ascorbate, the buffering agent is sodium acetate and the surfactant is Polysorbate 20.Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]The process of any one of embodiments 1-16, wherein the reaction buffer comprises sodium ascorbate at about 0.1% to about 5% (w / v), sodium acetate at about 0.01 M to about 1 M, and Polysorbate 20 at about 0.01% to about 1% (v / v).17a. The process of any one of embodiments 1- 17, wherein the reaction buffer comprises sodium ascorbate at about 0.5% (w / v), sodium acetate at about 25 mM, and Polysorbate 20 at about 0.04% (v / v).17b. The process of any one of embodiments 1- 17, wherein the pH of the reaction mixture is adjusted to about 5.0 to about 7.0, or about 5.5 to about 6.5, or about 6.0 to about 6.5 or about 6.0 to about 6.2, or about 6.2 to about 6.5 before incubation.The process of any one of embodiments 1 -17b, wherein the pH of the reaction mixture is adjusted to about 5.5, or about 5.6, or about 5.7, or about 5.8, or about 5.9, or about 6.0, or about 6.1 or about 6.2, or about 6.3, or about 6.4 or about 6.5 before incubation.The process of any one of embodiments 1- 18, wherein pH of the reaction mixture is adjusted to about 6.0-6.5 before incubation.19a. The process of any one of embodiments 1- 18, wherein the reaction buffer comprises sodium ascorbate at about 0.5% (w / v), sodium acetate at about 25 mM, and Polysorbate 20 at about 0.04% (v / v), and the pH of the reaction mixture is about 6.0-6.5 before incubation.The process of any one of embodiments 1- 19a, wherein in step (a), or before step (b), the molar ratio of the immunoconjugate to223Ac is at least about 440 to 1.The process of any one of embodiments 1- 19, wherein in step (a), or before step (b), the specific activity is at or below 200 pCi / mg, e.g., of about 100 pCi / mg to about 200 pCi / mg. The process of any one of embodiments 1- 21, wherein in step (a), or before step (b), the molar ratio of the immunoconjugate to225Ac is of at least about 440 to 1 and the specific activity is at or below 200 pCi / mg; or wherein the molar ratio of the immunoconjugate to225Ac is of at least about 440: 1 and the specific activity is of about 100 pCi / mg to about 200 pCi / mg, or wherein the molar ratio of the immunoconjugate to225Ac is of at least about 440: 1 and the specific activity is of about 100 pCi / mg; or wherein the molar ratio of the immunoconjugate to225Ac is of at least about 440: 1 and the specific activity is of about 200 pCi / mg.The process of any one of embodiments 1- 21, wherein in step (a), or before step (b), the molar ratio of the immunoconjugate to223Ac is about 500: 1 to about 1000: 1 and the specific activity is at or below 200 pCi / mg; or wherein the molar ratio of the immunoconjugate to225Ac is aboutAttorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]500:1 to about 1000:1 and the specific activity is of about 100 pCi / mg to about 200 pCi / mg; or wherein the molar ratio of the immunoconjugate to225Ac is about 500: 1 to about 1000: 1 and the specific activity is of about 100 pCi / mg; or wherein the molar ratio of the immunoconjugate to225Ac is about 500:1 to about 1000:1 and the specific activity is of about 200 pCi / mg.The process of any one of embodiments 1- 21, wherein in step (a), or before step (b), the molar ratio of the immunoconjugate to225Ac is of about 880: 1 and the specific activity is at or below 200 pCi / mg; or wherein the molar ratio of the immunoconjugate to225Ac is of about 880: 1 and the specific activity is of about 100 pCi / mg to about 200 pCi / mg; or wherein the molar ratio of the immunoconjugate to225Ac is of about 880: 1 and the specific activity is of about 100 pCi / mg; or wherein the molar ratio of the immunoconjugate to225Ac is of about 880: 1 and the specific activity is of about 200 pCi / mg.The process of any one of embodiments 1- 24, wherein the incubation is performed at a temperature of about 20°C to about 50°C, or about 20°C to 30°C, or about 20°C to 25°C, or about 25°C to 30°C, or about 30°C to 40°C, or about 30°C to 35°C, or about 35°C to 40°C, or about 40°C to 50°C, or about 40°C to 45°C, or about 45°C to 50°C, or about 20°C, or about 21 °C, or about 22°C, or about 23°C, or about 24°C, or about 25°C or about 26°C, or about 27°C, or about 28°C, or about 29°C, or about 30 °C, or about 31°C, or about 32°C, or about 33°C, or about 34°C, or about 33°C or about 35°C, or about 37°C or about 38°C, or about 39°C, or about 40°C, or about 41°C, or about 42°C, or about 43°C, or about 44°C, or about 45°C, or about 46°C, or about 47°C, or about 48°C, or about 49°C, or about 50°C.The process of any one of embodiments 1- 25, wherein the incubation is performed at about 37 °C.The process of any one of embodiments 1- 26, wherein the incubation is carried out for about 5 minutes to about 5 hours, or about 5 minutes to 60 minutes, or about 5 minutes to 30 minutes, or about 30 minutes to 60 minutes, or about 60 minutes to 120 minutes, or about 60 minutes to 90 minutes, or about 90 minutes to 120 minutes, or about 120 minutes to 300 minutes, or about 120 minutes to 180 minutes, or about 180 minutes to 240 minutes, or about 240 minutes to 300 minutes, or about 5 minutes, or about 6 minutes, or about 7 minutes, or about 8 minutes, or about 9 minutes, or about 10 minutes, or about 11 minutes, or about 12 minutes, or about 13 minutes, or about 14 minutes, or about 15 minutes, or about 16 minutes, or about 17 minutes, or about 18 minutes, or about 19 minutes, or about 20 minutes, or about 21 minutes, or about 22Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]minutes, or about 23 minutes, or about 24 minutes, or about 25 minutes, or about 26 minutes, or about 27 minutes, or about 28 minutes, or about 29 minutes, or about 35 minutes, or about 40 minutes, or about 45 minutes, or about 50 minutes, or about 60 minutes (1 hour), or about 70 minutes, or about 80 minutes, or about 90 minutes (1.5 hours), or about 100 minutes, or about 110 minutes, or about 120 minutes (2 hours), or about 130 minutes, or about 140 minutes, or about 150 minutes (2.5 hours) or about 160 minutes, or about 170 minutes, or about 180 minutes (3 hours) or about 190 minutes, or about 200 minutes, or about 220 minutes, or about 240 minutes (4 hours) or about 260 minutes, or about 280 minutes, or about 300 minutes (5 hours). The process of any one of embodiments 1- 27, wherein the incubation is carried out for about 2 hours.28a. The process of any one of embodiments 1- 28, wherein the incubation is carried out at about 37 °C for about 2 hours.The process of any one of embodiments 1- 28a, wherein the sterile filter has a pore size from about 0.1 pm to about 1.0 pm, for example of about 0.1 pm, or about 0.2 pm or about 0.22 pm, or about 0.3 pm, or about 0.45 pm, or about 0.5 pm, or about 0.6 pm, or about 0.8 pm, or about 1.0 pm.The process of any one of embodiments 1- 29, wherein the sterile filter has a pore size of about 0.22 pm.The process of any one of embodiments 1- 30, wherein the radioimmunoconjugate is directly filtered into a sterile container.The process of any one of embodiments 1- 31, wherein the radioimmunoconjugate is prepared at a scale of about 0.1 mCi to about 1,000 mCi.The process of any one of embodiments 1- 32, wherein the process is scaled for large-scale production with automated or semi-automated systems.The process of any one of embodiments 1- 33, wherein the radioimmunoconjugate has a radiochemical purity of at least 95% as determined by instant thin-layer chromatography (iTLC).The process of any one of embodiments 1- 34, further comprising performing a stability challenge using diethylenetriamine pentaacetate (DTP A) to confirm chelation stability of the radi oi mmunoconj ugate .Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]The process of embodiment 35, wherein the DTPA challenge shows at least 95% of the223Ac remains chelated after 30 minutes of incubation with DTPA.The process of any one of embodiments 1- 36, further comprising analyzing the radioimmunoconjugate using size-exclusion high-performance liquid chromatography (SEC-HPLC) to confirm protein integrity and radiochemical purity.The process of any one of embodiments 1- 37, wherein the radiochemical purity remains at least about 90% after 96 hours of storage at 4 °C.The process of any one of embodiments 1- 38, which does not comprise a step of mixing the radioimmunoconjugate recovered at step (c) with unlabeled immunoconjugate.The process of any one of embodiments 1-39, wherein the immunoconjugate comprises an antibody or an antigen binding fragment thereof that specifically binds to human kallikrein-related peptidase 2 (hK2).The process of any one of embodiments 1-40, wherein the antibody or an antigen-binding fragment thereof, comprises:a heavy chain CDR1 (HCDR1) of SEQ ID NO: 5;a heavy chain CDR2 (HCDR2) of SEQ ID NO: 6;a heavy chain CDR3 (HCDR3) of SEQ ID NO: 7;a light chain CDR1 (LCDR1) of SEQ ID NO: 8;a light chain CDR2 (LCDR2) of SEQ ID NO: 9; anda light chain CDR3 (LCDR3) of SEQ ID NO: 10.The process of any one of embodiments 1-41, wherein the antibody or an antigen-binding fragment thereof, comprises a variable heavy chain (VH) sequence of SEQ ID NO: 1 and a variable light chain (VL) sequence of SEQ ID NO: 2.The process of any one of embodiments 1-42, wherein the antibody or an antigen-binding fragment thereof, comprises a heavy chain (HC) sequence of SEQ ID NO: 3 and a light chain (LC) sequence of SEQ ID NO: 4.43a. The process of any one of embodiments 1-42, wherein the antibody or an antigen-binding fragment thereof, comprises a heavy chain (HC) sequence of SEQ ID NO: 12 and a light chain (LC) sequence of SEQ ID NO: 4The process of any one of embodiments 1- 43a, wherein the antibody is or comprises a humanized antibody, e.g., a humanized monoclonal antibody, such as hl 1B6.Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]45. The process of any one of embodiments 1- 44, wherein the process is a GMP-compliant process.46. The process of any one of embodiments 1- 45, wherein the process is for producing a radioimmunoconjugate for the treatment of prostate cancer, for example metastatic prostate cancer, for example metastatic castration-resistant prostate cancer (mCRPC).47. A radioimmunoconjugate obtained or obtainable by the process of any one of embodiments 1- 46.48. A composition comprising a radioimmunoconjugate obtained or obtainable by the process of any one of embodiments 1 -46.49. An intermediate composition, which is obtained or obtainable by implementing the process of any one of embodiments 1 - 46, wherein the intermediate composition comprises a radioimmunoconjugate and a buffer solution, and wherein the intermediate composition is obtained or obtainable at step (b) or (c) of the process.50. A method of treating prostate cancer (for example metastatic prostate cancer, for example metastatic castration-resistant prostate cancer (mCRPC)), comprising administering a therapeutically effective amount of a radioimmunoconjugate produced by the process of any one of embodiments 1-46 to a patient in need thereof.

[0309] The disclosure provided herein further provides the following non-limiting numbered embodiments.51. A process for producing a radioimmunoconjugate comprising Actinium-225 (225Ac) chelated to an immunoconjugate, wherein the immunoconjugate comprises a chelating agent conjugated to an antibody or an antigen-binding fragment thereof, the process comprising:(a) mixing the immunoconjugate with225Ac in a reaction buffer, wherein the reaction buffer comprises:(i) a radioprotectant,(ii) a buffering agent, and(iii) a surfactant, to form a reaction mixture;(b) incubating the reaction mixture of (a) at a temperature of 33°C to 39°C for up to 4 hours to form the radioimmunoconjugate.52. The process of embodiment 51, further comprising:Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT](c) filtering the reaction mixture through a sterile filter to produce a sterile radioimmunoconjugate.The process of embodiment 51 or 52, wherein the process excludes intermediate purification steps between mixing, incubation, and filtration.The process of any one of embodiments 51 to 53, wherein the chelating agent is or comprises DOTA, NOTA, TETA, PCTA, DO3A, DFO or DTPA, for example DOTA, DFO, DTP A, NOTA or TETA.The process of any one of embodiments 51-54, wherein the chelating agent is or comprises DOTA or a derivative thereof.The process of any one of embodiments 51-55, wherein the antibody or antigen-binding fragment thereof is conjugated to the chelating agent (e.g., to DOTA) via a linker.The process of embodiment 56, wherein the linker is or comprises 2-isothiocyanatobenzyl. The process of any one of embodiments 51-57, wherein the antibody or antigen-binding fragment thereof is conjugated to 2-(4-isothiocyanatobenzyl)-l,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, and wherein the conjugation comprises attachment of one or more molecules of 2-(4-isothiocyanatobenzyl)-l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid.The process of embodiment 58, wherein the one 2-(4-isothiocyanatobenzyl)-l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid group is covalently linked to an amino acid residue in the Fc region of the antibody or antigen-binding fragment thereof, or wherein each of the more than one 2-(4-isothiocyanatobenzyl)-l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid groups are covalently linked to (different) amino acid residues in the Fc region of the antibody or antigen-binding fragment thereof.The process of any one of embodiments 51- 59, wherein the radioimmunoconjugate comprises an average of from about 1 to about 4 chelator molecules conjugated to the antibody or antigen binding fragment thereof.The process of any one of embodiments 51- 60, wherein the radioprotectant is sodium ascorbate, gentisic acid, L-methionine or N-acetyl-cysteine.The process of any one of embodiments 51- 61, wherein the radioprotectant is sodium ascorbate.Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]The process of any one of embodiments 51- 62, wherein the buffering agent is sodium acetate, HEPES or TRIS.The process of any one of embodiments 51- 63, wherein the buffering agent is sodium acetate. The process of any one of embodiments 51- 64, wherein the surfactant is Polysorbate 20, Polysorbate 80, or Triton™ X.The process of any one of embodiments 51- 65, wherein the surfactant is Polysorbate 20.The process of any one of embodiments 51-66, wherein the radioprotectant is sodium ascorbate, the buffering agent is sodium acetate and the surfactant is Polysorbate 20.The process of any one of embodiments 51- 67, wherein the reaction buffer comprises sodium ascorbate at about 1% to about 8% (w / v), sodium acetate at about 0.01 M to about 1 M, and Polysorbate 20 at about 0.01% to about 1% (v / v).68a. The process of embodiment 68, wherein the reaction buffer comprises sodium ascorbate at about 6% (w / v), sodium acetate at about 0.5 M, and Polysorbate 20 at about 0.04% (v / v).The process of any one of embodiments 51- 68a, wherein the pH of the reaction mixture is adjusted to about 5.0 to about 7.0, or about 5.5 to about 6.5, or about 6.0 to about 6.5 or about 6.0 to about 6.2, or about 6.2 to about 6.5 before incubation.The process of any one of embodiments 51- 68a, wherein pH of the reaction mixture is adjusted to about 6.0-6.5 before incubation.70a. The process of embodiment 70, wherein the reaction buffer comprises sodium ascorbate at about 6% (w / v), sodium acetate at about 0.5 M, and Polysorbate 20 at about 0.04% (v / v), and the pH of the reaction mixture is about 6.0-6.5 before incubation.The process of any one of embodiments 51- 70a, wherein in step (a), or before step (b), the molar ratio of the immunoconjugate to225Ac is at least about 440 to 1.The process of any one of embodiments 51-71, wherein in step (a), or before step (b), the specific activity is at or below 200 pCi / mg, e.g., of about 100 pCi / mg to about 200 pCi / mg. The process of any one of embodiments 51- 72, wherein in step (a), or before step (b), the molar ratio of the immunoconjugate to22’Ac is of at least about 440 to 1 and the specific activity is at or below 200 pCi / mg; or wherein the molar ratio of the immunoconjugate to22?Ac is of at least about 440: 1 and the specific activity is of about 100 pCi / mg to about 200 pCi / mg, or wherein the molar ratio of the immunoconjugate to225Ac is of at least about 440: 1 and the specificAttorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]activity is of about 100 pCi / mg; or wherein the molar ratio of the immunoconjugate to225Ac is of at least about 440: 1 and the specific activity is of about 200 pCi / mg.The process of any one of embodiments 51- 72, wherein in step (a), or before step (b), the molar ratio of the immunoconjugate to225Ac is about 500: 1 to about 1000: 1 and the specific activity is at or below 200 pCi / mg; or wherein the molar ratio of the immunoconjugate to225Ac is about 500:1 to about 1000:1 and the specific activity is of about 100 pCi / mg to about 200 pCi / mg; or wherein the molar ratio of the immunoconjugate to225Ac is about 500: 1 to about 1000: 1 and the specific activity is of about 100 pCi / mg; or wherein the molar ratio of the immunoconjugate to225AC is about 500:1 to about 1000:1 and the specific activity is of about 200 pCi / mg.The process of any one of embodiments 51-72, wherein in step (a), or before step (b), the molar ratio of the immunoconjugate to223Ac is of about 880: 1 and the specific activity is at or below 200 pCi / mg; or wherein the molar ratio of the immunoconjugate to225Ac is of about 880: 1 and the specific activity is of about 100 pCi / mg to about 200 pCi / mg; or wherein the molar ratio of the immunoconjugate to225Ac is of about 880: 1 and the specific activity is of about 100 pCi / mg; or wherein the molar ratio of the immunoconjugate to225Ac is of about 880: 1 and the specific activity is of about 200 pCi / mg.The process of any one of embodiments 51- 75, wherein the incubation is performed at a temperature of about 33°C to about 39°C, such as about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, or about 39°C, or any temperature in between.The process of any one of embodiments 51- 76, wherein the incubation is performed at about 35°C ± 2°C.The process of any one of embodiments 51- 77, wherein the incubation is carried out for about 5 minutes to about 4 hours, or about 5 minutes to 60 minutes, or about 5 minutes to 30 minutes, or about 30 minutes to 60 minutes, or about 60 minutes to 120 minutes, or about 60 minutes to 90 minutes, or about 90 minutes to 120 minutes, or about 120 minutes to 300 minutes, or about 120 minutes to 180 minutes, or about 180 minutes to 240 minutes, or about 5 minutes, or about 6 minutes, or about 7 minutes, or about 8 minutes, or about 9 minutes, or about 10 minutes, or about 11 minutes, or about 12 minutes, or about 13 minutes, or about 14 minutes, or about 15 minutes, or about 16 minutes, or about 17 minutes, or about 18 minutes, or about 19 minutes, or about 20 minutes, or about 21 minutes, or about 22 minutes, or about 23 minutes, or about 24 minutes, or about 25 minutes, or about 26 minutes, or about 27 minutes, or about 28 minutes, orAttorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]about 29 minutes, or about 35 minutes, or about 40 minutes, or about 45 minutes, or about 50 minutes, or about 60 minutes (1 hour), or about 70 minutes, or about 80 minutes, or about 90 minutes (1.5 hours), or about 100 minutes, or about 110 minutes, or about 120 minutes (2 hours), or about 130 minutes, or about 140 minutes, or about 150 minutes (2.5 hours) or about 160 minutes, or about 170 minutes, or about 180 minutes (3 hours) or about 190 minutes, or about 200 minutes, or about 220 minutes, or about 240 minutes (4 hours).The process of any one of embodiments 51-78, wherein the incubation is carried out for at least about 3 hours up to 4 hours.The process of any one of embodiments 51- 78, wherein the incubation is carried out at 35°C ± 2°C for at least about 3 hours up to 4 hours.The process of any one of embodiments 51- 80, wherein sterile filtration is performed on the reaction mixture, on a diluted reaction mixture, or on a final formulation prior to administration or filling.The process of any one of embodiments 51-81, wherein the sterile filter has a pore size from about 0.1 pm to about 1.0 pm, for example of about 0.1 pm, or about 0.2 pm or about 0.22 pm, or about 0.3 pm, or about 0.45 pm, or about 0.5 pm, or about 0.6 pm, or about 0.8 pm, or about 1.0 pm.The process of any one of embodiments 51- 82, wherein the sterile filter has a pore size of about 0.22 pm.The process of any one of embodiments 51- 83, wherein the radioimmunoconjugate is directly filtered into a sterile container.The process of any one of embodiments 51- 84, wherein the radioimmunoconjugate is prepared at a scale of about 0.01 mCi to about 1,000 mCi, about 0.1 mCi to about 1 mCi, about 0.1 mCi, 0.2 mCi, 0.3 mCi, 0.4 mCi, 0.5 mCi, 0.6 mCi, 0.7 mCi, 0.8 mCi, 0.9 mCi or 1 mCi.The process of any one of embodiments 51- 85, wherein the process is scaled for large-scale production with automated or semi-automated systems.The process of any one of embodiments 51- 86, further comprising diluting the reaction mixture after step (b) or step (c) with a dilution buffer.87a. The process of embodiment 87, wherein the reaction mixture is diluted after step (c) with the dilution buffer.Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]87b. The process of embodiment 87, wherein the reaction mixture is diluted with the dilution buffer, after step (b) before the filtering step (c).The process of any one of embodiments 87 - 87b, wherein the dilution buffer comprises a second radioprotectant, a second buffering agent and a second surfactant.The process of embodiment 88, wherein the second radioprotectant is sodium ascorbate, gentisic acid, L-methionine or N-acetyl-cysteine.The process of embodiment 89, wherein the second radioprotectant is sodium ascorbate.The process of any one of embodiments 87- 90, wherein the second buffering agent is acetic acid.The process of any one of embodiments 87- 91, wherein the second surfactant is Polysorbate 20, Polysorbate 80, or Triton™ X.The process of any one of embodiments 87- 92, wherein the second surfactant is Polysorbate 20. The process of any one of embodiments 87-93, wherein the second radioprotectant is sodium ascorbate, the second buffering agent is acetic acid and the second surfactant is Polysorbate 20. The process of any one of embodiments 87- 94, wherein the dilution buffer comprises sodium ascorbate at about 0.1% to about 5% (w / v), acetic acid at about 0.5 - 10 mM, and Polysorbate 20 at about 0.01% to about 1% (v / v).The process of embodiment 95, wherein the dilution buffer comprises sodium ascorbate at about 3% (w / v), acetic acid at about 3 mM, and Polysorbate 20 at about 0.04% (v / v).The process of any one of embodiments 51-96, wherein the radioimmunoconjugate has a radiochemical purity of at least 95% as determined by instant thin-layer chromatography (iTLC).The process of any one of embodiments 51- 97, further comprising performing a stability challenge using diethylenetriamine pentaacetate (DTP A) to confirm chelation stability of the radi oimmunoconj ugate.The process of embodiment 98, wherein the DTPA challenge shows at least 95% of the225Ac remains chelated after 30 minutes of incubation with DTPA.. The process of any one of embodiments 51- 99, further comprising analyzing the radioimmunoconjugate using size-exclusion high-performance liquid chromatography (SEC-HPLC) to confirm protein integrity and radiochemical purity.Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]. The process of any one of embodiments 51-100, wherein the radiochemical purity remains at least about 90% after 96 hours of storage at 4 °C.. The process of any one of embodiments 51-101, which does not comprise a step of mixing the radioimmunoconjugate recovered at step (c) with unlabeled immunoconjugate.. The process of any one of embodiments 51-102, wherein the immunoconjugate comprises an antibody or an antigen binding fragment thereof that specifically binds to human kallikrein-related peptidase 2 (hK2).. The process of any one of embodiments 51-103, wherein the antibody or an antigen-binding fragment thereof, comprises:a heavy chain CDR1 (HCDR1) of SEQ ID NO: 5;a heavy chain CDR2 (HCDR2) of SEQ ID NO: 6;a heavy chain CDR3 (HCDR3) of SEQ ID NO: 7;a light chain CDR1 (LCDR1) of SEQ ID NO: 8;a light chain CDR2 (LCDR2) of SEQ ID NO: 9; anda light chain CDR3 (LCDR3) of SEQ ID NO: 10.. The process of any one of embodiments 51-104, wherein the antibody or an antigen-binding fragment thereof, comprises a variable heavy chain (VH) sequence of SEQ ID NO: 1 and a variable light chain (VL) sequence of SEQ ID NO: 2.. The process of any one of embodiments 51-105, wherein the antibody or an antigen-binding fragment thereof, comprises a heavy chain (HC) sequence of SEQ ID NO: 3 and a light chain (LC) sequence of SEQ ID NO: 4.106a. The process of any one of embodiments 51-105, wherein the antibody or an antigenbinding fragment thereof, comprises a heavy chain (HC) sequence of SEQ ID NO: 12 and a light chain (LC) sequence of SEQ ID NO: 4.. The process of any one of embodiments 51-106a, wherein the antibody is or comprises a humanized antibody, e.g., a humanized monoclonal antibody, such as hl 1B6.. The process of any one of embodiments 51-107, wherein the process is a GMP-compliant process.Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]109. The process if any one of embodiments 51-108, wherein the process is performed under conditions suitable for commercial manufacture, including closed, system handling, in-process quality control, and preparation of batch quantities suitable for clinical or commercial use.109a. The process of any one of embodiments 51- 109, wherein the process further comprises performing one or more in-process quality control assays during or after incubation.109b. The process of any one of embodiments 51- 109a, wherein the process is performed in a closed or semi-closed manufacturing system.109c. The process of any one of embodiments 51- 109b, wherein the process is performed at a radioactive concentration suitable for clinical or commercial manufacture.110. The process of any one of embodiments 51- 109c, wherein the process is for producing a radioimmunoconjugate for the treatment of prostate cancer, for example metastatic prostate cancer, for example metastatic castration-resistant prostate cancer (mCRPC).111. A radioimmunoconjugate obtained or obtainable by the process of any one of embodiments 51- 110.112. A composition comprising a radioimmunoconjugate obtained or obtainable by the process of any one of embodiments 51- 110.113. An intermediate composition, which is obtained or obtainable by implementing the process of any one of embodiments 51-110, wherein the intermediate composition comprises a radioimmunoconjugate and a buffer solution, and wherein the intermediate composition is obtained or obtainable at step (b) or (c) of the process.114. A method of treating prostate cancer (for example metastatic prostate cancer, for example metastatic castration-resistant prostate cancer (mCRPC)), comprising administering a therapeutically effective amount of a radioimmunoconjugate produced by the process of any one of embodiments 51-110 to a patient in need thereof.EXAMPLES

[0310] The following examples are provided to supplement the prior disclosure and to provide a better understanding of the subject matter described herein. These examples should not be considered to limit the described subject matter. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications orAttorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]changes in light thereof will be apparent to persons skilled in the art and are to be included within, and can be made without departing from, the true scope of the invention.EXAMPLE 1 : PREPARATION AND CONJUGATION OF A HUMANIZED MONOCLONAL ANTIBODY SPECIFIC FOR HUMAN KALLIKREIN-RELATED PEPTIDASE 2 (HK2) WITH DOTA CHELATORS FOR RADIOLABELING APPLICATIONS

[0311] A humanized monoclonal antibody (hl 1B6), specific for human kallikrein-related peptidase 2 (hK2), was expressed and purified using standard methodologies in Chinese hamster ovary (CHO) cells. The expression and purification methods are described in WO2015075445 and U.S. Patent No. 10,100,125, both of which are incorporated herein by reference in their entirety. Alternatively, the antibody hl 1B6 can be prepared using the methods detailed in U.S. Patent No. 9,873,991, which is also incorporated herein by reference. These methods utilize a CHO DG44-derived cell line and an hEFla promoter-driven double gene vector, commercially available from Fujifilm Diosynth Biotechnologies.

[0312] The humanized monoclonal antibody, hl 1B6, is of the IgGl kappa isotype. The sequences for the variable heavy (VH), variable light (VL), heavy chain (HC), light chain (LC), and complementarity-determining regions (CDRs) are specified as follows:(1) Variable Heavy (VH) Sequence (SEQ ID NO: 1):QVQLQESGPGLVKPSDTLSLTCAVSGNSITSDYAWNWIRQPPGKGLEWIGYISYSGS TTYNPSLKSRVTMSRDTSKNQFSLKLSSVTAVDTAVYYCATGYYYGSGFWGQGTL VTVSS(2) Variable Light (VL) Sequence (SEQ ID NO: 2):DIVLTQSPDSLAVSLGERATINCKASESVEYFGTSLMHWYQQKPGQPPKLLIYAASN RESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQTRKVPYTFGQGTKLEIK(3) Heavy Chain (HC) Sequence (SEQ ID NO: 3):QVQLQESGPGLVKPSDTLSLTCAVSGNSITSDYAWNWIRQPPGKGLEWIGYISYSGS TTYNPSLKSRVTMSRDTSKNQFSLKLSSVTAVDTAVYYCATGYYYGSGFWGQGTL VTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPA VLQ S SGLYSL S S V VTVP S S SLGTQTYICNVNHKP SNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(4) Heavy Chain (HC) Sequence (SEQ ID NO: 12):QVQLQESGPGLVKPSDTLSLTCAVSGNSITSDYAWNWIRQPPGKGLEWIGYISYSGS TTYNPSLKSRVTMSRDTSKNQFSLKLSSVTAVDTAVYYCATGYYYGSGFWGQGTL VTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPA VLQ S SGLYSL S S VVTVP S S SLGTQTYICNVNHKP SNTK VDKRVEPK SCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAttorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]AKGQPREPQ VYTLPP SREEMTKNQ VSLTCL VKGF YP SDIAVEWE SNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(5) Light Chain (LC) Sequence (SEQ ID NO: 4):DIVLTQSPDSLAVSLGERATINCKASESVEYFGTSLMHWYQQKPGQPPKLLIYAASN RESGVPDRF SGSGSGTDFTLTIS SLQAEDVAVYYCQQTRKVPYTFGQGTKLEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ D SKD S T Y SL S STLTL SK AD YEKHK VYACE VTHQGL S SP VTK SFNRGEC(6) Complementarity-Determining Regions (CDRs):HCDR1 (SEQ ID NO: 5) SDYAWNHCDR2 (SEQ ID NO: 6) YISYSGSTTYNPSLKSHCDR3 (SEQ ID NO: 7) GYYYGSGFLCDR1 (SEQ ID NO: 8): KASES VEYFGTSLMHLCDR2 (SEQ ID NO: 9): AASNRESLCDR3 (SEQ ID NO: 10): QQTRKVPYT

[0313] To prepare the immunoconjugate, hl 1B6 was first diluted to a concentration of 10 mg / mL in 50 mM sodium acetate buffer, pH 8.5. A 5-fold molar excess of 2-(4-isothiocyanatobenzyl)-l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid (DOTA-linker) was dissolved in dimethyl sulfoxide (DMSO) and added to the antibody solution. The conjugation reaction was incubated at room temperature for 2 hours with gentle mixing, and the pH was maintained at 8.5 by adding 0.1 N NaOH as necessary. Covalent attachment of the DOTA-linker to amino acid residues, predominantly in the Fc region of the antibody, was facilitated through the isothiocyanate functional group. Excess unreacted DOTA-linker was removed by diafiltration using a 10-kDa molecular weight cut-off (MWCO) ultrafiltration membrane, and the buffer was exchanged to 50 mM sodium acetate, pH 5.5. The chelator-to-antibody ratio (CAR) was determined to be approximately 3.5 using UV spectrophotometry and mass spectrometry.

[0314] Following preculture and expansion, the cell culture may be clarified using known filtration techniques. The filtrate is concentrated and diafiltered to a target final concentration of 10 g / L in buffer (25 mM NaOAc, pH 5.5). The hl 1B6 is filtered through a 0.2-pm filter, filled into sterilized bags, and may be frozen at < -65 °C for long-term storage, prior to conjugation.

[0315] The thawed hl 1B6 is then diafiltered (buffer exchanged) to 50 mM Bicine, 120 mM NaCl, pH 8.5 for the subsequent conjugation ofDOTA (1,4,7, 10-tetraazacyclododecane-l, 4, 7,10-tetraacetic acid) to hl 1B6. The retentate from the prior step is transferred to a reactor and stirred while warming to 25 °C. A solution of / ?-SCN-Bn-DOTA in water is prepared and added to the reactor. The reaction is maintained at 25 °C for 20 hours. The product of the conjugation reaction (DOTA-hl 1B6) is transferred directly to the retentate vessel for the final diafiltration with 25 mMAttorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]NaOAc, pH 5.5. Next, DOTA-hl 1B6 conjugate intermediate is filtered through a 0.2-pm filter, filled into sterilized polycarbonate containers, and may be frozen at < -65 °C for long-term storage.

[0316] The conjugation reaction results in addition of multiple DOTA molecules to the epsilon amino group of lysine side chains of the hl 1B6 mAb. The conjugate-to-antibody ratio (CAR), which designates the number of DOTA molecules per hl 1B6 mAb molecule, can be measured by intact mass analysis using RP-HPLC with online mass analysis. Based on the molecular structure of p-SCN-Bn-DOTA, each DOTA residue adds 552 Da mass to the antibody, which can be readily detected by intact mass analysis.EXAMPLE 2. ACTINIUM-225 RADIOLABELING OF DOTA-H11B6 AT 0.553 MCI SCALE

[0317] Preparation of Radiolabeling Reaction: Actinium-225 nitrate (225Ac(NO3)3), received in a glass vial, was dissolved in 0.1 N HC1 to achieve a target specific concentration of approximately 20 mCi / mL. To a 2-mL plastic vial containing 0.5 mL of purification buffer (25 mM sodium acetate (NaOAc), 0.04% Polysorbate 20, 0.5% sodium ascorbate w / v, pH 5.5), the following reagents were sequentially added:• 30 pL of the prepared225Ac solution in 0.1 N HC1,• 50 pL ofO.I NNaOH,• 0.5 mL of DOTA-hl lb6 antibody solution (10 mg / mL in 25 mM sodium acetate, pH 5.5).

[0318] The reaction mixture was gently mixed by pipetting up and down, ensuring thorough homogenization. The pH of the reaction mixture was confirmed to be between 6.0 and 6.5 using pH paper. The radioactivity in the reaction vial at the start of synthesis (SOS) was measured to be 0.553 mCi. The reaction was incubated at 37 °C for 2 hours without agitation.

[0319] Labeling Efficiency Analysis by iTLC: After 2 hours, the radiolabeling efficiency was analyzed by instant thin-layer chromatography (iTLC). A 0.5-pL aliquot of the reaction solution was spotted on a silica gel-impregnated iTLC-SG plate (3 cm x 15 cm), at approximately 2 cm from the bottom (baseline). The plate was developed using 10 mM EDTA (pH 6.0) as the mobile phase, allowing the solvent front to migrate to 2 cm from the top of the plate. The plate was dried and allowed to equilibrate at room temperature for over 16 hours to achieve secular equilibrium of225Ac whereby the rate of production of its radioactive daughter isotopes equal their rate of decay, resulting in a constant amount of each isotope.

[0320] The dried iTLC plate was scanned using an AR-2000 TLC scanner, generating a chromatogram (FIG. 1). The chromatogram showed two distinct radioactive regions of interestAttorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT](ROIs): the baseline (representing the radiolabeled conjugate) and the solvent front (representing free225Ac). The iTLC analysis indicated that 99.6% of the radioactivity was localized at the baseline, confirming successful chelation of225Ac to the DOTA-hl 1 b6 antibody, with only 0.4% detected at the solvent front.

[0321] Sterile Filtration and Formulation: The radiolabeled reaction mixture was transferred to a 6-mL syringe fitted with a 0.22-pm sterile filter. The reaction vial was washed with 4.0 mL of formulation buffer, and the washings were added to the syringe. The solution was filtered into a sterile Crystal Zenith® (CZ) vial, and the reaction vial was further washed with an additional 4.9 mL of purification buffer. The second washing was also filtered into the CZ vial. After filtration, air was pushed through the filter to collect any remaining solution. The filtrate, designated as the drug product, marked the end of synthesis (EOS), which contains the diluted radiolabeled reaction mixture. The total volume of the radiolabeled product was approximately 10 mL.

[0322] DTPA Challenge for Radiochemical Stability: To confirm the stability of the radiolabeled product, a DTPA challenge was performed. A 10-pL aliquot of the drug product was mixed with 15 pL of 10 mM DTPA (pH 6.5) and incubated at room temperature for 30 minutes. A 10-pL aliquot of the mixture was spotted on an iTLC-SG plate and developed with 10 mM EDTA as described earlier. The developed plate was scanned after three days of equilibration (FIG. 2). The iTLC chromatogram showed no detectable radioactivity at the solvent front, confirming that the radiometal (225Ac) was fully chelated to the DOTA-hl 1 b6 conjugate, with no free or loosely bound225Ac present.

[0323] Stability Testing by SEC-HPLC: The drug product was stored in a lead-shielded container at 4 °C, and its stability was assessed at t = 0 hours and t = 96 hours post-EOS using size-exclusion high-performance liquid chromatography (SEC-HPLC) with the following HPLC Conditions:• Column: Tosoh TSKgel G3000SWxl (7.8 mm x 30 cm, 5 pm).• Mobile phase: 0.2 M phosphate buffer solution (pH 6.8) containing 5% acetonitrile (v / v). • Flow rate: 1 mL / min.• Injection volume: 40 pL.• Run time: 25 minutes.• Detection: UV at 280 nm and off-line gamma-well counter.Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]

[0324] Fractions were collected at 0.5-minute intervals, allowed to reach secular equilibrium (>16 hours), and analyzed for radioactivity. Chromatograms were reconstructed based on the radioactivity measured in each fraction.

[0325] At t = 0 hours, the SEC-HPLC analysis showed a protein purity of 97.9% (UV chromatogram, FIG. 3A) and a radiochemical purity of 96.4% (radiochromatogram, FIG. 3B). Minor high molecular weight species (HMWS, 2.2%) and low molecular weight species (LMWS, 1.4%) were observed.

[0326] At t = 96 hours, the protein purity was 97.4% (UV chromatogram, FIG. 4A), and the radiochemical purity was 95.1% (radiochromatogram, FIG. 4B). These results demonstrated the stability of the drug product under clinical storage conditions.EXAMPLE 3: ACTINIUM-225 LABELING OF DOTA-H11B6 AT 1.173 MCI SCALE

[0327] The process was scaled up to produce >1 mCi batches.

[0328] Preparation of Reaction Mixture: Actinium-225 nitrate received in a glass vial was dissolved in 0.1 N HC1 to achieve a target concentration of approximately 20 mCi / mL.

[0329] To a 2-mL plastic vial containing 0.8 mL of purification buffer (25 mM sodium acetate, 0.04% Polysorbate 20, 0.5% sodium ascorbate w / v, pH 5.5) were added:• 60 pL of the223Ac solution (dissolved in 0.1 N HC1),• 102 pL ofO.l NNaOH, and• 0.8 mL of DOTA-hl 1 b6 (10 mg / mL in 25 mM sodium acetate at pH 5.5).

[0330] The reaction mixture was gently pipetted to ensure proper mixing. The pH of the solution was confirmed to be between 6.0 and 6.5 (close to 6.0) using pH paper. The radioactivity of the reaction mixture at the start of synthesis (SOS) was 1.173 mCi. The vial was left standing still at 37 °C for 2 hours to complete the reaction.

[0331] Instant Thin Layer Chromatography (iTLC) Analysis: To evaluate labeling efficiency, 0.5 pL of the reaction mixture was spotted onto an iTLC-SG plate (silica gel) approximately 2 cm from the edge of the plate. The plate was developed with 10 mM EDTA (pH 6.0) as the mobile phase until the solvent front reached 2 cm from the opposite edge. The developed plate was air-dried and scanned 3 days after development using an AR-2000 TLC scanner.

[0332] The iTLC results showed that 100% of225Ac was bonded to the chelator (FIG. 5), indicating successful chelation.Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]

[0333] Sterile Filtration and Formulation: The labeling reaction mixture was transferred into a 6-mL syringe fitted with a 0.22 gm filter. The reaction vial was washed with 2.5 mL of formulation buffer (25 mM sodium acetate, 0.04% Polysorbate 20, 0.5% sodium ascorbate w / v, pH 5.5), and the washings were transferred into the syringe.

[0334] The solution in the syringe was filtered into a centrifuge tube. The reaction vial was subsequently washed twice with 6 mL of purification buffer, and these washings were also filtered into the same centrifuge tube. Air was pushed through the filter to ensure complete transfer. The filtrate collected in the centrifuge tube constituted the drug product, marking the end of synthesis (EOS).

[0335] DTPA Challenge for Chelation Stability: To evaluate the stability of the chelated225Ac, a DTPA challenge test was performed. A mixture of 10 pL of the drug product and 15 pL of 10 mM DTPA (pH 6.5) was incubated at room temperature for 30 minutes.

[0336] After incubation, 10 pL of the mixture was spotted on an iTLC-SG plate and developed with 10 mM EDTA. The plate was dried and scanned 3 days later using an AR-2000 TLC scanner.Under these conditions, fully chelated Ac-225 remained at the baseline, while free or loosely bound225Ac migrated with the solvent front.

[0337] The iTLC analysis showed 99.5% chelated Ac-225, confirming that a stable complex was formed (FIG. 6).

[0338] Stability Testing of Drug Product: The drug product solution was stored in a lead-shielded container at 4 °C and its stability was monitored using size-exclusion high-performance liquid chromatography (sec-HPLC) at t = 0 h and t = 96 h after EOS, with the following sec-HPLC Method:• Column: Bioassist Tosoh TSKgel G3000SWxl (7.8 mm x 30 cm, 5 pm)• Column temperature: Room temperature• Elution buffer: 0.2 M phosphate buffer (pH 6.8)• Flow rate: 1 mL / min• Injection volume: 40 pL• Run time: 25 minutes

[0339] Fractions were collected every 30 seconds post-HPLC. After allowing the fractions to equilibrate at room temperature for >16 hours, the activity in each fraction was measured using aAttorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]gamma-well counter. A radio-chromatogram was reconstructed based on the activities of the collected fractions.

[0340] Results: At t = 0 h, the sec-HPLC UV chromatogram showed a protein purity of 98.3%, with 1.7% high molecular weight species (HMWS). The radiochemical purity of the drug product was 96.6%, with 2.1% radioactive HMWS and 1.3% low molecular weight species (LMWS) (FIG. 7A and FIG. 7B).

[0341] At t = 96 h, the sec-HPLC UV chromatogram indicated a protein purity of 97.4%. The radiochemical purity was 95.1%, confirming the stability of the drug product over time (FIG. 8A and 8B).

[0342] Conclusion: The labeling of DOTA-hl lb6 with225Ac at a 1.173 mCi scale achieved high chelation efficiency, robust stability under DTPA challenge, and maintained protein integrity and radiochemical purity over 96 hours of storage at 4 °C. These results demonstrate the efficacy of the labeling and purification process for producing clinical-grade radiopharmaceuticals.EXAMPLE 4: ACTINIUM-225 RADIOLABELING OF DOTA-H11B6 AT DIFFERENT RATIOS AND SPECIFIC ACTIVITIES

[0343] This example illustrates the labeling of DOTA-hl 1B6 with Actinium-225 (225Ac) at varying specific activities, evaluates the chelation efficiency, and confirms the stability of the radiolabeled product under different conditions. FIGs. 9-18 illustrate the labeling efficiencies and stability profiles across the tested ratios and specific activities.

[0344] Actinium-225 nitrate (225Ac(NO3)s) was received in a glass vial and dissolved in 0.1 N HC1 to achieve a target concentration of 10 mCi / mL. Labeling reactions were conducted at specific activities ranging from 100 pCi / mg to 1600 pCi / mg by varying the ratio of DOTA-hl lb6 to225Ac while maintaining other reaction conditions constant.

[0345] For a specific activity of 100 pCi / mg, corresponding to a DOTA-hl lb6:225Ac molar ratio of approximately 880:1, the reaction was conducted by adding 5 pL of225Ac solution (50 pCi, 10 mCi / mL in 0.1 N HC1) to 20 pL of 3 M sodium acetate (NaOAc) buffer in a plastic vial, followed by 50 pL of DOTA-hl 1B6 solution (10 mg / mL in 25 mM NaOAc, pH 5.5, CAR = 2.5) and 2 pL of 0.1 M NaOH. The pH of the mixture was approximately 6.5 using pH paper, and the reaction was incubated at 37 °C for 2.5 hours. Analysis by instant thin-layer chromatography (iTLC) demonstrated a chelation efficiency of 99.6%, as shown in FIG. 9. To assess stability, a 0.5 pL aliquot of the reaction mixture was mixed with 15 pL of 10 mM DTPA (pH 6.5) and incubated forAttorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]30 minutes at room temperature. iTLC analysis of the DTPA-challenged sample showed that 98.0% ofA225Ac remained chelated to the mAh (FIG. 10).

[0346] For a specific activity of 200 pCi / mg, the procedure was repeated with a reduced amount of DOTA-hl 1B6 (25 pL, 0.25 mg), resulting in a DOTA-hl lb6:225Ac molar ratio of approximately 440: 1. The reaction mixture was incubated under the same conditions as before, yielding a chelation efficiency of 99.5%, as shown in FIG. 11. The DTPA challenge confirmed the stability of the radiolabeled product, with 98.8% of225Ac remaining chelated (FIG. 12).

[0347] At a specific activity of 400 pCi / mg, the volume of DOTA-hl 1B6 was further reduced to 12.5 pL (0.125 mg), corresponding to a DOTA-hl lb6:225Ac molar ratio of approximately 220: 1. The reaction achieved a chelation efficiency of 95.3%, as determined by iTLC (FIG. 13). However, the stability of the product under DTPA challenge showed a reduction, with 88.1% ofA225Ac remaining chelated (FIG. 14).

[0348] For a specific activity of 800 pCi / mg, 6.25 pL of DOTA-hl 1B6 solution (0.0625 mg) was used, resulting in a DOTA-hl lb6:22:Ac molar ratio of approximately 110:1. The reaction achieved a chelation efficiency of approximately 20%, as shown in FIG. 15. Following the DTPA challenge, only 13% ofA225Ac remained chelated (FIG. 16), indicating significantly reduced stability at this specific activity.

[0349] At the highest specific activity of 1600 pCi / mg, 3.13 pL of DOTA-hl 1B6 solution (0.0313 mg) was added to the reaction mixture, corresponding to a DOTA-hl lb6:225Ac molar ratio of approximately 55:1. The chelation efficiency was approximately 29%, as shown in FIG. 17, and the stability under DTPA challenge was further reduced, with only 11% of225Ac remaining chelated (FIG. 18).

[0350] These results demonstrate that the labeling efficiency and stability of225Ac-labeled DOTA-hl 1B6 are highly dependent on the specific activity and the mAb-to-225Ac molar ratio. Lower specific activities (100-200 pCi / mg) yielded high chelation efficiencies (>99%) and robust stability under DTPA challenge, while higher specific activities (>800 pCi / mg) resulted in significantly lower efficiencies and reduced stability. These findings highlight the critical importance of optimizing the antibody-to-radionuclide ratio for reliable and scalable production of radioimmunoconjugates.

[0351] Actinium-225 sources contain non-radioactive metal ions and the level of non-radioactive metals varies from batch to batch and vendor to vendor. The non-radioactive metal ions competeAttorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]with225AC for chelation with DOTA. In the initial chelation step of the phase 1 process, the ratio of [mAb] / [Ac-225] is slightly below the 440:1 cut off, a ratio which is close to the limit of the achievable specific activity according to the above results. In practice, at this <440: 1 [mAb] / [Ac-225] ratio, the labeling yield post PD10 column and before blending is between 75% and 85% because the non-radioactive metal levels vary among the Ac-225 sources used. In the phase 3 process, the ratio of [mAb] / [ Ac-225] is further increased, e.g. to near 880: 1. The increased amount of mAb guarantees there is enough DOTA for Ac-225 and non-radioactive metal ions and ensures near quantitative chelation of Ac-225. It results in a high yielding, reliable and robust labeling process and moreover it negates purification step.EXAMPLE 5: SCALE VARIATION

[0352] Preparation of Reaction Mixture: Actinium-225 nitrate received in a glass vial will be dissolved in 0.1 NHC1 to achieve a target concentration of approximately 20 mCi / mL.

[0353] To a plastic vial containing 0.68 mL to 68 mL of purification buffer (25 mM sodium acetate, 0.04% Polysorbate 20, 0.5% sodium ascorbate w / v, pH 5.5), the following will be added:• 50 pL to 5 mL of225Ac solution (1 mCi to 100 mCi, dissolved in 0.1 NHC1),• 85 pL to 8.5 mL of 0.1 N NaOH, and• 0.68 mL to 68 mL of DOTA-hl lb6 (10 mg / mL in 25 mM sodium acetate at pH 5.5).

[0354] The reaction solution will be mixed gently by pipetting up and down. The pH of the solution will be verified using pH paper to be between 6 and 6.5. The reaction mixture will then be allowed to stand at 37 °C for 2 hours to complete the labeling.

[0355] iTLC Analysis: After the reaction, a 0.5 pL aliquot of the reaction mixture will be spotted onto an iTLC-SG plate. The plate will be developed with 10 mM EDTA (pH 6.0) as the mobile phase. The developed plate will be dried and scanned using an AR-2000 TLC scanner after standing for more than 16 hours. The iTLC will confirm successful chelation of225Ac to the antibody.

[0356] Sterile Filtration and Formulation: The labeling reaction solution will be transferred into a syringe fitted with a 0.22 pm filter. The reaction vial will be washed with 3 mL to 300 mL of formulation buffer, and the washings will be added to the syringe. The solution will then be filtered into a plastic tube.

[0357] The reaction vial will be washed further with 4.5 mL * 2 to 450 mL x 2 of purification buffer, and the washings will also be filtered into the plastic tube. Air will be pushed through theAttorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]filter to ensure complete transfer. The filtrate collected in the plastic tube will be the drug product, marking the end of synthesis (EOS).

[0358] DTPA Challenge: To evaluate chelation stability, 10 pL of the drug product solution will be mixed with 15 pL of 10 mM DTPA (pH 6.5). The mixture will be incubated at room temperature for 30 minutes. A 10 pL aliquot will then be spotted on an iTLC-SG plate, developed with 10 mM EDTA, dried, and scanned using an AR-2000 TLC scanner after standing for more than 16 hours. The iTLC will confirm the stability of the223Ac chelation.

[0359] Stability Testing: The drug product will be shielded in a lead container and stored at 4 °C.Its stability will be monitored by sec-HPLC analysis at t = 0 h and t = 96 h after EOS, with the following sec-HPLC Methodology:• Column: Bioassit Tosoh TSKgel G3000SWxl (7.8 mm * 30 cm, 5 pm)• Injection volume: 40 pL• Elution buffer: 0.2 M phosphate buffer (pH 6.8)• Flow rate: 1 mL / min• Run time: 25 minutes

[0360] Fractions will be collected every 30 seconds post-HPLC and allowed to stand for >16 hours. The radioactivity in each fraction will be measured using a gamma-well counter, and a radiochromatogram will be reconstructed. The HPLC will confirm the radiochemical purity of the drug product.EXAMPLE 6: CONCENTRATION VARIATION

[0361] Preparation of Reaction Mixture: Actinium-225 nitrate received in a glass vial will be dissolved in 0.05 to 0.2 N HC1 to achieve a target concentration of approximately 5 to 50 mCi / mL.

[0362] To a plastic vial containing 0.5 to 1.5 mL of purification buffer (25 mM sodium acetate, 0.04% Polysorbate 20, 0.5% sodium ascorbate w / v, pH 5.5), the following will be added:• 20 pL to 200 pL of Ac-225 solution (1 mCi, dissolved in 0.05 to 0.2 N HC1),• 34 pL to 340 pL of 0.05 to 0.2 NNaOH, and• 0.5 to 1.5 mL of DOTA-hl lb6 (10 mg / mL in 25 mM sodium acetate at pH 5.5).

[0363] The reaction mixture will be gently mixed by pipetting up and down. If the pH is not between 6 and 6.5, it will be adjusted using 0.05 to 0.2 N HC1 or 0.05 to 0.2 NNaOH. The reaction will then be left standing at 37 °C for 2 hours.Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]

[0364] iTLC Analysis: After the reaction, a 0.5 pL aliquot of the reaction mixture will be spotted on an iTLC-SG plate. The plate will be developed with 10 mM EDTA (pH 6.0), dried, and scanned using an AR-2000 TLC scanner after standing for more than 16 hours. The iTLC will confirm successful chelation of225Ac.

[0365] Sterile Filtration and Formulation: The reaction mixture will be diluted to the required concentration of the conjugate in the drug product. Using a syringe fitted with a 0.22 pm filter, the reaction mixture will be filtered into a plastic tube.

[0366] The reaction vial will be washed with the calculated volume of formulation buffer, and the washings will also be filtered into the plastic tube. Air will be pushed through the filter to ensure complete transfer. The filtrate collected in the plastic tube will be the drug product, marking the end of synthesis (EOS).

[0367] DTPA Challenge: To evaluate chelation stability, 10 pL of the drug product solution will be mixed with 15 pL of 10 mM DTPA (pH 6.5). The mixture will be incubated at room temperature for 30 minutes. A 10 pL aliquot will then be spotted on an iTLC-SG plate, developed with 10 mM EDTA, dried, and scanned using an AR-2000 TLC scanner after standing for more than 16 hours. The iTLC will confirm the stability of the225Ac chelation.

[0368] Stability Testing: The drug product will be shielded in a lead container and stored at 4 °C.Its stability will be monitored by sec-HPLC analysis at t = 0 h and t = 96 h after EOS, using the same sec-HPLC method as described above. The HPLC will confirm the radiochemical purity and stability of the drug product over time.EXAMPLE 7: MANUFACTURE OF225AC-DOTA-H11B6

[0369] Dissolve225Ac(NO3)3 in 0.1 N HC1 to target a concentration of 25 mCi / mL by mixing at 300 RPM at RT for >30 mins to allow for complete dissolution. Transfer the dissolved Ac225 to new plastic vial and measure the activity. Allow the Ac225 in the plastic tube to equilibrate for >30 min. Measure and record the radioactivity of the solution. Adjust the activity to target concentration (e.g., 20 mCi / mL) with the addition of HC1, if needed. Measure and record the radioactivity of stock solution.

[0370] Based on the predetermined batch size (A) for the manufacturing, calculate the volume of a sterile reaction buffer to be added to a 50-mL plastic tube (B):Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]>wherein:A = Batch scale size (pCi)RAC TO = Radioactive concentration at manufacturing start (e.g., 70.5 pCi / mL)Ciarget = Target DOTA-hl 1B6 protein conjugate concentration in final drug product (DP) (e.g., 0.5 mg / mL)CDS = Incoming concentration of DOTA-hl 1B6 (e.g., 10 mg / mL) prior to the radiolabeling

[0371] Add the sterile reaction buffer to the 50-mL plastic tube at the calculated B volume. The sterile reaction buffer contains: 6% sodium ascorbate, 0.5M acetate, and 0.04% Tween-20. It is obtained by dissolving sodium ascorbate into a base reaction buffer (0.5 M acetate, 0.04% Tween-20, pH 7.0 ± 0.2, metal free water), followed by filtering with a 0.22 pm filter.

[0372] Add the Ac-225 stock solution to the 50-mL plastic tube at a volume calculated based on the measured radioactivity (RAC) of the Ac-225 stock solution and according to the batch size (A) using the following equation:Vol. to be added = Batch scale size (pCi) / 7 C of stock solution ( Ci / [iL).

[0373] Add incoming DOTA-hl 1B6 to the above 50-mL plastic tube at a volume equal to that of the reaction buffer (B). Measure the pH of the reaction mixture with pH strip, which should be about 6 - 6.5 (strip).

[0374] The reaction mixture is incubated at 35°C ± 2°C for at least 3 hours (180 minutes) and maximum up to 4 hours (240 minutes). No shaking / mixing to be applied to the reaction mixture during the incubation. At the end of incubation period, take 50 pL of reaction mixture and measure the pH using a pH probe. After sample collection, measure and record radioactivity in pCi.

[0375] Calculate the volume (G) of a sterile dilution buffer required to dilute the reaction mixture:G = F - Ewherein:F = total DP volume:A EF" RACTl0XCA = Batch size (mCi)RAC TO = Radioactive concentration at manufacturing start (e.g., 70.5 mCi / mL)Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]C = Vol. of reaction mixture (Vol. reaction buffer + Vol. Av225 solution + Vol.DOTA-hllB6)E = Vol. of reaction mixture avaialble for dilution:E = C - DVol of Samples Taken

[0376] The sterile dilution buffer contains 3% sodium ascorbate, 3 mM acetic acid, and 0.04% Tween-20. It is obtained by dissolving sodium ascorbate into a stock dilution buffer (3 mM acetic acid, 0.04% Tween-20, metal free water), followed by filtering with a 0.22 pm filter.

[0377] To dilute the reaction mixture, transfer the reaction mixture from the reaction tube to a dilution vial, add the calculated volume (G) of sterile dilution buffer to the reaction tube to ensure complete rinsing of the reaction tube, then transfer the dilution buffer from the reaction tube to the dilution vial containing the radiolabeled materials. After dilution, measure and record radioactivity of the diluted drug product (DP) in the vial.

[0378] The diluted DP is analyzed by iTLC with DTPA challenge. Mix 10 pL of DP solution with 15 pL of 10 mM DTPA (pH 6.5). Agitate the solution by inversion for 60 seconds until completely mixed. The mixture is left at 37°C for 30 min. Spot 2pL of the mixture on iTLC and developed with 90% 10 mM EDTA / 10% MeOH solution. The TLC strip is then dried and scanned at T=0 and >16 h after development on a TLC scanner.

[0379] A filtration step is conducted on the diluted DP for sterilization.

[0380] In this example, dilution following incubation facilitated sterile filtration and adjustment of specific activity while maintaining radiochemical purity, consistent with the process embodiments described herein.

[0381] The conditions employed in this example fall within the disclosed embodiments utilizing immunoconjugate-to-Ac-225 molar ratio of at least about 440: 1 and a specific activity at or below about 200 pCi / mg, thereby enabling exclusion of intermediate purification steps.

Claims

Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]CLAIMS1. A process for producing a radioimmunoconjugate comprising Actinium-225 (225Ac) chelated to an immunoconjugate, wherein the immunoconjugate comprises a chelating agent conjugated to an antibody, or an antigen-binding fragment thereof, the process comprising:(a) mixing the immunoconjugate with225Ac in a reaction buffer, wherein the reaction buffer comprises:(i) a radioprotectant,(ii) a buffering agent, and(iii) a surfactant, to form a reaction mixture, wherein the ratio of immunoconjugate to225Ac is at least about 440 to 1;(b) incubating the reaction mixture of (a) under conditions sufficient to form the radioimmunoconjugate; and(c) filtering the reaction mixture through a sterile filter to produce a sterile radioimmunoconjugate;wherein the specific activity of the radioimmunoconjugate is at or below about 200 pCi / mg, andwherein the process excludes intermediate purification steps between mixing, incubation, and filtration.

2. A process for producing a radioimmunoconjugate comprising Actinium-225 (225Ac) chelated to an immunoconjugate, wherein the immunoconjugate comprises a chelating agent conjugated to an antibody, or an antigen-binding fragment thereof, the process comprising:(a) mixing the immunoconjugate with225Ac in a reaction buffer to form a reaction mixture, wherein the reaction buffer comprises:(i) a radioprotectant,(ii) a buffering agent, and(iii) a surfactant, and(b) incubating the reaction mixture of (a) at a temperature of 33°C to 39°C for up to 4 hours to form the radioimmunoconjugate,optionally, the process further comprising:Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT](c) filtering the reaction mixture through a sterile filter to produce a sterile radioimmunoconjugate, wherein the process excludes intermediate purification steps between mixing, incubation, and filtration.

3. The process of claim 1 or 2, wherein the chelating agent is 1,4,7,10-tetraazacyclododecane- 1,4, 7,10-tetraacetic acid (DOTA) or a derivative thereof.

4. The process of any one of claims 1-3, wherein the chelating agent is conjugated to the antibody, or antigen-binding fragment thereof, via a linker, such as a linker that is or comprises 2- isothiocyanatobenzyl; preferably, the antibody or antigen-binding fragment thereof is conjugated to 2-(4-isothiocyanatobenzyl)- 1 ,4,7, 10-tetraazacyclododecane- 1 ,4,7, 10-tetraacetic acid, and wherein the conjugation comprises attachment of one or more molecules of 2-(4- isothiocyanatobenzyl)-!, 4, 7, 10-tetraazacyclododecane- 1,4, 7,10-tetraacetic acid.

5. The process of any one of claims 1 to 4, wherein:i. the radioprotectant is sodium ascorbate;ii. the buffering agent is sodium acetate; and / oriii. the surfactant is Polysorbate 20.

6. The process of any one of claims 1 to 5, wherein the reaction buffer comprises:i. sodium ascorbate at about 0.1% to about 8% (w / v) (such as about 0.1% to about 5% (w / v));ii. sodium acetate at about 0.01 M to about 1 M; andiii. Polysorbate 20 at about 0.01% to about 1% (v / v).

7. The process of any one of claims 1 to 6, wherein the pH of the reaction mixture is or is adjusted to about 6.0-6.5 before step (b).

8. The process of any one of claims 1 to 7, wherein the incubation is performed at about 37 °C, 36 °C, 35 °C, 34 °C or 33 °C.

9. The process of any one of claims 1 to 8, wherein the incubation is carried out for about 2 hours, about 3 hours, or about 4 hours.

10. The process of any one of claims 1 to 9, further comprising diluting the reaction mixture after step (b) or step (c) with a dilution buffer comprising a second radioprotectant, a second buffering agent and a second surfactant, such as a dilution buffer comprising sodium ascorbate, acetic acid, and / or Polysorbate 20, or a dilution buffer comprising about 0.1-5% (w / v) sodium ascorbate, about 0.5 - 10 mM acetic acid, and about 0.01% to about 1% (v / v) Polysorbate 20.Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]11. The process of any one of claims 1 to 10, wherein the sterile filter has a pore size of about 0.22 pm.

12. The process of any one of claims 1 to 11, wherein:i. the radioimmunoconjugate is directly filtered into a sterile container;ii. the radioimmunoconjugate is prepared at a scale of about 0.1 mCi to about 1,000 mCi;iii. the radioimmunoconjugate has a radiochemical purity of at least 95% as determined by instant thin-layer chromatography (iTLC);iv. the process further comprises performing a stability challenge using diethylenetriamine pentaacetate (DTP A) to confirm chelation stability of the radioimmunoconjugate;v. at least 95% of the225Ac remains chelated after 30 minutes of incubation with DTPA as measured by a DTPA challenge;vi. the process further comprises analyzing the radioimmunoconjugate using sizeexclusion high-performance liquid chromatography (SEC-HPLC) to confirm protein integrity and radiochemical purity;vii. the radiochemical purity remains at least about 90% after 96 hours of storage at 4 °C;and / orviii. the process does not comprise a step of mixing the radioimmunoconjugate recovered at step (c) with an unlabeled immunoconjugate.

13. The process of any one of claims 1 to 12, wherein:i. the process is scaled for large-scale production or direct sterile fill with automated or semi-automated systems; and / orii. the process is a GMP-compliant process.

14. The process of any one of claims 1 to 13, wherein the immunoconjugate comprises an antibody, or an antigen binding fragment thereof, that specifically binds to human kallikrein-related peptidase 2 (hK2).

15. The process of claim 14, wherein the antibody or an antigen-binding fragment thereof, comprises:a heavy chain CDR1 (HCDR1) of SEQ ID NO: 5;a heavy chain CDR2 (HCDR2) of SEQ ID NO: 6;Attorney Docket No. 065768.12722 / 220WO1[JBI6978 PCT]a heavy chain CDR3 (HCDR3) of SEQ ID NO: 7;a light chain CDR1 (LCDR1) of SEQ ID NO: 8;a light chain CDR2 (LCDR2) of SEQ ID NO: 9; anda light chain CDR3 (LCDR3) of SEQ ID NO: 10.

16. The process of claim 15, wherein the antibody or an antigen-binding fragment thereof, comprises a variable heavy chain (VH) sequence of SEQ ID NO: 1 and a variable light chain (VL) sequence of SEQ ID NO: 2.

17. The process of claim 16, wherein the antibody comprises a heavy chain (HC) sequence of SEQ ID NO: 3 or SEQ ID NO: 12, and a light chain (LC) sequence of SEQ ID NO: 4.

18. The process of any one of claims 1 to 17, wherein the process is for producing a radioimmunoconjugate for the treatment of a prostate cancer, such as a metastatic prostate cancer, or a metastatic castration-resistant prostate cancer (mCRPC).

19. A radioimmunoconjugate obtained or obtainable by the process of any one of claims 1 to 18.

20. A composition comprising a radioimmunoconjugate obtained or obtainable by the process of any one of claims 1 to 18.

21. An intermediate composition, which is obtained or obtainable by implementing the process of any one of claims 1 to 18, wherein the intermediate composition comprises a radioimmunoconjugate and a buffer solution, and wherein the intermediate composition is obtained or obtainable at step (b) or (c) of the process.

22. A method of treating a prostate cancer comprising administering a therapeutically effective amount of a radioimmunoconjugate produced by the process of any one of claims 1-18 to a patient in need thereof.

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