Dimeric immunoconjugates for the treatment of cancer and methods of use thereof

Abstract

The present disclosure relates generally to dimeric immunoconjugates for the treatment of cancer, in particular to dimeric immunoconjugates that bind human sperm protein 17 (Sp17). The dimeric immunoconjugates of the present disclosure generally comprise a first monomeric antibody carrying a first payload and a second monomeric antibody carrying a second payload, wherein the first and second payloads are different. Such dimeric immunoconjugates advantageously allow for the simultaneous delivery of two chemotherapeutic agents to cancer cells, thereby enabling synergistic anti-tumor activity.

Description

Cross-references to related applications

[0001] This application claims the benefit of U.S. Patent Application No. 63 / 604,155, filed November 29, 2023, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0002] This disclosure generally relates to dimeric immunoconjugates for the treatment of cancer, and specifically to dimeric immunoconjugates that bind to human spermin 17 (Sp17). Background Technology

[0003] Therapeutic antibodies have significantly improved cancer treatment outcomes and provided new opportunities for at least a portion of patients to be cured of cancer. The first antibody-based cancer immunotherapy, Rituxan®, was approved in the United States in 1997 for the treatment of B-cell non-Hodgkin's lymphoma, and its lifetime sales have exceeded $100 billion. Despite widespread competition, Rituxan® still generates over $1 billion in annual sales. For example, the competitive antibody Zevalin® was approved in the United States in 2002. Both Rituxan® and Zevalin® target the B-cell antigen CD20, and both immunotherapies work by depleting B cells. When Rituxan® binds to CD20, it triggers antibody-dependent cytotoxicity and leukocyte-mediated cell death; while Zevalin® is chemically modified to chelate a radioactive isotope, thereby enabling additional radiation-induced cell death. Therapeutic antibodies can also be conjugated to cytotoxic drugs, for example, by constructing antibody-drug conjugates using cleavable linkers, allowing them to release their cytotoxic payload upon binding to the antigen. Many other antibody-based cancer treatment strategies also exist.

[0004] While therapeutic antibodies have revolutionized oncology, progress has been incremental. Cancer is not yet cured. Therefore, innovative strategies to improve upon existing antibody technologies remain a priority. Summary of the Invention

[0005] Several aspects of this disclosure relate to the discovery that dimeric immunoconjugates offer advantages in improving efficacy compared to conventional monomeric immunoconjugates and naked immunotherapies. The dimeric immunoconjugates of this disclosure advantageously carry different payloads on their two constituent monomers, allowing targeting a single cancer cell with the dimeric immunoconjugate to simultaneously deliver two different payloads to the cancer cell, thereby producing synergistic antitumor activity. Dimeric immunoconjugates typically also contain two Fc fragments, thereby enabling leukocytes, such as natural killer cells, to target cancer cells, thus providing a third independent cancer cell neutralization mechanism. Dimeric immunoconjugates can also more effectively mediate complement-mediated lysis of target cells.

[0006] Dimeric immunoconjugates containing two different loads can be prepared, for example, by crosslinking a first immunoconjugate containing a first load with a second immunoconjugate containing a second load.

[0007] Dimeric immunoconjugates exhibit pharmacokinetics, pharmacodynamics, and affinity that differ from their constituent monomers. These factors collectively and advantageously alter the administration route and efficacy of dimeric immunoconjugates in previously unrecognized and unpredictable ways. The inventors unexpectedly discovered that while the simultaneous administration of two different monomeric immunoconjugates can exhibit synergistic effects, cross-linking two different monomeric immunoconjugates to produce a dimeric immunoconjugate exhibits enhanced synergistic effects. For example, two different monomeric immunoconjugates may have different pharmacodynamics and / or pharmacokinetics, such that simultaneous administration does not correspond to simultaneous binding to the same cancer cell; and random variations in antigen binding events and / or endocytosis mean that even with similar pharmacodynamics and pharmacokinetics, two different monomeric immunoconjugates may not be able to simultaneously deliver their payloads to the same cancer cell. The dimeric immunoconjugates of this disclosure improve upon both of these problems, at least because they almost always deliver two payloads simultaneously. Furthermore, certain cancer-associated antigens exhibit low cell surface expression, low accessibility to antibodies, or other interfering characteristics that reduce the probability of multiple immunotherapeutic agents binding to the same cancer cell. The dimer immunoconjugates disclosed herein are particularly effective for targeting such antigens because: (1) the dimer immunoconjugates typically carry a payload approximately twice that of the monomeric immunoconjugates, thus delivering twice the payload in a single binding event; and (2) a synergistic combination of different payloads can be delivered in a single binding event.

[0008] Without limiting this disclosure or any patent claims formed based on this disclosure, the inventors have found that dimeric immunoconjugates that bind to cancer antigens are more advantageous than conventional immunoconjugates because they can cross-link cancer cells, thereby inhibiting metastasis that allows cancer to spread to other parts of the body, and can also inhibit cancer from escaping local interventions or immune responses.

[0009] Conventional naked therapeutic antibodies typically induce Fc receptor-mediated cell death, while conventional immunoconjugates can simultaneously achieve Fc receptor-mediated cell death and antitumor activity based on their payload. However, the payload risks masking the Fc moiety of the antibody, thereby limiting Fc receptor-mediated activity. Dimeric immunotherapeutic agents typically further mask the Fc moiety of the antibody, further limiting Fc receptor-based antitumor activity. The weakened Fc receptor-mediated cytotoxicity might have been expected to reduce the efficacy of the dimer immunoconjugate, but the inventors discovered the opposite. The dimer immunoconjugate exhibited unexpectedly improved efficacy compared to the monomeric immunoconjugate, for example, given the weakened Fc receptor-mediated cytotoxicity of the dimer immunoconjugate.

[0010] Without limiting this disclosure or any patent claims formed based on this disclosure, the inventors have discovered that limiting Fc receptor-based antitumor activity can be utilized to reduce nonspecific Fc binding and improve safety relative to conventional immunotherapeutic agents. Such advantages may be particularly useful in the reticuloendothelial system, including benefiting the liver by reducing the nonspecific side effects of antibody-drug conjugates. Therefore, eliminating the dependence of immunoconjugates on the helper Fc receptor pathway enables the development of immunoconjugates with higher drug-to-antibody ratios and also allows for the administration of higher doses of dimeric immunoconjugates while minimizing the risks associated with nonspecific Fc-related toxicity.

[0011] Without limiting this disclosure or any patent claims formed based on this disclosure, the inventors have discovered that dimer immunoconjugates binding to cancer antigens are more advantageous than conventional immunoconjugates, for example, because the additional binding sites facilitate the aggregation of cell surface antigens and thereby endocytosis, which retains the immunoconjugate within cancer cells, thus enhancing antitumor efficacy. Retaining immunoconjugates containing radioactive isotopes within cancer cells can both accelerate cell death and protect other cells from radiation. Many immunoconjugates containing pharmaceutical agents include cleavable linkers engineered to release the pharmaceutical agent after endocytosis, for example, in response to the pH of the endocytic vesicle or in response to hydrolases or other enzymes within the vesicle. Dimeric immunoconjugates accelerate the release of pharmaceutical agents that become cleavable after endocytosis by promoting aggregation that leads to endocytosis.

[0012] Another unexpected advantage of dimeric immunoconjugates is that improvements in one, two, or all of their efficacy, pharmacokinetics, and pharmacodynamics enable shorter infusion times compared to monomeric immunoconjugates. Therapeutic antibodies used as cancer immunotherapies are typically administered via intravenous infusion over a period of time, such as several hours. With the increasing development of therapeutic antibodies and other infusion-based therapeutic interventions, the capacity of infusion centers has become a limiting factor. Therefore, pharmacological interventions that can be administered in a shorter time compared to comparable interventions can reduce the burden on infusion centers, lower costs, and improve patient experience, and these benefits are independent of the improvements in clinical outcomes described in this article.

[0013] Without limiting the scope of this disclosure or any patent claims formed based on this disclosure, the dimer immunoconjugate can also enhance the cytotoxicity of cancer cells by increasing the number of immunoconjugates binding to cancer cells, since the binding domains of the immunoconjugates constituting the dimer may not be fully occupied when binding antigens. This can overcome some of the obstacles encountered when targeting copy number antigens on cancer cells by increasing the number of immunoconjugate units in the dimer that are close to and / or bind to cancer cells. Therefore, the dimer immunoconjugate may be ideal for targeting cancers showing low expression density of cell surface antigens. The size of the dimer immunoconjugate is also approximately twice that of the monomeric immunoconjugate, thus carrying approximately twice the payload to improve efficacy. Therefore, compared to the monomeric immunoconjugate, a single antigen binding event of the dimer immunoconjugate can localize twice the amount of payload to the vicinity of the target cell.

[0014] The following disclosure broadly describes various other novel aspects of the use of dimeric immunoconjugates in oncology. Nothing in the Summary, Background and Technical Field sections, or the following brief description of the drawings, detailed description, and examples should limit any patent claims formed based on this disclosure; any such patent claims should be interpreted in conjunction with the text of the claims and their dependent relationships. Brief description of the attached figures

[0015] Figure 1 These are SDS-PAGE gel images of 2 micrograms of recombinant SP17 protein loaded with molecular weight standards (lane M) and under reducing conditions (lane 1) and non-reducing conditions (lane 2).

[0016] Figure 2The image consists of two subplots. The left subplot is an SDS-PAGE gel stained with Coomassie Brilliant Blue, loaded with molecular weight standards (lane M), anti-SP17 antibody chAB2 under reducing conditions (lane 1), and anti-SP17 antibody chAB2 under non-reducing conditions (lane 2). The right subplot is a Western blot of the SDS-PAGE gel loaded with molecular weight standards (lane M) and anti-SP17 antibody chAB2 under reducing conditions (lane E), where chAB2 was detected by enhanced chemiluminescence (ECL) using anti-mouse secondary antibody.

[0017] Figure 3 It is a protein blot of an SDS-PAGE gel loaded with molecular weight standards (lane M) and SP17 protein (lanes 1, 2 and 3), in which SP17 protein was detected using anti-SP17 antibody chAB2.

[0018] Figure 4 The results consist of three subplots. The left subplot shows the flow cytometry results of ID8 cells incubated with anti-mouse fluorescein isothiocyanate (FITC) antibody but without primary antibody. The middle subplot shows the flow cytometry results of ID8 cells labeled with anti-SP17 antibody chAB2 and anti-mouse FITC antibody. The right subplot is a histogram showing that, compared to the ID8 cells incubated only with anti-mouse FITC antibody in the left subplot, the mean fluorescence intensity of ID8 cells labeled with chAB2 and anti-mouse FITC antibody in the middle subplot is increased.

[0019] Figure 5 The chromatogram is a high-performance liquid chromatography (HPLC) chromatogram, indicating that the anti-SP17 antibody chAB2 formulation can be manufactured with a purity of 93%.

[0020] Figure 6 This is a surface plasmon resonance (SPR) plot of the anti-SP17 antibody chAB2 binding to recombinant 6His-SP17 protein. The lines from highest to lowest correspond to the fitted cycles of 6His-SP17 concentrations at 300 nmol, 100 nmol, 50 nmol, 25 nmol, 12.5 nmol, and 6.25 nmol. This data was used to calculate the binding rate constant (ka) of chAB2 to SP17 as 62,600 mol / s, the dissociation rate constant (kd) as 0.0001298 s / s, and the dissociation constant (KD) as 2.073 nmol.

[0021] Figure 7 These are SDS-PAGE gel images of anti-SP17 antibody SP17-AB2 loaded with molecular weight standards (lane M) and under reducing conditions (lane 1) and non-reducing conditions (lane 2).

[0022] Figure 8It is a Western blot of protein on an SDS-PAGE gel loaded with molecular weight standard (lane M), SP17 protein (lane 1) and tumor cell lysate (lane 2), in which SP17 protein is detected using anti-SP17 antibody SP17-AB2.

[0023] Figure 9A , 9B 9C is a graph showing the immunotherapy concentration (x-axis, nanomolar) versus cytotoxicity against SK-OV-3 cells (y-axis, percentage of cytotoxicity), with the immunotherapy agents selected from the monomeric antibody SP17-AB2 (circle, ●), the monomeric immunoconjugate SP17-AB2-DXD (square, ■), the control human IgG4 antibody (positive triangle, ▲), and deruxtecan (inverted triangle, ▼). Figure 10 This is a graph showing the relationship between the concentration of immunotherapy drugs (x-axis, nanomolar) and their cytotoxicity to ID8 cells (y-axis, percentage of cytotoxicity). The immunotherapy drugs compared in the graph include: the monomeric antibody SP17-AB2 (dot, ●), the immunoconjugate SP17-AB2-DXD (square, ■), hIgG4 (upper triangle, ▲), and deruxtecan (lower triangle, ▼). Detailed Implementation

[0024] Several aspects of this disclosure relate to a method of treating cancer in a human subject, including administering a therapeutically effective amount of an immunotherapeutic agent to the subject. The term "immunotherapeutic agent," unless otherwise contextualized in this disclosure, refers to a dimeric immunoconjugate. The immunotherapeutic agent typically comprises two covalently cross-linked antibodies, each conjugated to a different payload.

[0025] Several aspects of this disclosure relate to an immunotherapeutic agent comprising a first immunoconjugate and a second immunoconjugate, the first immunoconjugate and the second immunoconjugate being covalently cross-linked.

[0026] In this disclosure, "immunoconjugate" refers to an antibody conjugated to either a pharmaceutical agent or a radioisotope. Specific conjugation strategies are not limiting. Nevertheless, immunoconjugates conjugated to pharmaceutical agents are typically covalently cross-linked to the pharmaceutical agent via a linker as described below. Immunoconjugates conjugated to radioisotopes are typically covalently cross-linked to a chelating agent that chelates the radioisotope as described below.

[0027] The term "antibody" includes immunoglobulins (Ig) of different classes (e.g., IgA, IgG, IgM, IgD, and IgE) and subclasses (e.g., IgG2a and IgG4), and includes fully human antibodies, chimeric antibodies, and their engineered variants. For example, this specification describes a chimeric human-mouse IgG (chAB2) and a fully human IgG engineered to contain the Ser228Pro mutation (SP17-AB2). The dimeric immunoconjugates disclosed herein generally contain IgG antibodies, but the specific class of immunoglobulin is not limiting.

[0028] There are various strategies for preparing dimeric immunoconjugates, and the specific linking strategy should not limit this disclosure or any claims formed based on this disclosure. For example, a first monomeric immunoconjugate can be mixed with an excess molar amount of 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfosuccinimide ester (sulfo-SMCC) or 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfosuccinimide ester (SMCC) to modify one or more amino groups of the immunoconjugate, such as the ε-amino group of lysine, using a maleimide that can react with a thiol group. This conjugation strategy is generally applicable to antibodies and is used, for example, to crosslink maytansine with trastuzumab to prepare the antibody-drug conjugate trastuzumab emtansine, i.e., Kadcyla®. The second monomeric immunoconjugate can be mixed, for example, with an excess molar amount of S-acetylmercaptopropionic acid N-succinimide ester (SATP) or S-acetylmercaptoacetic acid N-succinimide ester (SATA), thereby modifying one or more amino groups of the immunoconjugate, such as the ε-amino group of lysine, with an acetyl-protected thiol group. The thiol group can then be deprotected, for example, with hydroxylamine under acidic conditions. Approximately equimolar amounts of the first and second monomeric immunoconjugates can then be crosslinked. The resulting dimer immunoconjugate can be purified, for example, by dialysis and / or size exclusion chromatography.

[0029] Antibodies contain dozens of lysine residues; therefore, chemical cross-linking based on strategies that primarily modify the ε-amino group of lysine often results in heterogeneous mixtures of various dimers exhibiting different efficacies. For example, two different antibodies can be covalently cross-linked using disulfide bonds by introducing a thiol group by cloning a cysteine ​​residue into a specific position within the antibody, thus ensuring a precise quaternary structure. Such strategies are known and, for example, documented in Shopes, B., “A genetically engineered human IgG mutant with enhanced cytolytic activity,” *Journal of Immunology*, 1992, 148(9):2918-22. Cloning a thiol group into the antibody to form a disulfide bond in the monomeric component of the cross-linked dimer immunoconjugate also advantageously preserves the ε-amino group of lysine in the monomeric component for use in the conjugation load.

[0030] In this disclosure, the term "cancer" refers to any type of carcinoma, sarcoma, lymphoma, leukemia, germ cell tumor, and blastoma. In some embodiments, the cancer is brain cancer, ovarian cancer, breast cancer, vaginal cancer, vulvar cancer, uterine cancer, cervical cancer, endometrial cancer, prostate cancer, testicular cancer, penile cancer, liver cancer, intrahepatic bile duct cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, bronchial cancer, mesothelioma, pancreatic cancer, gallbladder cancer, non-melanoma skin cancer, melanoma, Kaposi's sarcoma, thyroid cancer, head and neck cancer, nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer, laryngeal cancer, oral cancer, tongue cancer, oral cancer, salivary gland cancer, esophageal cancer, gastric cancer, colorectal cancer, colon cancer, rectal cancer, anal cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, bladder cancer, urethral cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, myeloma, multiple myeloma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, osteosarcoma, or soft tissue cancer. In some specific embodiments, the cancer is multiple myeloma, ovarian cancer, or non-small cell lung cancer.

[0031] In this disclosure, the term "treatment" means at least one of the following: curing cancer; increasing the probability of cancer being cured; shortening the time required to cure cancer; increasing the probability of shortening the time required to cure cancer; reducing cancer severity; increasing the probability of reducing cancer severity; shortening the time required to reduce cancer severity; increasing the probability of shortening the time required to reduce cancer severity; inhibiting cancer progression; increasing the probability of cancer not progressing; delaying cancer progression; increasing the probability of delaying cancer progression; inhibiting cancer occurrence or recurrence; reducing the probability of cancer occurrence or recurrence; delaying cancer onset; increasing the probability of delaying cancer onset; alleviating at least one symptom of cancer; increasing the probability of alleviating at least one symptom of cancer; shortening the time required to cure at least one symptom of cancer. The time required for relief; increasing the probability of shortening the time required for relief of at least one cancer symptom; reducing the severity of at least one cancer symptom; increasing the probability of reducing the severity of at least one cancer symptom; shortening the time required for reducing the severity of at least one cancer symptom; increasing the probability of shortening the time required for reducing the severity of at least one cancer symptom; inhibiting the deterioration of at least one cancer symptom; increasing the probability of not deteriorating at least one cancer symptom; delaying the deterioration of at least one cancer symptom; increasing the probability of delaying the deterioration of at least one cancer symptom; inhibiting the occurrence or recurrence of at least one cancer symptom; reducing the probability of the occurrence or recurrence of at least one cancer symptom; delaying the onset of at least one cancer symptom; and increasing the probability of delaying the onset of at least one cancer symptom.

[0032] In this disclosure, the term "therapeutic effective amount" refers to an amount that is effective in treating cancer, which is typically determined empirically first based on preclinical data and then confirmed through pilot and / or phase I-II clinical trials.

[0033] In some embodiments, the administration is performed by injection. In some specific embodiments, the injection is intravenous, intradermal, subcutaneous, intramuscular, intraperitoneal, or intratumoral. In some very specific embodiments, the injection is intravenous.

[0034] The immunotherapeutic agents disclosed herein typically comprise a first immunoconjugate and a second immunoconjugate covalently cross-linked. The first immunoconjugate typically comprises a first antibody binding to a first cancer-associated antigen and a first payload selected from a first radioisotope and a first pharmaceutical agent. The second immunoconjugate typically comprises a second antibody binding to a second cancer-associated antigen and a second payload selected from a second radioisotope and a second pharmaceutical agent. In some embodiments, the first cancer-associated antigen and the second cancer-associated antigen are the same.

[0035] The first and second antibodies in an immunotherapeutic agent are not necessarily identical and may differ, for example, in post-translational modifications, which typically result in heterogeneous glycosylation patterns. In some embodiments, the first and second antibodies in the immunotherapeutic agent are encoded by the same nucleotide sequence; such antibodies typically have the same amino acid sequence. In some embodiments, the first and second antibodies in the immunotherapeutic agent are encoded by different nucleotide sequences that encode different amino acid sequences; these different amino acid sequences may, for example, ensure a specific quaternary structure, generate complementary stereostructures, and / or provide complementary electrostatic charges via one or more inter-IgG disulfide bonds to promote a favorable relative orientation between the first and second antibodies, thereby increasing affinity relative to random or other unoptimized orientations.

[0036] In some embodiments, the first antibody and the second antibody have amino acid sequences with at least 90% sequence homology. In some specific embodiments, the first antibody and the second antibody have amino acid sequences with at least 95% sequence homology. In some very specific embodiments, the first antibody and the second antibody have amino acid sequences with at least 98% sequence homology.

[0037] As used in this disclosure, the term "sequence homology" refers to the percentage of "positive matches" determined across the full length of the sequence by Standard Protein BLAST®. Standard Protein BLAST® is available at https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastp. BLAST® is commonly described in Altschul et al. (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs," *Nucleic Acids Res.*, 25:3389-3402, and Altschul et al. (2005), "Protein database searches using compositionally adjusted substitution matrices," *FEBS J.*, 272:5101-5109. As used in this disclosure, the term "sequence identity" refers to the percentage of a complete match across the entire length of the sequence.

[0038] In some embodiments, the first antibody and the second antibody have amino acid sequences with at least 90% sequence identity. In some specific embodiments, the first antibody and the second antibody have amino acid sequences with at least 95% sequence identity. In some very specific embodiments, the first antibody and the second antibody have amino acid sequences with at least 98% sequence identity.

[0039] In some implementations, the first antibody and the second antibody have the same amino acid sequence.

[0040] In some embodiments, the immunotherapy agent is a dimer of a first immunoconjugate and a second immunoconjugate of the same antibody. In some specific embodiments, the immunotherapy agent is a dimer of a first immunoconjugate and a second immunoconjugate, wherein the first immunoconjugate and the second immunoconjugate are immunoconjugates selected from the following antibodies: 3F8 monoclonal antibody (3F8), abagovomab, abituzumab, adecatumumab, amatuximab, andecaliximab, anrukinzumab, apolizumab, and astragalusumab. crinvacumab, atezolizumab, avelumab, basiliximab, bavituximab, belimumab, bemarituzumab, bermekimab, bevacizumab, bivatuzumab, bleselumab, blontuvetmab (brontictuzumab), cabiralizumab, camrelizumab, capromab, carotuximab, cemiplimab, cetrelimab, cetuximab, cibisatamab, cirmtuzumab, cixutumumab, clazakizumab Codrituzumab, conatumumab, cusatuzumab, dacetuzumab, daclizumab, dalotuzumab, daratumumab, decttrekumab, demcizumab, detumomab, dinutuximab, dinutuximab beta, dostarlimab.drozitumab, duligotuzumab, durvalumab, dusigitumab, ecroximab, edrecolomab, eldelumab, elgemtumab, elotuzumab, elsilimomab, emactuzumab, emibetuzumab, enavatuzumab ab), Enoblituzumab, Enoticumab, Enctituximab, Epratuzumab, Etaracizumab, Etiglimab, Faricimab, Farletuzumab, Fibatuzumab, Ficlatuzumab, Figitumumab, Flanvotumab, Fres olimumab, futuximab, galiximab, ganitumab, gatipotuzumab, gevokizumab, gilvetmab, gimsilumab, girentuximab, gomiliximab, icrucumab, ifabotuzumab, imalumab, imaprelimab Imgatuzumab, Inebilizumab, Inomomab, Intetumumab, Ipilimumab, Iratumumab, Isatuximab, Iscalimab, Istiratumab, Labetuzumab, Lacnotuzumab, Lebrikizumab, LenzilumabLeronlimab, lexatumumab, lintuzumab, lirilumab, lucatumumab, lulizumab pegol, lumiliximab, lumretuzumab, lutikizumab, mapatumumab, margetuximab, matuzumab, milatuzumab, mitumomab, modotuximab, mogamulizumab, monalizumab. Namilumab, Narnatumab, Navicixizumab, Naxitamab, Necitumumab, Nesvacumab, Nimotuzumab, Nivolumab, Obinutuzumab, Ocaratuzumab, Ocrelizumab, Ofatu mumab, olaratumab, oleclumab, olokizumab, omburtamab, ontuxizumab, oregovomab, otlertuzumab, panitumumab, pankomab, parsatuzumab, pastotuxizumab, pertuxumab (p atritumab, pembrolizumab, pemtumomab, pertuzumab, pidilizumab, pritumumab, prolgolimab, racotumomab, radretumab, ramucirumab, ravagalimab, and relatlimab.Retifanlimab, rilotumumab, rinucumab, rituximab, robatumumab, romilkimab, rosmantuzumab, samalizumab, sarilumab, selicrelumab, seribantumab, sibrotuzumab, and siltuximab. Sintilimab, sirukumab, spartalizumab, tabalumab, tafasitamab, talacotuzumab, tarextumab, tabolimab, telisotuzumab, tenatumomab, teneliximab, tepoditamab, and teprotumumab are all listed. ), theralizumab, tigatuzumab, timigutuzumab, tiragotumab, tislelizumab, tomouzotuximab, tositumomab, tovetumab, tralokinumab, trastuzumab, tremelimumab, ublituximab, ulurocurumab Ocuplumab, urelumab, utomilumab, vanalimab, vantictumab, vanucizumab, varisacumab, varlilumab, veltuzumab, volociximab, vonlerolizumab, votumumab, xentuzumab, zalutumumab.Zatuximab, Zenocutuzumab, and Zolbetuximab. The dimer forms of the immunoconjugates of these antibodies can be prepared, for example, using the SATP / SATA and sulfo-SMCC / SMCC crosslinking strategies described herein.

[0041] In some embodiments, the immunotherapeutic agent is a dimer of a first and a second immunoconjugate of a bispecific IgG antibody. The bispecific IgG antibody contains two antigen-binding sites, each of which specifically binds to a different cancer-associated antigen, such that the immunotherapeutic agent contains exactly four antigen-binding sites, each of which specifically binds to two different cancer-associated antigens. In some specific embodiments, the immunotherapeutic agent comprises two different immunoconjugates of amivantamab, glofitamab, talquetamab, or teclistamab, all of which are bispecific antibodies.

[0042] In some implementations, the first cancer-associated antigen and the second cancer-associated antigen are the same.

[0043] In some embodiments, the cancer-associated antigens are 4-1BB, 5'-nucleotidase, 5T4, activin receptor-like kinase 1, alpha-fetoprotein, angiopoietin 2, AXL, B7-H3, B cell activating factor (BAFF), B cell maturation antigen (BCMA), c-Met, C242, CA-125, CanAg, carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen, CCR4, CCR5, CD3, CD4, CD19, CD20, CD45. 22, CD23, CD25, CD27, CD28, CD30, CD33, CD37, CD38, CD40, CD44, CD51, CD56, CD70, CD74, CD79B, CD80, CD123, CD134, CD152, CD200, CD276, CD319, CEACAM5, Tight junction protein 18, Coagulation factor III, Connective tissue growth factor (CTGF), Colony-stimulating factor 1 (C SF1), Colony-stimulating factor 1 receptor (CSF1R), Colony-stimulating factor 2 (CSF2), CTLA-4, CXCR4, Dendritic cell-associated lectin 2, DLL3, DLL4, DR5, EGFL7, EGFR, Endothelial glycoprotein, EpCAM, Hepatic glycoside receptor A3 (EPHA3), Epidermal growth factor receptor (EGFR), ERBB3 (HER3), Fibroblast activator protein α (FAP), FGFR2, Fibronectin extradomain B, Folate hydrolase, Folate receptor 1, Coil receptor, GD2 ganglioside, GD3 ganglioside, Gelatinase B, Glycoprotein 100 (gp100), Phosphatidylinositol proteoglycan 3, GPNMB, G protein-coupled receptor 5D (GPRC5D), GUCY2C, Hepatocyte growth factor (HGF), HER1, HER2, HGFR, Histone complex, HLA-DR, Human discrete factor receptor kinase, IGF-1 receptor (IGF-1R);CD221), IGF-2, interleukin-1α, interleukin-2, interleukin-6, interleukin-13, integrin α5β1, integrin αVβ3, KIR2D, LAG3, Lewis-Y antigen, LIV-1, LRRC15, macrophage migration inhibitory factor (MIF), MCP-1, melanoma cell adhesion molecule (MCAM), mesothelin, MUC1, MUC5AC, nectin-4, NGNA gangliosides, Notch 1, Notch receptor, NRP1, PCDC1, PD-1, PD-L1, PDGFRA, phosphate-sodium cotransporter, phosphatidylserine, PTK7, rootplate-specific spondin 3. ROR1, SDC1, SLAMF7, SLITRK6, Sp17, STEAP1, TEM1, tendon glycoprotein C, TGF-β, TIGIT, TRAIL-R1, TRAIL-R2, tumor-associated calcium signaling transducer 2 (TROP-2), tumor antigen CTAA16.88, tumor-specific glycosylated MUC1, tumor-associated glycoprotein 72 (TAG-72), TWEAK receptor, TYRP1, VEGF-A, VEGFR-1, VEGFR-2, or vimentin. In some specific embodiments, the cancer-associated antigen is Sp17.

[0044] In some implementations, the cancer comprises cells expressing Sp17.

[0045] In some embodiments, the first antibody and the second antibody each contain two antigen-binding sites, each of which specifically binds to Sp17, such that the immunotherapeutic agent contains exactly four antigen-binding sites, each of which specifically binds to Sp17.

[0046] In some embodiments, the first antibody and the second antibody each include a first variable domain and a second variable domain, wherein the first variable domain of the first antibody and the first variable domain of the second antibody have the same amino acid sequence, and the second variable domain of the first antibody and the second variable domain of the second antibody have the same amino acid sequence.

[0047] In some embodiments, the first variable domain of the first antibody comprises a VH CDR1 region, a VH CDR2 region, and a VHCDR3 region, wherein the VH CDR1 region contains the same amino acid sequence as SEQ ID NO:5, the VH CDR2 region contains the same amino acid sequence as SEQ ID NO:6, and the VH CDR3 region contains the same amino acid sequence as SEQ ID NO:7. In some embodiments, the first variable domain of the second antibody comprises a VH CDR1 region, a VH CDR2 region, and a VH CDR3 region, wherein the VHCDR1 region contains the same amino acid sequence as SEQ ID NO:5, the VH CDR2 region contains the same amino acid sequence as SEQ ID NO:6, and the VH CDR3 region contains the same amino acid sequence as SEQ ID NO:7. In some specific embodiments, the first variable domain of the first antibody and the first variable domain of the second antibody each comprise a VH CDR1 region, a VHCDR2 region, and a VH CDR3 region, wherein the VH CDR1 region comprises the same amino acid sequence as SEQ ID NO:5, the VH CDR2 region comprises the same amino acid sequence as SEQ ID NO:6, and the VH CDR3 region comprises the same amino acid sequence as SEQ ID NO:7.

[0048] In some embodiments, the second variable domain of the first antibody comprises a VL CDR1 region, a VL CDR2 region, and a VLCDR3 region, wherein the VL CDR1 region contains the same amino acid sequence as SEQ ID NO:8, the VL CDR2 region contains the same amino acid sequence as SEQ ID NO:9, and the VL CDR3 region contains the same amino acid sequence as SEQ ID NO:10. In some embodiments, the second variable domain of the second antibody comprises a VL CDR1 region, a VL CDR2 region, and a VL CDR3 region, wherein the VL CDR1 region contains the same amino acid sequence as SEQ ID NO:8, the VL CDR2 region contains the same amino acid sequence as SEQ ID NO:9, and the VL CDR3 region contains the same amino acid sequence as SEQ ID NO:10. In some specific embodiments, the second variable domain of the first antibody and the second variable domain of the second antibody each include a VL CDR1 region, a VLCDR2 region, and a VL CDR3 region, wherein the VL CDR1 region contains the same amino acid sequence as SEQ ID NO:8, the VL CDR2 region contains the same amino acid sequence as SEQ ID NO:9, and the VL CDR3 region contains the same amino acid sequence as SEQ ID NO:10.

[0049] In some embodiments, the first antibody comprises a heavy chain having at least 90% sequence homology with SEQ ID NO:11 and a light chain having at least 90% sequence homology with SEQ ID NO:12. In some embodiments, the second antibody comprises a heavy chain having at least 90% sequence homology with SEQ ID NO:11 and a light chain having at least 90% sequence homology with SEQ ID NO:12. In some specific embodiments, the first antibody comprises a heavy chain having at least 90% sequence homology with SEQ ID NO:11 and a light chain having at least 90% sequence homology with SEQ ID NO:12, and the second antibody comprises a heavy chain having at least 90% sequence homology with SEQ ID NO:11 and a light chain having at least 90% sequence homology with SEQ ID NO:12.

[0050] In some embodiments, the first antibody comprises a heavy chain having at least 90% sequence identity with SEQ ID NO:11 and a light chain having at least 90% sequence identity with SEQ ID NO:12. In some embodiments, the second antibody comprises a heavy chain having at least 90% sequence identity with SEQ ID NO:11 and a light chain having at least 90% sequence identity with SEQ ID NO:12. In some specific embodiments, the first antibody comprises a heavy chain having at least 90% sequence identity with SEQ ID NO:11 and a light chain having at least 90% sequence identity with SEQ ID NO:12, and the second antibody comprises a heavy chain having at least 90% sequence identity with SEQ ID NO:11 and a light chain having at least 90% sequence identity with SEQ ID NO:12.

[0051] In some implementations, the first load and the second load are different.

[0052] In some embodiments, the first load is a first radioactive isotope. In some embodiments, the first radioactive isotope is selected from actinium-225, astatine-211, bismuth-212, bismuth-213, copper-67, gallium-68, holmium-166, iodine-124, iodine-131, lutetium-177, samarium-153, technetium-99, terbium-149, and yttrium-90.

[0053] In some embodiments, the first radioactive isotope is an alpha particle emitter. In some specific embodiments, the first radioactive isotope is selected from actinium-225, astatine-211, bismuth-212, bismuth-213, and terbium-149.

[0054] In some embodiments, the second load is a second radioactive isotope. In some embodiments, the second radioactive isotope is selected from actinium-225, astatine-211, bismuth-212, bismuth-213, copper-67, gallium-68, holmium-166, iodine-124, iodine-131, lutetium-177, samarium-153, technetium-99, terbium-149, and yttrium-90.

[0055] In some embodiments, the second radioactive isotope is a beta particle emitter. In some specific embodiments, the second radioactive isotope is selected from copper-67, gallium-68, holmium-166, iodine-124, iodine-131, lutetium-177, technetium-99, samarium-153, and yttrium-90.

[0056] In some embodiments, (1) the first load is a first radioactive isotope, (2) the first radioactive isotope is an alpha particle emitter, (3) the second load is a second radioactive isotope, and (4) the second radioactive isotope is a beta particle emitter.

[0057] Examples of antibodies conjugated with radioisotopes include tositumomab (also known as BEXXAR®) and ibritumomab tiuxetan (also known as ZEVALIN®). Those skilled in the art can design antibodies conjugated with radioisotopes using known strategies, such as those for conjugating iodine-131 to tositumomab and yttrium-90 or indium-111 to ibritumomab tiuxetan (see, for example, U.S. Patent Nos. 6,565,827 and 7,422,739, the entire contents of which are incorporated herein by reference). Other examples of antibodies conjugated with radioisotopes include clivatuzumab tetraxetan, lilotomab satetraxetan, and tacatuzumab tetraxetan. In some implementations, the first immunotherapy agent is selected from clivatuzumab tetraxetan, ibritumomab tiuxetan, lilotomab satetraxetan, tacatuzumab tetraxetan, and tositumomab.

[0058] The immunotherapeutic agents disclosed herein can advantageously be prepared, for example, by using the same chelating agent to bind different radioisotopes separately. For example, a batch of antibodies can be coupled to a 1,4,7,10-tetrazacyclododecane-1,4,7,10-tetraacetic acid (tetraxetan) moiety capable of chelating cations. A first portion of this batch can be used to chelate a first radioisotope, a second portion can be used to chelate a second radioisotope, and the first and second portions can subsequently be crosslinked to produce the immunotherapeutic agent. For example, this strategy results in a substantially equal distribution of the first and second radioisotopes on the immunotherapeutic agent compared to simultaneously chelating the first and second radioisotopes; if the first and second radioisotopes are simultaneously chelated, it results in different proportions of the first and second radioisotopes being chelated in the individual molecules of the immunotherapeutic agent. Taking tiemomab (ZEVALIN®) chelating two different radioisotopes as an example, this can lead to some tiemomab molecules being enriched in one radioisotope, some in the other, and some in only one. The immunotherapeutic agent disclosed herein advantageously avoids this distribution, instead ensuring that all molecules chelate both radioisotopes.

[0059] In some embodiments, (1) the first load is a first pharmaceutical agent, (2) the second load is a second pharmaceutical agent, (3) the first and second pharmaceutical agents are selected from calicheamicin, camptothecin, deruxtecan, doxorubicin, emtansine, exatecan, irinotecan, maleimidocaproyl monomethyl auristatin F, mertansine, monomethyl auristatin F, paclitaxel, PE38, pyrrolobenzodiazepine, SN-38, and vedotin; and (4) the first and second pharmaceutical agents are different pharmaceutical agents.

[0060] Examples of antibodies conjugated to drug formulations include gemtuzumab ozogamicin (MYLOTARG®) and trastuzumab emtansine (KADCYLA®). Those skilled in the art can design antibodies conjugated to drug formulations using known strategies, such as those for conjugating calicheamicin to gemtuzumab and emtansine to trastuzumab (see, for example, U.S. Patent Nos. 5,877,296 and 8,088,387, the entire contents of which are incorporated herein by reference).

[0061] Other examples of antibodies conjugated to drug formulations include anetumabravtansine, aprotumab ixadotin, azintuxizumab vedotin, belantamab mafodotin, brentuximab vedotin, camidanlumab tesirine, cantuzumab mertansine, cantuzumab ravtansine, CBR96-doxorubicin immunoconjugate, cergutuzumab amunaleukin, and cofetuzumab peridotine. Pelidotin, coltuximab ravtansine, denintuzumab mafodotin, depatuxizumab mafodotin, derbyotuximab biotin, enapotamab vedotin, enfortumab vedotin, glembatumumab vedotin, iladatuzumab vedotin, indatuximab ravtansine, indusatumab vedotin, inotuzumab ozogamicin, ladiratuzumab vedotin Vedotin, Lapituximab Emtansine, Lifastuzumab Vedotin, Loncastuximab Tesirine, Lovotuzumab Mertansine, Losatuxizumab Vedotin, Mirvetuximab Soravtansine, MoxetumomabPasudotox, naratuximab mtansine, pinatuzumab vedotin, polotuzumab vedotin, rovalpituzumab tesirine, sacituzumab govitecan, sarotamab vedotin, sirtratumab vedotin, sofituzumab vedotin, tapitutumomab paptox, telisotuzumab vedotin, tisotumab vedotin, trastuzumab The drugs include deruxtecan, trastuzumab duocarmazine, tucotuzumab celmoleukin, vadastuximab talirine, vandortuzumab vedotin, and vorsetuzumab mafodotin.

[0062] In some embodiments, the first immunoconjugate is selected from anetumabravtansine, aprotumab ixadotin, azintuxizumab vedotin, belantamab mafodotin, brentuximab vedotin, camidanlumab tesirine, cantuzumab mertansine, cantuzumab ravtansine, CBR96-doxorubicin immunoconjugate, cergutuzumab amunaleukin, and cofetuzumab peridotine. Pelidotin, coltuximab ravtansine, denintuzumab mafodotin, depatuxizumab mafodotin, derbyotuximab biotin, enapotamab vedotin, enfortumab vedotin, gemtuzumab ozogamicin, glembatumumab vedotin, iladutuzumab vedotin, indatuximab ravtansine, indusatumab vedotin, inotuzumab ozogamicin ozogamicin, ladiratuzumab vedotin, lapituximab emtansine, lidostuzumab vedotin, loncastuximab tesirine, lorvotuzumab metansine, losatuxizumab vedotin, mirvetuximabSoravtansine, Moxetumomab Pasudotox, Naratuximab Emtansine, Pinatuzumab Vedotin, Polotuzumab Vedotin, Rovalpituzumab Tesirine, Sacituzumab Ggovitecan, Samrotamab Vedotin, Sirtratumab Vedotin, Sofituzumab Vedotin, Tapitutumomab Paptox, Telisotuzumab Vedotin, Tisotumab Vedotin The following are listed: trastuzumab, deruxtecan, duocarmazine, emtansine, celmoleukin, vadastuximab, vandortuzumab, and mafodotin.

[0063] In some embodiments, the immunotherapeutic agent has a drug-antibody ratio of at least 5. In some specific embodiments, the immunotherapeutic agent has a drug-antibody ratio equal to the sum of the number of first and second loads in a therapeutically effective amount of the immunotherapeutic agent divided by the number of molecules of the immunotherapeutic agent in the therapeutically effective amount. In some specific embodiments, the drug-antibody ratio is at least 8. In some specific embodiments, the drug-antibody ratio is at least 10. The drug-antibody ratios for monomeric immunodrug conjugates range from approximately 2 to 3 for gemtuzumab ozogamicin (MYLOTARG®) to approximately 7.6 for sacituzumab govitecan (TRODELVY®); most FDA-approved therapeutic antibodies have drug-antibody ratios of approximately 3.5 to 4, including brentuximab vedotin (ADCETRIS®, approximately 4), trastuzumab emtansine (KADCYLA®, approximately 3.5), polotuzumab vedotin (POLIVY®, approximately 3.5), enfortumab vedotin (PADCEV®, approximately 4), and belantamab mafodotin (BLENREP®, approximately 4). Compared to monomeric immunoconjugates, the immunotherapeutic agents of this disclosure advantageously carry additional payload, thereby allowing for the delivery of more payload per binding event.

[0064] In some embodiments, (1) the combination of equal concentrations of the first and second immunoconjugates has a reference IC50, which is a calculated concentration of the first and second immunoconjugates required to observe 50% cytotoxicity in an in vitro assay against a cancer cell line expressing the antigen; (2) the immunotherapy has an improved IC50 against the cancer cell line; and (3) the improved IC50 is less than the reference IC50. In some specific embodiments, (1) the combination of equal concentrations of the first and second immunoconjugates has a reference IC50, which is a calculated concentration of the first and second immunoconjugates required to observe 50% cytotoxicity in an in vitro assay against a cancer cell line expressing the antigen, wherein the calculated concentration is determined based on equal concentrations of the first and second immunoconjugates, rather than on the sum of equal concentrations of the first and second immunoconjugates; (2) the immunotherapy has an improved IC50 against the cancer cell line; and (3) the improved IC50 is less than the reference IC50.

[0065] In some embodiments, the improved IC50 is less than 100% of the reference IC50. In some specific embodiments, the improved IC50 is less than 95% of the reference IC50. In some very specific embodiments, the improved IC50 is less than 95% of the reference IC50.

[0066] In some embodiments, (1) the first immunoconjugate has a first reference efficacy against the cancer per mole of the first immunoconjugate, (2) the second immunoconjugate has a second reference efficacy against the cancer per mole of the second immunoconjugate, (3) the immunotherapy agent has an improved efficacy against the cancer per mole of the immunotherapy agent, and (4) the improved efficacy is greater than the sum of the first reference efficacy and the second reference efficacy.

[0067] In some implementations, the therapeutic effect is determined in vitro, for example by identifying the survival rate of cells incubated with the first immunoconjugate, the second immunoconjugate, or the immunotherapeutic agent.

[0068] In some implementations, efficacy is determined in vivo during preclinical studies, for example by identifying the survival rate of animals treated with any of the first immunoconjugate, the second immunoconjugate, or the immunotherapeutic agent, wherein the animals have cancer expressing the cancer-associated antigen.

[0069] In some implementations, efficacy is determined in vivo during clinical studies, for example by identifying the survival rate of human subjects treated with any of the first immunoconjugate, the second immunoconjugate, or the immunotherapeutic agent, wherein the human subjects have cancer expressing the cancer-associated antigen.

[0070] In some embodiments, the improved therapeutic effect is at least 10% higher than the sum of the first and second reference therapeutic effects. In some specific embodiments, the improved therapeutic effect is at least 20% higher than the sum of the first and second reference therapeutic effects. In some very specific embodiments, the improved therapeutic effect is at least 50% higher than the sum of the first and second reference therapeutic effects.

[0071] In some embodiments, (1) the first immunoconjugate and the second immunoconjugate have a combined reference efficacy against the cancer per mole of the first immunoconjugate and the second immunoconjugate, (2) the immunotherapy agent has an improved efficacy against the cancer per mole of the immunotherapy agent, and (3) the improved efficacy is greater than the combined reference efficacy. The term "combined reference efficacy" refers to the efficacy of the combination of the first immunoconjugate and the second immunoconjugate. For example, (1) the first immunoconjugate and the second immunoconjugate can be combined at the same molar concentration, and this combination can be used to evaluate the combined reference efficacy of the combination in vitro or in vivo. The immunotherapy agent can be used at the same molar concentration to evaluate the improved efficacy in vitro or in vivo. According to various aspects of the invention, the improved efficacy is greater than the combined efficacy.

[0072] In some embodiments, the efficacy is determined in vitro, for example by identifying the survival rate of cells incubated with either (1) the first immunoconjugate or the second immunoconjugate, or (2) the immunotherapeutic agent.

[0073] In some implementations, efficacy is determined in vivo during preclinical studies, for example by identifying the survival rate of animals treated with either (1) the first and second immunoconjugates or (2) the immunotherapeutic agent, wherein the animals have cancer expressing the cancer-associated antigen.

[0074] In some implementations, efficacy is determined in vivo during clinical studies, for example by identifying the survival rate of human subjects treated with either (1) the first immunoconjugate and the second immunoconjugate or (2) the immunotherapeutic agent, wherein the human subjects have cancer expressing the cancer-associated antigen.

[0075] In some embodiments, the improved therapeutic effect is at least 10% higher than the combined reference therapeutic effect. In some specific embodiments, the improved therapeutic effect is at least 20% higher than the combined reference therapeutic effect. In some very specific embodiments, the improved therapeutic effect is at least 50% higher than the combined reference therapeutic effect.

[0076] In some embodiments, (1) the first immunoconjugate has a first reference infusion time per mole of the first immunoconjugate; (2) the second immunoconjugate has a second reference infusion time per mole of the second immunoconjugate; (3) the immunotherapy agent has a shorter infusion time per mole of the immunotherapy agent; and (4) the shorter infusion time is less than the sum of the first reference infusion time and the second reference infusion time.

[0077] In this specification, the term "infusion time" refers to the amount of time required to infuse a therapeutically effective amount of the disclosed immunoconjugate or immunotherapeutic agent for the treatment of a human cancer expressing the cancer-associated antigen.

[0078] In some embodiments, the shorter infusion time is at least 30 minutes shorter than the sum of the first reference infusion time and the second reference infusion time. In some specific embodiments, the shorter infusion time is at least 60 minutes shorter than the sum of the first reference infusion time and the second reference infusion time.

[0079] In some embodiments, the shorter infusion time is shorter than the sum of the first reference infusion time and the second reference infusion time because the immunotherapy agent has superior efficacy relative to the combination of the first immunoconjugate and the second immunoconjugate, wherein the immunoconjugate and the second immunoconjugate are not cross-linked. In some specific embodiments, the shorter infusion time is shorter than the reference infusion time because the immunotherapy agent has superior efficacy per mole relative to the first immunoconjugate and the second immunoconjugate.

[0080] In some embodiments, the shorter infusion time is shorter than the reference infusion time because the immunotherapeutic agent has a higher mass / volume concentration relative to the combination of the first and second immunoconjugates, wherein the immunoconjugates and the second immunoconjugate are not cross-linked in the combination. For example, the immunotherapeutic agent of this disclosure can have a higher mass / volume concentration relative to a monomeric immunotherapeutic agent because the molecular weight of the dimer immunotherapeutic agent is approximately twice that of the monomeric immunotherapeutic agent.

[0081] In some embodiments, the infusion time of the immunotherapy agent is no more than 4 hours. In some specific embodiments, the infusion time of the immunotherapy agent is no more than 3 hours. In some more specific embodiments, the infusion time of the immunotherapy agent is no more than 2 hours. In some very specific embodiments, the infusion time of the immunotherapy agent is no more than 1 hour.

[0082] In some embodiments, each antigen-binding site of the first and second immunoconjugates comprises: (1) a first variable domain comprising a VH CDR1 region, a VH CDR2 region, and a VH CDR3 region, wherein the VH CDR1 region comprises the same amino acid sequence as SEQ ID NO: 5, the VH CDR2 region comprises the same amino acid sequence as SEQ ID NO: 6, and the VH CDR3 region comprises the same amino acid sequence as SEQ ID NO: 7; and (2) a second variable domain comprising a VL CDR1 region, a VL CDR2 region, and a VL CDR3 region, wherein the VL CDR1 region comprises the same amino acid sequence as SEQ ID NO: 8, the VL CDR2 region comprises the same amino acid sequence as SEQ ID NO: 9, and the VL CDR3 region comprises the same amino acid sequence as SEQ ID NO: 10. Each antigen-binding site comprising a dimer immunoconjugate containing the first and second variable domains typically binds Sp17 with high affinity.

[0083] In some embodiments, the first immunoconjugate and the second immunoconjugate each comprise a heavy chain having at least 90% sequence homology with SEQ ID NO: 11 and a light chain having at least 90% sequence homology with SEQ ID NO: 12. Dimeric immunoconjugates each comprising the heavy and light chains described in this paragraph are generally capable of binding Sp17 specifically with high affinity.

[0084] In some embodiments, the dissociation constant (KD) of the immunotherapeutic agent with the cancer-associated antigen is no greater than 25 nanomolars. In some specific embodiments, the KD of the immunotherapeutic agent with the cancer-associated antigen is no greater than 10 nanomolars. In some very specific embodiments, the KD of the immunotherapeutic agent with the cancer-associated antigen is no greater than 2.5 nanomolars. In some embodiments, the KD is determined by surface plasmon resonance, for example using a Biacore™ instrument.

[0085] In some embodiments, the first immunoconjugate and the second immunoconjugate each comprise a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region and a human κ or λ light chain constant region. In some specific embodiments, one or both of the first immunoconjugate and the second immunoconjugate comprise a human IgG1 or IgG4 heavy chain constant region and a human κ or λ light chain constant region. In some very specific embodiments, one or both of the first immunoconjugate and the second immunoconjugate comprise a human IgG4 heavy chain constant region and a human κ light chain constant region.

[0086] In some embodiments, one or both of the first and second immunoconjugates comprise the IgG2 heavy chain constant region. In some specific embodiments, one or both of the first and second immunoconjugates comprise the IgG2a heavy chain constant region.

[0087] In some embodiments, one or both of the first and second immunoconjugates contain the IgG4 heavy chain constant region.

[0088] In some embodiments, one or both of the first and second immunoconjugates contain a κ or λ light chain constant region. In some specific embodiments, one or both of the first and second immunoconjugates contain a κ light chain constant region.

[0089] In some embodiments, one or both of the first and second immunoconjugates comprise a human heavy chain constant region and a human light chain constant region. In some specific embodiments, one or both of the first and second immunoconjugates comprise a human IgG heavy chain constant region and a human κ or λ light chain constant region. In some very specific embodiments, one or both of the first and second immunoconjugates comprise a human IgG4 heavy chain constant region and a human κ light chain constant region.

[0090] In some embodiments, the first immunoconjugate and the second immunoconjugate are covalently cross-linked in the immunotherapeutic agent via engineered disulfide bonds.

[0091] In some embodiments, the first and second immunoconjugates are covalently cross-linked in the immunotherapeutic agent via a synthetic linker. For example, in a cross-linking process mediated by sulfo-SMCC and SATP, the synthetic linker is 1-oxo-S-{N-[4-formyl-cyclohexyl(methyl)]-3,4-dihydro-2,5-dioxo-1H-pyrrolo-3-yl}-3-thiopropyl, wherein the carbon atom of the formyl group is covalently bonded to the primary amino group of the first IgG antibody of the two IgG antibodies, and the carbon atom at position 1 of the propyl group is bonded to the primary amino group of the second IgG antibody of the two IgG antibodies.

[0092] In some embodiments, each antigen-binding site comprises: (1) a first variable domain comprising a VH CDR1 region, a VH CDR2 region, and a VH CDR3 region, wherein the VH CDR1 region comprises the same amino acid sequence as SEQ ID NO:5, the VH CDR2 region comprises the same amino acid sequence as SEQ ID NO:6, and the VH CDR3 region comprises the same amino acid sequence as SEQ ID NO:7; and (2) a second variable domain comprising a VLCDR1 region, a VL CDR2 region, and a VL CDR3 region, wherein the VL CDR1 region comprises the same amino acid sequence as SEQ ID NO:8, the VL CDR2 region comprises the same amino acid sequence as SEQ ID NO:9, and the VL CDR3 region comprises the same amino acid sequence as SEQ ID NO:10.

[0093] Several aspects of this disclosure relate to immunotherapeutic agents exhibiting high affinity for Sp17. As described in Example 1 below, these antibodies were developed using a Fab phage display biopanning strategy on a library generated from peripheral blood of 120 healthy donors. This library contained approximately one trillion different combinations. The best-performing Fab in this library had an immunoglobulin heavy chain variable region (VH) and an immunoglobulin light chain variable region (VL), wherein the immunoglobulin heavy chain variable region has the nucleotide sequence shown in SEQ ID NO:1, and the immunoglobulin light chain variable region has the nucleotide sequence shown in SEQ ID NO:2, which are shown in Table 1 below. The VH amino acid sequence is shown in SEQ ID NO:3, and the VL amino acid sequence is shown in SEQ ID NO:4. The VH and VL amino acid sequences are shown in Table 2 below, where CDRs are shown in bold and underlined, and framed areas are shown in plain text. CDR sequences are shown in SEQ ID NO:5-10 and are shown in Table 3 below, respectively. Those skilled in the art will recognize that, at least for some CDRs, the precise boundary between a CDR and a frame region is not clear, and therefore the CDRs shown in Tables 2 and 3 and SEQ ID NO: 5-10 may contain one or more amino acids that may be more appropriately classified as a frame region rather than a CDR.

[0094] The nucleotide sequences encoding the best-performing Fab variable regions (SEQ ID NO: 1 and 2) were initially cloned into the mouse IgG2a heavy chain gene and the mouse κ light chain gene to express a chimeric monoclonal antibody (chAB2). The affinity of this chimeric antibody, measured by surface plasmon resonance, was approximately 2 nanomolar. Immunohistochemical analysis of this chimeric antibody against 33 normal human tissues showed that the variable regions exhibited no detectable cross-reactivity.

[0095] The human variable region was then cloned into the human IgG4 heavy chain gene and the human κ light chain gene to generate a human monoclonal antibody (SP17-AB2) suitable for use as a therapeutic antibody. The heavy chain constant region was mutated to include the Ser228Pro mutation to reduce non-specific interaction with the Fc receptor γ (FcγR). The full-length heavy chain has the amino acid sequence shown in SEQ ID NO: 10, and the full-length light chain has the amino acid sequence shown in SEQ ID NO: 11, as shown in Table 4 below.

[0096] Several aspects of this disclosure relate to a pharmaceutical composition comprising the immunotherapeutic agent described in any part of this disclosure and a pharmaceutically acceptable carrier. A suitable pharmaceutically acceptable carrier typically comprises water containing a dissolved solute, which buffers the pH and provides the metal cation and ionic strength to stabilize the immunotherapeutic agent of this disclosure. Such formulations are typically sterile, and the selection and preparation of such pharmaceutically acceptable carriers are well known in the art. Solid dosage forms (including lyophilized therapeutic agents) typically comprise, for example, metal cations, anions, and optionally polyols such as sugars (e.g., trehalose or glucose), which stabilize the therapeutic agent in the solid phase and during reconstitution into an aqueous formulation. General guidance on selecting pharmaceutically acceptable carriers can be found, for example, in REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY, 22nd edition (edited by Allen Jr., Loyd V.), Pharmaceutical Press, 2012; and those skilled in the art will also refer to formulations of the IgG antibodies described in this disclosure and formulations of other existing therapeutic agents when selecting pharmaceutically acceptable carriers. Such guidance is generally available from scientific literature and existing product labels.

[0097] In some embodiments, the pharmaceutical composition is suitable for administration to a subject. In some specific embodiments, the pharmaceutical composition is suitable for administration to a human patient. In some very specific embodiments, the pharmaceutical composition is suitable for intravenous administration to a human patient.

[0098] Various aspects of this disclosure relate to a kit comprising (1) a sealed container containing a pharmaceutical composition as described in any part of this disclosure, and (2) instructions for use of the pharmaceutical composition.

[0099] Various aspects of this disclosure relate to medical devices that include pharmaceutical compositions described in any part of this specification. In some embodiments, the medical device is a syringe, intravenous cannula, or drug-eluting implant.

[0100] Several aspects of this disclosure relate to a method of treating or preventing cancer in a subject, comprising identifying the subject as containing cells ectopically expressing the cancer-associated antigen, and administering a pharmaceutical composition as described in any part of this disclosure. Identifying the subject as containing cells ectopically expressing the cancer-associated antigen includes, for example, identifying mRNA encoding the cancer-associated antigen by RT-PCR, and identifying the expression of the cancer-associated antigen by flow cytometry and / or immunohistochemistry. Such methods can advantageously allow the determination of cancer cell expression of the cancer-associated antigen, for example based on analysis of one or more pre-selected cancer cells, or based on the co-identification of the expression of the cancer-associated antigen and cancer phenotype, such as the co-expression of the cancer-associated antigen with another cancer antigen.

[0101] Several aspects of this disclosure relate to a method for modulating cells expressing the cancer-associated antigen in a human subject, including administering an immunotherapeutic agent as described in any part of this disclosure to the human subject. In some embodiments, the method is a method for treating or preventing cancer in a human subject. In some specific embodiments, the method is a method for treating or preventing cancer in a human subject who has cancer, and at least a portion of the cells expressing the cancer-associated antigen are cancer cells.

[0102] In some embodiments, the immunotherapeutic agent is administered in an effective amount to induce cell death in at least a portion of the cells expressing the cancer-associated antigen. In some specific embodiments, the immunotherapeutic agent is coupled with a radioactive isotope, and the method induces cell death by emitting radiation within or in a region adjacent to the cells expressing the cancer-associated antigen. In some specific embodiments, the immunotherapeutic agent is coupled with a cytotoxic drug agent, and the method induces cell death by releasing the drug agent within or in a region adjacent to the cells expressing the cancer-associated antigen. In some very specific embodiments, the immunotherapeutic agent is coupled with a cleavable linker, the drug agent being cytotoxic, and the method induces cell death by releasing the drug agent within or in a region adjacent to the cells expressing the cancer-associated antigen.

[0103] In some embodiments, the subject is a mammal. In some specific embodiments, the subject is a rodent, lagomorph, feline, canine, pig, sheep, goat, llama, cattle, horse, or primate. In some very specific embodiments, the subject is a human patient.

[0104] In some embodiments, the subject is male or female. In some specific embodiments, the subject is female. In some specific embodiments, the subject is male.

[0105] In some embodiments, the subject exhibits ectopic expression of the cancer-associated antigen. In some specific embodiments, the subject has cancer, and the cancer cells express the cancer-associated antigen.

[0106] In some embodiments, the method includes identifying that the subject contains cells expressing the cancer-associated antigen.

[0107] In some embodiments, the method includes identifying that the subject contains cells expressing the cancer-associated antigen, wherein the cells expressing the cancer-associated antigen include leukocytes, and the cancer is lymphoma, leukemia, or myeloma. In some specific embodiments, the method includes identifying that the subject contains cells expressing the cancer-associated antigen, wherein the cells expressing the cancer-associated antigen include plasma cells, and the cancer is multiple myeloma. In some specific embodiments, the method includes identifying that the subject contains cells expressing the cancer-associated antigen, wherein the cells expressing the cancer-associated antigen include lymphocytes, and the cancer is lymphoma. In some specific embodiments, the method includes identifying that the subject contains cells expressing the cancer-associated antigen, wherein the cells expressing the cancer-associated antigen include ovarian cells, and the cancer is ovarian cancer. In some specific embodiments, the method includes identifying that the subject contains cells expressing the cancer-associated antigen, wherein the cells expressing the cancer-associated antigen include lung epithelial cells, and the cancer is non-small cell lung cancer.

[0108] In some embodiments, the method includes determining, prior to administration, that a tissue sample from the human subject contains RNA encoding the cancer-associated antigen or a protein containing the cancer-associated antigen.

[0109] In some implementations, the tissue sample is a blood sample.

[0110] In some embodiments, the tissue sample is a biopsy sample. In some specific embodiments, the tissue sample is a tumor biopsy sample. In some very specific embodiments, the tissue sample is a bone marrow biopsy sample. In some very specific embodiments, the tissue sample is an ovarian biopsy sample. In some very specific embodiments, the tissue sample is a lung biopsy sample.

[0111] In some embodiments, the method includes identifying that the tissue sample contains a cancer biomarker, wherein the cancer biomarker is neither RNA encoding the cancer-associated antigen nor the cancer-associated antigen.

[0112] In some embodiments, the cells expressing the cancer-associated antigen include leukocytes, and the cancer is lymphoma, leukemia, or myeloma. In some specific embodiments, the cells expressing the cancer-associated antigen include leukocytes, and the cancer is multiple myeloma or lymphoma. In some very specific embodiments, the cells expressing the cancer-associated antigen include plasma cells, and the cancer is multiple myeloma. In some very specific embodiments, the cells expressing the cancer-associated antigen include lymphocytes, and the cancer is lymphoma.

[0113] In some embodiments, the cells expressing the cancer-associated antigen include ovarian cells, and the cancer is ovarian cancer.

[0114] In some embodiments, the cells expressing the cancer-associated antigen include lung epithelial cells, and the cancer is non-small cell lung cancer.

[0115] In some embodiments, the administration is selected from intravenous, intramuscular, subcutaneous, intradermal, intraocular, parenteral, intraperitoneal, intrathecal, intralesional, intralesional, and intratumoral administration. In some specific embodiments, the administration is intravenous. Example Example 1. Identification of human variable regions of anti-SP17 antibodies based on human diversity

[0116] A Fab phage display library was constructed from peripheral blood obtained from 120 healthy human donors. In short, the library was constructed by randomly combining nucleotide sequences encoding the variable regions of the immunoglobulin heavy chain and the immunoglobulin light chain. The library exhibits approximately one trillion possible combinations. Biopanning of the library identified 12 positive clones, and nucleotide sequencing of these clones identified two distinct clones. A single clone was identified by ELISA as binding to SP17. The nucleotide sequences of the human VH and VL regions of this antibody are listed in Table 1 below. The variable regions have the amino acid sequences shown in Table 2 and the CDRs shown in Table 3 below. Table 1. Nucleotide sequences of the anti-SP17 VH and VL regions identified in a Fab phage display library of approximately one trillion human Fab samples developed from peripheral blood samples of 120 human subjects via biopanning. Table 2. Amino acid sequences of the anti-SP17 VH and VL regions identified in a Fab phage display library of approximately one trillion human Fab samples developed from peripheral blood samples of 120 human subjects via biopanning. Table 3. Amino acid sequences of CDRs in the VH and VL regions identified in a Fab phage display library of approximately one trillion personal proto-Fab cells developed from peripheral blood samples of 120 human subjects via biopanning. Example 2. Modified mouse-human chimeric anti-SP17 IgG2a / KAPPA monoclonal antibody

[0117] The nucleotide sequences encoding the VH and VL regions of Example 1 were cloned into the mouse IgG2a heavy chain gene and the mouse κ light chain gene, respectively, and expressed in CHO-S cells to generate a mouse-human chimeric antibody, named chAB2. Successful antibody generation was confirmed by SDS-PAGE and Western blotting using anti-mouse heavy chain and light chain antibodies and recombinant SP17 protein. Figure 2 and 3 The chAB2 antibody is reactive to the mouse SP17 protein expressed on the surface of the mouse ovarian cancer cell line ID8. Figure 4 HPLC analysis of the chAB2 antibody preparation showed a purity of 93%. Figure 5 The KD of chAB2 and SP17, determined using surface plasmon resonance with Biacore™ T200, was 2.073 nanomolar. Figure 6 ).

[0118] The specificity of the chAB2 antibody against SP17 was determined by immunohistochemistry using a normal tissue microarray composed of 33 normal tissues from 2-3 human donors for each tissue. Normal tissues included the brain, eye, adrenal gland, pituitary gland, thyroid gland, parathyroid gland, tonsils, thymus, spleen, heart, lung, larynx, esophagus, stomach, small intestine, colon, liver, pancreas, salivary glands, kidney, bone, skeletal muscle, skin, peripheral nerves, mesothelial cell cartilage, breast, ovary, endometrium, cervix, testis, and prostate. The chAB2 antibody bound to the testis but not to normal tissues. Example 3. Modified human anti-SP17 IgG4 / KAPPA monoclonal antibody

[0119] The VH and VL nucleotide sequences from Example 1 were cloned into the human IgG4 heavy chain gene and the human κ light chain gene, respectively. A Ser228Pro mutation was introduced into the heavy chain to reduce non-specific Fc receptor γ binding. The resulting antibody was named SP17-AB2. Successful cloning was confirmed by sequence analysis. The amino acid sequences of the heavy and light chains are shown in Table 4, with the Ser228Pro mutation underlined in SEQ ID NO: 11. Successful expression of the SP17-AB2 antibody was confirmed by SDS-PAGE. Figure 7 The ability of the SP17-AB2 antibody to bind to the SP17 protein was confirmed by Western blotting. Figure 8 ). Table 4. Amino acid sequence of human anti-SP17 antibody SP17-AB2 Based on the above results, SP17-AB2 antibodies may be particularly suitable as therapeutic antibodies for treating cancers expressing SP17. Example 4. Modification of anti-SP17 immunoconjugate with deruxtecan The SP17-AB2 antibody described in Example 3 was cross-linked with deruxtecan using the conjugation strategy described in U.S. Patent No. 10,729,782 (the entire contents of which are incorporated herein by reference) to generate the immunoconjugate SP17-AB2-DXD. Deructetane, a drug that exhibits antitumor cytotoxicity based on its exatecan moiety, is a topoisomerase inhibitor covalently cross-linked with SP17-AB2 via a linker and disulfide bond, as described in U.S. Patent No. 10,729,782. Successful preparation of the immunoconjugate was confirmed by reducing it with excess dithiothreitol followed by mass spectrometry analysis. The results showed that the deruxtecan to SP17-AB2 ratio of SP17-AB2 in SP17-AB2-DXD was approximately 7.93, indicating that, on average, a single SP17-AB2-DXD molecule contains approximately eight deruxtecan moieties. Notably, this drug-to-antibody ratio is significantly higher than that of most FDA-approved antibodies. Prior to cytotoxicity assays, SK-OV-3 cells were seeded at 1000, 2000, or 3000 cells per well and cultured overnight. SK-OV-3 cells are SP17-expressing human ovarian cancer cells resistant to multiple chemotherapeutic agents, including cisplatin, doxorubicin, and diphtheria toxin. SK-OV-3 cells were treated with different concentrations of SP17-AB2 antibody, SP17-AB2-DXD immunoconjugate, control human IgG4 antibody (hIgG4), or drutecan (DXD) alone. Cells were cultured for 5 or 6 days, and then cytotoxicity was measured using a luminescence assay and an EnVision™ plate reader (PerkinElmer, Massachusetts, USA). The SP17-AB2-DXD immunoconjugate showed superior cytotoxicity compared to the naked SP17-AB2 antibody and the human IgG4 control. Figures 9A-9C (Table 5). Table 5. The IC50 and cytotoxicity of the immunoconjugate SP17-AB2-DXD against human ovarian cancer cells are superior to those of the naked antibody SP17-AB2. Prior to the second cytotoxicity assay, ID8 cells were seeded at 250 or 300 cells per well and cultured overnight to establish approximately 90% confluence. Cells were then treated with different concentrations of SP17-AB2 antibody, SP17-AB2-DXD immunoconjugate, hIgG4, or deruxtecan alone. Cells were cultured for another 3 days before cytotoxicity was measured. The SP17-AB2-DXD immunoconjugate showed superior cytotoxicity compared to the naked SP17-AB2 antibody and human IgG4 control. Figure 10 (Table 6). Table 6. The immunoconjugate SP17-AB2-DXD showed superior cytotoxicity against mouse ovarian cancer cells compared to the naked antibody SP17-AB2. Example 5. Modification of anti-Sp17 immunoconjugates using ozogamicin The SP17-AB2 antibody described in Example 3 was crosslinked with calicheamicin using the conjugation strategy described in U.S. Patent No. 5,877,296 (the entire contents of which are incorporated herein by reference) to produce the immunoconjugate SP17-AB2-ozogamicin. Example 6. Modified dimeric anti-Sp17 immunoconjugate

[0120] 1.1 g of SP17-AB2-DXD immunoconjugate was diluted to a concentration of 10 mg / mL in phosphate-buffered saline (PBS; 0.1 mol phosphate and 0.15 mol sodium chloride, pH 7.4) to obtain 110 mL of first-buffered antibody solution.

[0121] 10 mg of SATP (Pierce Biotechnology, Illinois, USA) was dissolved in 1 mL of dimethyl sulfoxide (DMSO) to obtain an SATP solution, which was immediately added to the first buffer antibody to form an SATP reaction system. The SATP reaction system was stirred at room temperature for 30 minutes to produce a protected immunoconjugate. Subsequently, excess SATP and DMSO were removed from the protected immunoconjugate by dialyzing with PBS as the dialysate.

[0122] 1.1 g of SP17-AB2-ozogamicin immunoconjugate was diluted in PBS to a concentration of 10 mg / mL to obtain 110 mL of second buffer antibody solution.

[0123] 20 mg of sulfo-SMCC (Thermo Scientific, Massachusetts, USA) was dissolved in 4 mL of distilled deionized water to obtain a sulfo-SMCC solution, which was immediately added to the second buffer antibody to form a sulfo-SMCC reaction system. The sulfo-SMCC reaction system was stirred at 4°C for 2 hours to generate an activated immunoconjugate. Subsequently, excess sulfo-SMCC was removed from the activated immunoconjugate by dialyzing with PBS as the dialysate.

[0124] During the sulfo-SMCC reaction incubation, a 20 mL deacetylation solution was prepared by dissolving 0.7 g of hydroxylamine hydrochloride and 0.2 g of tetrasodium ethylenediaminetetraacetate (EDTA) in 20 mL of PBS and adjusting the pH to 7.2–7.5 with a 10 mol / L sodium hydroxide aqueous solution. Subsequently, 10 mL of the deacetylation solution was added to the protected immunoconjugate to produce a deprotected solution, which was stirred at room temperature for 2 hours to produce a thiol-modified immunoconjugate. Excess hydroxylamine and EDTA were then removed from the thiol-modified immunoconjugate by dialyzing with PBS as the dialysate.

[0125] Subsequently, the activated immunoconjugate and the thiol-modified immunoconjugate were mixed and stirred at 4°C for 2 hours to obtain the dimer immunoconjugate dSP17-AB2-DXD / ozogamicin. The dimer immunoconjugate dSP17-AB2-DXD / ozogamicin was then purified by size exclusion chromatography to remove byproducts and monomeric antibodies. Example 7. Cross-linking of dimeric immunoconjugates with cells in vitro

[0126] ID8 cells were labeled with the monomeric antibody SP17-AB2, the monomeric immunoconjugate SP17-AB2-DXD, the monomeric immunoconjugate SP17-AB2-ozogamicin, and the dimeric immunoconjugate dSP17-AB2-DXD / ozogamicin, and cell cross-linking was assessed by flow cytometry. Forward and side scattering results showed a large cell population in ID8 cells labeled with dSP17-AB2-DXD / ozogamicin, consistent with cross-linked cells. This large cell population was absent in ID8 cells labeled with SP17-AB2, SP17-AB2-DXD, and SP17-AB2-ozogamicin. These data indicate that the dimeric immunoconjugate can cross-link cells. Example 8. Homodimeric antibodies showed higher cellular uptake compared to monomeric antibodies.

[0127] A homodimeric form of SP17-AB2, named hdSP17-AB2, was prepared according to the strategy described in Example 6 and was not conjugated with any drug. The monomeric antibody SP17-AB2 and the homodimeric antibody hdSP17-AB2 were labeled with fluorescein isothiocyanate (FITC) to generate FITC-SP17-AB2 and FITC-hdSP17-AB2. ID8 cells of different cell densities were incubated with the FITC-labeled antibody, washed, and observed under a fluorescence microscope. Cells incubated with FITC-SP17-AB2, regardless of confluence, showed FITC-related fluorescence primarily localized in the periphery. Cells incubated with FITC-hdSP17-AB2 at near or 100% confluence showed FITC-related fluorescence localized between different cells. Cells incubated with FITC-hdSP17-AB2 at less than 50% confluence showed significant FITC-related fluorescence in the cytoplasm. These results suggest that homodimeric antibodies exhibit increased cellular uptake compared to monomeric antibodies. Example 9. Dimeric immunoconjugate dSP17-AB2-DXD / ozogamicin relative to monomer The combination of SP17-AB2-DXD and SP17-AB2-ozogamicin showed lower IC50 and higher cytotoxicity.

[0128] Prior to the cytotoxicity assay, SK-OV-3 cells were seeded at 2000 cells per well and cultured overnight. SK-OV-3 cells were then treated with different concentrations of the following treatments: (1) SP17-AB2 antibody; (2) SP17-AB2-DXD immunoconjugate; (3) SP17-AB2-ozogamicin immunoconjugate; (4) both SP17-AB2-DXD and SP17-AB2-ozogamicin; (5) dSP17-AB2-DXD / ozogamicin immunoconjugate; (6) hdSP17-AB2; (7) hIgG4; or (8) deruxtecan. Cells were cultured for an additional 5 days, after which cytotoxicity was measured using a luminescence assay and an EnVision™ plate reader (PerkinElmer, Massachusetts, USA). IC50 and maximum cytotoxicity were then determined.

[0129] (4) The IC50 of the SP17-AB2-DXD and SP17-AB2-ozogamicin combination treatment group was determined based on the individual concentrations of SP17-AB2-DXD and SP17-AB2-ozogamicin, rather than based on the combined concentrations of the two. For example, when SK-OV-3 cells are incubated with SP17-AB2-DXD and SP17-AB2-ozogamicin at concentrations of 128 nanomoles each, the IC50 is calculated based on a concentration of 128 nanomoles rather than a combined concentration of 256 nanomoles, such that the 128 nanomoles of (4) SP17-AB2-DXD and SP17-AB2-ozogamicin combination contains approximately the same concentrations of deruxtecan, ozogamicin, and antigen-binding sites as the 128 nanomoles of (5) described dSP17-AB2-DXD / ozogamicin immunotherapy agent.

[0130] The dimer immunotherapy agent dSP17-AB2-DXD / ozogamicin showed a lower IC50 and higher cytotoxicity compared to the combination of SP17-AB2-DXD and SP17-AB2-ozogamicin (Table 7). Table 7. The IC50 and cell counts of the dimeric immunoconjugate dSP17-AB2-DXD / ozogamicin. Superior to monomeric immunoconjugates and naked dimers in terms of cytotoxicity

Claims

1. A method of treating cancer in a human subject, comprising administering a therapeutically effective amount of an immunotherapeutic agent to the subject, wherein: The immunotherapeutic agent comprises a covalently cross-linked first immunoconjugate and a second immunoconjugate; The first immunoconjugate comprises a first antibody that binds to a first cancer-associated antigen and a first payload selected from a first radioactive isotope and a first pharmaceutical agent; The second immunoconjugate comprises a second antibody that binds to a second cancer-associated antigen and a second payload selected from a second radioisotope and a second pharmaceutical agent; and The first load and the second load are different.

2. The method according to claim 1, wherein the first antibody and the second antibody have the same amino acid sequence.

3. The method according to claim 1 or 2, wherein: The cancer contains cells that express human sperm protein 17 (Sp17); The first antibody and the second antibody each contain two antigen-binding sites, and each antigen-binding site specifically binds to Sp17, such that the immunotherapeutic agent contains exactly four antigen-binding sites that specifically bind to Sp17. The first antibody and the second antibody each contain a first variable domain and a second variable domain; The first variable domain of the first antibody and the first variable domain of the second antibody have the same amino acid sequence; The second variable domain of the first antibody and the second variable domain of the second antibody have the same amino acid sequence; The first variable domain of the first antibody and the second antibody comprises a VH CDR1 region, a VH CDR2 region, and a VH CDR3 region, wherein the VH CDR1 region contains the same amino acid sequence as SEQ ID NO: 5, the VH CDR2 region contains the same amino acid sequence as SEQ ID NO: 6, and the VH CDR3 region contains the same amino acid sequence as SEQ ID NO: 7; and The second variable domain of the first antibody and the second antibody includes a VL CDR1 region, a VL CDR2 region, and a VLCDR3 region, wherein the VL CDR1 region contains the same amino acid sequence as SEQ ID NO: 8, the VL CDR2 region contains the same amino acid sequence as SEQ ID NO: 9, and the VL CDR3 region contains the same amino acid sequence as SEQ ID NO:

10.

4. The method according to any one of claims 1 to 3, wherein: The cancer contains cells that express human sperm protein 17 (Sp17); The first antibody and the second antibody each contain two antigen-binding sites, and each antigen-binding site specifically binds to Sp17, such that the immunotherapeutic agent contains exactly four antigen-binding sites that specifically bind to Sp17. The first antibody comprises a heavy chain having at least 90% sequence homology to SEQ ID NO: 11 and a light chain having at least 90% sequence homology to SEQ ID NO: 12; and The second antibody comprises a heavy chain having at least 90% sequence homology with SEQ ID NO: 11 and a light chain having at least 90% sequence homology with SEQ ID NO:

12.

5. The method according to any one of claims 1 to 4, wherein the first load is a first radioactive isotope.

6. The method according to claim 5, wherein the first radioactive isotope is an alpha particle emitter.

7. The method according to claim 5 or 6, wherein the first radioactive isotope is selected from actinium-225, astatine-211, bismuth-212, bismuth-213 and terbium-149.

8. The method according to any one of claims 1 to 7, wherein the second load is a second radioactive isotope.

9. The method of claim 8, wherein the second radioactive isotope is a β-particle emitter.

10. The method according to claim 8 or 9, wherein the second radioactive isotope is selected from copper-67, gallium-68, holmium-166, iodine-124, iodine-131, lutetium-177, samarium-153, technetium-99, and yttrium-90.

11. The method according to any one of claims 1 to 10, wherein: The first load is a first radioactive isotope; The first radioactive isotope is an alpha particle emitter; The second load is a second radioactive isotope; and The second radioactive isotope is a beta particle emitter.

12. The method according to any one of claims 1 to 4, wherein: The first load is the first drug agent; The second load is the second drug agent; The first and second drug agents are selected from calicheamicin, camptothecin, deruxtecan, doxorubicin, emtansine, exatecan, irinotecan, maleimidocaproyl monomethyl auristatin F, mertansine, monomethyl auristatin F, paclitaxel, PE38, pyrrolobenzodiazepine, SN-38, and vedotin; and The first drug agent and the second drug agent are different drugs.

13. The method according to any one of claims 1 to 12, wherein: The immunotherapy agent has a drug-antibody ratio, which is equal to the sum of the number of the first load and the second load in the therapeutically effective amount of the immunotherapy agent divided by the number of molecules of the immunotherapy agent in the therapeutically effective amount; and The drug-antibody ratio is at least 5.

14. The method according to any one of claims 1 to 13, wherein: A combination of the first and second immunoconjugates at equal concentrations has a reference IC50, which is the calculated concentration of the first and second immunoconjugates required to observe 50% cytotoxicity in an in vitro assay against a cancer cell line expressing the antigen. The immunotherapy agent has an improved IC50 against the cancer cell line; and The improved IC50 is smaller than the reference IC50.

15. The method according to any one of claims 1 to 14, wherein: The first immunoconjugate has a first reference therapeutic effect against the cancer, calculated per mole of the first immunoconjugate. The second immunoconjugate has a second reference therapeutic effect against the cancer, calculated per mole of the second immunoconjugate; The immunotherapy agent has an improvement in efficacy against the cancer per mole of the immunotherapy agent; and The improvement in therapeutic effect is greater than the sum of the first reference therapeutic effect and the second reference therapeutic effect.

16. The method according to any one of claims 1 to 15, wherein: The first immunoconjugate has a first reference infusion time per mole of the first immunoconjugate; The second immunoconjugate has a second reference infusion time per mole of the second immunoconjugate; The immunotherapy agent has a shortened infusion time per mole of the immunotherapy agent; and The shortened infusion time is shorter than the sum of the first reference infusion time and the second reference infusion time.

17. An immunotherapeutic agent comprising a covalently cross-linked first immunoconjugate and a second immunoconjugate, wherein: The first immunoconjugate comprises a first antibody that binds to a first cancer-associated antigen and a first payload selected from a first radioactive isotope and a first pharmaceutical agent; The second immunoconjugate comprises a second antibody that binds to a second cancer-associated antigen and a second payload selected from a second radioisotope and a second pharmaceutical agent; and The first load and the second load are different.

18. The immunotherapeutic agent of claim 17, wherein the first antibody and the second antibody have the same amino acid sequence.

19. The immunotherapeutic agent according to claim 17 or 18, wherein: The first antibody and the second antibody each contain two antigen-binding sites, and each antigen-binding site specifically binds to Sp17, such that the immunotherapeutic agent contains exactly four antigen-binding sites that specifically bind to Sp17. The first antibody and the second antibody each contain a first variable domain and a second variable domain; The first variable domain of the first antibody and the first variable domain of the second antibody have the same amino acid sequence; The second variable domain of the first antibody and the second variable domain of the second antibody have the same amino acid sequence; The first variable domain of the first antibody and the second antibody comprises a VH CDR1 region, a VH CDR2 region, and a VH CDR3 region, wherein the VH CDR1 region contains the same amino acid sequence as SEQ ID NO: 5, the VH CDR2 region contains the same amino acid sequence as SEQ ID NO: 6, and the VH CDR3 region contains the same amino acid sequence as SEQ ID NO: 7; and The second variable domain of the first antibody and the second antibody includes a VL CDR1 region, a VL CDR2 region, and a VLCDR3 region, wherein the VL CDR1 region contains the same amino acid sequence as SEQ ID NO: 8, the VL CDR2 region contains the same amino acid sequence as SEQ ID NO: 9, and the VL CDR3 region contains the same amino acid sequence as SEQ ID NO:

10.

20. The immunotherapeutic agent according to any one of claims 17 to 19, wherein: The first antibody and the second antibody each contain two antigen-binding sites, and each antigen-binding site specifically binds to Sp17, such that the immunotherapeutic agent contains exactly four antigen-binding sites that specifically bind to Sp17. The first antibody comprises a heavy chain having at least 90% sequence homology to SEQ ID NO: 11 and a light chain having at least 90% sequence homology to SEQ ID NO: 12; and The second antibody comprises a heavy chain having at least 90% sequence homology with SEQ ID NO: 11 and a light chain having at least 90% sequence homology with SEQ ID NO:

12.

21. The immunotherapeutic agent according to any one of claims 17 to 20, wherein the first load is a first radioisotope.

22. The immunotherapeutic agent according to claim 21, wherein the first radioactive isotope is an alpha particle emitter.

23. The immunotherapeutic agent according to claim 21 or 22, wherein the first radioisotope is selected from actinium-225, astatine-211, bismuth-212, bismuth-213 and terbium-149.

24. The immunotherapeutic agent according to any one of claims 17 to 23, wherein the second load is a second radioisotope.

25. The immunotherapeutic agent according to claim 24, wherein the second radioisotope is a beta particle emitter.

26. The immunotherapeutic agent according to claim 24 or 25, wherein the second radioisotope is selected from copper-67, gallium-68, holmium-166, iodine-124, iodine-131, lutetium-177, samarium-153, technetium-99, and yttrium-90.

27. The immunotherapeutic agent according to any one of claims 17 to 26, wherein: The first load is a first radioactive isotope; The first radioactive isotope is an alpha particle emitter; The second load is a second radioactive isotope; and The second radioactive isotope is a beta particle emitter.

28. The immunotherapeutic agent according to any one of claims 17 to 27, wherein: The first load is the first drug agent; The second load is the second drug agent; The first and second drug agents are selected from calicheamicin, camptothecin, deruxtecan, doxorubicin, emtansine, exatecan, irinotecan, maleimidocaproyl monomethyl auristatin F, mertansine, monomethyl auristatin F, paclitaxel, PE38, pyrrolobenzodiazepine, SN-38, and vedotin; and The first drug agent and the second drug agent are different drugs.

29. The immunotherapeutic agent according to any one of claims 17 to 28, wherein: A combination of the first and second immunoconjugates at equal concentrations has a reference IC50, which is the calculated concentration of the first and second immunoconjugates required to observe 50% cytotoxicity in an in vitro assay against a cancer cell line expressing the antigen. The immunotherapy agent has an improved IC50 against the cancer cell line; and The improved IC50 is smaller than the reference IC50.

30. The immunotherapeutic agent according to any one of claims 17 to 29, wherein: The first immunoconjugate has a first reference therapeutic effect against the cancer, calculated per mole of the first immunoconjugate. The second immunoconjugate has a second reference therapeutic effect against the cancer, calculated per mole of the second immunoconjugate; The immunotherapy agent has an improvement in efficacy against the cancer per mole of the immunotherapy agent; and The improvement in therapeutic effect is greater than the sum of the first reference therapeutic effect and the second reference therapeutic effect.

31. The method according to any one of claims 17 to 30, wherein: The first immunoconjugate has a first reference infusion time per mole of the first immunoconjugate; The second immunoconjugate has a second reference infusion time per mole of the second immunoconjugate; The immunotherapy agent has a shorter infusion time per mole of the immunotherapy agent; and The shorter infusion time is shorter than the sum of the first reference infusion time and the second reference infusion time.

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