Anti-cd38 monoclonal antibodies and uses thereof

Abstract

Provided are humanized monoclonal antibodies targeting CD38 for use in treating immune thrombocytopenia (ITP). The anti-CD38 monoclonal antibodies stand out by their unique mechanism of action, which includes reducing autoantibody-mediated platelet destruction and modulating the immune system, thereby restoring hemostatic balance, which addresses both the symptoms of ITP and the underlying immune dysregulation features.

Description

Technical Field

[0001] This disclosure pertains to the field of medical technology and specifically relates to antibodies targeting CD38. Background Technology

[0002] CD38 is a type II transmembrane glycoprotein widely expressed on the surface of many immune cells, including B lymphocytes, natural killer cells, and certain types of T cells. Its functions are multifaceted, encompassing roles in cell adhesion, signal transduction, and calcium signaling. The enzymatic activity of CD38 also affects the metabolism of nicotinamide adenine dinucleotide (NAD) and cyclic ADP-ribose, influencing various cellular processes. CD38 plays a crucial role in the regulation of immune responses, and its presence on plasma cells (particularly those involved in autoimmune responses) makes it a target of interest in immunotherapy.

[0003] The development of anti-CD38 monoclonal antibodies marks a significant advance in the treatment of autoimmune diseases and certain hematologic malignancies. These antibodies modulate the immune response by targeting CD38, providing a new approach to understanding the role of CD38 expression in disease pathology.

[0004] In the context of immune thrombocytopenic purpura (ITP), traditional treatments primarily include glucocorticoids, intravenous immunoglobulins, and splenectomy, as well as second-line therapies such as rituximab and thrombopoietin receptor agonists (TPO-RAs). However, these treatments often have limitations in terms of efficacy, duration of response, and side effects. This poses a significant treatment challenge, especially for patients with refractory or relapsed ITP.

[0005] The synthesis and application of anti-CD38 monoclonal antibodies, as described in WO2021104052A1, provides insights into their unique structure and potential therapeutic benefits. Because anti-CD38 monoclonal antibodies target underlying immune dysregulations, they differ from conventional ITP treatment by offering a dual mechanism of action: firstly, by reducing platelet destruction mediated by autoantibodies; and secondly, by modulating the immune system to restore hemostatic balance. Therefore, this dual action addresses both the symptoms and the underlying cause of ITP, providing a more comprehensive treatment approach. Summary of the Invention

[0006] This disclosure covers antibodies targeting CD38 or their antigen-binding portions, characterized by a unique complementarity-determining region sequence that distinguishes them from existing anti-CD38 antibodies (WO2021104052A1). It exhibits potent cell-killing ability and favorable safety profile via an Fc receptor-dependent mechanism.

[0007] In one implementation, an antibody targeting CD38 or its antigen-binding portion is used to treat ITP, targeting both short-lived and long-lived plasma cells (LLPCs) responsible for producing autoantibodies against platelets. This approach not only reduces platelet destruction mediated by autoantibodies but also modulates the immune system to restore hemostatic balance.

[0008] Immune thrombocytopenic purpura (ITP) is an autoimmune disease of the blood system, characterized by a platelet count below 100 × 10⁻⁶. 9 It increases the risk of bleeding. It affects approximately 9–20 individuals per 10,000 adults annually. Patients with ITP are more likely to experience hospitalization, fatigue, and decreased quality of life. Clinical manifestations are primarily skin and mucous membrane bleeding, the severity of which varies depending on the platelet count and can be life-threatening, especially when the platelet count drops below 30 × 10⁹ / L. 9 In the case of / L.

[0009] Corticosteroids are the primary first-line treatment for ITP. Although many patients initially respond to corticosteroid regimens, only about 20-40% maintain a sustained platelet response after discontinuation of treatment. Long-term use of corticosteroids is not recommended due to associated adverse reactions. Patients who do not respond to first-line treatment or relapse are often switched to second-line therapy, such as splenectomy, rituximab, and thrombopoietin receptor agonists (TPO-RAs). However, responses to these treatments vary, highlighting the urgent need for safer and more effective treatment strategies.

[0010] The pathophysiology of ITP involves loss of immune tolerance to platelet autoantigens, leading to the production of antiplatelet autoantibodies and abnormal activation of humoral and cellular immunity. This results in platelet destruction and insufficient platelet production by megakaryocytes. Notably, antiplatelet-specific plasma cells have been identified in the spleen of ITP patients receiving rituximab treatment, revealing resistance to this type of therapy. These cells, including long-lived plasma cells (LLPCs), are a major source of natural antibodies and play a crucial role in the immune response. Therefore, strategies targeting B cell elimination alone may be ineffective due to the persistent antibody production by LLCPCs.

[0011] CD38 is a type II glycoprotein highly expressed in plasmablasts, short-lived plasma cells, and low-density leukocytes (LLPCs). Its role in autoimmune diseases has become a focus of clinical research, particularly the use of antibodies targeting CD38 or their antigen-binding moieties. The complementarity-determining region (CD-determining region) of humanized monoclonal anti-CD38 antibodies differs from that of other antibodies (such as daratumumab). Preclinical studies have shown that antibodies targeting CD38 or their antigen-binding moieties effectively induce potent cytotoxicity via an Fc receptor-dependent mechanism, and are well-tolerated with no significant drug-related adverse reactions.

[0012] This disclosure also covers methods for administering CD38-targeting antibodies or their antigen-binding portions based on clinical trials, including dosage and treatment regimens. These trials demonstrated the safety and efficacy of anti-CD38 antibodies in treating ITP, focusing on rapid increases in platelet counts and sustained responses. Anti-CD38 antibodies offer a promising new strategy for ITP treatment by targeting and eliminating short-lived plasma cells and LLPCs. Phase 1 / 2 trials of anti-CD38 antibodies showed promising results in terms of efficacy and treatment of ITP. This study also aims to elucidate the mechanisms underlying the rapid increase in platelet counts observed with anti-CD38 antibody treatment and to investigate changes in peripheral blood immune status in patient and passive ITP mouse models.

[0013] The unique aspect of anti-CD38 antibodies lies in addressing the underlying immune dysregulation in ITP. By targeting the CD38 pathway, anti-CD38 antibodies not only reduce platelet destruction mediated by autoantibodies but also modulate the immune system to restore hemostatic balance. This dual mechanism of action provides a comprehensive treatment approach, representing a significant improvement over conventional therapies. The rapid increase in platelet count and sustained efficacy, along with a favorable safety profile, positions anti-CD38 antibodies as a potential breakthrough in ITP treatment, potentially transforming the treatment prospects for patients who have exhausted other options.

[0014] Anti-CD38 antibodies have shown remarkable efficacy as immunomodulators in the treatment of ITP and other potential autoimmune diseases. Unlike traditional treatments that primarily focus on symptom management, anti-CD38 antibodies directly target the dysregulated immune cells that contribute to disease pathology.

[0015] In one implementation, anti-CD38 antibodies are used as novel therapeutic agents for immune thrombocytopenic purpura (ITP) by targeting and eliminating short-lived and long-lived plasma cells (LLPCs). This approach is based on the premise that these cell types are key contributors to the autoimmune response visible in ITP, where they produce autoantibodies against platelets, leading to platelet destruction and thus causing thrombocytopenia.

[0016] In one implementation, the anti-CD38 monoclonal antibody was administered intravenously weekly at a dose of 16 mg / kg for 8 weeks. During the treatment period, prophylactic medications as specified in Table 1 below were provided.

[0017] Table 1. Prophylactic drugs for anti-CD38 monoclonal antibody therapy In one implementation, a phase 1 / 2 trial of an anti-CD38 monoclonal antibody has demonstrated promising results regarding the safety and efficacy of this treatment in patients with ITP. This trial involved patients with at least a three-month history of ITP and a baseline platelet count below 30 × 10⁻⁶. 9 Adult patients with a platelet count of ≥50 × 10⁹ / L were treated. Patients received intravenous anti-CD38 monoclonal antibody weekly at a dose of 16 mg / kg for eight weeks, followed by a 16-week follow-up period. The primary endpoint of this trial included achieving two consecutive platelet counts ≥ 50 × 10⁹ / L within eight weeks of treatment initiation. 9 The proportion of patients with a negative blood alcohol content ( / L), as well as the incidence and severity of adverse events.

[0018] In one implementation, this study also seeks to elucidate the mechanism underlying the rapid increase in platelet count observed with treatment using anti-CD38 monoclonal antibodies. This involves investigating changes in the peripheral blood immune status of ITP patients receiving anti-CD38 monoclonal antibody therapy. Key endpoints include monitoring CD38 expression on various immune cells, serum immunoglobulin levels, and the composition of immune cell subsets (such as T cells, B cells, natural killer cells, and monocytes).

[0019] In one implementation, passive ITP mouse models are used to further understand the effects of anti-CD38 treatment on the mononuclear phagocyte system. These models involve inducing ITP in mice, followed by administration of anti-CD38 monoclonal antibodies, to allow for a detailed examination of the effects of this treatment on splenic macrophage populations, NK cell counts, and other relevant immune parameters.

[0020] The mechanism of action of anti-CD38 monoclonal antibodies involves several key aspects: 1. Reduced production of autoantibodies: Anti-CD38 monoclonal antibodies help reduce the production of autoantibodies that target and destroy platelets by binding to CD38 on plasma cells.

[0021] 2. Immune system regulation: Anti-CD38 monoclonal antibodies affect various aspects of the immune system. They regulate the activity of B cells and T cells, potentially restoring immune tolerance and reducing autoimmune attacks on platelets.

[0022] 3. Effects on NK cells and monocytes: Anti-CD38 monoclonal antibody treatment has been observed to reduce the number of CD56dimCD16+ NK cells and alter the expression of CD32b on monocytes. These play a role in antibody-dependent cell cytotoxicity (ADCC) and phagocytosis, which are involved in platelet destruction in ITP.

[0023] The advantages of anti-CD38 monoclonal antibodies over traditional ITP treatment are multifaceted: Rapid and sustained response: Clinical trials have shown that anti-CD38 monoclonal antibodies can rapidly increase platelet counts within weeks and maintain a response for months.

[0024] Safety profile: Anti-CD38 monoclonal antibodies have demonstrated a manageable safety profile, with most adverse events being mild to moderate and resolvable.

[0025] Potential for treating refractory cases: Anti-CD38 monoclonal antibodies offer hope to patients who have not responded to conventional treatment or have relapsed.

[0026] Comprehensive immune modulation: By targeting the CD38 pathway, anti-CD38 monoclonal antibodies address both the symptoms and immune aspects of ITP, providing a more comprehensive treatment approach.

[0027] In summary, anti-CD38 monoclonal antibodies represent a significant advance in the treatment of ITP and other potential autoimmune diseases, providing a novel mechanism of action with substantial therapeutic benefits. Attached Figure Description

[0028] Figure 1 A flowchart of a human clinical trial of an anti-CD38 monoclonal antibody in the treatment of ITP is shown.

[0029] Figure 2 The platelet count dynamics of patients 009 and 014 are shown.

[0030] Figure 3 This demonstrates that the platelet count in patients who completed anti-CD38 monoclonal antibody therapy decreased to below 50 × 10 for the first time. 9 The cumulative incidence of / L.

[0031] Figure 4 The dynamic changes of peripheral blood CD19+ B cells in ITP patients before and after infusion of anti-CD38 monoclonal antibody are shown.

[0032] Figure 5 This study illustrates the dynamic changes in peripheral blood T cells in ITP patients before and after infusion of anti-CD38 monoclonal antibody.

[0033] Figure 6The changes in plasma cytokines and T cell proliferation before and after anti-CD38 monoclonal antibody infusion are shown.

[0034] Figure 7 The changes in peripheral blood mononuclear cell subsets and Fcγ receptor expression levels on the membrane surface are shown before and after anti-CD38 monoclonal antibody treatment.

[0035] Figure 8 The study demonstrates the establishment of a passive ITP mouse model and the changes in spleen volume after intervention with an anti-mouse CD38 monoclonal antibody.

[0036] definition As used in this article, the term "immune thrombocytopenic purpura (ITP)" refers to a significantly reduced platelet count in the blood (below 100 × 10⁻⁶). 9 An autoimmune disease characterized by platelet destruction or inhibition of platelet production ( / L) often leads to an increased tendency for bleeding and bruising. This is primarily caused by the immune system destroying or inhibiting platelet production.

[0037] As used herein, the term "monoclonal antibody" refers to a group of identical antibodies, meaning that each individual antibody molecule in a monoclonal antibody group is identical to every other antibody molecule. This characteristic contrasts with that of polyclonal antibody groups, which contain antibodies with many different sequences. Monoclonal antibodies can be prepared using a variety of well-known methods (Smith et al. (2004) J. Clin. Pathol. 57, 912-917; and Nelson et al., J Clin Pathol (2000), 53, 111-117). For example, monoclonal antibodies can be prepared by immortalizing B cells, such as by fusing B cells with myeloma cells to generate hybridoma cell lines, or by infecting B cells with a virus such as EBV. Recombinant techniques can also be used to prepare antibodies in vitro from a clonal population of host cells by transforming host cells with plasmids carrying artificial sequences encoding antibody nucleotides.

[0038] As used herein, the term "bispecific antibody (BsAb)" refers to an antibody and / or antigen-binding molecule capable of specifically binding to two different antigenic determinants. Typically, bispecific antibodies and / or antigen-binding molecules contain two antigen-binding sites, each specific to a different antigenic determinant. In some embodiments, bispecific antibodies and / or antigen-binding molecules are capable of binding to two antigenic determinants simultaneously, particularly two antigenic determinants expressed on two different cells.

[0039] As used herein, the term "hybridoma" or "hybridoma cell" refers to a cell or cell line (typically myeloma or lymphoma cells) resulting from the fusion of antibody-producing lymphocytes with non-antibody-producing cancer cells. As is known to those skilled in the art, hybridomas can be proliferated and continuously supplied to produce specific monoclonal antibodies. Methods for producing hybridomas are known in the art (see, for example, Harlow & Lane, 1988). When referring to the term "hybridoma" or "hybridoma cell," subclones and progeny cells of the hybridoma are also included.

[0040] As used herein, a “full-length antibody” is an antibody having two full-length heavy chains (e.g., VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH4) and two full-length light chains (VL-CL) as well as a hinge region, such as antibodies naturally produced by antibody-secreting B cells, and synthetic antibodies having the same domains.

[0041] As used herein, the term "chimeric antibody" refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, such as an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody.

[0042] As used herein, a “humanized” antibody refers to a non-human (e.g., mouse) antibody form that is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding sequence of an antibody) containing a very small amount of sequence derived from a non-human immunoglobulin. Preferably, the humanized antibody is a human immunoglobulin (receptor antibody) in which residues of the complementarity-determining region (CDR) of the receptor antibody are replaced by CDR residues of a non-human animal species (donor antibody) (such as mouse, rat, or rabbit) having the desired specificity, affinity, and capability.

[0043] Furthermore, in humanization, amino acid residues in the CDR1, CDR2, and / or CDR3 regions of VH and / or VL may be mutated to improve one or more binding properties of the antibody (e.g., affinity). Mutations can be introduced, for example, by PCR-mediated mutagenesis, and their effect on antibody binding or other functional properties can be assessed using the in vitro or in vivo assays described herein. Typically, conserved mutations are introduced. Such mutations can be amino acid substitutions, additions, or deletions. Furthermore, mutations within the CDRs generally do not exceed one or two. Therefore, the humanized antibodies of this invention also cover antibodies containing one or two amino acid mutations within the CDRs.

[0044] As used herein, an "antibody fragment" or "antigen-binding fragment" of an antibody refers to any portion of a full-length antibody that is shorter than the full length, but contains at least a portion of the antibody's variable region that binds an antigen (e.g., one or more CDRs and / or one or more antibody binding sites), thereby retaining the binding specificity of the full-length antibody and at least a portion of its specific binding capacity. Therefore, an antigen-binding fragment refers to an antibody fragment containing an antigen-binding portion that binds to the same antigen from which the antibody fragment is derived. Antibody fragments include antibody derivatives produced by enzymatic treatment of a full-length antibody, as well as synthetic derivatives, such as recombinant derivatives. Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, single-chain Fv (scFv), Fv, dsFv, biantibodies, Fd and Fd' fragments, and other fragments, including modified fragments (see, for example, Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov). These fragments may contain multiple chains linked together, for example, by disulfide bonds and / or by peptide linkers. Antibody fragments typically contain at least or about 50 amino acids, and generally at least or about 200 amino acids. Antigen-binding fragments include any antibody fragment that, when inserted into an antibody framework (e.g., by translocation of a corresponding region), produces an antibody that specifically binds to an antigen (i.e., exhibits a Ka of at least or at least about 107-108 M⁻¹). As used herein, a “functional fragment” or “anti-CD38 antibody analogue” is a fragment or analogue that prevents or significantly reduces the receptor’s ability to bind ligands or initiate signal transduction. As used herein, “functional fragment” generally has the same meaning as “antibody fragment”, and in the case of an antibody, it can refer to a fragment that prevents or significantly reduces the receptor’s ability to bind ligands or initiate signal transduction, such as Fv, Fab, F(ab')2, etc. An “Fv” fragment consists of a dimer (VH-VL dimer) formed by the non-covalent association of variable domains of the heavy chain and variable domains of the light chain. In this configuration, the three CDRs of each variable domain interact to define a target-binding site on the surface of the VH-VL dimer, as in the case of a complete antibody. These six CDRs collectively confer the target-binding specificity of a complete antibody. However, even a single variable domain (or half of an Fv containing only three target-specific CDRs) can still have the ability to recognize and bind to a target.

[0045] As used herein, the term "CDR" refers to the complementary determinant region (CDR) of three CDRs on each of the heavy and light chains of a known antibody molecule. CDRs, also known as hypervariable regions, are located in the variable regions of both the heavy and light chains and are highly variable sites in the primary structure of the antibody. In this specification, the CDRs of the heavy chain are represented by CDR1, CDR2, and CDR3 from the N-terminal sequence of the heavy chain, and the CDRs of the light chain are represented by CDR1, CDR2, and CDR3 from the N-terminal sequence of the light chain. These sites are adjacent to each other in the tertiary structure and determine the specificity of the antigen to which the antibody binds.

[0046] As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which the complementary site of an antibody binds. Epitope determinants typically comprise molecules of chemically active surface types (such as amino acid or sugar side chains) and usually possess specific three-dimensional structural features as well as specific charge features.

[0047] As used herein, “expression” refers to the process by which a polypeptide is produced through the transcription and translation of polynucleotides. The expression level of a polypeptide can be assessed using any method known in the art, including, for example, methods for determining the amount of polypeptide produced by the host cell. Such methods may include, but are not limited to, quantifying polypeptides in cell lysates using ELISA, gel electrophoresis followed by Coomassie brilliant blue staining, Lowry protein assays, and Bradford protein assays.

[0048] As used herein, a “host cell” is a cell used to receive, maintain, replicate, and amplify a vector. Host cells can also be used to express polypeptides encoded by the vector. The nucleic acids contained in the vector replicate during host cell division, thereby amplifying the nucleic acids. Host cells can be eukaryotic or prokaryotic cells. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, and HEK cells (such as HEK 293 cells).

[0049] As used herein, "vector" refers to a reproducible nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into a suitable host cell. References to vectors include those that can typically introduce nucleic acids encoding polypeptides or fragments thereof through restriction digestion and ligation. References to vectors also include those that contain nucleic acids encoding polypeptides. Vectors are used to introduce nucleic acids encoding polypeptides into host cells, amplify nucleic acids, or express / display polypeptides encoded by nucleic acids. Vectors are typically kept in a free state, but can also be designed to integrate genes or fragments thereof into chromosomes of the genome. Vectors for artificial chromosomes, such as yeast artificial vectors and mammalian artificial chromosomes, are also envisioned. The selection and use of such solvents are well known to those skilled in the art.

[0050] As used in this article, vectors also include "virus vectors" or "viral vectors". Viral vectors are engineered viruses that are functionally linked to a foreign gene to transfer the foreign gene (as a solvent or shuttle) into cells.

[0051] As used herein, "expression vector" includes a vector capable of expressing DNA operatively linked to regulatory sequences, such as promoter regions capable of influencing the expression of such DNA fragments. Such additional fragments may include promoter and terminator sequences and optionally include one or more origins of replication, one or more selection markers, enhancers, polyadenylation signals, etc. Expression vectors are typically derived from plasmid or viral DNA, or may contain elements of both. Therefore, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, bacteriophage, recombinant virus, or other vector, which, when introduced into a suitable host cell, results in the expression of cloned DNA. Suitable expression vectors are well known to those skilled in the art, including expression vectors that can replicate in eukaryotic and / or prokaryotic cells, as well as expression vectors that remain free or are integrated into the host cell genome.

[0052] As used in this article, the term "efficacy" refers to the ability of a treatment (such as a drug or therapy) to produce the desired or anticipated outcome. In clinical trials, this often refers to the effectiveness of a treatment in achieving a specific outcome, such as increasing platelet counts in patients with ITP.

[0053] As used in this article, the term "safety" in the context of clinical trials refers to the monitoring and assessment of various adverse events and side effects associated with or related to treatment, with the aim of determining the risk profile of the treatment and the overall tolerability of the patients.

[0054] As used in this article, the term "pharmacodynamics" refers to the study of the biochemical and physiological effects of drugs (especially pharmaceuticals) on the body. This includes the mechanisms of drug action and the relationship between drug concentration and effect.

[0055] As used in this article, the term "immunogenicity" refers to the ability of a particular substance (such as a therapeutic agent or vaccine) to induce an immune response in the body. This term is frequently used to evaluate a patient's immune system response to biological therapies, including monoclonal antibodies.

[0056] As used herein, the term “treatment-related adverse event (TEAE)” refers to any unintended experience related to the use of a medical product or drug that occurs or worsens during treatment.

[0057] As used herein, the term "intention-to-treat population" refers to the principle in clinical trial analyses in which participants who were randomly assigned to each treatment were included in the analysis, regardless of whether they completed the treatment according to the study protocol.

[0058] As used herein, the term "Fc receptor-dependent mechanism" refers to the biological process involving the interaction between the Fc (crystallizable fragment) region of an antibody and Fc receptors on various immune cells. This interaction is crucial for triggering immune responses such as phagocytosis and antibody-dependent cytotoxicity (ADCC).

[0059] As used in this article, the term "plasma cell" refers to a type of white blood cell derived from B lymphocytes and primarily responsible for antibody production. In the context of ITP, certain plasma cells contribute to the pathology of the disease by producing autoantibodies against platelets.

[0060] As used in this article, the term "autoantibody" refers to antibodies produced by the immune system that mistakenly target and react with the body's own tissues or organs, leading to autoimmune diseases. In ITP, autoantibodies target and destroy platelets.

[0061] As used in this article, the term "thrombopoietin receptor agonist (TPO-RA)" refers to a class of drugs that stimulate platelet production by mimicking the action of the natural hormone thrombopoietin in the body. They are used to treat conditions such as ITP to increase platelet count.

[0062] As used herein, “treatment” for a person suffering from a disease or condition means that the person’s symptoms are partially or completely relieved, or remain unchanged, after treatment. Therefore, treatment includes prevention, treatment, and / or cure. Prevention refers to preventing underlying disease and / or preventing the worsening of symptoms or disease progression. Treatment also includes any antibodies or antigen-binding fragments thereof provided, and any pharmaceutical use of the compositions provided herein.

[0063] As used in this article, "therapeutic effect" refers to the effect of changing (usually improving or alleviating) the symptoms of a disease or condition or curing a disease or condition as a result of treatment of the subject.

[0064] As used herein, "therapeutic effective amount" or "therapeutic effective dose" refers to an amount of a substance, compound, material, or composition containing a compound that is at least sufficient to produce a therapeutic effect when applied to a subject. Therefore, it is the amount required to prevent, cure, alleviate, block, or partially block the symptoms of a disease or condition.

[0065] As used herein, "preventative effective amount" or "preventative effective dose" refers to the amount of a substance, compound, material, or composition containing a compound that, when applied to a subject, has the desired preventative effect, such as preventing or delaying the onset or recurrence of a disease or condition, or reducing the likelihood of the onset or recurrence of a disease or condition. A complete preventative effective dose does not need to be achieved by administering a single dose and can be achieved only after administering a series of doses. Therefore, a preventative effective amount can be administered once or more.

[0066] As used herein, the term "cytotoxic dose" refers to the amount of a substance (usually a drug or therapeutic agent) sufficient to cause cell death. In the context of medical treatment, a cytotoxic dose is an amount sufficient to kill cells (such as cancer cells or other pathologically proliferating cells) or inhibit their growth without causing unacceptable damage to healthy cells or tissues. Specific cytotoxic doses can vary depending on factors such as the type of cells targeted, the specific drug or agent used, the method of administration, and the individual characteristics of the patient.

[0067] As used in this article, the term "patient" refers to mammals, such as humans. Detailed Implementation

[0068] This trial of the anti-CD38 monoclonal antibody was designed as an investigator-initiated, open-label phase 1 / 2 trial. The primary objective was to evaluate the efficacy, safety, pharmacodynamics, and immunogenicity of the anti-CD38 monoclonal antibody in patients with immune thrombocytopenic purpura (ITP).

[0069] The trial protocol was approved by the Ethics Committee of the Institute of Hematology, Chinese Academy of Medical Sciences (CAMS). The trial was conducted in accordance with the principles of the Declaration of Helsinki. Written informed consent was provided to all patients. All animal protocols were approved by the Animal Care and Use Committee (IACUC) of the Institute of Hematology, Chinese Academy of Medical Sciences (CAMS). All procedures were performed under pentobarbital anesthesia, and every effort was made to minimize pain.

[0070] The experimental design and procedure are as follows: Figure 1 As shown: 1. Screening period (4 weeks): Eligibility screening of patients is conducted, including those aged 18 years and older, diagnosed with ITP for at least 3 months, and with a platelet count below 30 × 10⁻⁶. 9 Adults with a platelet count of / L. Exclusion criteria included secondary thrombocytopenia, prior anti-CD38 antibody therapy, and significant organ dysfunction.

[0071] 2. Treatment period (8 weeks): Eligible patients received anti-CD38 monoclonal antibody intravenously at a dose of 16 mg / kg weekly. Concurrent treatment with thrombopoietin receptor agonists (TPO-RA) or glucocorticoids was permitted. In addition, all patients received prophylactic medications, including glucocorticoids, antihistamines, and acetaminophen, before and after the anti-CD38 monoclonal antibody infusion.

[0072] 3. Follow-up period (16 weeks): After the 8-week treatment period, patients will be monitored for another 16 weeks to observe long-term effects and any delayed adverse reactions.

[0073] Participants: The detailed inclusion and exclusion criteria for the selected patients are listed below: Inclusion criteria: (1) Age ≥ 18 years, male or female.

[0074] (2) According to the American Society of Hematology 2011 Evidence-Based Practice Guidelines (Neunert et al., Blood (2011), 117, 4190-4207) or the International Consensus Report on the Study and Management of Primary Immune Thrombocytopenic-Pearl Disease (Provanet al., Blood (2010), 115, 168-186), the subjects had been clinically diagnosed with primary immune thrombocytopenic-pearl disease for at least three months prior to enrollment.

[0075] (3) Failure of glucocorticoid therapy (due to ineffectiveness, inability to maintain efficacy, or relapse), and failure of at least one second-line ITP therapy (including rituximab and / or platelet-producing stimulants such as rhTPO or eltrombopag) as specified in the guidelines (due to ineffectiveness, inability to maintain efficacy, or relapse). Ineffectiveness is defined as a platelet count <30 × 10⁹ / L after 28 days of use of rhTPO at 300 U / kg / day, eltrombopag at 75 mg / day, eltrombopag at 7.5 mg / day, or avatrombopag at 60 mg / day. Previous ITP therapy (e.g., methylprednisolone, platelet transfusion, gamma globulin transfusion, etc.) must have ended at least two weeks prior to the first dose.

[0076] (4) Subjects with a platelet count <30×10⁹ / L within 48 hours prior to the first dose of the study drug (if observed during screening visits and / or before administration of the study drug, with at least two consecutive platelet counts <30×10⁹ / L at at least two separate assessments, with a minimum interval of one day between the two assessments).

[0077] (5) ECOG physical condition score ≤2.

[0078] (6) Subjects receiving stable doses of maintenance therapy, including glucocorticoids (≤ 0.5 mg / kg prednisone or equivalent) or TPO receptor agonists, are permitted to enroll. However, at enrollment, subjects may only use one stable dose of concomitant medication, and that concomitant medication must have been stable for at least 4 weeks prior to the initial infusion of the study drug.

[0079] (7) For female patients of childbearing potential, a negative pregnancy test result is required. Both male and female patients of childbearing potential must use effective contraception during the study period and for 4 or 6 months after discontinuation of study drug treatment.

[0080] (8) The subjects must fully understand and be able to comply with the requirements of the research protocol and voluntarily sign the informed consent form.

[0081] 2.1.2 Exclusion Criteria (1) Subjects who are known to be allergic to anti-CD38 monoclonal antibodies or excipients, or subjects who have previously received anti-CD38 monoclonal antibodies but whose treatment outcomes were ineffective.

[0082] (2) Individuals diagnosed with the following conditions: autoimmune hemolytic anemia, various secondary and hereditary thrombocytopenic diseases, including leukemia, lymphoma, multiple myeloma, aplastic anemia, myelodysplastic syndrome, Evans syndrome, common variant immunodeficiency diseases, systemic lupus erythematosus, cirrhosis, antiphospholipid syndrome, pseudothrombocytopenia, and drug-induced thrombocytopenia (such as quinine, heparin, antibacterial agents, and antiepileptic drugs).

[0083] (3) Subjects with the following medical history within 12 months prior to starting the first dose of the study drug: any history of thrombotic or embolic events, or extensive and severe bleeding (such as hemoptysis, severe upper gastrointestinal bleeding, intracranial hemorrhage), or sepsis or other irregular bleeding.

[0084] (4) Subjects who have participated in any other investigational drug study (including vaccine study) or have been exposed to other investigational drugs within 4 weeks or 5 half-lives (whichever is longer) before the first dose of the investigational drug.

[0085] (5) Subjects who have used anticoagulants or any medication with antiplatelet effects (such as aspirin) within 3 weeks prior to the first dose of the study drug.

[0086] (6) Subjects who have received emergency treatment for ITP (e.g., methylprednisolone, platelet transfusion, intravenous immunoglobulin transfusion or thrombopoietin receptor agonist treatment) within 2 weeks prior to the first dose of the study drug.

[0087] (7) Subjects who have received drug treatment (including azathioprine, danazol, dapsone, cyclosporine A, tacrolimus, and sirolimus) within 4 weeks prior to the first dose of the study drug. Subjects who have received anti-CD20 monoclonal antibodies (such as rituximab) or drugs (including cyclophosphamide and vindesine) within 3 months prior to the first dose.

[0088] (8) Subjects who have undergone splenectomy within 6 months prior to the first dose of the study drug.

[0089] (9) Subjects who have received a live vaccine within 4 weeks prior to the first dose of the study drug, or who are scheduled to receive any live vaccine during the study.

[0090] (10) Individuals who have undergone allogeneic stem cell transplantation or organ transplantation.

[0091] (11) A clinically significant medical history within the six months prior to screening, which the investigator deems to be a risk to the safety of the subjects during the study period or may affect safety or efficacy analysis. This includes major clinical history such as cardiovascular events (e.g., acute myocardial infarction, heart failure, unstable angina, severe arrhythmias (e.g., frequent premature ventricular contractions, ventricular tachycardia, fibrillation) or poorly controlled hypertension); New York Heart Association (NYHA) class III-IV heart failure; poorly controlled diabetes, peptic ulcer, liver or kidney dysfunction; and various connective tissue diseases.

[0092] (12) Screening subjects with a history of malignant tumors within the past 5 years (excluding those with completely cured in situ cervical cancer and non-metastatic squamous cell carcinoma or basal cell carcinoma of the skin).

[0093] (13) Subjects with the following medical history: severe recurrent or chronic infection or acute infection requiring systemic treatment with antibiotics, antiviral drugs, antiparasitic drugs, antiamoebic drugs or antifungal drugs within 4 weeks prior to the first dose and during the screening period, or superficial skin infection requiring systemic treatment within one week prior to the first dose of the study drug. It is worth noting that subjects may be rescreened after the infection has subsided.

[0094] (14) Subjects with a known or suspected history of immunosuppression, including invasive opportunistic infections (such as histoplasmosis, listeriosis, coccidioidomycosis, Pneumocystis pneumonia and aspergillosis, even if the infection has subsided); or unusually frequent, recurrent or long-term infections (as determined by the investigator).

[0095] (15) Significant laboratory abnormalities during screening: a) Alanine aminotransferase or aspartate aminotransferase equal to or greater than the upper limit of normal (ULN). b) Total bilirubin equal to or greater than 1.2 times ULN (Note: Based on this criterion, individuals diagnosed with Gilbert's syndrome based on medical records should not be excluded). c) Creatinine and blood urea nitrogen equal to or greater than ULN.

[0096] (16) Positive for HIV antibody or syphilis antibody.

[0097] (17) Individuals who test positive for hepatitis B surface antigen (HBsAg), hepatitis B core antibody, HBV-DNA (by polymerase chain reaction), or hepatitis C virus antibody during the screening period. Individuals who test positive for hepatitis B core antibody but negative for HBV-DNA may be enrolled, and HBV-DNA will be monitored every 4 weeks.

[0098] (18) Pregnant or lactating women, or women who intend to conceive or lactate during the study; and male patients who intend to impregnate their partners during the study.

[0099] (19) Individuals with mental disorders who are unable to provide informed consent or participate in the trial and follow-up.

[0100] (20) Subjects whose toxic symptoms caused by previous treatment have not yet subsided were excluded from the trial.

[0101] (21) Any other medical condition deemed unsuitable for participation in this study by the investigators.

[0102] A total of 27 ITP patients were initially screened, of whom 22 met the eligibility criteria and were enrolled in the trial. Four patients were excluded based on eligibility criteria (three due to misdiagnosis: myelodysplastic syndrome (n=1), aplastic anemia (n=1), and common variant immunodeficiency (n=1); one patient was a hepatitis B virus carrier), and one patient withdrew their informed consent. Ultimately, 22 patients were enrolled in the study. During the entire anti-CD38 monoclonal antibody treatment period, one patient died of sudden cerebral hemorrhage following a single dose of anti-CD38 monoclonal antibody infusion. All 22 patients were included in efficacy and TRAE evaluations, and peripheral blood immune status was dynamically monitored in 21 patients before and after anti-CD38 monoclonal antibody infusion. Nineteen patients were included in the immunogenicity set for anti-antibody analysis. The patient group consisted primarily of adults with a median age of 35.5 years, predominantly female (72.7%). The median duration of ITP in these participants was 27 months, and they had previously received multiple ITP treatments, including glucocorticoids, intravenous immunoglobulin, TPO-RA, rituximab, and in some cases, splenectomy.

[0103] Outcome indicators: 1. Primary outcome: The primary focus is on safety / tolerability, and achieving a platelet count of ≥ 50 × 10⁶ on ≥ 2 consecutive occasions within 8 weeks after the first dose. 9 / L. Closely monitor adverse events and classify them according to severity.

[0104] 2. Secondary outcomes: These include the definition of response, changes in immune cell populations, cytokine levels, and other biomarkers, analyzed by flow cytometry, enzyme-linked immunosorbent assay (ELISA), and other methods.

[0105] Statistical analysis: The statistical analysis plan for this trial was designed to test the hypothesis that the primary endpoint (increased platelet count) would exceed the target value using descriptive statistics and confidence interval calculations. Both efficacy and safety data were analyzed, with efficacy assessed based on intention-to-treat (ITT), including all enrolled patients, and safety data considered for patients receiving at least one dose of the anti-CD38 monoclonal antibody.

[0106] Ethical considerations: The trial was conducted in accordance with ethical standards and was approved by the relevant ethics committees and institutional review committees. Written informed consent was provided to all patients, and any related animal studies strictly adhered to animal care protocols.

[0107] Implementation Plan 1: Human Clinical Trial of Anti-CD38 Monoclonal Antibody in ITP Patients To evaluate the efficacy, safety, pharmacodynamics, and immunogenicity of anti-CD38 monoclonal antibody as monotherapy in patients with immune thrombocytopenic purpura (ITP), a study was conducted based on eligibility criteria (diagnosis of ITP ≥ 3 months, platelet count < 30 × 10⁻⁶). 9 Twenty-seven patients were initially screened. Exclusion criteria included prior anti-CD38 antibody therapy and severe organ dysfunction. Twenty-two patients were enrolled. An investigator-initiated, open-label phase 1 / 2 trial (NCT05694767) was conducted, comprising a 4-week screening period, an 8-week treatment period, and a 16-week follow-up period. Anti-CD38 monoclonal antibody was administered intravenously at 16 mg / kg weekly. Concomitant therapy with TPO-RA or glucocorticoids was permitted. The median age was 35.5 years, and the predominantly female population (72.7%). Patients had diverse histories and treatments for ITP, including rituximab and splenectomy. Patients received anti-CD38 monoclonal antibody therapy, along with prophylactic medications such as glucocorticoids, antihistamines, and acetaminophen before and after infusion.

[0108] Between January 22, 2023 and December 25, 2023, based on eligibility criteria (diagnosis of ITP ≥ 3 months, platelet count < 30 × 10⁻⁶), [the following criteria were met]. 9 A total of 27 patients were screened. Exclusion criteria included prior anti-CD38 antibody therapy and severe organ dysfunction. 22 patients were enrolled. An investigator-initiated open-label phase 1 / 2 trial (NCT05694767) was conducted, comprising a 4-week screening period, an 8-week treatment period, and a 16-week follow-up period. During the trial, patients received anti-CD38 monoclonal antibody therapy at a dose of 16 mg / kg (L). Figure 1 Table 2 summarizes the patients' demographic data and baseline characteristics.

[0109] Table 2. Patient demographics and baseline characteristics * Gender was self-reported by the participants, with the option of "male" or "female". Platelet GP autoantibodies were detected using a commercially available kit (PakAutoassay, Immucor GTI Diagnostics, USA).

[0110] The median age of the 22 patients was 35.5 years (IQR: 25.0–41.0), of whom 72.7% were female. The median duration of ITP was 27 months (IQR: 13–110), and the median baseline platelet count was 12 × 10⁻⁶. 9 / L (IQR: 6-23× 10 9 / L). Nearly half (45.5%) of the patients had a baseline platelet count <10 × 10⁹ / L. 9 / L. Patients had received four (median) different treatments for ITP (range: 3–7). All patients had received glucocorticoids, intravenous immunoglobulin, and TPO-RA, with 36.4% (8 / 22) and 18.2% (4 / 22) having received rituximab and splenectomy, respectively. Platelet glycoprotein (GP) autoantibody testing revealed positive results in 14 of the 22 patients (63.6%): 4.5% were positive only for anti-GP Ib / IX, 27.3% were positive only for anti-GP IIb / IIIa, 9.1% were positive for both anti-GP IIb / IIIa and anti-GP Ib / IIa, 4.5% were positive for both anti-GP IIb / IIIa and anti-GP Ib / IX, and 18.2% were positive for anti-GP IIb / IIIa, anti-GP Ia / IIa, and anti-GP Ib / IX.

[0111] analyze: In the trials conducted, it was assumed that a platelet count ≥ 50 × 10⁶ would be achieved within 8 weeks post-treatment. 9 The proportion of patients with a mean weight of 55% was set at 20%, with a target value of 20%. The Type I error margin was set at 0.05 for two-sided cases. Using PASS software, a cohort of 16 subjects was determined to provide 85.6% confidence, used to test the hypothesis that the primary endpoint exceeded the target value. Considering an expected dropout rate of 20%, a total enrollment of 20 subjects was ultimately determined.

[0112] Efficacy analyses were based on intention-to-treat, covering all enrolled patients. On the other hand, safety analyses were limited to the safety population, which included all participants who had received at least one dose of the study drug.

[0113] The statistical analyses performed were primarily descriptive and were conducted using SAS version 9.4. For the purposes of these analyses, categorical variables were summarized by frequency and percentage, while continuous variables were presented as their median along with their interquartile range (IQR) or the observed minimum and maximum values. The 95% confidence intervals (CIs) for binary endpoints were calculated using the Clopper-Pearson method. For the event occurrence endpoints, the Kaplan-Meier method was used, with the median (IQR) and 95% confidence intervals (CIs) calculated using the Brookmeyer-Crowley method, which included a log-log transformation. Changes in immune function and biomarkers are expressed as mean versus standard deviation (SD). Paired t-tests and independent samples t-tests were used for statistical testing.

[0114] In data processing, binary data recorded after emergency medication were considered non-responders. Continuous data after emergency medication were considered missing and were not included in the analysis of the event occurrence time endpoint. Any missing efficacy data were imputed using the Last Observation Carryover (LOCF) method before being used to derive relevant endpoints. Other missing data were not addressed in the analysis.

[0115] Results and Discussion: 1. Security Analysis: Adverse events were monitored and graded based on severity. Of the 22 patients, 16 (72.7%) experienced at least one treatment-emergent adverse event (TEAE), with most events being grade 1 or 2 in severity. Infusion-related reactions (IRRs) were reported as drug-related TEAEs, with 31.8% of patients experiencing grade 2 reactions, as shown in Table 3. All IRRs occurred during the initial infusion. The most common symptom of IRR reported during the initial anti-CD38 monoclonal antibody infusion was… The incidence rate (IRR) was 10%, including chills (22.7%), nausea (22.7%), and elevated blood pressure (13.6%). All IRRs resolved on the day of antibody infusion by temporarily discontinuing anti-CD38 monoclonal antibody infusion and reducing the infusion rate after glucocorticoid administration.

[0116] Table 3. Summary of Adverse Events *Includes Grade 2 fever (n=2), Grade 2 COVID-19 (n=1), and fever combined with COVID-19 (n=3, all of which are Grade 2).

[0117] # Patient 021 developed a grade 3 perianal infection with fever, which improved after anti-infective treatment.

[0118] Excluding IRR, the most common treatment-associated adverse events (TEAEs) of any grade were upper respiratory tract infection (31.8%), fatigue (18.2%), and hyperuricemia (13.6%), with upper respiratory tract infection (31.8%) being the most common drug-related TEAE. As shown in Table 4, one patient (4.5%) experienced a grade 3 drug-related TEAE (fever and perianal infection). Patient 009 had elevated levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT), and alkaline phosphatase (ALP), which were considered unrelated to anti-CD38 monoclonal antibody.

[0119] Table 4. Summary of Treatment-Related Adverse Events One death occurred (Patient 022) due to a grade 5 intracranial hemorrhage TEAE, which was considered drug-independent (Table 3). This 39-year-old female patient had received seven different prior treatments and initially responded to eltrombopag (75 mg / day) for two months. After subsequently losing response, she continued to receive eltrombopag (75 mg / day) in combination with antibody infusions. Unfortunately, intracranial hemorrhage occurred the day after the first antibody infusion, and rescue therapy could not save her life.

[0120] 2. Treatment outcome: Treatment outcomes are listed in Table 5 below. During the 8-week treatment period, 21 out of 22 patients (95.5%) successfully achieved a platelet count ≥ 50 × 10⁻⁶. 9 / L, with a median cumulative response duration of 23 weeks (IQR: 17–24) throughout the study (Table 5). Initial platelet count ≥ 50 × 10⁹ / L. 9The median time to platelet count was 1 week (range: 1–3 weeks) (Table 5). Furthermore, during the 8-week treatment period, these 21 patients achieved ≥ 2 consecutive platelet counts ≥ 30 × 10⁹ / L. 9 A platelet count of ≥30 × 10⁹ / L and an increase of ≥2-fold from baseline, with a median cumulative response duration of 24 weeks (IQR: 17–24) throughout the study (Table 5). 9 The median time for a ≥2-fold increase in count from baseline was 1 week (range: 1–3 weeks). Throughout the trial, an overall response rate was observed in 95.5% of patients (n=20 for CR; n=1 for PR) (Table 5), with nine patients achieving CR and five achieving PR at week 24.

[0121] Table 5. Treatment Outcomes Patients 009 and 014, who responded to the anti-CD38 monoclonal antibody, both experienced a transient increase in platelet count after the first antibody infusion, which returned to baseline levels at weeks 3 and 4, respectively. However, a subsequent increase occurred, reaching ≥ 50 × 10⁻⁶ at weeks 9 and 7, respectively. 9 / L, and maintain these levels during the follow-up period, such as Figure 2 As shown.

[0122] Throughout the trial, the proportion of patients experiencing bleeding (WHO bleeding score > 0) decreased from 68.2% (15 / 22) at baseline to 4.8% (1 / 21) at weeks 8 and 24 (Table 6).

[0123] Table 6. WHO bleeding scores before and after anti-CD38 monoclonal antibody treatment When the platelet count is ≥ 50 × 10 9 Of the 21 patients with / L, 7 (31.8%) experienced a relapse. Figure 3 Two patients relapsed at weeks 14 and 17, respectively, but did not require rescue medication; the remaining five patients received rescue medication, including glucocorticoids, intravenous immunoglobulin, platelet transfusion, and TPO-RA (Table 7).

[0124] Table 7. Rescue Drugs During the Trial Period Nine patients (40.1%) in this study used concomitant medication, most of whom discontinued it after their platelet counts returned to normal or safe levels following antibody therapy (Table 8). All patients, except for one (Patient 022), met the primary endpoint, regardless of their platelet GP autoantibody results. At week 24, 53.3% (8 / 15) of patients with positive GP autoantibodies and 46.7% (7 / 15) of patients with negative GP autoantibodies were observed to have platelet counts ≥50 × 10⁻⁶. 9 / L (Table 9).

[0125] Table 8. Combined medications used during the trial period Table 9. Treatment outcomes for different types of platelet glycoprotein autoantibodies 3. Immunological Insights Of the 22 patients, 19 had at least one valid ADA assessment. Table 10 shows the results of the 19 patients who tested negative for ADA.

[0126] Table 10. MSD analysis of ADA before and after antibody infusion MSD: Meso Scale Discovery; ADA: Anti-drug antibody; RLU: Relative light unit; SIR: Signal inhibition rate; NA: Not available.

[0127] * For patients 001-019, the cutoff value for screening was 63.5; for patients 020-021, the cutoff value for screening was 62.3. ADA data are missing for the following patients: patients 010, 016, and 022.

[0128] This trial marks a significant advance in ITP treatment, demonstrating a manageable safety profile and remarkable efficacy. The anti-CD38 monoclonal antibody rapidly and sustainably increases platelet counts in a multi-treatment patient population, representing a significant achievement in ITP management. The dual action of anti-CD38 monoclonal antibodies in increasing and maintaining platelet counts, combined with a manageable safety profile, makes it a potentially transformative treatment option for ITP patients.

[0129] Achieving an initial platelet response sufficient to prevent bleeding and ensuring a durable, treatment-free remission remains a challenge in managing ITP. Although TPO-RA typically achieves an overall response rate of over 70% in patients, only up to one-third of patients can maintain this response.18 Prior to the option of splenectomy, rituximab has become increasingly common in cases of persistent or chronic ITP. Approximately 40% to 50% of patients with persistent or chronic ITP report an initial response to rituximab monotherapy, with estimated response rates of 38%, 31%, and 21% at 1, 2, and 5 years, respectively. 19, 20 In our study, 95.5% (21 / 22) of ITP patients who had received a median of four different treatments achieved a platelet count ≥ 50 × 10⁻⁶ after receiving anti-CD38 monoclonal antibody therapy. 9 / L. This response was rapid, observed in all but one patient within the first week, with a median cumulative response duration of 23 weeks. Following treatment, bleeding symptoms were completely resolved in 86.7% (13 / 15) of patients, highlighting the potential of anti-CD38 monoclonal antibodies as an adjunct or emergency treatment option. Anti-CD38 monoclonal antibodies also demonstrated efficacy in patients with a history of splenectomy or rituximab failure or relapse, and in patients who were negative for GP autoantibodies. The overall response rates in patients who had previously undergone splenectomy or rituximab treatment were 75% (3 / 4) and 100% (8 / 8), respectively. Furthermore, all patients who were negative for GP autoantibodies (n=8) met the primary endpoint.

[0130] The analysis of changes in immune function aims to gain a deeper understanding of the mechanism of action of anti-CD38 antibodies. This is consistent with previous findings regarding daratumumab. 21 Infusion of anti-CD38 monoclonal antibody effectively inhibited the proliferation of peripheral blood lymphocytes in ITP patients and reduced plasma IgG levels; this indicates that the drug can downregulate autoantibody production by clearing plasma cells, thereby maintaining a long-term response in patients. Correspondingly, the reduction of platelet destruction and the recovery of platelet count can gradually restore the immune activation state to equilibrium.

[0131] Notably, patients experienced a rapid increase in platelet count within one week of receiving antibody infusion. Considering the metabolic cycle of existing endogenous antibodies in the body, this suggests that the rapid recovery of platelet count involves other mechanisms. Therefore, we examined various immune markers in patients before and after receiving anti-CD38 monoclonal antibody therapy, with particular attention to changes within one week. Interestingly, CD56, as a type of ADCC effector cell, showed a significant increase. dim CD16 + The number of NK cells decreased significantly on day 7 after antibody treatment. Simultaneously, CD32b surface expression on monocytes was significantly reduced. This decrease may be related to the activation of CD32b by the anti-CD38 monoclonal antibody, leading to subsequent CD32b internalization. 22Inhibitory signal transduction to suppress platelet phagocytosis. Therefore, we hypothesize that anti-CD38 monoclonal antibodies similar to IVIG may achieve rapid platelet recovery by downregulating ADCC. 23, 24 As key effector cells in phagocytosis, we also evaluated the effects of anti-CD38 targeted therapy on macrophages in the spleen of a passive ITP mouse model. Similar to results in patients, anti-CD38 antibody injection significantly downregulated the number of CD38+ monocytes and macrophages (especially CD38+ macrophages). Given that CD38 can serve as a surface marker for M1 macrophages, which primarily perform phagocytic functions, 25, 26 Anti-CD38 targeted therapy primarily works by eliminating M1 macrophages, thereby downregulating platelet phagocytosis and reducing macrophage numbers. In summary, based on monitoring results of immunological markers in ITP patients and mouse models, we hypothesize that, in addition to maintaining a durable response in ITP patients by eliminating antibody-secreting cells, similar to IVIG, this treatment can also rapidly increase platelet counts by inhibiting ADCC mediated by NK cells and the mononuclear phagocytic system. Therefore, anti-CD38 monoclonal antibody therapy achieves a dual effect of rapidly increasing and maintaining platelet counts.

[0132] In this study, the anti-CD38 monoclonal antibody demonstrated a manageable safety profile, with most treatment-emergent adverse events (TEAEs) occurring during treatment being grade 1 or 2. IRR and upper respiratory tract infection were the most frequently reported TEAEs, all of which were grade 2 and subsided while patients continued antibody infusions. A significant decrease in immunoglobulin levels may increase the risk of infection, as demonstrated in nine infection events observed in eight patients, with only one event classified as grade 3. Despite the limited severity of infection, given the potential risks, patients may benefit from intermittent intravenous immunoglobulin administration to reduce the incidence of infection.

[0133] Implementation Plan 2: Immunological Analysis of ITP Patients After Treatment with Anti-CD38 Monoclonal Antibody The aim was to evaluate the immunological changes in ITP patients after treatment with anti-CD38 monoclonal antibodies.

[0134] Peripheral blood from patients receiving anti-CD38 monoclonal antibody therapy was analyzed at different time points. This involved staining peripheral blood cells with fluorescently conjugated antibodies, followed by analysis by flow cytometry. Additional assays were performed on peripheral blood helper T cell subsets, as well as to evaluate T and B lymphocyte proliferation and apoptosis.

[0135] (1) Flow cytometry of peripheral blood cells Peripheral blood was obtained from healthy volunteers and patients receiving anti-CD38 monoclonal antibody therapy at multiple observation time points (d0, d7, d21, d35, d56, d77, d105, d133, and d161). Peripheral blood cells were then stained at room temperature with fluorescently conjugated mouse anti-human antibodies (listed in the table below) for 15 minutes, protected from light. Cells were treated with FACS lysis buffer (BD Biosciences) to remove erythrocytes, washed twice with phosphate-buffered saline (PBS), evaluated using Canto II (BD Biosciences), and analyzed using FlowJo version 10.4 (Windows).

[0136] (2) Measurement of peripheral blood helper T cell (Th) subsets Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood by density gradient centrifugation. After washing twice with PBS, the PBMCs were resuspended at 1 × 10⁶ cells / mL. Subsequently, the PBMCs were cultured for 5.5 h (37°C, 5% CO₂) in complete RPMI 1640 medium with or without iomycin (1 μg / mL, Sigma), phorbol 12-myristate 13-acetate (PMA, 75 ng / mL; Sigma), and a protein transport inhibitor (BD GolgiStop™; BD biosciences). After incubation, the PBMCs were collected and stained according to the manufacturer's instructions (BD Biosciences) using fluorescently conjugated mouse anti-human CD4, interleukin-4 (IL-4), interleukin-17A (IL-17A), and interferon-γ (IFN-γ) antibodies. The proportions of Th1, Th2, and Th17 cells were then analyzed by flow cytometry as described above.

[0137] (3) Measurement of peripheral blood T and B lymphocyte proliferation and apoptosis function PBMCs were isolated and resuspended as described above, and cultured for 72 hours (37°C, 5% CO2) in complete RPMI 1640 medium containing recombinant human interleukin-2 (rhIL-2; 5 ng / mL; PeproTech) and phytohemagglutinin (PHA; 5 μg / mL; Sigma), or recombinant human CD40L (1 μg / mL; BioLegend), recombinant human interleukin-4 (rhIL-4; 20 ng / mL; PeproTech), and F(ab')2 fragment goat anti-human IgG + IgM secondary antibody (10 μg / mL; Invitrogen). The incubated cells were then harvested and stained using the FITC BrdU flow cytometry kit and the FITC Annexin V apoptosis detection kit (BioLegend), respectively, according to the manufacturer's instructions. Finally, T and B lymphocyte proliferation and apoptosis functions were analyzed by flow cytometry as described above.

[0138] According to the manufacturer's instructions, plasma transforming growth factor β1 (TGFβ1) levels in ITP patients were assessed before and after receiving anti-CD38 monoclonal antibody infusions (d0, d7, d21, and d105) using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (NeoBioscience). The detection limit of the assay kit is 15.6 pg / ml.

[0139] Compared to healthy controls, patients exhibited significantly elevated CD38 expression levels on various immune cells, including T lymphocytes, B lymphocytes, monocytes, and NK cells, prior to antibody administration. Following anti-CD38 monoclonal antibody treatment, a significant decrease in CD38 surface expression on these immune cells was observed.

[0140] Key findings include: 1. Serum IgG levels and plasma granzyme B (GZMB) concentrations decreased.

[0141] 2. Decreased proliferation of T and B cells.

[0142] 3. Increased plasma TGF-β1 concentration.

[0143] 4. The ratio, distribution, and apoptosis of B and T cells showed no significant changes.

[0144] 5. After treatment, CD56dimCD16+ NK cells decreased rapidly, and the expression of CD32b on monocytes decreased.

[0145] Compared with healthy controls, ITP patients exhibited significantly elevated CD38 expression on various immune cells (T lymphocytes, B lymphocytes, monocytes, and natural killer cells) prior to anti-CD38 monoclonal antibody infusion, along with a higher proportion of antibody-secreting cells in peripheral blood. Following CD38-targeted therapy, CD38 surface expression on immune cells significantly decreased. Furthermore, serum IgG levels, plasma GZMB concentrations, and T and B cell proliferation persistently decreased, while plasma TGF-β1 concentrations increased. No significant changes in the proportion, distribution, or apoptosis of B and T cells were observed after treatment. Figure 4-6 ).

[0146] Notably, consistent with the rapid increase in platelet count, NK cell counts (particularly CD56dimCD16+ NK cells) showed a rapid downregulation within one week of receiving anti-CD38 monoclonal antibody treatment. Correspondingly, CD32b expression on residual monocytes was significantly reduced compared to pre-treatment levels, while the distribution of monocytes and the expression of other activated Fcγ receptors remained unchanged. Figure 7 Since macrophages are rarely found in peripheral blood, a passive ITP mouse model was constructed to further investigate the effects of anti-CD38 targeted therapy on macrophages. Figure 8 The results showed that the number of monocytes and macrophages decreased after treatment. Notably, the proportion of CD38+ monocytes and CD38+ macrophages decreased significantly, with CD38+ macrophages almost completely disappearing.

[0147] These results suggest that the production of autoantibodies may be downregulated and the immune response modulated after antibody treatment.

[0148] Implementation Plan 3: Passive ITP Mouse Model Experiment Purpose: To investigate the effects of anti-CD38 targeted therapy on immune cells in a passive ITP mouse model.

[0149] Methods and steps: 1. Induction of passive ITP in mice: Female BALB / c mice were intraperitoneally injected with anti-mouse CD41 antibody to induce antibody-mediated platelet clearance.

[0150] 2. Intervention: When the platelet count dropped to its lowest level, mice were given an intraperitoneal injection of anti-CD38 monoclonal antibody or saline (control group).

[0151] 3. Spleen Sample Harvesting and Analysis: Spleens were collected for analysis. Spleen cells were stained with various antibodies and analyzed by flow cytometry.

[0152] On day +3, fresh mouse spleens were harvested and ground on ice to obtain a spleen cell suspension. The spleen cells were then stained at 4°C with CD45, Ly6G, Ly6C, F4 / 80, and other fluorescently conjugated rat anti-mouse antibodies (listed in the table below) for 30 minutes in the dark. After removing red blood cells and washing twice with PBS, the stained cells were analyzed by flow cytometry as described above.

[0153] Analysis and Discussion: 1. Spleen cell analysis: The study revealed a decrease in monocytes and macrophages after treatment. Notably, the number of CD38+ monocytes and macrophages was significantly reduced, with CD38+ macrophages almost completely disappearing.

[0154] 2. Effects on the immune response: Data showed that anti-CD38 treatment in mice led to significant changes in the composition of spleen immune cells, similar to changes observed in human patients. This included downregulation of phagocytes, which may have promoted a rapid increase in platelet count.

[0155] 3. Relevance to human ITP: This mouse model provides insights into the mechanism of action of anti-CD38 monoclonal antibodies, demonstrating their potential to achieve rapid platelet recovery and sustained response in ITP patients by modulating immune cell function and reducing autoantibody-mediated platelet destruction.

[0156] In both implementation schemes, the outcomes demonstrated the efficacy of anti-CD38 monoclonal antibodies in modulating immune responses in human ITP patients and passive ITP mouse models, highlighting their therapeutic potential for treating ITP via the immune pathway.

[0157] Treatment mechanism: This study provides valuable insights into the immunological mechanisms of anti-CD38 monoclonal antibodies. It was found that these antibodies effectively inhibit the proliferation of peripheral blood lymphocytes and reduce plasma IgG levels, indicating their potential to downregulate autoantibody production. The rapid increase in platelet count within one week after antibody infusion suggests that, in addition to clearing antibody-secreting cells, there are other mechanisms involved, which may be related to the regulation of antibody-dependent cytotoxicity (ADCC).

[0158] Interestingly, a significant reduction in CD56dimCD16+ NK cells and decreased CD32b expression on monocytes were observed after treatment, indicating downregulation of ADCC. Furthermore, studies in a passive ITP mouse model showed that anti-CD38 treatment led to a significant reduction in the number of CD38+ monocytes and macrophages, particularly CD38+ macrophages known for their phagocytic function. These findings suggest that anti-CD38 monoclonal antibodies can rapidly increase platelet counts by inhibiting NK cell-mediated ADCC and the mononuclear phagocytic system.

Claims

1. A method for treating an autoimmune disease in a subject, wherein the method includes the step of administering to the subject a therapeutically effective amount of an antibody or antigen-binding moiety thereof that has binding specificity to CD38. The antibody or antigen-binding portion thereof includes the antibody or antigen-binding portion thereof shown in WO2021104052A1.

2. The method of claim 1, wherein the autoimmune disease comprises the group consisting of: systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), multiple sclerosis (MS), and immune thrombocytopenic purpura (ITP).

3. The method of claim 1 or 2, wherein the autoimmune disease is immune thrombocytopenic purpura (ITP).

4. The method of claim 3, wherein the object is a mammal, preferably a human patient.

5. The method of claim 4, wherein the patient has failed glucocorticoid treatment or relapsed after glucocorticoid treatment, and has received at least one second-line ITP treatment.

6. The method of claim 5, wherein the patient has previously shown resistance or intolerance to rituximab and / or thrombopoietin receptor agonist (TPO-RA) treatment.

7. The method of any one of claims 1-6, wherein the therapeutically effective amount of the antibody or its antigen-binding portion is administered intravenously.

8. The method of any one of claims 1-7, wherein the therapeutically effective amount of the antibody or its antigen-binding portion is administered at a dose of 10-20 mg / kg, preferably 14-18 mg / kg, more preferably 16 mg / kg.

9. The method of any one of claims 1-8, wherein the therapeutically effective amount of the antibody or its antigen-binding portion is administered according to a dosage regimen comprising an 8-week treatment period and a 16-week follow-up period, preferably the dosage regimen further comprising a 4-week screening period.

10. The method of any one of claims 3-9, wherein the treatment results in the patient achieving a platelet count ≥ 50 × 10⁻⁶ during an 8-week treatment period. 9 / L.

11. The method of any one of claims 3-10, wherein the method is particularly suitable for baseline platelet counts <30 × 10⁻⁶. 9 Patients with / L.

12. The method of any one of claims 3-11, wherein the patient has been diagnosed with ITP for at least 3 months.

13. The method of any one of claims 3-12, wherein the method further comprises the steps of monitoring the platelet count of the patient and adjusting the dose of the antibody or its antigen-binding moiety based on the patient's response; Preferably, the method further includes a maintenance period, wherein the dose of the antibody or its antigen-binding portion is adjusted based on the patient's stable platelet count or clinical response. Preferably, the method further includes performing genetic, immunological, or biochemical tests before treatment begins to identify patients most likely to benefit from treatment with the antibody or its antigen-binding portion.

14. The method of any one of claims 1-13, wherein the method comprises administering the antibody or its antigen-binding portion in combination with a second therapeutic agent to produce a synergistic effect; Preferably, the second therapeutic agent is selected from one or more of the group consisting of: thrombopoietin receptor agonists (TPO-RA), corticosteroids, and immunosuppressants; Optionally, the patient receives prophylactic medication before and after infusion of the antibody or its antigen-binding portion, the prophylactic medication including glucocorticoids, antihistamines, acetaminophen, etc.

15. The method of any one of claims 1-14, wherein the antibody or its antigen-binding portion is contained in a pharmaceutical preparation suitable for intravenous infusion; Preferably, the pharmaceutical preparation is a lyophilized powder for intravenous infusion.

16. The method of any one of claims 1-15, wherein the method further comprises a pre-treatment screening step targeting CD38 expression on plasma cells.

17. The method of any one of claims 1-16, wherein the method reduces the number of adverse events occurring during treatment compared to conventional treatment.

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