Methods of treating cancer with anti-TMEFF2xCD3 bispecific antibodies

Abstract

A method of treating cancer in a patient comprises administering an anti-TMEFF2xCD3 bispecific antibody as disclosed herein.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Serial No. 63 / 405,188, filed on September 9, 2022, and U.S. Serial No. 63 / 420,146, filed on October 28, 2022, the disclosures of each of which are incorporated herein by reference in their entirety.

[0003] Reference sequence listing submitted electronically

[0004] This application contains a sequence listing that has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. The XML copy was created on July 7, 2023, named JBI6759WOPCT1_SL, and is 32,707 bytes in size. Background Art

[0005] Prostate cancer is the second most common cancer in men worldwide and the sixth leading cause of cancer-related death. Globally, there are approximately 1,100,000 new cases and 300,000 deaths each year, accounting for 4% of all cancer deaths. It is estimated that 1 in 6 men will be diagnosed with the disease in his lifetime. Prostate cancer risk is strongly associated with age: approximately three-quarters of cases occur in men over the age of 65, with the highest number of cases occurring in men aged 70-74 years. Based on estimates from autopsy data, approximately half of men in their 50s and 80% of men in their 80s have histological evidence of prostate cancer. In the early stages, the 5-year survival rate is close to 100%. However, when the cancer has metastasized, the 5-year survival rate drops to 28%, and effective treatments for advanced prostate cancer are still needed.

[0006] TMEFF2 is a conserved cell membrane-bound proteoglycan, also known as TENB2, HPP1 or tomoregulin-2. It is a 41kDa transmembrane protein composed of two follistatin-like domains (FS1 and FS2) and one epidermal growth factor (EGF)-like domain.

[0007] TMEFF2 expression is retained at all stages of prostate cancer disease, but expression is limited in extraprostatic tissues. The direct correlation between increased TMEFF2 expression levels and high-grade tumors strongly suggests that TMEFF2 is associated with disease progression and possible androgen independence in the setting of advanced prostate cancer disease (Afar DE, Bhaskar V, Ibsen E, et al., “Preclinical validation of anti-TMEFF2-auristatin E–conjugated antibodies in the treatment of prostate cancer.” Mol Cancer Ther. 2004;3(8):921–932). TMEFF2 RNA and protein expression was observed in both brain and prostate. Low levels of transcript and protein expression were detected in the retina and conjunctival epithelium, while only transcripts but no protein were detected in the non-pigmented ciliary epithelium and ganglion cells of the myenteric plexus of the colon in the eye.

[0008] Treatment for prostate cancer currently includes surgery, radiotherapy and hormone therapy. The treatment for the purpose of eradicating tumors is unsuccessful in 30% of males, and they develop recurrent diseases, which are usually first manifested as plasma prostate-specific antigen (PSA) rising, and then spread to distal sites. Because prostate cancer cells rely on androgen receptor (AR) to proliferate and survive, the medicament (such as GnRH agonist) produced by blocking testosterone is used to treat the male with advanced prostate cancer individually or in combination with the antiandrogen (such as bicalutamide) of the effect of antagonizing any residual testosterone on AR. These treatments reduce serum testosterone to castration levels, which usually slows down disease progression over a period of time. However, androgen depletion is usually effective in a limited duration, and prostate cancer evolves to restore growth capacity despite circulating androgens at low levels. Therefore, most patients eventually die due to cancer recurrence.

[0009] There remains a high need for more improved treatments and effective therapies for patients with advanced prostate cancer whose disease has proven resistant to current therapies. Summary of the invention

[0010] Provided herein are methods of treating cancer in a subject comprising administering to the subject at least one dose of an anti-TMEFF2xCD3 bispecific antibody of the present disclosure.

[0011] In some embodiments, the cancer is metastatic castration-resistant prostate cancer (mCRPC), including adenocarcinoma with small cell or neuroendocrine features.

[0012] In some embodiments, the mCRPC subject has received prior treatment with at least 1 prior novel AR-targeted therapy or chemotherapy.

[0013] In some embodiments, the administration is subcutaneous and the dose is about 0.3 mg to about 6.0 mg of the anti-TMEFF2xCD3 bispecific antibody of the present disclosure.

[0014] In some embodiments, the administration is subcutaneous and the dose is 0.3 mg, 1.0 mg, 1.5 mg, 2.0 mg, 3.0 mg, 4.0 mg or 6.0 mg of the anti-TMEFF2xCD3 bispecific antibody of the present disclosure.

[0015] In some embodiments, the administration is subcutaneous, and the dose is given once a week (Q1W).

[0016] In some embodiments, the administration is subcutaneous, and the dose is given once every two weeks (Q2W).

[0017] In some embodiments, the administration is subcutaneous and the dose is about 0.3 mg to about 6.0 mg and is given Q1W.

[0018] In some embodiments, the administration is subcutaneous and the dose is 0.3 mg, 1.0 mg, 1.5 mg, 2.0 mg, 3.0 mg, 4.0 mg, or 6.0 mg and is given Q1W.

[0019] In some embodiments, the administration is subcutaneous and the dose is 0.3 mg, 1.0 mg, 1.5 mg, 3.0 mg, or 6.0 mg and is given Q1W.

[0020] In some embodiments, the administration is subcutaneous and the dose is about 0.3 mg to about 6.0 mg and is given Q2W.

[0021] In some embodiments, the administration is subcutaneous and the dose is 0.3 mg, 1.0 mg, 1.5 mg, 2.0 mg, 3.0 mg, 4.0 mg, or 6.0 mg and is given Q2W.

[0022] In some embodiments, the administration is subcutaneous and the dose is 2.0 mg, 3.0 mg, 4.0 mg, or 6.0 mg and is given Q2W.

[0023] In some embodiments, the methods of the present disclosure comprise more than one repetition of the administering step.

[0024] In some embodiments, before the first administration of the anti-TMEFF2xCD3 bispecific antibody of the present disclosure, corticosteroids, antihistamines and antipyretics are administered in advance to minimize the risks associated with cytokine release syndrome (CRS) and infusion-related reactions (IRR). For subsequent doses, the pre-medication dose or schedule can be reduced or omitted. For subjects who experience grade 2 or higher CRS or IRR, for at least 1 subsequent dose administered to the subject, corticosteroids are pretreated or can be administered.

[0025] In some embodiments, the anti-TMEFF2xCD3 antibody administered in the disclosed method of treatment comprises a first binding domain that binds to TMEFF2 and a second binding domain that binds to CD3, wherein (a) the first binding domain that binds to TMEFF2 comprises a HCDR of a VH having an amino acid sequence of SEQ ID NO: 13 and a LCDR of a VL having an amino acid sequence of SEQ ID NO: 14, and the second binding domain that binds to CD3 comprises a HCDR of a VH having an amino acid sequence of SEQ ID NO: 17 and a LCDR of a VL having an amino acid sequence of SEQ ID NO: 18; (b) the first binding domain that binds to TMEFF2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively, and the second binding domain that binds to CD3 comprises HCDRs of SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively. NO:7, 8, 9, 10, 11 and 12 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3; (c) the first binding domain that binds TMEFF2 comprises VH and VL of SEQ ID NO:13 and 14, respectively, and the second binding domain that binds CD3 comprises VH and VL of SEQ ID NO:17 and 18, respectively; and / or (d) the first binding domain that binds TMEFF2 comprises HC1 and LC1 of SEQ ID NO:15 and 16, respectively; and the second binding domain that binds CD3 comprises HC2 and LC2 of SEQ ID NO:19 and 20, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 A schematic overview of the study is shown.

[0027] Figure 2 Shown are mean study drug serum concentrations in humans predicted by the PK model following a single IV dose of 4 u / kg or SC administration of 300 μg assuming a median body weight of 75 kg. Abbreviations: F = bioavailability; IV = intravenous, MABEL = minimum anticipated biological effect level, SC = subcutaneous

[0028] Figure 3 Mean serum concentration-time profiles of study drug after the first SC injection of study drug are shown.Abbreviations: n = total number of subjects in each cohort; SC = subcutaneous.

[0029] Figure 4 Mean serum concentration-time profiles of study drug following Q1W SC dosing are shown.Abbreviations: n = total number of subjects in each cohort; SC = subcutaneous.

[0030] Figure 5 Mean serum concentration-time profiles of study drug following Q2W SC dosing are shown.Abbreviations: n = total number of subjects in each cohort; SC = subcutaneous.

[0031] Figure 6 A trend was shown for lower body weight subjects to have higher exposure when compared to higher body weight subjects.

[0032] Fig. 7A The relationship between study drug volume distribution and body weight is shown. Figure 7B The relationship between study drug clearance and body weight is shown.

[0033] Figure 8 A waterfall plot of the maximum percent decrease in PSA from baseline is shown. Abbreviations: PSA = prostate specific antigen; Q1W = once a week; Q2W = once every 2 weeks; SC = subcutaneous. Reference lines represent 30% and 50% decreases. Increases greater than 100% are set to 100%.

[0034] Fig. 9 Waterfall plot showing the maximum percent reduction from baseline in the sum of target lesion diameters. Abbreviations: Q1W = once a week; Q2W = once every 2 weeks; SC = subcutaneous; SoD = sum of diameters. The reference line represents a 30% reduction. Increases greater than 100% were set to 100%.

[0035] Fig.10 Study drug concentrations following different dosing regimens are shown.

[0036] Fig.11A and Fig. 11B Shown are the effects of study drug dosing regimens on CD8+ T cell infiltration.

[0037] Fig. 12A and Fig. 12B Shown are the effects of study drug dosing regimens on CD4+ T cell infiltration.

[0038] FIG. 13A to FIG. 13F Shown are the effects of study drug dosing regimens on T cell activation and proliferation. FIG. 13A to FIG. 13CIt is an activation marker of CD4+ prostate infiltrating lymphocytes (PIL). FIG. 13D to FIG. 13F It is an activation marker for CD8+ prostate infiltrating lymphocytes (PIL).

[0039] FIG. 14A to FIG. 14F Shown are the effects of study drug dosing regimens on suppressive T cell markers. FIG. 14A to FIG. 14C is a CD4+ prostate infiltrating lymphocyte (PIL) cell activation marker, and FIG. 14D to FIG. 14F It is a CD8+ prostate infiltrating lymphocyte (PIL) cell activation marker.

[0040] FIG. 15A to FIG. 15C Shown are the effects of study drug dosing regimens on suppressive T cell markers. FIG. 15A to FIG. 15B It is an activation marker of CD4+ prostate infiltrating lymphocytes (PIL). Fig. 15C It is an activation marker for CD8+ prostate infiltrating lymphocytes (PIL).

[0041] FIG. 16A to FIG. 16B Shown are the effects of study drug dosing regimens on myeloid cell infiltration.

[0042] 17A to 17C Shown are the effects of study drug dosing regimens on myeloid cell infiltration. DETAILED DESCRIPTION

[0043] The method disclosed in the present invention can be more easily understood by referring to the following detailed description in conjunction with the accompanying drawings which form a part of the present disclosure. It should be understood that the method disclosed in the present invention is not limited to the specific method described and / or shown herein, and the terms used herein are only used to describe specific embodiments by way of example and are not intended to limit the method protected by the claims.

[0044] Unless specifically stated otherwise, any description of possible mechanisms or modes of action or reasons for improvement is intended for exemplary purposes only, and the methods disclosed herein are not bound by the correctness or incorrectness of any such suggested mechanisms or modes of action or reasons for improvement.

[0045] When enumerating or establishing a numerical range herein, the range includes its end values ​​and all individual integers and fractions within the range, and also includes each of the narrower ranges formed by all various possible combinations of those end values ​​and internal integers and fractions, to form a subset of the larger numerical group within the range to the same extent, as if each of those narrower ranges were explicitly enumerated. When stating a numerical range greater than a specified value herein, the range is however limited, and its upper limit is limited by a value operable in the context of the method as described herein. When stating a numerical range less than a specified value herein, the lower limit of the range is however limited by a non-zero value. It is not intended that the scope of the method be limited to the specific values ​​enumerated when the range is limited. All ranges include the end values ​​and can be combined.

[0046] When "about" is used above to express a value as an approximation, it should be understood that the specific value constitutes another embodiment. A reference to a specific value includes at least that specific value unless the context clearly dictates otherwise.

[0047] It should be understood that certain features of the methods disclosed herein are described in the context of each separate embodiment for clarity, but may also be provided in combination in a single embodiment. Conversely, various features of the methods disclosed herein are described in the context of a single embodiment for simplicity, and may also be provided separately or in any sub-combination.

[0048] As used herein, the singular forms "a," "an," and "the" include plural forms.

[0049] Various terms related to various aspects of the specification are used throughout the specification and claims. Unless otherwise indicated, such terms are given the ordinary meaning of the art. Other specifically defined terms should be understood in a manner consistent with the definitions provided herein.

[0050] The term "about" is intended to encompass variations of ±10% or less, ±5% or less, ±1% or less, ±0.5% or less, or ±0.1% or less from the specified value.

[0051] The transitional terms "comprising," "consisting essentially of," and "consisting of" are intended to suggest their recognized meanings in patent parlance; that is, (i) "comprising" is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps; (ii) "consisting of" excludes any element, step, or ingredient not specified in the claim; and (iii) "consisting essentially of" limits the scope of the claim to the specified materials or steps "and those that do not materially affect the basic and novel characteristics of the disclosure protected by the claims." Embodiments described with the phrase "comprising" (or its equivalent) are also provided, such as those embodiments described independently with "consisting of" and "consisting essentially of." Embodiments described with the phrase "consisting essentially of" (or its equivalent) are also provided, such as those embodiments described independently with "consisting of."

[0052] Treatment

[0053] Provided herein are methods of treating cancer in a subject, comprising administering to the subject at least one dose of an anti-TMEFF2xCD3 bispecific antibody of the present disclosure, wherein the dose is a safe and therapeutically effective amount of the anti-TMEFF2xCD3 antibody.

[0054] "Subject" or "patient" includes any human or non-human animal. "Non-human animals" include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. The terms "subject" and "patient" can be used interchangeably herein. In some embodiments, the subject or patient is a human. Specifically, the subject according to the present invention is a human. In some embodiments, the subject according to the present invention suffers from or is susceptible to prostate cancer.

[0055] "Treating" a subject with a pathological condition such as cancer refers not only to ameliorating the condition by killing cancer cells, but also to achieving one or more of the following effects: reducing the severity and / or duration of the condition, delaying the progression of the condition, slowing the progression of the condition, inhibiting the worsening of symptoms characteristic of the condition being treated, limiting or preventing the recurrence of the condition in a subject who previously had the condition, or limiting or preventing the recurrence of symptoms in a subject who previously had symptoms of the condition. Treatment as a preventative measure (i.e., prevention) is also included.

[0056] As used herein, the terms "delaying the progression of" and "slowing the progression of" shall include: (a) delaying or slowing the development of one or more symptoms or complications of a disease, condition, or disorder; (b) delaying or slowing the development of one or more new / additional symptoms or complications of a disease, condition, or disorder; and / or (c) delaying or slowing the progression of a disease, condition, or disorder to a subsequent stage or more severe form of the disease, condition, or disorder.

[0057] As used in accordance with the present disclosure, the term "treating" means treating a mammal suffering from prostate cancer by providing an effective amount of an anti-TMEFF2xCD3 bispecific antibody, with the purpose of reducing or eradicating cancer cells and / or prolonging the survival of the mammal.

[0058] As used herein, the terms "effective amount" and "therapeutically effective amount" refer to an amount sufficient to achieve a concentration of the compound that can prevent or slow down the disease to be treated. Such concentrations can be determined by those skilled in the art in a routine manner. The amount of polypeptide actually administered is usually determined by a physician or veterinarian based on relevant circumstances, including the condition to be treated, the selected route of administration, the actual compound administered, the age, weight and response of the patient, the severity of the subject's symptoms, etc. It should also be understood by those skilled in the art that the dosage may depend on the stability of the administered antibody.

[0059] The therapeutically effective amount may vary depending on a variety of factors, such as the disease state, the age, sex and weight of the individual, the patient's physical condition, the duration of treatment, the nature of concurrent treatment (if any), the specific formulation used, the structure of the compound or its derivative, and the ability of the therapeutic agent or combination of therapeutic agents to elicit the desired response in the individual. The therapeutically effective dose of the antibody polypeptide administered also depends on the type and severity of the cancer to be treated, as well as the route of administration of the antibody polypeptide or pharmaceutical composition of the antibody polypeptide.

[0060] In some embodiments, the subject to be treated includes subjects with metastatic castration-resistant prostate cancer (mCRPC), including adenocarcinoma with small cell or neuroendocrine features.

[0061] In some embodiments, the effectiveness of treatment of prostate cancer can be assessed using CT scans, MRI, or whole-body PET scans, or by measuring PSA levels.

[0062] In some embodiments, the therapeutic efficiency of prostate cancer can be assessed by measuring the progress of soft tissue lesions by CT or MRI using the RECIST standard. As used herein, the term "solid tumor efficacy evaluation criteria (RECIST)" refers to a set of public rules that define when cancer patients improve ("reaction"), remain unchanged ("stable") or deteriorate ("progress") during treatment. The initial standard was announced in February 2000 by international cooperative organizations including the European Organization for Research and Treatment of Cancer (EORTC), the National Cancer Institute of the United States (NCI) and the National Cancer Institute of Canada Clinical Trials Group. RECIST 1.1 was announced in January 2009, which is an update to the original standard. Usually, those skilled in the art draw conclusions by conventional imaging methods such as computed tomography (CT), when the sum of the longest diameters of the target lesions increases by at least 20% (with the minimum sum of the longest diameters recorded since the start of treatment or the appearance of one or more new lesions as a reference), disease progression (therefore the patient is resistant to treatment or becomes resistant to treatment).

[0063] In some embodiments, the therapeutic efficacy of prostate cancer can be assessed according to the PCWG3 (Prostate Cancer Working Group 3) criteria.

[0064] The term "safety" when it relates to a dose, dosage regimen or treatment method performed with the anti-TMEFF2xCD3 bispecific antibody of the present disclosure refers to a relatively low or reduced frequency and / or low or reduced severity of adverse events (referred to as AEs or TEAEs) occurring during treatment from a clinical trial conducted (e.g., a Phase 1 clinical trial) compared to the standard of care or another comparator. Adverse events are adverse medical events that occur in patients receiving drug administration, including adverse vital signs (heart rate, systolic and diastolic blood pressure, temperature), adverse standard clinical laboratory tests (hematology, clinical chemistry, urinalysis, lipids, coagulation), allergic reactions / hypersensitivity reactions, adverse local injection site reactions or adverse EKGs. Specifically, when it relates to a dose, dosage regimen or treatment performed with the anti-TMEFF2xCD3 bispecific antibody of the present disclosure, "safety" means that if it is considered that the attribution is likely, likely or very likely to be due to the use of the anti-TMEFF2xCD3 bispecific antibody, the frequency of adverse events associated with the administration of the antibody is relatively low or reduced, and / or the severity is low or reduced.

[0065] Route of administration

[0066] The methods of the present invention may include any means of administration to achieve the intended purpose. Any suitable route of administration may be used to administer the antibody polypeptide or pharmaceutical composition used in the therapeutic methods disclosed herein. For example, administration may be achieved by a variety of different routes, including but not limited to subcutaneous routes.

[0067] Prostate cancer

[0068] The disclosed methods can treat any cancer associated with TMEFF2. An exemplary cancer associated with TMEFF2 is prostate cancer. As used herein, the term "cancer" refers to an abnormal growth of cells that tend to proliferate in an uncontrolled manner, and in some cases refers to metastasis (spread).

[0069] In some embodiments, the methods of the present disclosure can treat or slow the progression of prostate cancer.

[0070] As used herein, the term "prostate cancer" refers to histologically or cytologically confirmed prostate adenocarcinoma and neuroendocrine prostate cancer, advanced manifestations of prostate adenocarcinoma, and hormone-refractory subtypes of prostate cancer caused by prostate cancer treatment. Based on the extent of the disease, hormonal status, and the presence or absence of detectable metastases, the process of prostate cancer from diagnosis to death is most appropriately classified into a series of clinical states: localized disease, elevated levels of prostate-specific antigen (PSA) after radiation therapy or surgery but without detectable metastases, and clinical metastases in the non-castrated or castrated state.

[0071] In the early stages of prostate cancer, cancer is confined to the prostate gland. In these early stages, treatment usually involves surgical resection of the prostate gland or radiotherapy to the prostate gland, or only conservative observation and no active intervention therapy is taken for some patients. In the early stages when prostate cancer is localized and requires intervention, surgery or radiotherapy are effective by eradicating cancer cells. However, in about 30% of the cases, these treatment procedures fail, and prostate cancer continues to progress, as usually confirmed by increased PSA levels. Therefore, a large part of patients treated by surgery, radiation or a combination of the two suffer from recurrent disease, which may lead to the development of metastasis, particularly in high-risk populations-transformed to the lethal phenotype of the disease. Men whose prostate cancer still progresses after receiving these early treatment strategies are referred to as having advanced or recurrent prostate cancer.

[0072] The terms "locally advanced prostate cancer," "advanced prostate cancer," or "advanced disease" refer to prostate cancer that has spread beyond the prostate capsule, where all active cancer cells appear to be confined to the prostate and associated organs or adjacent organs (e.g., seminal vesicles, bladder neck, and rectal wall). Advanced prostate cancer includes stage C disease under the American Urological Association (AUA) system, stage C1-C2 disease under the Whitmore-Jewett system, and stage T3-T4 and N+ disease under the TNM (tumor, node, metastasis) system. Generally, surgery is not recommended for patients with locally advanced disease, and these patients have significantly poorer treatment outcomes than patients with clinically localized (organ-confined) prostate cancer. The clinical diagnosis of locally advanced disease is based on the presence of palpable induration beyond the lateral margins of the prostate, or asymmetry or induration above the base of the prostate. If the tumor invades or penetrates the prostate capsule, extends to the surgical margins, or invades the seminal vesicles, the pathological diagnosis of locally advanced prostate cancer is currently made after radical prostatectomy.

[0073] The term "high-risk localized prostate cancer" refers to locally advanced prostate cancer with the possibility of developing metastasis or recurrent disease after initial treatment with treatment attempt. In some embodiments, the high risk of metastasis development is defined as prostate specific antigen doubling time (PSADT) <12 months, or <11 months, <10 months, <9 months, <8 months, <7 months, <6 months, <5 months, <4 months, <3 months, <2 months or <1 month. In some embodiments, the high risk of metastasis development is defined as prostate specific antigen doubling time (PSADT) <10 months. In some embodiments, the high risk of metastasis development is defined as having a high Gleason score or a large tumor.

[0074] The terms "metastatic prostate cancer" and "metastatic disease" refer to prostate cancer that has spread to regional lymph nodes or distant sites, and are intended to include stage D disease under the AUA system and stage TxNxM+ disease under the TNM system. As with locally advanced prostate cancer, patients with metastatic disease are generally not suitable for surgery, and hormone (androgen ablation) therapy or androgen deprivation therapy (ADT) is the preferred treatment. Patients with metastatic prostate cancer eventually develop into androgen refractory status within 12 to 18 months of the start of treatment, and about half of these patients die within 6 months thereafter. The most common site of prostate cancer metastasis is bone. In general, prostate cancer bone metastasis is characterized by osteogenic rather than osteolytic (ie, resulting in net bone formation). Bone metastasis is most common in the spine, followed by the femur, pelvis, thorax, skull and humerus. Other common metastatic sites include lymph nodes, lungs, liver and brain. Metastatic prostate cancer is usually diagnosed by open or laparoscopic pelvic lymph node dissection, whole-body radionuclide scanning, bone radiography, and / or biopsy of bone lesions.

[0075] Androgen receptor (AR) is a member of the steroid and nuclear receptor superfamily, and its function is regulated by androgen binding. AR is mainly expressed in androgen target tissues such as prostate, skeletal muscle, liver and central nervous system (CNS), wherein the highest expression level is observed in prostate, adrenal gland and epididymis. AR can be activated by the combination of endogenous androgens including testosterone and 5-dihydrotestosterone (5a-DHT). After androgen activation, AR mediates the transcription of target genes regulating prostate epithelial cell growth and differentiation. AR signals are crucial to the development and maintenance of male reproductive organs including prostate, because genetically males with loss-of-function AR mutations and engineered mice with AR defects do not develop prostate or prostate cancer. This dependence of prostate cells on AR signals continues even after neoplastic transformation.

[0076] The term "androgen deprivation therapy (ADT)" refers to the reduction of androgen levels to castrate levels of testosterone (<50 ng / dL) in patients with prostate cancer. ADT includes surgical castration (orchiectomy) and / or administration of a gonadotropin-releasing hormone (also known as luteinizing hormone-releasing hormone ["LHRH") agonist or antagonist to a person. Examples of LHRH agonists include, but are not limited to, goserelin acetate, histrelin acetate, leuprolide acetate, and triptorelin palmitate.

[0077] Antiandrogens can be used for the early treatment of prostate cancer. These treatments reduce serum testosterone to castration levels, which usually slows down disease progression over a period of time. However, prostate cancer often develops into a "hormone refractory" state, in which the disease still progresses in the presence of persistent androgen ablation or antiandrogen therapy, and most patients eventually die from cancer recurrence. The situation of antiandrogen withdrawal syndrome after long-term treatment with antiandrogens has also been reported. Molecular profiling studies of castration-resistant prostate cancer often show that androgen receptor (AR) expression increases, and this expression increase can occur by AR gene amplification or other mechanisms.

[0078] The term "castration-sensitive prostate cancer" is a cancer that responds to androgen deprivation therapy (ADT) as localized disease, biochemical recurrence, or in the metastatic setting. Castration-sensitive prostate cancer is classified as non-metastatic or metastatic, depending on whether the prostate cancer has metastasized to other parts of the body.

[0079] The term "metastatic castration-sensitive prostate cancer" refers to cancer that has spread (metastasized) to other areas of the body, such as bones, lymph nodes, or other sites in a man's body and that responds to androgen deprivation therapy (ADT).

[0080] The term "non-metastatic castration-sensitive prostate cancer" refers to cancer that has not spread (metastasized) in men and that responds to androgen deprivation therapy (ADT). In some embodiments, non-metastatic castration-sensitive prostate cancer is assessed using a bone scan and computed tomography (CT), magnetic resonance imaging (MRI) scan, or positron emission tomography (PET).

[0081] The term "CRPC" or "castration-resistant prostate cancer" refers to prostate cancer that continues to grow despite the suppression of androgens, which provide energy for the growth of prostate cancer cells. Castration-resistant prostate cancer (CRPC) is classified as non-metastatic or metastatic, depending on whether the prostate cancer has metastasized to other parts of the body. Castration-resistant prostate cancer (CRPC) is a lethal phenotype, and almost all patients will die from prostate cancer. Interestingly, although a small number of CRPCs do bypass the need for AR signals, the vast majority of CRPCs, although often referred to as "androgen-independent prostate cancer" or "hormone-refractory prostate cancer," still maintain their lineage dependence on AR signals. The term "metastatic castration-resistant prostate cancer" or "mCRPC" refers to castration-resistant prostate cancer that has metastasized to other parts of the human body.

[0082] The term "NM-CRPC" or "non-metastatic castration-resistant prostate cancer" refers to cancer that has not spread (metastasized) in males and is resistant to androgen deprivation therapy ADT, i.e., cancer that continues to grow despite the suppression of male hormones. In some embodiments, non-metastatic castration-sensitive prostate cancer is evaluated using bone scans and computed tomography (CT), magnetic resonance imaging (MRI) scans, or positron emission tomography (PET).

[0083] In some embodiments, non-metastatic castration-resistant prostate cancer is high-risk non-metastatic castration-resistant prostate cancer. The term "high-risk nm-CRPC" refers to the high probability of metastasis in men with nm-CRPC. In some embodiments, the high risk of metastatic development is defined as prostate-specific antigen doubling time (PSADT) <10 months, <9 months, <8 months, <7 months, <6 months, <5 months, <4 months, <3 months, <2 months or <1 month. In some embodiments, the high risk of metastatic development is defined as having local regional recurrence (e.g., primary tumor bed, bladder neck, anastomotic area, pelvic lymph nodes).

[0084] The term "chemotherapy-naive metastatic castration-resistant prostate cancer" refers to metastatic castration-resistant prostate cancer that has not been previously treated with chemotherapeutic agents.

[0085] The term "neuroendocrine prostate cancer (NEPC)", also known as therapy-related NEPC (tNEPC), is an aggressive androgen-independent variant of prostate cancer, most commonly seen in the late stages of mCRPC as a mechanism of treatment resistance (Wang HT et al., J. of Clinical Oncology, 2014, Vol. 32, No. 30, pp. 3383-3390). This tumor does not secrete prostate-specific antigen (PSA) and is a highly aggressive subtype of prostate cancer characterized by the following clinical features: no response to hormone therapy, presence of osteolytic bone lesions, rapid disease progression, presence of visceral metastases, significant enlargement of the prostate, and abnormally low PSA levels in the setting of metastatic disease. In NEPC, androgen receptor (AR) expression is usually low or absent, and the Aurora kinase A (AURKA) and N-Myc (MYCN) genes are often amplified.

[0086] It is estimated that the development of tNEPC causes approximately 25% of the nearly 34,000 fatal prostate cancers in the United States each year (AJemal, F Bray, MM Center, et al., Global cancer statistics CA Cancer J Clin 61:69–90, 2011). However, data from autopsy studies suggest that the incidence of NEPC may be severely underestimated (PN Brawn, VO Spights: The dedifferentiation of metastatic prostate carcinoma Br J Cancer 59:85–88, 1989). The amount of neuroendocrine differentiation increases as the disease progresses and is associated with patients receiving long-term androgen deprivation therapy. Preclinical studies also support the view that androgen deprivation therapy promotes the transformation of PCa to tNEPC and may appear as a resistance mechanism.

[0087] The present disclosure provides a method for treating or slowing down the prostate cancer progress of a subject by administering an anti-TMEFF2xCD3 bispecific antibody. The method for treating or slowing down the progress of prostate cancer includes but is not limited to treating castration-resistant prostate cancer (CRPC), metastatic castration-resistant prostate cancer (mCRPC), non-metastatic castration-resistant prostate cancer (NM-CRPC), androgen receptor (AR) targeted therapy after recurrence of prostate cancer disease, locally advanced prostate cancer, high-risk local prostate cancer, castration-sensitive prostate cancer, non-metastatic castration-sensitive prostate cancer, metastatic castration-sensitive prostate cancer, metastatic castration-resistant prostate cancer without chemotherapy or neuroendocrine prostate cancer.

[0088] In some embodiments, the therapeutic methods of the present disclosure include methods of treating or slowing progression of prostate cancer in a subject with an anti-TMEFF2xCD3 bispecific antibody, wherein the prostate cancer is metastatic castration-resistant prostate cancer (mCRPC).

[0089] Antibody

[0090] The methods of the present disclosure are implemented by administering a bispecific antibody that specifically binds to human TMEFF2 and human CD3. Anti-TMEFF2xCD3 bispecific antibodies can be used to treat cancers associated with TMEFF2, such as metastatic castration-resistant prostate cancer (mCRPC).

[0091] "TMEFF2" refers to a human transmembrane protein with an EGF-like and two follistatin-like domains 2, also known as tomoregulin 2. The amino acid sequence of full-length human TMEFF2 is shown in SEQ ID NO: 27. The extracellular domain of TMEFF2 is shown in SEQ ID NO: 28 and spans residues 40-320 of full-length TMEFF2. The TMEFF2 extracellular domain carries three different subdomains: Kazal-like 1 (residues 85-137), Kazal-like 2 (residues 176-229) and EGF domain (residues 261-301). The TMEFF2 EGF domain is shown in SEQ ID NO: 29. The "membrane proximal region" of TMEFF2 refers to the TMEFF2 region of SEQ ID NO: 21, which covers the EGF domain and the NC-terminal linker region (e.g., residues 230-320 of the full-length human TMEFF2 of SEQ ID NO: 27). Unless explicitly indicated as being from a non-human species, all references herein to proteins, polypeptides and protein fragments are intended to refer to the human form of the corresponding protein, polypeptide or protein fragment. Therefore, unless indicated as being from a non-human species, such as "mouse TMEFF2" or "monkey TMEFF2", "TMEFF2" means human TMEFF2.

[0092] SEQ ID NO:27 (full length human TMEFF2)

[0093] MVLWESPRQCSSWTLCEGFCWLLLLLPVMLLIVARPVKLAAFPTSLSDCQTPTGWNCSGYDDRENDLFLCDTNTCKFDGECLRIGDTVTCVCQFKCNNDYVPVCGSNGESYQNECYLRQAACKQQSEILVVSEGSCATDAGSGSGDGVHEGSGETSQKETSTCDICQFGAECDEDAEDVWCVCNIDCS QTNFNPLCASDGKSYDNACQIKEASCQKQEKIEVMSLGRCQDNTTTTTKSEDGHYARTDYAENANKLEESAREHHIPCPEHYNGFCMHGKCEHSINMQEPSCRCDAGYTGQHCEKKDYSVLYVVPGPVRFQYVLIAAVIGTIQIAVICVVVLCITRKCPRSNRIHRQKQNTGHYSSDNTTRASTRLI

[0094] SEQ ID NO: 28 (extracellular domain of human TMEFF2)

[0095] FPTSLSDCQTPTGWNCSGYDDRENDLFLCDTNTCKFDGECLRIGDTVTCVCQFKCNNDYVPVCGSNGESYQNECYLRQAACKQQSEILVVSEGSCATDAGSGSGDGVHEGSGETSQKETSTCDICQFGAECDEDAEDVWC VCNIDCSQTNFNPLCASDGKSYDNACQIKEASCQKQEKIEVMSLGRCQDNTTTTTKSEDGHYARTDYAENANKLEESAREHHIPCPEHYNGFCMHGKCEHSINMQEPSCRCDAGYTGQHCEKKDYSVLYVVPGPVRFQYV

[0096] TMEFF2 EGF domain SEQ ID NO:29

[0097] HHIPCPEHYNGFCCMHGKCEHSINMQEPSCRCDAGYTGQHCE

[0098] TMEFF2 membrane proximal region SEQ ID NO: 21

[0099] NTTTTTKSEDGHYARTDYAENANKLEESAREHHIPCPEHYNGFCCMHGKCEHSINMQEPSCRCDAGYTGQHCEKKDYSVLYVVPGPVRFQYV

[0100] "CD3" refers to an antigen expressed on T cells as part of a multimolecular T cell receptor (TCR) complex and composed of homodimers or heterodimers, which are formed by the association of two or four receptor chains: CD3ε, CD3δ, CD3ζ and CD3γ. Human CD3ε comprises the amino acid sequence of SEQ ID NO:22. The extracellular domain spans residues 23-126 of the full-length CD3. Unless explicitly indicated as being from a non-human species, all references to proteins, polypeptides and protein fragments herein are intended to refer to the human version of the corresponding protein, polypeptide or protein fragment. Therefore, unless indicated as being from a non-human species, such as "mouse CD3" or "monkey CD3", etc., "CD3" means human CD3.

[0101] SEQ ID NO:22 (human CD3ε)

[0102] MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGS KPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI

[0103] As used herein, the term "antibody" refers broadly to and includes immunoglobulin molecules, including monoclonal antibodies, including murine monoclonal antibodies, human monoclonal antibodies, humanized monoclonal antibodies, and chimeric monoclonal antibodies; antigen-binding fragments; multispecific antibodies, such as bispecific antibodies, trispecific antibodies, tetraspecific antibodies; dimeric antibodies, tetrameric antibodies, or multimeric antibodies; single-chain antibodies; domain antibodies; and any other modified configuration of immunoglobulin molecules that contain an antigen-binding site with the desired specificity. The term antibody includes full-length antibodies, whole antibodies, intact antibodies, antibody fragments, antigen-binding fragments, and antigen-binding domains.

[0104] Generally speaking, an antibody is a protein or peptide chain that exhibits binding specificity for a specific antigen. Antibody structure is well known. According to the heavy chain constant domain amino acid sequence, immunoglobulins can be designated as five major classes (i.e., IgA, IgD, IgE, IgG, and IgM). IgA and IgG are further subclassified into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Therefore, the antibody of the present invention can be any one of the five main classes or corresponding subclasses. Preferably, the antibody of the present invention is IgG1, IgG2, IgG3, or IgG4. Based on the amino acid sequence of its constant domain, the antibody light chain of a vertebrate species can be designated as one of two completely different types, i.e., κ and λ. Therefore, the antibody of the present invention can contain κ or λ light chain constant domains. According to some embodiments, the antibody of the present invention includes a heavy chain and / or light chain constant region from a rat or human antibody. In addition to the heavy and light chain constant domains, the antibody also contains an antigen binding region consisting of a light chain variable region and a heavy chain variable region, wherein each variable region contains three domains (i.e., complementary determining regions 1-3; CDR1, CDR2, and CDR3). The light chain variable region domains are alternatively referred to as LCDR1, LCDR2, and LCDR3, and the heavy chain variable region domains are alternatively referred to as HCDR1, HCDR2, and HCDR3.

[0105] The term "variable region" or "variable domain" refers to the domain of the heavy or light chain involved in binding an antibody to an antigen. The variable domain of a heavy or light chain (VH and VL, respectively) comprises four framework regions (FR) and three complementarity determining regions (CDR).

[0106] "Complementarity determining regions" (CDRs) are regions of antibodies that bind antigen. There are three CDRs in VH (HCDR1, HCDR2, HCDR3) and three CDRs in VL (LCDR1, LCDR2, LCDR3). CDRs can be defined using various depictions, such as Kabat (Wu et al., (1970) J Exp Med 132:211-50; Kabat et al., "Sequences of Proteins of Immunological Interest", 5th Ed., Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia (Chothia et al., (1987) J Mol Biol 196:901-17), IMGT (Lefranc et al., (2003) Dev Comp Immunol 27:55-77) and AbM (Martin and Thornton, J Bmol Biol 263:800-15, 1996). The correspondence between various delineations and variable region numbers is described (see, e.g., Lefranc et al., (2003) Dev Comp Immunol 27:55-77; Honegger and Pluckthun (2001), J Mol Biol 309:657-70; International ImMunoGeneTics (IMGT) database; Web resources, http: / / www_imgt_org). Available programs (such as abYsis of UCL Business PLC) can be used to delineate CDRs. Unless otherwise expressly stated in the specification, as used herein, the terms "CDR", "HCDR1", "HCDR2", "HCDR3", "LCDR1", "LCDR2" and "LCDR3" include CDRs defined by any of the above methods (Kabat, Chothia, IMGT or AbM). The correspondence between numbering systems, including, for example, the Kabat numbering and the IMGT unique numbering system, is well known to those skilled in the art (see, for example, Kabat, supra; Chothia, supra; Martin, supra; Lefranc et al., supra).

[0107] Table 1 .

[0108] IMGT Kabat AbM Chothia <![CDATA[V H CDR1]]> 27-38 31-35 26-35 26-32 <![CDATA[V H CDR2]]> 56-65 50-65 50-58 53-55 <![CDATA[V H CDR3]]> 105-117 95-102 95-102 96-101 <![CDATA[V L CDR1]]> 27-38 24-34 24-34 26-32 <![CDATA[V L CDR2]]> 56-65 50-56 50-56 50-52 <![CDATA[V L CDR3]]> 105-117 89-97 89-97 91-96

[0109] "Specific binding" or "binding" refers to the binding of a protein molecule to an antigen or an epitope within an antigen with a greater affinity than to other antigens. Typically, a protein molecule binds to an antigen or an epitope within an antigen with an equilibrium dissociation constant (K D ) is about 1×10 -7 M or less, for example, about 5×10 -8 M or less, about 1×10 -8 M or less, about 1×10 -9 M or less, about 1×10 -10 M or less, about 1×10 -11 M or less or about 1×10 -12 M or lower, usually K D It is the K of its binding to non-specific antigens (such as BSA, casein) D The term "K D ” refers to D The ratio of Kd to Ka (i.e., Kd / Ka) is obtained and expressed as the dissociation constant of molar concentration (M). According to the present disclosure, the K of an antibody D The K value can be determined using methods known in the art. For example, the K value of an antibody D This can be accomplished by using surface plasmon resonance, such as by using a biosensor system (e.g. The K of the antibody can be determined by using biolayer interferometry techniques such as the Octet RED96 system. D The smaller the value, the higher the affinity of the antibody for binding to the target antigen.

[0110] As used herein, an antibody that "binds to TMEFF2" or "specifically binds to TMEFF2" refers to an antibody that binds to TMEFF2 at a concentration of 1×10 -7 M or less, preferably 1×10 -8 M or less, more preferably 5×10 -9 M or smaller, 1×10 -9 M or smaller, 5×10 -10 M or smaller or 1×10 -10 M, 5×10 -11 M, 1×10 -11 M, 5×10 -12 M or 1×10 -12 M or smaller K D An antibody that binds to TMEFF2 (preferably human TMEFF2).

[0111] As used herein, an antibody that "binds to CD3" or "specifically binds to CD3" refers to an antibody that binds to CD3 at a rate of 1×10-7 M or less, preferably 1×10 -8 M or less, more preferably 5×10 -9 M or smaller, 1×10 -9 M or smaller, 5×10 -10 M or smaller or 1×10 -10 M, 5×10 -11 M, 1×10 -11 M, 5×10 -12 M or 1×10 -12 M or smaller K D An antibody that binds to CD3, preferably human CD3.

[0112] "Bispecific" refers to an antibody that specifically binds to two different antigens or two different epitopes within the same antigen. Bispecific antibodies may have cross-reactivity to other related antigens, for example, to the same antigen from other species (homologous) (such as humans or monkeys, e.g., cynomolgus (cyno) or chimpanzees), or may bind to epitopes shared between two or more different antigens.

[0113] “Bispecific anti-TMEFF2 / anti-CD3 antibody,” “TMEFF2 / CD3 antibody,” “anti-TMEFF2xCD3 bispecific antibody,” and the like refer to antibodies that bind to TMEFF2 and CD3.

[0114] In some embodiments, the anti-TMEFF2xCD3 bispecific antibody administered in the method of treatment comprises a whole antibody or a full-length antibody, an Fv fragment, a single-chain scFv fragment (scFv), Fab, F(ab) 2 or single-chain antibodies.

[0115] The terms "full-length antibody", "whole antibody" and "intact antibody" are used interchangeably herein and refer to antibodies with a structure similar to that of natural antibodies. "Intact antibodies" are composed of two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds, and their multimers (e.g., IgM). Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (composed of domains CH1, hinge, CH2, and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further subdivided into hypervariable regions, which are called complementarity determining regions (CDRs) and are interspersed with framework regions (FRs). Each VH and VL is composed of three CDRs and four FR segments, and are arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Immunoglobulins can be assigned to five major classes, IgA, IgD, IgE, IgG, and IgM, based on the amino acid sequence of the heavy chain constant domain. IgA and IgG are further subclassified into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. The antibody light chains of any vertebrate species can be assigned to one of two distinct types, kappa and lambda, based on the amino acid sequence of their constant domains.

[0116] As used herein, the terms "antibody fragment" and "antigen-binding fragment" refer to molecules other than intact antibodies. Antigen-binding fragments can be synthetic, enzymatically obtainable, or genetically engineered polypeptides, and include portions of immunoglobulins that bind to antigens, such as VH, VL, VH and VL, Fab, Fab', F(ab'), 2 , Fd and Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv) 2 , bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (dsdiabodies), single-chain antibody molecules (scFv), single domain antibodies (sdab), scFv dimers (bivalent diabodies), multispecific antibodies formed from a portion of an antibody comprising one or more CDRs, camelized single domain antibodies, nanobodies, domain antibodies, domain antibodies (dAbs) consisting of one VH domain or one VL domain, shark variable IgNAR domains, camelized VH domains, VHH domains, minimal recognition units consisting of amino acid residues that mimic the CDRs of antibodies (such as FR3-CDR3-FR4 portions), HCDR1, HCDR2 and / or HCDR3 and LCDR1, LCDR2 and / or LCDR3, alternative scaffolds that bind to antigens, bivalent domain antibodies, multispecific proteins comprising antigen-binding fragments or any other antibody fragment that binds to an antigen but does not comprise a full antibody structure.

[0117] In some embodiments, the methods of the present disclosure are implemented by administering a full-length TMEFF2xCD3 bispecific antibody comprising two heavy chains (HC1 and HC2) and two light chains (LC1 and LC2).

[0118] In some embodiments, the methods of the present disclosure are implemented by administering a TMEFF2xCD3 bispecific antibody comprising a heavy chain (HC1), a light chain (LC), and a single-chain Fv (scFV).

[0119] "Single-chain Fv" or "scFv" is a fusion protein comprising at least one antibody fragment containing a light chain variable region (VL) and at least one antibody fragment containing a heavy chain variable region (VH), wherein VL and VH are continuously connected via a polypeptide linker and can be expressed as a single-chain polypeptide. The scFv can have the VL variable region and the VH variable region in either order, for example, relative to the N-terminus and C-terminus of the polypeptide, the scFv can comprise VL-linker-VH or can comprise VH-linker-VL. The scFv can comprise a linker peptide connecting the heavy chain variable region and the light chain variable region, such as two to about eight glycine or other amino acid residues.

[0120] In some embodiments, the anti-TMEFF2xCD3 bispecific antibodies used in the therapeutic methods of the present disclosure include chimeric, humanized, or fully human antibodies that specifically bind to TMEFF2 and CD3.

[0121] "Human antibody" refers to an antibody optimized to have a minimal immune response when administered to a human subject. The variable region of a human antibody is derived from a human immunoglobulin sequence. If a human antibody comprises a constant region or a portion of a constant region, the constant region is also derived from a human immunoglobulin sequence. If the variable region of a human antibody is obtained by a system using human germline immunoglobulins or rearranged immunoglobulin genes, the human antibody comprises a heavy chain variable region and a light chain variable region "derived from" a sequence of human origin. Such exemplary systems are human immunoglobulin gene libraries displayed on phages, and transgenic non-human animals, such as mice or rats carrying human immunoglobulin loci. Because of the differences between the systems for obtaining human antibodies and human immunoglobulin loci, the introduction of somatic mutations or the intentional introduction of substitutions into the framework or CDR or both, "human antibodies" generally comprise amino acid differences compared to immunoglobulins expressed in humans.

[0122] Typically, the amino acid sequence of a "human antibody" is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence encoded by a human germline immunoglobulin gene or a rearranged immunoglobulin gene. In some cases, a "human antibody" may comprise a consensus framework sequence derived from human framework sequence analysis (e.g., as described in Knappik et al., (2000) J Mol Biol 296: 57-86), or a synthetic HCDR3 bound to a human immunoglobulin gene library displayed on a phage (e.g., as described in Shi et al., (2010) J Mol Biol 397: 385-96 and International Patent Publication No. WO2009 / 085462). Antibodies in which at least one CDR is derived from a non-human species are not included in the definition of "human antibody".

[0123] Transgenic animals (such as mice, rats or chickens) carrying human immunoglobulin (Ig) loci in their genomes can be used to produce antibodies used in the disclosed methods, as described, for example, in U.S. Pat. No. 6,150,584; International Patent Publication No. WO1999 / 45962, International Patent Publication No. WO2002 / 066630, WO2002 / 43478, WO2002 / 043478 and WO1990 / 04036. The endogenous immunoglobulin loci in such animals can be disrupted or deleted, and at least one complete or partial human immunoglobulin locus can be inserted into the genome of the animal by homologous or nonhomologous recombination using a transchromosome or minigene. Companies such as Regeneron (World Wide Web: regeneron.com), Harbour Antibodies (World Wide Web: harbourantibodies.com), Open Monoclonal Technology, Inc. (OMT) (World Wide Web: omtinc.net), KyMab (World Wide Web: kymab.com), Trianni (World Wide Web: trianni.com), and Ablexis (World Wide Web: ablexis.com) can be involved in providing human antibodies against the selected antigen.

[0124] Antibodies generated by immunizing non-human animals can be humanized using methods well known in the art. Generally speaking, humanized or engineered antibodies have one or more amino acid residues from non-human sources, such as, but not limited to, mice, rats, rabbits, non-human primates or other mammals. Exemplary humanization techniques including selection of human acceptor frameworks include CDR grafting (U.S. Patent No. 5,225,539), SDR grafting (U.S. Patent No. 6,818,749), resurfacing (Padlan, (1991) Mol Immunol 28: 489-499), resurfacing of specificity-determining residues (U.S. Patent Publication 2010 / 0261620), human framework remodeling (U.S. Patent No. 8,748,356) or superhumanization (U.S. Patent No. 7,709,226). In these methods, the CDRs or a subset of CDR residues of a parent antibody are transferred to a human framework, which can be selected based on its overall homology to the parent framework, based on similarity in CDR length or canonical structural identity, or a combination thereof.

[0125] The humanized antigen binding domain can be further optimized to improve its selectivity or affinity for the desired antigen by incorporating altered framework support residues to maintain binding affinity (back mutations) using techniques such as those described in International Patent Publication Nos. WO1090 / 007861 and WO1992 / 22653, or by introducing variations in any CDR to improve the affinity of the antigen binding domain.

[0126] The anti-TMEFF2xCD3 bispecific antibodies used according to the present disclosure can be prepared by recombinant means, including preparation from mammalian cells or transgenic preparations, or can be purified from other biological sources, as described herein or as known in the art. As is well known in the art, the antibodies used in the methods of the present disclosure can be prepared by cell lines, mixed cell lines, immortalized cells or clonal populations of immortalized cells. Cell lines can be engineered to express antibodies of the present disclosure, and the antibodies can be produced intracellularly, in the periplasmic space, or directly secreted into the culture medium.

[0127] Cell lysates or supernatants containing anti-TMEFF2xCD3 bispecific antibodies can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography. Other protein purification techniques can also be used, such as ion exchange column separation, ethanol precipitation, reversed-phase HPLC, silica gel chromatography, anion or cation exchange resin chromatography.

[0128] Treatment

[0129] In some embodiments, the methods of the present disclosure are implemented by administering a TMEFF2xCD3 bispecific antibody comprising a first variable domain that specifically binds to TMEFF2 and a second variable domain that specifically binds to CD3, wherein the first variable domain that specifically binds to TMEFF2 comprises VH of SEQ ID NO: 13 and VL of SEQ ID NO: 14; and the second variable domain that specifically binds to CD3 comprises VH of SEQ ID NO: 17 and VL of SEQ ID NO: 18.

[0130] In some embodiments, the method of the present disclosure is implemented by administering a TMEFF2xCD3 bispecific antibody comprising a first variable domain that specifically binds to TMEFF2 and a second variable domain that specifically binds to CD3, wherein the first variable domain that specifically binds to TMEFF2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 1, 2, 3, 4, 5 and 6, respectively; and the second variable domain that specifically binds to CD3 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 7, 8, 9, 10, 11 and 12, respectively.

[0131] In some embodiments, the method of the present disclosure is implemented by administering a TMEFF2xCD3 bispecific antibody comprising a first variable domain that specifically binds to TMEFF2 and a second variable domain that specifically binds to CD3, wherein the first variable domain that specifically binds to TMEFF2 comprises a heavy chain (HC) of SEQ ID NO: 15 and a light chain (LC) of SEQ ID NO: 16; and the second variable domain that specifically binds to CD3 comprises a heavy chain (HC) of SEQ ID NO: 19 and a light chain (LC) of SEQ ID NO: 20.

[0132] In some embodiments, the methods of the present disclosure are implemented by administering an anti-TMEFF2xCD3 bispecific antibody comprising a first binding domain that binds to TMEFF2 and a second binding domain that binds to CD3, wherein (a) the first binding domain that binds to TMEFF2 comprises a HCDR of a VH having an amino acid sequence of SEQ ID NO: 13 and a LCDR of a VL having an amino acid sequence of SEQ ID NO: 14, and the second binding domain that binds to CD3 comprises a HCDR of a VH having an amino acid sequence of SEQ ID NO: 17 and a LCDR of a VL having an amino acid sequence of SEQ ID NO: 18; (b) the first binding domain that binds to TMEFF2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively, and the second binding domain that binds to CD3 comprises HCDRs of SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively. NO:7, 8, 9, 10, 11 and 12 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3; (c) the first binding domain that binds TMEFF2 comprises VH and VL of SEQ ID NO:13 and 14, respectively, and the second binding domain that binds CD3 comprises VH and VL of SEQ ID NO:17 and 18, respectively; and / or (d) the first binding domain that binds TMEFF2 comprises HC1 and LC1 of SEQ ID NO:15 and 16, respectively; and the second binding domain that binds CD3 comprises HC2 and LC2 of SEQ ID NO:19 and 20, respectively.

[0133] In some embodiments, the methods of the present disclosure induce T cell activation and proliferation in a target-specific manner without significant activation or proliferation of T cells in the periphery.

[0134] In some embodiments, the methods of the present disclosure induce T cell activation and proliferation in a target-specific manner.

[0135] In some embodiments, the methods of the present disclosure induce infiltration of activated and proliferating T cells and inflammatory cells in the prostate.

[0136] In some embodiments, the methods of the present disclosure increase the number of T cells within the prostate.

[0137] In some embodiments, the methods of the present disclosure do not induce significant numbers of suppressive T cell markers (PD1+Ki-67- and CD25+FoxP3).

[0138] In some embodiments, the methods of the present disclosure induce myeloid cell infiltration.

[0139] Composition

[0140] The methods of the present disclosure are also implemented by administering a composition comprising an anti-TMEFF2xCD3 bispecific antibody described herein.

[0141] In the methods of the present disclosure, the antibodies may also be administered as a pharmaceutical composition comprising a therapeutically effective amount of an anti-TMEFF2xCD3 bispecific antibody and an optional pharmaceutically acceptable carrier.

[0142] Example

[0143] The following examples are provided to further describe some of the embodiments disclosed herein. These examples are intended to illustrate, but not to limit, the embodiments disclosed herein.

[0144] Example 1. Generation and description of study drugs

[0145] The exemplary TMEFF2xCD3 bispecific antibody Ab1 will be used as the study drug in the Examples.

[0146] Ab1 is immunoglobulin (Ig) G4-proline, alanine, alanine (IgG4 PAA) Bispecific antibodies that simultaneously bind to the cluster of differentiation (CD) 3 receptor complex on T lymphocytes (T cells) and a transmembrane protein (TMEFF2) with epidermal growth factor-like and 2 follistatin-like domains on tumor cells. It is speculated that through this binding activity, the bispecific antibody mediates synapse formation between T cells and TMEFF2-expressing cells, leading to T cell activation and subsequent lysis of TMEFF2-positive cells by perforin and granzymes secreted by cytotoxic T cells. Due to the mutation of leucine at positions 234 and 235 to alanine, the binding of Ab1 to the crystallizable fragment (Fc) γ receptor is reduced, and due to the mutation of serine at position 228 to proline, the exchange of Ab1 with the fragment antigen binding arm is reduced.

[0147] Ab1 was developed to evaluate the therapeutic potential of targeting TMEFF2 for CD3-mediated T cell redirection. The bispecific antibody was generated by controlled fragment antigen binding (Fab) arm exchange from 2 antibodies: TMEB762 and CD3B376. TMEB762 was generated by immunizing OmniRats (OMT TM ) to produce anti-TMEFF2 antibody. CD3B376 was produced by immunizing OmniRats (OMT TM ) produced anti-CD3ε antibodies.

[0148] The generation of Ab1 has been described in U.S. Patent Publication 16,417,889, entitled "Monospecific and multispecific anti-TMEFF2 antibodies and their uses", published on November 28, 2019, which is incorporated herein by reference in its entirety. Assays for evaluating the functional activity and structural properties (e.g., amino acid sequence) of Ab1 are also described in detail in U.S. Patent Publication 16,417,889, which is incorporated herein by reference in its entirety.

[0149] Using Kabat delineation, the TMEFF2 binding domain of Ab1 (TMCB150) comprises HCDR1 of the amino acid sequence SYSMS (SEQ ID NO: 1), HCDR2 of the amino acid sequence VISGSGGFTDYADSVKG (SEQ ID NO: 2), and HCDR3 of the amino acid sequence MPLNSPHDY (SEQ ID NO: 3), as well as LCDR1 of the amino acid sequence RASQGIRNDLG (SEQ ID NO: 4), LCDR2 of the amino acid sequence AASSLQS (SEQ ID NO: 5), and LCDR3 of the amino acid sequence LQDYNYPLT (SEQ ID NO: 6).

[0150] Using Kabat delineation, the CD3 binding domain of Ab1 (CD3B376) comprises HCDR1 of the amino acid sequence NNNAAWS (SEQ ID NO: 7), HCDR2 of the amino acid sequence RTYYRSKWLYDYAVSVKS (SEQ ID NO: 8), and HCDR3 of the amino acid sequence GYSSSFDY (SEQ ID NO: 9), as well as LCDR1 of the amino acid sequence TGTSSNIGTYKFVS (SEQ ID NO: 10), LCDR2 of the amino acid sequence EVSKRPS (SEQ ID NO: 11), and LCDR3 of the amino acid sequence VSYAGSGTLL (SEQ ID NO: 12).

[0151] The VH, VL, HC and LC sequences of the TMEFF2 and CD3 binding domains of Ab1 (TMCB150) are described below.

[0152] Ab1 VH, VL, HC, LC of TMEFF2 binding domain

[0153] VH amino acid sequence of the TMEFF2 binding domain of TMCB150; SEQ ID NO: 13EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYSMSWVRQAPGKGLEWVSVISGSGGFTDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARMPLNSPHDYWGQGTLVTVSS

[0154] VL amino acid sequence of TMCB150 TMEFF2 binding domain; SEQ ID NO: 14

[0155] DIQMTQSPSSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDYNYPLTFGGGTKVEIK

[0156] HC amino acid sequence of TMCB150 TMEFF2 binding domain; SEQ ID NO: 15

[0157] LC amino acid sequence of the TMCB150 TMEFF2 binding domain; SEQ ID NO: 16 DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDYNYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0158] HC nucleic acid sequence of the TMCB150 TMEFF2 binding domain; SEQ ID NO: 23

[0159]

[0160] LC nucleotide sequence of the TMCB150 TMEFF2 binding domain; SEQ ID NO:24

[0161] GACATCCAGATGACCCAGAGCCCTAGCAGCCTGAGCGCTAGCGTGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGGGCATCAGAAACGACCTGGGCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGAGTGCCTAGCAGGTTCAGCGGAAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCTGCAGGACTACAACTACCCCCTGACATTCGGCGGCGGCACCAAGGTGGAGATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

[0162] VH, VL, HC and LC of Ab1 CD3 binding domain

[0163] VH amino acid sequence of the TMCB150 CD3 binding domain; SEQ ID NO:17

[0164] QVQLQQSGPRLVRPSQTLSLTCAISGDSVFNNNAAWSWIRQSPSRGLEWLGRTYYRSKWLYDYAVSVKSRITVNPDTSRNQFTLQLNSVTPEDTALYYCARGYSSSFDYWGQGTLVTVSS

[0165] VL amino acid sequence of the CD3-binding domain of TMCB150; SEQ ID NO: 18 QSALTQPASVSGSPGQSITISCTGTSSNIGTYKFVSWYQQHPDKAPKVLLYEVSKRPSGVSSRFSGSKSGNTASLTISGLQAEDQADYHCVSYAGSGTLLFGGGTKLTVL

[0166] HC amino acid sequence of the CD3-binding domain of TMCB150; SEQ ID NO: 19

[0167] QVQLQQSGPRLVRPSQTLSLTCAISGDSVFNNNAAWSWIRQSPSRGLEWLGRTYYRSKWLYDYAVSVKSRITVNPDTSRNQFTLQLNSVTPEDTALYYCARGYSSSFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0168] LC amino acid sequence of the CD3-binding domain of TMCB150; SEQ ID NO: 20

[0169] QSALTQPASVSGSPGQSITISCTGTSSNIGTYKFVSWYQQHPDKAPKVLLYEVSKRPSGVSSRFSGSKSGNTASLTISGLQAEDQADYHCVSYAGSGTLLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0170] HC nucleic acid sequence of TMCB150 CD3 binding domain; SEQ ID NO: 25

[0171]

[0172] LC nucleic acid sequence of TMCB150 CD3 binding domain; SEQ ID NO: 26

[0173] CAGTCTGCTCTGACCCAGCCTGCTCCGTGTCTGGCTCTCCCGGCCAGTCCATCACCATCAGCTGTACCGGCACCTCCTCCAACATCGGCACCTACAAGTTCGTGTCCTGGTATCAGCAGCACCCCGACAAGGCCCCCAAAGTGCTGCTGTACGAGGTGTCC AAGCGGCCCTCTGGCGTGTCCTCCAGATTCTCCGGCTCCAAGTCTGGCAACACCGCCTCCCTGACCATCAGCGGACTGCAGGCTGAGGACCAGGCCGACTACCACTGTGTGTCCTACGCTGCCTCTGGCACCCTGCTGTTTGGCGGAGGCACCAAGCTGACC GTGCTGGGTCAGCCCAGGCTGCACCCAGTGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCCGATAGCAGCCCCGTCAAGGCGGGA GTGGAGACCACCACAACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCA

[0174] The term "study drug" refers to Abl described herein, and refers to the study drug used in the pharmacodynamic studies described in Example 2 and the clinical trial protocol described in Example 3.

[0175] Example 2. Preclinical pharmacodynamic study in cynomolgus monkeys

[0176] The pharmacodynamics of the study drug was evaluated in cynomolgus monkeys to understand the dose and targeting effects of the study drug in the prostate.

[0177] Study Design

[0178] Two dosing regimens of intravenous study drug were evaluated in male cynomolgus monkeys (3 to 8 years old): a single fixed dose (0.075 mg / kg) and an ascending dose (0.075 mg / kg, 0.3 mg / kg after 1 week). Each dosing regimen included 12 male cynomolgus monkeys, which were evenly divided into 4 groups to receive study drug or control. Prostate tissue, blood and serum were collected for further analysis 24 hours, 72 hours and 168 hours after administration of a fixed dose of 0.075 mg / kg and 24 hours, 72 hours and 168 hours after administration of a 0.3 mg / kg dose of an ascending dosing regimen.

[0179] Sample collection

[0180] For fixed dosing of study drug, whole blood was collected before dosing, 6 hours after dosing, and immediately after necropsy at all other time points (24 hours, 72 hours, and 168 hours). Prostate tissue was collected at three necropsy time points.

[0181] For research drug incremental administration, collect sample at the time point similar to fixed administration, and make some slight adjustments.Before the initial administration and after 6 hours, collect whole blood, and before the incremental administration after one week and after 6 hours, collect whole blood.Collect whole blood immediately when carrying out autopsy in 24 hours, 72 hours and 168 hours after the incremental administration.As described for fixed administration, collect prostate tissue.

[0182] All samples were processed by flow cytometry.

[0183] Cell surface staining

[0184] Whole blood samples were collected, prepared, and stained for CD4, CD8, CD25, CD45, CD69, PD-1, Ki-67, FoxP3, and Granzyme B according to standard procedures. Prostate tissue was cut into smaller pieces and processed for further analysis according to standard procedures. Data analysis was performed using FlowJo version 10 to determine frequencies and counts. Cell counting was facilitated by adding a constant volume of absolute counting beads. The obtained cell events were converted to cells / mL as follows:

[0185]

[0186] Study drug concentration

[0187] exist The validated and applicable ECLIA (electrochemiluminescence immunoassay) method on the platform was used to quantify the concentration of study drug in serum and prostate tissue, respectively. The lowest quantifiable concentrations in serum and prostate tissue lysate samples were 0.01 μg / mL and 1.25 ng / mL, respectively. An approximately dose-proportional increase in study drug was observed after escalating dosing ( Fig.10 ). The median peak study drug exposure in the prostate was 11.0% of serum for the first dose and 8.7% of serum after the second dose.

[0188] T cell infiltration

[0189] Study drug administration increased prostate infiltration of CD8+ T cells and CD4+ T cells and decreased peripheral T cells ( FIG. 11A to FIG. 11B , FIG. 12A to FIG. 1 2F). With fixed dosing of the study drug, the reduction in peripheral CD8+ T cells was more significant.

[0190] T cell activation and proliferation

[0191] After study drug administration, both CD4+ and CD8+ prostate T cells expressed markers of activation and proliferation ( FIG. 13A to FIG. 13F ). The extent of T cell activation appeared to decrease slightly with increasing dosing of the study drug.

[0192] Suppressor T cell markers

[0193] The study drug had minimal effect on inhibitory T cell markers (PD-1+Ki67-, CD4+CD25+Foxp3) FIG. 14A to FIG. 14C and FIG. 15A to FIG. 15B ).

[0194] Myeloid cell infiltration

[0195] Study drug administration resulted in an influx of proinflammatory cells (dendritic cells, myeloid cells, B cells) ( FIG. 16A to FIG. 16B , FIG. 17A to FIG. 17C ). Compared with fixed dosing, escalating dosing of study drug showed a reduction in immature and proinflammatory myeloid cell subsets in prostate tissue. Total B cell counts were lower with escalating dosing of study drug compared with fixed dosing.

[0196] in conclusion

[0197] Administration of the study drug increased the number of T cells within the prostate. T cell activation and proliferation were observed in a target-specific manner, while no significant activation or proliferation of T cells was detected in the periphery. No significant number of inhibitory T cell markers (PD1+Ki-67- and CD25+FoxP3) were observed after study drug administration. Myeloid cell infiltration supports an active immune microenvironment in the prostate after study drug administration.

[0198] Example 3.1 Phase Clinical Study

[0199] Program Overview

[0200] This is a first-in-human (FIH), open-label, multicenter, Phase 1 dose-escalation study designed to evaluate the safety, pharmacokinetics (PK), pharmacodynamics (PD), and preliminary clinical activity of investigational drug monotherapy in patients with metastatic castration-resistant prostate cancer (mCRPC). Figure 1 A diagram of the study design is provided in .

[0201] The modified continuous reassessment method (mCRM) supports dose escalation. The goal is to determine the maximum tolerated dose (MTD) of the study drug.

[0202] During the study, the Study Evaluation Team (SET) monitored safety regularly, particularly at each dose escalation step. The study began with a once-weekly (QW) subcutaneous (SC) dosing schedule. Based on new data identified by the SET, an alternative dosing schedule of once every 2 weeks [Q2W] was explored.

[0203] Approximately 73 participants were treated in this study.

[0204] Table 22 .

[0205]

[0206] Goal, end point

[0207] The research objectives and endpoints of this study are listed in Table 33.

[0208] Table 33. Study objectives and endpoints

[0209]

[0210] Efficacy evaluation

[0211] Clinical activity will be assessed using the following evaluations: Computed tomography (CT) scan of the chest, abdomen, and pelvis with contrast as clinically indicated; alternatively, magnetic resonance imaging (MRI) may be used as clinically indicated (ie, for sites not adequately imaged using CT). Additional evaluations for participants with mCRPC include serum prostate-specific antigen (PSA) and whole-body bone scan ( 99m Tc). Treatment response will be evaluated according to the Prostate Cancer Working Group 3 (PCWG3) criteria and Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 to assess the progression of soft tissue lesions (CT or MRI).

[0212] Pharmacokinetics, biomarkers, and immunogenicity assessments

[0213] Blood samples will be collected to characterize serum pharmacokinetics of study drug and anti-drug antibodies. Blood samples will also be collected to evaluate pharmacodynamics, safety, and biomarkers predicting response or resistance to study drug treatment. To evaluate TMEFF2 expression as well as T cell infiltration, archived tissue samples of metastatic (non-prostate) tumor lesions (collected at any time prior to recruitment) and archived prostate samples collected ≤15 months after providing informed consent (if available) will be requested. In addition, selected participants in the Part 1 PK / PD cohort and all participants in Part 2 will undergo a fresh tumor biopsy to evaluate pharmacodynamic markers in tumor tissue. Selected PK / PD cohorts may receive additional blood sample collection.

[0214] Safety Assessment

[0215] The safety of the study drug will be assessed by physical examination (including neurologic examination), vital signs, Eastern Cooperative Oncology Group (ECOG) performance status, clinical laboratory tests, electrocardiogram (ECG), ophthalmologic examination, and adverse event (AE) monitoring (including dose-limiting toxicity [DLT], serious adverse events [SAE], and adverse events of special interest [AESI]). Concomitant medication use will be recorded. The severity of adverse events will be evaluated using the National Cancer Institute Common Terminology Criteria for Evaluation of Adverse Events (version 5.0), with the exception of cytokine release syndrome (CRS), which will be graded according to the American Society for Transplantation and Cellular Therapy (ASTCT) guidelines. Central and peripheral neurotoxicity (≥Grade 3) and new retinal abnormalities or noninfectious inflammatory eye disease (≥Grade 2) have been identified as adverse events of special interest that require enhanced reporting and data collection.

[0216] Starting dose rationale

[0217] The 300 μg first-in-human (FIH) starting dose administered SC approximates a 4 μg / kg dose (assuming a median body weight of 75 kg and 100% bioavailability).

[0218] In vitro cytotoxicity assays were performed to characterize the T cell activation, TMEFF2 positive tumor cell killing and cytokine release induced by the study drug. These assays were performed using purified human T cells and LNCaP-AR from healthy donors, which are human prostate cancer cell lines expressing TMEFF2 and showing sensitivity to T cell-mediated killing. Median cytotoxicity was shown to be the most sensitive and was considered to be the most predictive of T cell-mediated killing (Table 4). From the median EC of cytotoxicity 20 The values ​​were determined for a MABEL concentration of 1.0 nM (0.15 μg / mL).

[0219] Table 4. T cell-mediated cytotoxicity and T cell activation assays for study drugs using LNCaP-AR cells Summary of exposure-response analyses

[0220]

[0221] Assuming a median body weight of 75 kg, the PK model MABEL predicts the mean serum concentration of the study drug in humans after a single dose of 300 μg as follows: Figure 2 shown.

[0222] Based on the overall evaluation of the in vitro and in vivo data, and the selection of the FIH starting dose based on MABEL, 300 μg weekly SC doses of study drug should result in drug exposures that are minimally bioactive in participants treated in this study.

[0223] Study Design

[0224] Overall Design

[0225] This is a FIH, open-label, multicenter, Phase 1 study to evaluate the safety, pharmacokinetics, pharmacodynamics, and preliminary clinical activity of study drug monotherapy in participants with mCRPC. Approximately 73 participants will be treated in this study. Once a participant is determined to be eligible (i.e., inclusion / exclusion criteria) for the study and has provided informed consent for study participation, the study drug will be administered as a SC injection.

[0226] The pharmacodynamics of the study drug was further characterized in selected PK / PD cohorts determined by the Study Evaluation Team (SET).

[0227] Figure 1 A diagram of the study design is provided in .

[0228] Dose escalation

[0229] The study aims to select an RP2D and regimen and to determine the MTD of study drug in participants with mCRPC, if possible. Dose escalation begins with a starting dose of 300 μg (0.3 mg) based on MABEL and is continued as Figure 1 Continue as indicated. Only the starting dose is pre-specified. After the starting dose, subsequent doses are selected based on a review of all available data (including but not limited to pharmacokinetics, pharmacodynamics, safety, and preliminary clinical activity). Dose escalation decisions are guided by the modified continuous reassessment method (mCRM).

[0230] Multiple dose levels are recruited in parallel with each new dose level / schedule recommended by the SET and supported by a statistical model with overdose escalation control (EWOC) principles. Additional participants are recruited into one or more dose cohorts (called PK / PD cohorts) at doses equal to or below the dose deemed safe by the SET in order to better understand safety, tolerability, pharmacokinetics, pharmacodynamics, or preliminary antitumor activity.

[0231] Dose escalation is performed in single or multiple participant consecutive cohorts at the dose specified by the SET. The following guidelines apply during dose escalation:

[0232] If more than 1 participant was treated at a given dose level, the first participant treated at that given dose level was observed for at least 2 days before subsequent participants were treated.

[0233] Escalation was performed with dose increases not exceeding half-log increments.

[0234] Dose escalation decisions are made by the SET based on the mCRM using all available data such as safety, pharmacodynamics, pharmacokinetic and other biomarker data for all previous dose levels. Preliminary clinical activity (if available) is reviewed by the SET during dose escalation.

[0235] Part 2 (dose expansion)

[0236] In Part 2, the RP2D / regimen of study drug as determined in Part 1 will be administered to additional participants with mCRPC to confirm the safety, pharmacokinetics, pharmacodynamics, and preliminary clinical activity of study drug. Additional histologies (besides mCRPC) will be considered for dose expansion based on new data from Part 1.

[0237] Treatment dosage schedule

[0238] The study was initiated with Q1W treatment dosing. An alternative dosing schedule of Q2W was evaluated based on emerging safety and pharmacokinetic data following SET approval.

[0239] Prior to the first dose of study drug, premedication with corticosteroids, antihistamines, and antipyretics was administered to minimize the risk associated with cytokine release syndrome (CRS) and infusion-related reactions. Premedication doses or schedules for subsequent doses were reduced or omitted based on the SET's review of available data; premedication with corticosteroids was reduced or omitted as appropriate. For participants who experience Grade 2 or higher CRS or IRR, pretreatment with corticosteroids will be required for at least 1 subsequent dose administered to that participant.

[0240] Determination of the recommended phase 2 dose (RP2D)

[0241] The RP2D will be determined after reviewing all available data, including safety, pharmacokinetics, pharmacodynamics, and clinical activity from at least 6 participants treated at the dose level considered in the RP2D submission. In addition, pharmacokinetic data from at least 12 participants at all dose levels will be considered and the recommended dose derived by Bayesian logistic regression modeling (BLRM).

[0242] Definition of Dose-Limiting Toxicity (DLT)

[0243] The DLT evaluation period was defined as the first 21 days of treatment.

[0244] Table 5. Dose-limiting toxicity criteria

[0245]

[0246] Route of administration

[0247] This study explored the SC administration route.

[0248] Treatment discontinuation / follow-up

[0249] Participants received study drug until radiographic disease progression, unequivocal clinical progression, unacceptable toxicity, or any other treatment discontinuation criteria were met. Following treatment discontinuation, participants clinically able to return for assessment had an end-of-treatment (EOT) visit within 30 (±7) days after the last dose of study drug.

[0250] Study Group

[0251] The inclusion and exclusion criteria for recruiting participants in this study are described below.

[0252] Inclusion criteria

[0253] Each potential participant met all of the following study recruitment criteria:

[0254] Aged ≥18 years;

[0255] Histology: metastatic CRPC (mCRPC) with histological confirmation of adenocarcinoma. Adenocarcinoma with small cell or neuroendocrine features is allowed;

[0256] Measurable or evaluable disease;

[0257] ·Previous treatment with at least 1 novel AR-targeted therapy (i.e., abiraterone acetate, apalutamide, enzalutamide, darolutamide) or chemotherapy (e.g., docetaxel);

[0258] Eastern Cooperative Oncology Group (ECO) performance status of 0 or 1;

[0259] Participants must be transfusion independent within 7 days or growth factor independent within 3 weeks prior to the first dose of study drug, with hematology laboratory parameters within the following ranges. Participants must be transfusion independent (hemoglobin ≥9 g / dL, absolute neutrophil count ≥1.5 × 10 9 / L, platelet count ≥75×10 9 / L);

[0260] Chemistry laboratory parameters within the following ranges (serum albumin ≥3.0 g / dL, calculated or measured creatinine clearance >50 mL / min / 1.73 m 2 , serum total bilirubin ≤1.5× upper limit of normal (ULN); in participants with Gilbert's syndrome, direct and indirect bilirubin were measured if total bilirubin was ≥1.5×ULN, and participants were eligible if direct bilirubin was within normal limits, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) ≤2.5×ULN (or ≤4×ULN for participants with liver tumors).

[0261] Exclusion criteria

[0262] Any potential participant who met any of the following criteria was excluded from participation in this study: · Known brain metastases.

[0263] Concurrent use of any other anticancer treatment (including non-palliative radiotherapy) or investigational agent; prior anticancer treatment (including non-palliative radiotherapy) must be discontinued for at least 2 weeks prior to the first dose of study drug.

[0264] Toxicity related to prior anticancer therapy that has not recovered to ≤ Grade 1 or baseline, except for alopecia and vitiligo.

[0265] Symptomatic diabetic retinopathy with macular edema or active wet age-related macular degeneration (AMD) and any symptomatic ocular inflammation or optic nerve disease (e.g., uveitis, optic neuritis) within the previous 12 months.

[0266] Solid organ or bone marrow transplant.

[0267] Epileptic seizures or known conditions that may predispose to seizures or intracranial masses causing edema or mass effect, such as schwannomas and meningiomas.

[0268] Any of the following within 6 months before signing the informed consent form:

[0269] a. Myocardial infarction

[0270] b. Severe or unstable angina

[0271] c. Clinically significant ventricular arrhythmias

[0272] d. Congestive heart failure (New York Heart Association class III to IV)

[0273] e. Transient ischemic attack

[0274] f. Cerebrovascular events.

[0275] Venous thromboembolic events (i.e., pulmonary embolism) within 1 month before the first dose of study drug; uncomplicated (≤ Grade 2) deep vein thrombosis was not considered exclusionary.

[0276] ≥ Grade 2 peripheral neuropathy or neuropathic pain;

[0277] Clinically significant lung impairment, particularly requiring supplemental oxygen use (>2 L via nasal cannula) to maintain adequate oxygenation.

[0278] Known allergy, hypersensitivity or intolerance to the study drug or its excipients

[0279] Concurrent use of any other anticancer treatment or investigational agent for the treatment of advanced disease.

[0280] Active infection or condition requiring treatment with systemic anti-infective drugs within 7 days prior to the first dose of study drug.

[0281] Receipt of immunosuppressive doses of systemic medications, such as corticosteroids (doses >10 mg / day prednisone or equivalent) within 3 days prior to the first dose of study drug. A single course of corticosteroids is permitted as prophylaxis for imaging contrast (i.e., for participants who are allergic to contrast). If corticosteroids are used to treat an immune-related adverse event related to prior therapy, ≥7 days must have passed since the last dose of corticosteroids.

[0282] Active autoimmune disease requiring systemic immunosuppressive medication (i.e., chronic corticosteroids, methotrexate, or tacrolimus) within 12 months prior to signing the consent form.

[0283] · Major surgery (e.g., requiring general anesthesia). Participants must have recovered adequately without sequelae for at least 3 weeks prior to the first dose of study drug.

[0284] Active or chronic hepatitis B or hepatitis C infection. Hepatitis B infection is defined as a positive hepatitis B surface antigen (HBsAg) test. Hepatitis C infection is defined as a positive hepatitis C antibody test.

[0285] · Known history of positive human immunodeficiency virus (HIV) antibodies.

[0286] Plan to be a father at the time of enrollment in this study or within 90 days after the last dose of study drug.

[0287] Any condition that the investigator believes is not in the best interest of the participant to participate in the study (e.g., would compromise health) or that may prevent, limit, or confound protocol-specified assessments.

[0288] Receipt of a live or live-attenuated vaccine within 4 weeks before the first dose of study drug, during the study, or within 4 weeks after the last dose of study drug. Vaccines approved or authorized for emergency use (e.g., COVID-19) and non-live vaccines (e.g., influenza) are permitted.

[0289] Study Drug Administration

[0290] Study Drug Administration

[0291] The study started with SC injection as the route of administration, administered on a once-weekly dosing schedule. An alternative study drug administration schedule of Q2W was also explored. Dose escalation began with a starting dose of 300 μg administered SC.

[0292] Research Assessment and Procedures

[0293] Efficacy evaluation

[0294] Clinical activity was estimated using the following assessments:

[0295] The same methods (CT scan or MRI and 99m Disease assessment is performed by Tc bone scan to characterize each lesion identified and reported to document disease status. Ultrasound, 18 F]-fluorodeoxyglucose positron emission tomography (PET) and conventional X-ray are unacceptable methods for assessing disease response. Imaging was not delayed due to delays in study drug administration. Efficacy assessments included the following: PSA, whole body bone scan ( 99m Tc), CT scan, or MRI. The response to prostate cancer treatment was evaluated according to the Prostate Cancer Working Group 3 (PCWG3) criteria.

[0296] Participants with an objective response per RECIST v1.1 must have a confirmatory scan after 4 weeks. If a participant is assessed as having a partial response (PR) or complete response (CR) at any time during study drug treatment but it is not confirmed after ≥4 weeks, the participant's best response is classified as stable disease / progressive disease / not evaluable based on the participant's next immediate assessment. During the study, CT or MRI scans of known lesion locations will be used to assess disease response.

[0297] Assessment of disease response and progressive disease

[0298] Evaluation of soft tissue lesions (CT or MRI, physical examination)

[0299] Baseline disease burden was assessed using CT scans of the chest, abdomen, and pelvis plus other areas as appropriate with IV contrast. Participants who were intolerant of IV contrast had their CT scans performed with oral contrast and the reason for not using IV contrast was documented in the source document. Subsequent efficacy assessments during the study included radiographic imaging of all disease sites documented at baseline.

[0300] Magnetic resonance imaging is used to evaluate disease sites that cannot be adequately imaged using CT (in any case where MRI is required, it must be the imaging technique used to evaluate disease at baseline and all subsequent response assessments). For all other disease sites, MRI evaluation does not replace the required CT scans of the chest, abdomen, and pelvis unless CT scanning is contraindicated. Brain MRI is only required if clinically indicated. If MRI is contraindicated, a CT scan of the head is used.

[0301] For participants with palpable / superficial lesions, clinical disease assessment was performed by physical examination at baseline and throughout study drug treatment as clinically indicated. Irradiated or resected lesions were considered non-measurable and monitored only for disease progression.

[0302] Bone lesion assessment in prostate cancer

[0303] Bone disease in participants with prostate cancer was assessed according to PCWG3 (ie, assessing duration of response) as follows:

[0304] • Progression of soft tissue lesions measured by CT or MRI as defined in RECIST v1.1.

[0305] Progression of bone lesions as determined by bone scan and based on PCWG3 observations. Under these criteria, any bone progression must be confirmed by a follow-up scan ≥ 6 weeks later. The Week 8 scan (first post-treatment scan) should be used as the reference scan to which all subsequent scans are compared to determine progression. Bone scan is defined as one of the following:

[0306] -1. Participants observed to have ≥2 new bone lesions on the Week 8 scan compared to the baseline scan will need to undergo a confirmatory scan ≥6 weeks later and will fall into one of the following 2 categories:

[0307] -a. Participants whose confirmatory scan (performed ≥6 weeks later) shows ≥2 new lesions compared to the Week 8 scan (i.e., a total of ≥4 new lesions compared to the baseline scan) will be considered to have bone scan progression at Week 8.

[0308] -b. Participants whose confirmatory scan does not show ≥2 new lesions compared to the Week 8 scan will not be considered to have bone scan progression at that time. The Week 8 scan will be considered the reference scan to which subsequent scans will be compared.

[0309] -2. For participants who do not have ≥2 new bone lesions on the Week 8 scan compared to the baseline scan, if these new lesions are confirmed by a follow-up scan ≥6 weeks later, the first scan time point showing ≥2 new lesions compared to the Week 8 scan will be considered the bone scan progression time point.

[0310] Pharmacokinetic and immunogenicity evaluation

[0311] Collect venous blood samples for measuring serum concentrations of study drugs and anti-study drug antibodies. When the study drug is administered by peripheral vein, a blood sample is obtained from the contralateral arm of the arm infused with the study drug. At the time point of assessing serum concentrations and immunogenicity, collect blood once and divide the serum sample into separate aliquots. Samples collected for analysis of study drug serum concentrations and antibodies to the study drug can be used in addition to evaluate the safety or efficacy of solving problems arising during or after the study period, for further characterization of immunogenicity or for evaluating related biomarkers (e.g., the possible presence of soluble TMEFF2).

[0312] Populations used for analysis

[0313] For the purpose of analysis, the following groups were defined:

[0314] All-Treatment Analysis Group: This group consists of participants who received at least 1 dose of study drug. This analysis group will be considered primary and will be used for all safety and efficacy summaries.

[0315] DLT-evaluable analysis group: This group is a subgroup of the "All As-Treatment Analysis" group. Participants who received at least 75% of the planned doses of study drug during the DLT observation period will be included in this analysis.

[0316] Biomarker Analysis Group: This group consisted of all participants who received at least 1 dose of study drug and had at least 1 pre- or post-treatment biomarker measurement.

[0317] · Pharmacokinetic Analysis Group: This group consisted of all participants who received at least 1 dose of study drug and had at least 1 evaluable study drug concentration measurement.

[0318] Statistical analysis

[0319] Efficacy analysis

[0320] End point definition

[0321] PSA response rate (RR), defined as the proportion of participants with a PSA decrease of at least 30% or more from baseline. The maximum change at any time during the study for each participant was reported using a waterfall plot. To reflect changes in clinical status, participants were to remain on the study whenever possible until radiographic or symptomatic progression. The primary endpoint analysis was performed for the all-treated population, and PSA RR with 90% two-sided exact CIs are presented.

[0322] The overall response rate (ORR) was defined as the proportion of participants with a PR or better according to RECIST v1.1 response criteria without evidence of bone progression according to PCWG3. Response to treatment was assessed by the investigator.

[0323] Duration of response (DOR) was calculated from the date of initial documented response (PR or better) to the date of first documented evidence of disease progression as defined in PCWG3 or RECISTv1.1 response criteria or death from any cause, whichever occurred first. For participants who did not progress and were alive and responded to disease treatment (CR or PR), data were censored at the last disease assessment before starting any subsequent anticancer therapy.

[0324] Time to response (TTR) was defined as the time from the date of the first dose of study drug to the date of the first recorded response.

[0325] Analytical methods

[0326] The overall response rate was tabulated along with its two-sided 90% exact confidence interval. In addition, the number and percentage of participants in each response category were tabulated. For TTR, descriptive statistics were used to summarize the results, including the mean, median, standard deviation, and range of participants with a response. For DOR, descriptive summaries were performed using the Kaplan-Meier method.

[0327] Security Analysis

[0328] All safety analyses were performed on data from the "All Treatment Analysis Group". Baseline values ​​for safety assessments were defined as values ​​collected at the time closest to but prior to the start of the first study drug administration. Safety parameters to be assessed were the incidence, severity, and type of adverse events, clinically significant changes in participant physical examination findings, vital sign measurements, clinical laboratory and other clinical test results (e.g., ECG). Exposure to study drug and reasons for discontinuation of study drug were tabulated. Adverse events were summarized by system organ class, preferred term, worst grade experienced by the participant, and dose level. Safety was summarized by dose, route, and schedule as appropriate.

[0329] Adverse Events

[0330] The verbatim terms used by the investigator in the CRF to identify adverse events were coded using the Medical Dictionary for Regulatory Activities (MedDRA). Study drug-emergent adverse events were adverse events that had an onset during the study drug period or were the result of a pre-existing condition that worsened since baseline. All reported treatment-emergent adverse events were included in the analysis. For each treatment-emergent adverse event, the percentage of participants who experienced at least 1 occurrence of a given event was summarized by dose level / dose cohort.

[0331] For participants who died, discontinued study drug due to an adverse event, or experienced a severe or serious adverse event, provide a summary, listing, data set, or participant narrative, as appropriate. Listing of DLTs Use the DLT-evaluable analysis group. List DLTs and rates are summarized by major system organ class, preferred term, worst grade and type of adverse event, and dose level.

[0332] Example 4. Safety and initial efficacy of investigational drugs for the treatment of metastatic castration-resistant prostate cancer (mCRPC) Step clinical activity

[0333] The study drug was administered subcutaneously (SC) at a dose of 0.3 mg to 6 mg once a week (Q1W) and 2 mg to 6 mg once every 2 weeks (Q2W). A total of 9 dose levels were tested (Q1W: 300 ug, 1 mg, 1.5 mg, 3 mg and 6 mg; Q2W: 2 mg, 3 mg, 4 mg and 6 mg), and a total of 82 patients were given.

[0334] Pharmacokinetics

[0335] Preliminary pharmacokinetics

[0336] Preliminary PK of the study drug was evaluated in 71 subjects from the ongoing FIH study. Preliminary data after SC injection of the study drug are available in a dose range of 0.3 mg to 6.0 mg Q1W (cohorts 1 to 4 and 9) and a dose range of 2.0 mg to 6.0 mg every 2 weeks (Q2W; cohorts 5 to 8). Baseline characteristics are shown in Table 6.

[0337] Table 6. Baseline characteristics

[0338]

[0339]

[0340] Abbreviations: AR, antigen receptor; ECOG, Eastern Cooperative Oncology Group; PSA, prostate-specific antigen; SD, standard deviation.

[0341] a including the lungs, liver, adrenal glands, and central nervous system, b Including pelvic and extrapelvic areas, c The PSA of one patient was not entered into the database at the time of data censoring.

[0342] After the first SC injection of study drug ranging from 0.3 mg to 6.0 mg, C max and AUC 0-168h Slow increases in mean serum concentrations of study drug were observed for all dose levels ( Figure 3 ). Median T max Typically, it occurs between 72 and 168 hours (Table 7).

[0343] Table 7. Summary of Pharmacokinetic Parameters of Study Drugs after First SC Injection

[0344]

[0345]

[0346] Abbreviations: AUC(168h) = area under the serum concentration-time curve from 0 to 168 hours; AUC(336h) = area under the serum concentration-time curve from 0 to 336 hours; Cmax = maximum serum concentration; dn = dose normalized to 1 mg; n = number of subjects; QXW = every X weeks; SC = subcutaneous; SD = standard deviation; Tmax = time to maximum concentration.

[0347] Mean ± SD is shown if there were ≥ 3 evaluable subjects, and only mean is shown if there were < 3 evaluable subjects. Subjects with missing concentrations near expected Cmax were excluded from the descriptive statistics.

[0348] a For AUC(168h) and AUC(168h,dn), n=4.

[0349] b For AUC(168h), AUC(336h), AUC(168h,dn) and AUC(336h,dn), n=9.

[0350] c For AUC(336h) and AUC(336h,dn), n=9.

[0351] d For AUC(168h), AUC(336h), AUC(168h,dn) and AUC(336h,dn), n=4.

[0352] After multiple SC injections of study drug, steady-state was achieved after the seventh SC injection when given Q1W and the fourth SC injection when given Q2W ( Figure 4 and Figure 5 At steady state, after Q1W dosing, serum trough concentrations (C trough ) were increased in an approximately dose-proportional manner, whereas for the 2.0 mg to 4.0 mg Q2W cohorts, C trough The values ​​appear comparable (Table 8). The mean accumulation ratios of AUC were approximately 4.9 and 1.6 for Q1W dosing and Q2W dosing, respectively.

[0353] Table 8. Drug changes after the seventh SC injection given Q1W and the fourth SC injection given Q2W. Summary of Kinetic Parameters

[0354]

[0355] Abbreviation: AR AUC = Cumulative ratio; C trough = serum trough concentration; d n= dose normalized to 1 mg; n = number of subjects; QXW = every X weeks; SC = subcutaneous; SD = standard deviation.

[0356] C trough is the concentration observed immediately before the start of the dosing interval.

[0357] Mean ± SD is shown if there were ≥ 3 evaluable subjects, and only the mean is shown if there were < 3 evaluable subjects. a For AR AUC , n=1.

[0358] b For AR AUC , n=2.

[0359] c For AR AUC , n=1.

[0360] d For AR AUC , n = 2

[0361] Effect of body weight on pharmacokinetics

[0362] The effect of body weight on pharmacokinetics is shown in Figure 6 and Fig. 7A and Figure 7B Medium. Low body weight subjects may have lower volume of distribution and clearance compared with high body weight subjects, resulting in higher PK exposure.

[0363] effect

[0364] Efficacy / Pharmacodynamics

[0365] Preliminary PD data from 73 subjects were analyzed, including 38 in the Q1W SC dosing cohort and 35 in the Q2W SC dosing cohort. A maximum prostate-specific antigen (PSA) reduction of at least 50% was reported in 8 subjects: 6 subjects in the Q1W SC dosing cohort and 2 subjects in the Q2W SC dosing cohort ( Figure 8 ). An additional 7 subjects achieved a maximum PSA reduction of at least 30%, 4 of whom were dosed Q1W and 3 of whom were dosed Q2W.

[0366] Among the 38 subjects with available data, the maximum percentage reduction in the sum of target lesion diameters according to RECIST 1.1 was at least 30% (partial response or better) for 7 subjects ( Fig. 9 ); 5 subjects from the Q1W SC dosing cohort and 2 subjects from the Q2W SC dosing cohort. Confirmed partial responses were observed in 5 patients.

[0367] Summary of Preliminary Efficacy Data

[0368] Preliminary efficacy data (PSA and RECIST responses) are summarized in Table 9.

[0369] Table 9. Preliminary efficacy observed at the dose levels examined .

[0370]

[0371]

[0372] *Two patients had intra-patient dose escalation to 3 mg QW; one before confirmed PR and one after confirmed PR. **1 patient had uPR before dose reduction to 3 mg Q2W due to an AE (Day 127). PR was confirmed during a subsequent scan (Day 175). Same patient had PSA50 before dose reduction; another patient had PSA50 after dose reduction to 3 mg QW due to G3 decline (DLT) on Day 16.

[0373] Safety and Tolerability

[0374] Nature and frequency of adverse events

[0375] At least 10% of the subjects experienced AEs that occurred during treatment. Overall, 71 of 73 subjects (97.3%) reported at least 1 TEAE (Tables 10 and 11). The most commonly reported TEAEs were fatigue (45.2%), decreased appetite (43.8%), injection site erythema (37.0%), anemia (32.9%), back pain (24.7%), arthralgia (21.9%), and nausea (19.2%). The incidence of common TEAEs did not differ significantly between doses.

[0376] Table 10. Safety features

[0377]

[0378]

[0379] AE, adverse event; AST, aspartate aminotransferase; COVID, coronavirus disease; Q1W, weekly; Q2W, biweekly; SC, subcutaneous; TEAE, treatment-emergent adverse event.

[0380] Table 11. Safety features

[0381]

[0382] AE, adverse event; AST, aspartate aminotransferase; COVID, coronavirus disease; Q1W, weekly; Q2W, biweekly; SC, subcutaneous; TEAE, treatment-emergent adverse event.

[0383] Serious adverse events (SAEs) during treatment

[0384] Overall, serious adverse events (SAEs) that emerged during treatment were reported in 31 (42.5%) subjects (17 subjects in the Q1W SC dosing cohort and 14 subjects in the Q2W SC dosing cohort) in the clinical trial studies (Table 12). SAEs that the investigator considered to be related to study drug are described below. In the 1.0 mg and 3.0 mg study drug Q1W SC dosing cohorts, 1 subject each experienced an SAE of Grade 2 vomiting. In the 1.5 mg study drug Q1W SC dosing cohort, 1 subject experienced an SAE of Grade 2 CRS. In the 6.0 mg study drug Q1W SC dosing cohort, 1 subject experienced an SAE of Grade 3 asthenia, and 1 subject experienced SAEs of Grade 2 impaired balance, Grade 2 confusional state, and Grade 3 falls (TEAEs of falls occurred after the study drug dose was reduced to 3.0 mg). The subject also experienced an SAE of Grade 3 orthostatic hypotension when treatment with study drug was discontinued. In the 4.0 mg Q2W SC cohort, 1 subject experienced a Grade 3 SAE of increased aspartate aminotransferase (AST), and in the 6.0 mg study drug Q2W SC cohort, 1 subject experienced a SAE of Grade 2 pyrexia and Grade 2 confusional state, and 1 subject experienced a Grade 1 SAE of pyrexia.

[0385] Dose-limiting toxicity

[0386] During the study, 2 subjects experienced a total of 3 DLTs. One subject in the 6.0 mg study drug Q1W SC cohort experienced a DLT of Grade 3 fall, and one subject in the 6.0 mg study drug Q2W SC cohort experienced a DLT of Grade 3 orthostatic hypotension. At the time of reporting, the TEAE of orthostatic hypotension was reported as unrecovered / unresolved, and study treatment was withdrawn. Ten days later, the same subject experienced a Grade 3 DLT of syncope, which has now recovered / resolved.

[0387] Grade 3 or higher treatment-emergent adverse events

[0388] Grade 3 or higher TEAEs were reported in 40 (54.8%) subjects in the study. Grade 3 or higher TEAEs reported in more than 1 subject (≥5% incidence) were anemia (13 subjects, 18%), fatigue (8 subjects, 11%), lymphocytopenia (5 subjects, 7%), asthenia (4 subjects, 6%) and hypertension (4 subjects, 6%), back pain (3 subjects) and arthralgia, AST increased, atrial fibrillation, decreased appetite, dizziness, hypotension, muscle weakness, orthostatic hypotension, pelvic pain, spinal cord compression, and syncope (2 subjects each).

[0389] Seven patients discontinued treatment due to at least 1 TEAE (increased blood creatine [n=1], decreased weight [n=1], arthralgia [n=1], back pain [n=1], orthostatic hypotension [n=2], hypotension [n=1], pulmonary edema [n=1]). Of the 7 patients, 2 patients discontinued the study due to treatment-related AEs (both with orthostatic hypertension).

[0390] Cytokine release syndrome occurred in 4 of 73 patients (6%) and was considered grade 1 or 2.

[0391] Six deaths occurred during the study (disease progression [n=4]; pulmonary edema [n=1] and COVID-19 [n=1]) and were considered unrelated to study drug. Dose-limiting cytotoxicity was reported in two patients (both grade 3; falls requiring hospitalization [n=1]; orthostatic hypotension and syncope [n=1]).

[0392] In conclusion, in patients with mCRPC with PSA50 and RECIST responses, the study drugs demonstrated a tolerable safety profile at certain doses.

Claims

1. A method of treating or slowing the progression of cancer in a subject, the method comprising administering to the subject at least one dose of an anti-TMEFF2xCD3 bispecific antibody, wherein the administration is subcutaneous.

2. The method of claim 1, wherein the subcutaneous dose is about 0.3 mg to about 6 mg of the bispecific antibody.

3. The method of claim 2, wherein the subcutaneous dose is administered once a week or once every two weeks at a dose of about 1.5 mg.

4. The method of any one of claims 1 to 3, wherein the cancer is metastatic castration-resistant prostate cancer (mCRPC).

5. The method according to claim 4, wherein the anti-TMEFF2xCD3 bispecific antibody comprises a first binding domain that binds to TMEFF2 and a second binding domain that binds to CD3, wherein a. the first binding domain that binds to TMEFF2 comprises a HCDR of a VH having an amino acid sequence of SEQ ID NO: 13 and a LCDR of a VL having an amino acid sequence of SEQ ID NO: 14, and the second binding domain that binds to CD3 comprises a HCDR of a VH having an amino acid sequence of SEQ ID NO: 17 and a LCDR of a VL having an amino acid sequence of SEQ ID NO: 18; b. the first binding domain that binds to TMEFF2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 1, 2, 3, 4, 5 and 6, respectively, and the second binding domain that binds to CD3 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 7, 8, 9, 10, 11 and 12, respectively; c. the first binding domain that binds to TMEFF2 comprises VH and VL of SEQ ID NOs: 13 and 14, respectively, and the second binding domain that binds to CD3 comprises VH and VL of SEQ ID NOs: 17 and 18, respectively; and / or d. The first binding domain that binds to TMEFF2 comprises HC1 and LC1 of SEQ ID NOs: 15 and 16, respectively; and the second binding domain that binds to CD3 comprises HC2 and LC2 of SEQ ID NOs: 19 and 20, respectively.

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