Methods for treating cancer with anti-TMEFF2×CD3 bispecific antibodies

Abstract

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

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Patent Application No. 63 / 405,188, filed September 9, 2022, and U.S. Patent Application No. 63 / 420,146, filed October 28, 2022, the disclosures of each of which are incorporated herein by reference in their entireties.

[0002] (Reference to electronically submitted sequence listing) This application contains a Sequence Listing that has been submitted electronically in XML file format, which is incorporated herein by reference in its entirety. A copy of this XML was created on July 7, 2023, has the file name JBI6759WOPCT1_SL, and is 32,707 bytes in size. [Background technology]

[0003] Prostate cancer is the second most common cancer and the sixth leading cause of cancer-related deaths among men worldwide. Approximately 1.1 million new cases and 300,000 deaths are reported annually worldwide, accounting for 4% of all cancer deaths. It is estimated that one in six men will be diagnosed with the disease during their lifetime. Prostate cancer risk is strongly correlated with age, with approximately three-quarters of cases occurring in men over 65 years of age, with the highest number of cases occurring between 70 and 74 years of age. Autopsy data estimate that approximately half of men in their 50s and 80% of men aged 80 years have histological evidence of cancer in the prostate. In early-stage disease, the five-year survival rate is nearly 100%. However, if the cancer metastasizes, the five-year survival rate drops to 28%, and there remains a need for effective treatments for advanced prostate cancer.

[0004] TMEFF2, also known as TENB2, HPP1, or tomoregulin-2, is a conserved membrane-bound proteoglycan. It is a 41-kDa transmembrane protein consisting of two follistatin-like domains (FS1 and FS2) and one epidermal growth factor (EGF)-like domain.

[0005] TMEFF2 expression is maintained throughout all stages of prostate cancer disease, with limited expression in extraprostatic tissues. The direct correlation between increased TMEFF2 expression levels and aggressive tumors strongly suggests that TMEFF2 is associated with disease progression and potential androgen independence in 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 are observed in both the brain and prostate. Low levels of transcript and protein expression are detected in the retinal and conjunctival epithelium, and transcripts, but not protein, are detected in the nonpigmented ciliary epithelium of the eye and ganglion cells of the myenteric plexus of the colon.

[0006] Current treatments for prostate cancer include surgery, radiation therapy, and hormone therapy. Treatments aimed at eradicating tumors fail in 30% of men, who develop recurrent disease. This disease usually first manifests as elevated plasma prostate-specific antigen (PSA) levels, followed by invasion to distant sites. Because prostate cancer cells depend on the androgen receptor (AR) for growth and survival, men with advanced prostate cancer are treated with drugs that block testosterone production (e.g., GnRH agonists) alone or in combination with antiandrogens (e.g., bicalutamide), which antagonize the effects of any residual testosterone on the AR. These treatments reduce serum testosterone to castrate levels, which generally slows disease progression for some period of time. However, androgen ablation is usually effective for a limited period of time, and prostate cancer progresses and regains its growth potential even with low levels of circulating androgens. Therefore, most patients ultimately die from cancer regrowth.

[0007] There remains a high need for improved treatments and effective therapies for patients with advanced prostate cancer whose disease has proven resistant to current therapies. Summary of the Invention

[0008] Provided herein is a method 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.

[0009] In some embodiments, the cancer is metastatic castration-resistant prostate cancer (mCRPC), including those with small cell or adenocarcinoma with neuroendocrine features.

[0010] In some embodiments, the mCRPC subject has received prior treatment with at least one prior novel AR-targeted therapy or chemotherapy.

[0011] In some embodiments, administration is subcutaneous and the dose is about 0.3 mg to about 6.0 mg of an anti-TMEFF2xCD3 bispecific antibody of the disclosure.

[0012] In some embodiments, 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 an anti-TMEFF2xCD3 bispecific antibody of the disclosure.

[0013] In some embodiments, administration is subcutaneous and doses are given once weekly (Q1W).

[0014] In some embodiments, administration is subcutaneous and doses are given once every two weeks (Q2W).

[0015] In some embodiments, administration is subcutaneous and the dose is from about 0.3 mg to about 6.0 mg given Q1W.

[0016] In some embodiments, 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 given Q1W.

[0017] In some embodiments, administration is subcutaneous and the dose is 0.3 mg, 1.0 mg, 1.5 mg, 3.0 mg, or 6.0 mg given Q1W.

[0018] In some embodiments, administration is subcutaneous and the dose is from about 0.3 mg to about 6.0 mg given Q2W.

[0019] In some embodiments, 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 given Q2W.

[0020] In some embodiments, administration is subcutaneous and the dose is 2.0 mg, 3.0 mg, 4.0 mg, or 6.0 mg given Q2W.

[0021] In some embodiments, the methods of the present disclosure include repeating the administering step two or more times.

[0022] In some embodiments, corticosteroid, antihistamine, and antipyretic premedication are administered prior to the first dose of an anti-TMEFF2xCD3 bispecific antibody of the present disclosure to minimize risks associated with cytokine release syndrome (CRS) and infusion-related reactions (IRR). Premedication doses or schedules are or can be reduced or omitted for subsequent doses. For subjects who experience Grade 2 or higher CRS or IRR, pretreatment with corticosteroids is or can be administered for at least one subsequent dose administered to the subject.

[0023] In some embodiments, the anti-TMEFF2xCD3 antibody administered in the disclosed therapeutic methods 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 an HCDR of a VH having the amino acid sequence of SEQ ID NO: 13 and an LCDR of a VL having the amino acid sequence of SEQ ID NO: 14, and the second binding domain that binds to CD3 comprises an HCDR of a VH having the amino acid sequence of SEQ ID NO: 17 and an LCDR of a VL having the amino acid sequence of SEQ ID NO: 18, and (b) the first binding domain that binds to TMEFF2 comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively. , LCDR1, LCDR2, and LCDR3, 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. [Brief explanation of the drawings]

[0024] [Figure 1] A schematic overview of the test is shown. [Figure 2] PK model predicted mean test drug serum concentrations in humans after a single IV dose of 4u / kg or SC dose of 300µg, assuming a median body weight of 75kg. Abbreviations: F = bioavailability, IV = intravenous, MABEL = minimum expected biological effect level, SC = subcutaneous. [Figure 3] 1 shows the mean serum concentration-time curves of study drug after the first SC injection of study drug. Abbreviations: n = total number of subjects in each cohort, SC = subcutaneous. [Figure 4]

[0033] Figure 1 shows the mean serum concentration-time curves of study drug after Q1W SC dosing. Abbreviations: n = total number of subjects in each cohort, SC = subcutaneous. [Figure 5]

[0033] Figure 1 shows the mean serum concentration-time curves of study drug after Q2W SC dosing. Abbreviations: n = total number of subjects in each cohort, SC = subcutaneous. [Figure 6] Subjects with lower body weights tend to have higher exposures when compared to subjects with higher body weights. [Figure 7A] 1 shows the relationship between test drug volume distribution and body weight. [Figure 7B] 1 shows the relationship between test drug clearance and body weight. [Figure 8] Figure 1 shows a waterfall plot of maximum percent reduction from baseline in PSA. Abbreviations: PSA = prostate-specific antigen, Q1W = weekly, Q2W = every 2 weeks, SC = subcutaneous. Baseline represents a 30% and 50% reduction. Increases greater than 100% were set at 100%. [Figure 9] A waterfall plot of maximum percent reduction from baseline in target lesion sum diameter is shown. Abbreviations: Q1W = weekly, Q2W = every 2 weeks, SC = subcutaneous, SoD = sum diameter. Baseline represents a 30% reduction. Increases greater than 100% were set at 100%. [Figure 10] Test drug concentrations after different dosing regimens are shown. [Figure 11A] 1 shows the effect of test drug dosing regimens on CD8+ T cell infiltration. [Figure 11B] 1 shows the effect of test drug dosing regimens on CD8+ T cell infiltration. [Figure 12A] 1 shows the effect of test drug dosing regimens on CD4+ T cell infiltration. [Figure 12B] 1 shows the effect of test drug dosing regimens on CD4+ T cell infiltration. [Figure 13A] Figures 13A-C show the effect of test drug dosing regimens on T cell activation and proliferation. CD4+ Prostate Infiltration Lymphocyte (PIL) cell activation markers. [Figure 13B] Figures 13A-C show the effect of test drug dosing regimens on T cell activation and proliferation. CD4+ Prostate Infiltration Lymphocyte (PIL) cell activation markers. [Figure 13C] Figures 13A-C show the effect of test drug dosing regimens on T cell activation and proliferation. CD4+ Prostate Infiltration Lymphocyte (PIL) cell activation markers. [Figure 13D] Figures 13D-13F show the effect of test drug dosing regimens on T cell activation and proliferation. CD8+ prostate infiltrating lymphocyte (PIL) cell activation markers. [Figure 13E] Figures 13D-13F show the effect of test drug dosing regimens on T cell activation and proliferation. CD8+ prostate infiltrating lymphocyte (PIL) cell activation markers. [Figure 13F] Figures 13D-13F show the effect of test drug dosing regimens on T cell activation and proliferation. CD8+ prostate infiltrating lymphocyte (PIL) cell activation markers. [Figure 14A] Figures 14A-C show the effect of test drug dosing regimens on suppressor T cell markers: CD4+ prostate infiltrating lymphocyte (PIL) cell activation markers. [Figure 14B] Figures 14A-C show the effect of test drug dosing regimens on suppressor T cell markers: CD4+ prostate infiltrating lymphocyte (PIL) cell activation markers. [Figure 14C] Figures 14A-C show the effect of test drug dosing regimens on suppressor T cell markers: CD4+ prostate infiltrating lymphocyte (PIL) cell activation markers. [Figure 14D] Figures 14D-14F show the effect of test drug dosing regimens on suppressor T cell markers: CD8+ prostate infiltrating lymphocyte (PIL) cell activation markers. [Figure 14E]Figures 14D-14F show the effect of test drug dosing regimens on suppressor T cell markers: CD8+ prostate infiltrating lymphocyte (PIL) cell activation markers. [Figure 14F] Figures 14D-14F show the effect of test drug dosing regimens on suppressor T cell markers: CD8+ prostate infiltrating lymphocyte (PIL) cell activation markers. [Figure 15A] 15A-15B show the effect of test drug dosing regimens on suppressor T cell markers: CD4+ prostate infiltrating lymphocyte (PIL) cell activation markers. [Figure 15B] 15A-15B show the effect of test drug dosing regimens on suppressor T cell markers: CD4+ prostate infiltrating lymphocyte (PIL) cell activation markers. [Figure 15C] Figure 15C shows the effect of test drug dosing regimens on suppressor T cell markers: CD8+ prostate infiltrating lymphocyte (PIL) cell activation markers. [Figure 16A] 1 shows the effect of test drug dosing regimens on bone marrow cell infiltration. [Figure 16B] 1 shows the effect of test drug dosing regimens on bone marrow cell infiltration. [Figure 17A] 1 shows the effect of test drug dosing regimens on bone marrow cell infiltration. [Figure 17B] 1 shows the effect of test drug dosing regimens on bone marrow cell infiltration. [Figure 17C] 1 shows the effect of test drug dosing regimens on bone marrow cell infiltration. DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0025] The methods of the present disclosure may be understood more readily by reference to the following detailed description taken in conjunction with the accompanying drawings, which form a part of this disclosure: It is to be understood that the methods of the present disclosure are not limited to the specific methods described and / or illustrated herein, and further, the terminology used herein is for the purpose of describing particular embodiments, by way of example only, and is not intended to limit the methods claimed.

[0026] Unless otherwise stated, any description of possible mechanisms or modes of operation or reasons for improvement is intended to be illustrative only, and the methods of the present disclosure are not limited by the merits or demerits of the proposed mechanisms or modes of operation or reasons for improvement.

[0027] When a range of numerical values ​​is recited or established herein, the range includes its endpoints and all individual integers and fractions within the range, and also includes each of the narrower ranges formed by all the various possible combinations of these endpoints and internal integers and fractions, each of which forms a subgroup of the larger group of values ​​within the recited range, as if each of the narrower ranges were explicitly recited. When a range of numerical values ​​is recited herein as being greater than the recited value, the range is nevertheless finite, with its upper limit defined by a value operable within the context of the method described herein. When a range of numerical values ​​is recited herein as being less than the recited value, the range is nevertheless defined by a lower limit defined by a non-zero value. It is not intended that the scope of the method be limited to the specific values ​​recited when defining the range. All ranges are inclusive and combinable.

[0028] When values ​​are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. Reference to a particular numerical value is intended to include at least that particular value unless the context dictates otherwise.

[0029] It should be understood that certain features of the methods of the present disclosure, which are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the methods of the present disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any subcombination.

[0030] As used herein, the singular forms "a," "an," and "the" are intended to include plurals.

[0031] Various terms relating to aspects of the present specification are used throughout the specification and claims. Unless otherwise indicated, such terms shall be given their ordinary meaning in the art. Other specifically defined terms shall be construed in a manner consistent with the definition provided herein.

[0032] The term "about" is used to encompass a variation of no more than ±10%, a variation of no more than ±5%, a variation of no more than ±1%, a variation of no more than ±0.5%, or a variation of no more than ±0.1% from the stated value.

[0033] The transitional phrases "comprising," "consisting essentially of," and "consisting of" are intended to connote their generally accepted meanings in patent language, i.e., (i) "comprising" is synonymous with "comprising," "containing," or "characterized by" and is inclusive or open-ended, not excluding additional, unrecited elements or method steps; (ii) "consisting" 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 claimed disclosure. Embodiments described with the phrase "comprising" (or its equivalents) also provide as embodiments embodiments described separately with "consisting of" and "essentially consisting of." Embodiments described with the phrase "consisting essentially of" (or its equivalents) also provide as embodiments described separately with "consisting of."

[0034] Treatment method Provided herein is a method 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.

[0035] A "subject" or "patient" includes any human or non-human animal. A "non-human animal" includes 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" may be used interchangeably herein. In some embodiments, a subject or patient is a human. In particular, a subject according to the present invention is a human. In some embodiments, a subject according to the present invention has or is suspected of having prostate cancer.

[0036] "Treating," "treating," or "treatment" of a subject with a pathological condition such as cancer refers to the effect of alleviating the condition by killing cancer cells, but also hereinafter refers to achieving one or more of: reducing the severity and / or duration of the disorder; delaying the progression of the disorder; slowing the progression of the disorder; inhibiting the worsening of symptoms characteristic of the disorder being treated; limiting or preventing the recurrence of the disorder in a subject who previously had the disorder; or limiting or preventing the recurrence of symptoms in a subject who was previously symptomatic for the disorder. Treatment as a preventative measure (i.e., prophylaxis) is also included.

[0037] As used herein, the terms "delaying the progression of" and "slowing the progression of" are intended to include (a) delaying or slowing the onset of one or more symptoms or complications of a disease, condition, or disorder; (b) delaying or slowing the onset 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 later or more severe form of the disease, condition, or disorder.

[0038] As used in accordance with the present disclosure, the term "treating" means treating a mammal with prostate cancer by providing an effective amount of an anti-TMEFF2xCD3 bispecific antibody for the purpose of reducing or eradicating cancer cells and / or prolonging survival of the mammal.

[0039] As used herein, the terms "effective amount" and "therapeutically effective amount" refer to an amount sufficient to achieve a concentration of the compound capable of preventing or slowing the disease being treated. Such concentrations can be routinely determined by one skilled in the art. The amount of polypeptide actually administered is typically determined by a physician or veterinarian in light of the relevant circumstances (including the condition being treated, the selected route of administration, the actual compound being administered, the age, weight, and response of the patient, the severity of the subject's symptoms, etc.). It will also be understood by one skilled in the art that the dosage may depend on the stability of the antibody being administered.

[0040] The therapeutically effective amount may vary depending on factors such as the disease state, age, sex, and weight of the individual, the patient's health condition, the duration of treatment, the nature of concomitant therapy (if any), the particular formulation used, the structure of the compound or its derivatives, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual. The therapeutically effective amount of the antibody polypeptide to be administered will also depend on the type and severity of the cancer being treated and the route of administration of the antibody polypeptide or pharmaceutical composition of the antibody polypeptide.

[0041] In some embodiments, the subjects to be treated include those with metastatic castration-resistant prostate cancer (mCRPC), including adenocarcinoma with small cell or neuroendocrine features.

[0042] In some embodiments, the efficacy of treating prostate cancer can be assessed using a CT scan, MRI, or whole body PET scan, or by measuring PSA levels.

[0043] In some embodiments, the efficacy of prostate cancer treatment can be evaluated by measuring the progression of soft tissue lesions by CT or MRI using the RECIST criteria. As used herein, the term "Response Evaluation Criteria In Solid Tumors (RECIST)" refers to a set of published rules that define when a cancer patient improves ("responds"), stays the same ("stable"), or worsens ("progresses") during treatment. The original criteria were published in February 2000 by an international collaboration including the European Organization for Research and Treatment of Cancer (EORTC), the National Cancer Institute (NCI) of the United States, and the National Cancer Institute of Canada Clinical Trials Group. RECIST 1.1, published in January 2009, is an update to the original criteria. Typically, one skilled in the art will conclude that the disease has progressed (and therefore the patient is or becomes refractory to treatment) when the sum of the longest diameters of the target lesions increases by at least 20% (referenced to the smallest sum longest diameter recorded since treatment began or the appearance of one or more new lesions) by conventional imaging methods such as computed tomography (CT).

[0044] In some embodiments, the efficacy of treating prostate cancer can be evaluated according to PCWG3 (Prostate Cancer Working Group 3) criteria.

[0045] The term "safety," in relation to a dose, dosing regimen, or treatment method with an anti-TMEFF2xCD3 bispecific antibody of the present disclosure, refers to a relatively low or reduced frequency and / or a low or reduced severity of treatment-emergent adverse events (also referred to as AEs or TEAEs) from an conducted clinical trial, e.g., a Phase 1 clinical trial, compared to a standard of care or another comparator. An adverse event is an untoward medical occurrence in a patient administered a medicinal product, including adverse vital signs (heart rate, systolic and diastolic blood pressure, body temperature), adverse standard laboratory tests (hematology, clinical chemistry, urinalysis, lipids, coagulation), allergic reaction / hypersensitivity, adverse local injection site reaction, or adverse EKG. In particular, when referring to a dose, dosing regimen, or treatment with an anti-TMEFF2xCD3 bispecific antibody of the present disclosure, safety refers to a relatively low or reduced frequency and / or a low or reduced severity of adverse events associated with the administration of the antibody, when the cause is considered possible, probable, or highly likely to be due to the use of the anti-TMEFF2xCD3 bispecific antibody.

[0046] Administration route The methods of the present invention may include any means of administration that achieves the intended purpose. Any suitable route of administration may be used to administer the antibody polypeptide or pharmaceutical composition used in the therapeutic methods of the present disclosure. For example, administration may be achieved by several different routes, including, but not limited to, subcutaneous.

[0047] Prostate cancer The disclosed method can treat any cancer associated with TMEFF2.An exemplary cancer associated with TMEFF2 is prostate cancer.As used herein, the term "cancer" refers to the abnormal proliferation of cells, which tend to grow uncontrollably and sometimes tend to invade (spread).

[0048] In some embodiments, the methods of the present disclosure can treat or slow the progression of prostate cancer.

[0049] As used herein, the term "prostate cancer" refers to histologically or cytologically confirmed adenocarcinoma of the prostate, as well as neuroendocrine prostate cancer, late-stage manifestations of prostate cancer, and hormone-refractory subtypes of prostate cancer resulting from prostate cancer treatment. The course of prostate cancer from diagnosis to death is best classified as a series of clinical stages based on the extent of disease, hormonal status, and the presence or absence of detectable metastases, localized disease, elevated prostate-specific antigen (PSA) levels without detectable metastases after radiation therapy or surgery, and clinical metastases in the non-castrated or castrated stages.

[0050] In the early stages of prostate cancer, the cancer is confined to the prostate gland. At these early stages, treatment typically involves either surgical removal of the prostate gland or radiation therapy to the prostate, or in some patients, confirmation without active therapeutic intervention. In the early stages when prostate cancer is localized and requires intervention, surgery or radiation therapy is curative by eliminating cancer cells. However, in approximately 30% of cases, these treatments fail, and the prostate cancer continues to progress, typically as evidenced by rising PSA levels. Therefore, a significant proportion of patients treated with surgery, radiation, or a combination of both will have recurrent disease, which can lead to metastasis formation—a fatal phenotype of the disease, especially in high-risk groups. Men whose prostate cancer progresses after these early treatment regimens are said to have advanced or recurrent prostate cancer.

[0051] The term "locally advanced prostate cancer" refers to prostate cancer that has spread through the prostate capsule, with all active cancer cells confined to the prostate and associated 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. Surgery is generally not recommended for patients with locally advanced disease, and these patients have substantially less favorable outcomes compared with patients with clinically localized (organ-confined) prostate cancer. Locally advanced disease is clinically identified by palpable evidence of induration beyond the outer border of the prostate or asymmetry or induration above the prostate base. Locally advanced prostate cancer is currently diagnosed pathologically after radical prostatectomy when the tumor invades or penetrates the prostate capsule, extends to the surgical margin, or invades the seminal vesicles.

[0052] The term "high-risk localized prostate cancer" refers to locally advanced prostate cancer that is likely to develop metastasis or recurrent disease after primary therapy with curative intent. In some embodiments, a high risk for metastasis formation is defined as a prostate specific antigen doubling time (PSADT) of less than 12 months, or less than 11 months, or less than 10 months, or less than 9 months, or less than 8 months, or less than 7 months, or less than 6 months, or less than 5 months, or less than 4 months, or less than 3 months, or less than 2 months, or less than 1 month. In some embodiments, a high risk for metastasis formation is defined as a prostate specific antigen doubling time (PSADT) of less than 10 months. In some embodiments, a high risk for metastasis formation is defined as having a high Gleason score or bulky tumors.

[0053] The terms "metastatic prostate cancer" and "metastatic disease" refer to prostate cancer that has spread to regional lymph nodes or distant sites, including stage D disease under the AUA system and stage TxNxM+ disease under the TNM system. As with locally advanced prostate cancer, surgery is generally not indicated for patients with metastatic disease, and hormonal (androgen ablation) therapy or androgen-deprivation therapy (ADT) is the preferred treatment. Patients with metastatic prostate cancer eventually develop an androgen-refractory state within 12 to 18 months of initiating treatment, and approximately half of these patients die within the next six months. The most common site of prostate cancer metastasis is bone. Prostate cancer bone metastases are characteristically osteoblastic rather than osteolytic (i.e., result in net bone formation). Bone metastases are most frequently found in the spine, followed by the femur, pelvis, rib cage, skull, and humerus. Other common sites of metastasis include lymph nodes, lungs, liver, and brain. Metastatic prostate cancer is typically diagnosed by open or laparoscopic pelvic lymphadenectomy, whole-body radionuclide scan, skeletal radiography, and / or bone lesion biopsy.

[0054] The androgen receptor (AR) is a member of the steroid and nuclear receptor superfamily, whose function is regulated by androgen binding. AR is primarily expressed in androgen target tissues, such as the prostate, skeletal muscle, liver, and central nervous system (CNS), with highest expression levels in the prostate, adrenal glands, and epididymis. AR can be activated by binding of endogenous androgens, such as testosterone and 5-dihydrotestosterone (5a-DHT). Upon androgen activation, AR mediates target gene transcription and regulates the growth and differentiation of prostate epithelial cells. AR signaling is important for the development and maintenance of male reproductive organs, including the prostate, as evidenced by the failure of prostate development or prostate cancer progression in genetic males with loss-of-function AR mutations and in mice genetically engineered to lack AR. This dependence of prostate cells on AR signaling continues during neoplastic transformation.

[0055] The term "androgen deprivation therapy (ADT)" refers to the reduction of androgen levels in prostate cancer patients to castrate levels of testosterone (less than 50 ng / dL). ADT includes surgical castration (orchiectomy) and / or administration of gonadotropin-releasing hormone (luteinizing hormone-releasing hormone, also known as an LHRH agonist or antagonist) to humans. Examples of LHRH agonists include, but are not limited to, goserelin acetate, histrelin acetate, leuprolide acetate, and triptorelin palmoate.

[0056] Antiandrogens are useful in treating early-stage prostate cancer. These treatments reduce serum testosterone to castration levels, which generally slows disease progression for some period of time. However, prostate cancer often progresses to a "hormone-refractory" stage, where the disease progresses in the presence of continued androgen ablation or antiandrogen therapy, and most patients ultimately die from cancer regrowth. Cases of antiandrogen withdrawal syndrome have also been reported after long-term treatment with antiandrogens. Molecular profiling studies of castration-resistant prostate cancer generally show increased androgen receptor (AR) expression, which may result from AR gene amplification or other mechanisms.

[0057] The term "castration-sensitive prostate cancer" refers to cancer that responds to androgen deprivation therapy (ADT), either as localized disease, biochemical relapse, or in the metastatic setting. Castration-sensitive prostate cancer is classified as non-metastatic or metastatic, depending on whether the prostate cancer has spread to other parts of the body.

[0058] 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 parts of a man's body, and that responds to androgen deprivation therapy (ADT).

[0059] The term "non-metastatic castration-sensitive prostate cancer" refers to cancer in men that has not spread (metastasized) and that responds to androgen deprivation therapy (ADT). In some embodiments, non-metastatic castration-sensitive prostate cancer is assessed by bone scan and computed tomography (CT), magnetic resonance imaging (MRI) scan, or positron emission tomography (PET).

[0060] The term "CRPC" or "castration-resistant prostate cancer" refers to prostate cancer that continues to grow despite the suppression of male hormones, which stimulate 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 spread to other parts of the body. Castration-resistant prostate cancer (CRPC) is a terminal phenotype, and almost all patients die from prostate cancer. Interestingly, while a small proportion of CRPC cases circumvent the need for AR signaling, most CRPC cases retain their lineage dependence on AR signaling, often referred to as "androgen-independent prostate cancer" or "hormone-refractory prostate cancer." The term "metastatic castration-resistant prostate cancer" or mCRPC refers to castration-resistant prostate cancer that has metastasized to other parts of the body.

[0061] The term "NM-CRPC" or "non-metastatic castration-resistant prostate cancer" refers to cancer that has not spread (metastasized) in men 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 assessed by bone scan and computed tomography (CT), magnetic resonance imaging (MRI) scan, or positron emission tomography (PET).

[0062] In some embodiments, the non-metastatic castration-resistant prostate cancer is high-risk non-metastatic castration-resistant prostate cancer. The term "high-risk nm-CRPC" refers to a high probability that men with nm-CRPC will develop metastases. In some embodiments, a high risk for metastasis formation is defined as a prostate-specific antigen doubling time (PSADT) of less than 10 months, less than 9 months, less than 8 months, less than 7 months, less than 6 months, less than 5 months, less than 4 months, less than 3 months, less than 2 months, or less than 1 month. In some embodiments, a high risk of metastasis formation is identified as having a locoregional recurrence (e.g., primary tumor bed, bladder neck, anastomosis, pelvic lymph nodes).

[0063] The term "chemotherapy-naive metastatic castration-resistant prostate cancer" refers to metastatic castration-resistant prostate cancer that has not been previously treated with a chemotherapy drug.

[0064] Neuroendocrine prostate cancer (NEPC), also known as treatment-related NEPC (tNEPC), is an aggressive androgen-independent variant of prostate cancer that most commonly arises in the later stages of mCRPC as a mechanism of treatment resistance (Wang HT et al., J. of Clinical Oncology, 2014, Vol. 32, Issue 30, 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: non-responsiveness to hormonal therapy, presence of lytic bone lesions, rapid disease progression, presence of visceral metastases, significant prostate enlargement, and disproportionately low PSA levels in the setting of metastatic disease. Androgen receptor (AR) expression is typically low or absent in NEPC, and the genes Aurora kinase A (AURKA) and N-Myc (MYCN) are frequently amplified.

[0065] The development of tNEPC is estimated to cause approximately 25% of the nearly 34,000 fatal cases of prostate cancer per year in the United States (A Jemal, F Bray, M M 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 significantly underestimated (P N Brawn, V O Spights: The dedifferentiation of metastatic prostate carcinoma Br J Cancer 59:85-88, 1989). The amount of neuroendocrine differentiation increases with disease progression and correlates with patient exposure to long-term androgen deprivation therapy. Preclinical studies also support the idea that transformation of PCa to tNEPC can be promoted by androgen deprivation therapy and may arise as a mechanism of resistance.

[0066] The present disclosure provides methods for treating or slowing the progression of prostate cancer in a subject by administering an anti-TMEFF2xCD3 bispecific antibody. Methods for treating or slowing the progression of prostate cancer include, but are not limited to, treating castration-resistant prostate cancer (CRPC), metastatic castration-resistant prostate cancer (mCRPC), non-metastatic castration-resistant prostate cancer (NM-CRPC), recurrent prostate cancer disease after androgen receptor (AR)-targeted therapy, locally advanced prostate cancer, high-risk localized prostate cancer, castration-sensitive prostate cancer, non-metastatic castration-sensitive prostate cancer, metastatic castration-sensitive prostate cancer, chemotherapy-naive metastatic castration-resistant prostate cancer, or neuroendocrine prostate cancer.

[0067] In some embodiments, the therapeutic methods of the present disclosure include methods of treating or slowing the progression of prostate cancer in a subject with an anti-TMEFF2xCD3 bispecific antibody, wherein the prostate cancer is metastatic castration-resistant prostate cancer (mCRPC).

[0068] antibody The disclosed methods are carried out by administering a bispecific antibody that specifically binds human TMEFF2 and human CD3. The anti-TMEFF2xCD3 bispecific antibody is useful for treating TMEFF2-associated cancers, such as metastatic castration-resistant prostate cancer (mCRPC).

[0069] "TMEFF2" refers to a human transmembrane protein containing an EGF-like domain and two follistatin-like domains 2, also known as tomoregulin 2. The amino acid sequence of full-length human TMEFF2 is set forth in SEQ ID NO: 27. The extracellular domain of TMEFF2 is set forth in SEQ ID NO: 28 and spans residues 40-320 of full-length TMEFF2. The TMEFF2 extracellular domain possesses three distinct subdomains: Kazal-like 1 (residues 85-137), Kazal-like 2 (residues 176-229), and an EGF domain (residues 261-301). The TMEFF2 EGF domain is set forth in SEQ ID NO: 29. The TMEFF2 "membrane proximal region" refers to the TMEFF2 region of SEQ ID NO: 21, encompassing the EGF domain and the NC-terminal linker region (e.g., residues 230-320 of full-length human TMEFF2 of SEQ ID NO: 27). All references herein to proteins, polypeptides, and protein fragments are intended to refer to the human version of the respective protein, polypeptide, or protein fragment unless expressly specified as being from a non-human species. Thus, "TMEFF2" refers to human TMEFF2 unless specified as being from a non-human species, such as "mouse TMEFF2" or "monkey TMEFF2."

[0070] SEQ ID NO: 27 (full-length human TMEFF2) MVLWESPRQCSSWTLCEGFCWLLLLPVMLLIVARPKLAAFPTSLSDCQTPTGWNCSGYDDRENDLFLCDTNTCKFDGECLRIGDTVTCVCQFKCNNDYVPVCGSNGESYQNECYLRQAACKQQSEILVVSEGSCATDAGSGSGDGVHEGSGETSQKETSTCDICQFGAECDEDAEDVWCVCNIDCS QTNFNPLCASDGKSYDNACQIKEASCQKQEKIEVMSLGRCQDNTTTTTKSEDGHYARTDYAENANKLEESAREHHIPCPEHYNGFCMHGKCEHSINMQEPSCRCDAGYTGQHCEKKDYSVLYVVPGPVRFQYVLIAAVIGTIQIAVICVVVLCITRKCPRSNRIHRQKQNTGHYSSDNTTRASTRLI

[0071] SEQ ID NO: 28 (extracellular domain of human TMEFF2) FPTSLSDCQTPTGWNCSGYDDRENDLFLCDTNTCKFDGECLRIGDTVTCVCQFKCNNDYVPVCGSNGESYQNECYLRQAACKQQSEILVVSEGSCATDAGSGSGDGVHEGSGETSQKETSTCDICQFGAECDEDAEDVWC VCNIDCSQTNFNPLCASDGKSYDNACQIKEASCQKQEKIEVMSLGRCQDNTTTTTKSEDGHYARTDYAENANKLEESAREHHIPCPEHYNGFCMHGKCEHSINMQEPSCRCDAGYTGQHCEKKDYSVLYVVPGPVRFQYV

[0072] TMEFF2 EGF domain SEQ ID NO: 29 HHIPCPEHYNGFCMHGKCEHSINMQEPSCRCDAGYTGQHCE

[0073] TMEFF2 membrane proximal region SEQ ID NO: 21 NTTTTTKSEDGHYARTDYAENANKLEESAREHHIPCPEHYNGFCMHGKCEHSINMQEPSCRCDAGYTGQHCEKKDYSVLYVVPGPVRFQYV

[0074] "CD3" refers to an antigen expressed on T cells as part of the multimolecular T cell receptor (TCR) complex, consisting of a homodimer or heterodimer formed from the association of two or four receptor chains: CD3 epsilon, CD3 delta, CD3 zeta, and CD3 gamma. Human CD3 epsilon comprises the amino acid sequence of SEQ ID NO: 22. The extracellular domain spans residues 23-126 of full-length CD3. All references to proteins, polypeptides, and protein fragments herein are intended to refer to the human version of the respective protein, polypeptide, or protein fragment, unless explicitly specified as being from a non-human species. Thus, "CD3" refers to human CD3 unless specified as being from a non-human species, e.g., "mouse CD3," "simian CD3," etc.

[0075] SEQ ID NO: 22 (human CD3 epsilon) MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGS KPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI

[0076] As used herein, the term "antibody" has a broad meaning and includes immunoglobulin molecules, including monoclonal antibodies (including murine, human, humanized, and chimeric monoclonal antibodies), antigen-binding fragments, multispecific antibodies such as bispecific, trispecific, and tetraspecific antibodies, dimeric, tetrameric, or multimeric antibodies, single-chain antibodies, domain antibodies, and any other modified form of immunoglobulin molecule that contains an antigen-binding site of the required specificity. The term antibody includes full-length antibodies, whole antibodies, intact antibodies, antibody fragments, antigen-binding fragments, and antigen-binding domains.

[0077] In general, antibodies are proteins or peptide chains that exhibit binding specificity to a specific antigen. The structure of antibodies is well known. Immunoglobulins can be assigned to five major classes (i.e., IgA, IgD, IgE, IgG, and IgM) depending on the amino acid sequence of the heavy chain constant domain. IgA and IgG are further subdivided into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Thus, antibodies of the present invention can be of any of the five major classes or corresponding subclasses. Preferably, antibodies of the present invention are IgG1, IgG2, IgG3, or IgG4. Antibody light chains of vertebrate species can be assigned to one of two clearly distinct types, kappa and lambda, based on the amino acid sequence of their constant domains. Thus, antibodies of the present invention can contain either a kappa or a lambda light chain constant domain. According to some embodiments, antibodies of the present invention comprise heavy and / or light chain constant regions from a rat or human antibody. In addition to the heavy and light constant domains, antibodies contain an antigen-binding region consisting of a light chain variable region and a heavy chain variable region, each of which contains three domains (i.e., complementarity-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.

[0078] The term "variable region" or "variable domain" refers to the heavy or light chain domain involved in binding an antibody to an antigen. The heavy or light chain variable domain (VH and VL, respectively) contains four framework regions (FR) and three complementarity determining regions (CDR).

[0079] The "complementarity-determining region (CDR)" is the region of an antibody that binds to an antigen. VH has three CDRs (HCDR1, HCDR2, and HCDR3), and VL has three CDRs (LCDR1, LCDR2, and LCDR3). CDRs can be defined using various descriptions, 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). Correspondence between various descriptions and numbering of variable regions has been described (see, e.g., Lefranc et al. (2003) Dev Comp Immunol 27:55-77; Honegger and Pluckthun (2001), J Mol Biol 309:657-70; the International ImMunoGeneTics (IMGT) database; web resource, http: / / www_imgt_org). CDRs can be described using available programs such as abYsis by UCL Business PLC. As used herein, the terms "CDR," "HCDR1," "HCDR2," "HCDR3," "LCDR1," "LCDR2," and "LCDR3" include CDRs defined by any of the Kabat, Chothia, IMGT, or AbM methods described above, unless expressly stated otherwise in the specification. For example, the correspondence between numbering systems, including Kabat numbering and the IMGT specific numbering system, is well known to those skilled in the art (see, e.g., Kabat, supra; Chothia, supra; Martin, supra; Lefranc et al., supra).

[0080] [Table 1]

[0081] "Specifically binds," "specific binding," "specifically binding," or "binds" refers to a proteinaceous molecule binding to an antigen or an epitope within an antigen with an affinity higher than its affinity for other antigens. Typically, a proteinaceous molecule binds to an antigen with an affinity of about 1×10 -7 M or less, e.g., about 5 × 10 -8 M or less, approximately 1×10 -8 M or less, approximately 1×10 -9 M or less, approximately 1×10 -10 M or less, approximately 1×10 -11 M or less, or about 1 x 10 -12 The equilibrium dissociation constant (K D ) binds to an antigen or an epitope within an antigen, typically D is the K for binding to nonspecific antigens (e.g., BSA, casein) D It is at least 100 times smaller than "K D The term "K D The dissociation constant of an antibody is obtained from the ratio of its K to its K (i.e., K / Ka) and is expressed as a molar concentration (M). D Values ​​can be determined using methods in the art in light of the present disclosure. For example, the K D The K of an antibody can be determined by using surface plasmon resonance, for example, by using a biosensor system, such as a Biacore® system, or by using biolayer interferometry techniques, for example, an Octet RED96 system. D The smaller the value of , the higher the affinity with which the antibody binds to the target antigen.

[0082] As used herein, an antibody that "binds to TMEFF2" or "specifically binds to TMEFF2" is an antibody that binds to TMEFF2 in an amount of 1×10 -7 M or less, preferably 1 x 10 -8 M or less, preferably 5×10 -9 M or less, 1×10 -9 M or less, 5×10 -10 M or less, or 1 x 10 -10 M, 5 x 10 -11 M, 1 x 10 -11 M, 5 x 10 -12 M or 1 x 10 -12 K below M D and refers to an antibody that binds to TMEFF2, preferably human TMEFF2.

[0083] As used herein, an antibody that "binds to CD3" or "specifically binds to CD3" is an antibody that binds to CD3 in an amount of 1 x 10 -7 M or less, preferably 1 x 10 -8 M or less, preferably 5×10 -9 M or less, 1×10 -9 M or less, 5×10 -10 M or less, or 1 x 10 -10 M, 5 x 10 -11 M, 1 x 10 -11 M, 5 x 10 -12 M or 1 x 10 -12 K below M D and refers to an antibody that binds to CD3, preferably human CD3.

[0084] "Bispecific" refers to an antibody that specifically binds to two different antigens or two different epitopes on the same antigen. Bispecific antibodies may be cross-reactive to the same antigen (homologue) from other related antigens, e.g., other species such as humans or monkeys, e.g., cynomolgus monkeys (Macaca cynomolgus, cyno) or chimpanzees, or may bind to an epitope shared between two or more different antigens.

[0085] The terms "bispecific anti-TMEFF2 / anti-CD3 antibody," "TMEFF2 / CD3 antibody," "anti-TMEFF2xCD3 bispecific antibody," and the like refer to antibodies that bind to both TMEFF2 and CD3.

[0086] In some embodiments, the anti-TMEFF2xCD3 bispecific antibody administered in the therapeutic method comprises a whole or full-length antibody, an Fv fragment, a single-chain scFv fragment (scFv), an Fab, an F(ab)2, or a single-chain antibody.

[0087] The terms "full-length antibody," "whole antibody," and "intact antibody" are used interchangeably herein to refer to antibodies with a structure similar to that of a native antibody. An "intact antibody" is composed of two heavy chains (HC) and two light chains (LC), inter-connected by disulfide bonds, and multimers thereof (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 called complementarity-determining regions (CDRs), which are interspersed with framework regions (FR). Each VH and VL is composed of three CDR and four FR segments, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Immunoglobulins can be assigned to five major classes, namely IgA, IgD, IgE, IgG, and IgM, depending on the amino acid sequence of the heavy-chain constant domain. IgA and IgG are further subdivided into isotypes: IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. The antibody light chain of any vertebrate species can be assigned to one of two clearly distinct types, namely kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0088] As used herein, the terms "antibody fragment" and "antigen-binding fragment" refer to molecules other than intact antibodies. Antigen-binding fragments may be synthetic, enzymatically obtained, 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 (ds diabodies), single-chain antibody molecules (scFv), single-domain antibodies (sdab), scFv dimers (bivalent diabodies), multispecific antibodies formed from portions of antibodies comprising one or more CDRs. Examples of antibody fragments that bind to antigen include 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 an antibody, such as a FR3-CDR3-FR4 portion, HCDR1, HCDR2 and / or HCDR3 and LCDR1, LCDR2 and / or LCDR3, alternative scaffolds that bind to antigen, bivalent domain antibodies, multispecific proteins comprising antigen-binding fragments, or any other antibody fragment that binds to an antigen but does not comprise the complete antibody structure.

[0089] In some embodiments, the methods of the disclosure are carried out by administering a full-length TMEFF2xCD3 bispecific antibody composed of two heavy chains (HC1 and HC2) and two light chains (LC1 and LC2).

[0090] In some embodiments, the methods of the disclosure are carried out by administering a TMEFF2xCD3 bispecific antibody comprising a heavy chain (HC1), a light chain (LC), and a single-chain Fv (scFv).

[0091] A "single-chain Fv" or "scFv" is a fusion protein comprising at least one antibody fragment comprising a light chain variable region (VL) and at least one antibody fragment comprising a heavy chain variable region (VH), in which the VL and VH are contiguously linked via a polypeptide linker and can be expressed as a single-chain polypeptide. An scFv may have the VL and VH variable regions in either order, based on the N-terminus and C-terminus of the polypeptide, and may comprise either a VL-linker-VH or a VH-linker-VL. An scFv may comprise a linker peptide, such as 2 to about 8 glycine or other amino acid residues, connecting the heavy chain variable region and the light chain variable region.

[0092] In some embodiments, the anti-TMEFF2xCD3 bispecific antibody used in the therapeutic methods of the present disclosure comprises a chimeric, humanized, or fully human antibody that specifically binds to TMEFF2 and CD3.

[0093] A "human antibody" refers to an antibody optimized to elicit a minimal immune response when administered to a human subject. The variable regions of a human antibody are derived from human immunoglobulin sequences. If a human antibody contains a constant region or a portion of a constant region, the constant region is also derived from human immunoglobulin sequences. A human antibody contains heavy and light chain variable regions "derived" from sequences of human origin when the variable regions of the human antibody are obtained from a system using human germline immunoglobulins or rearranged immunoglobulin genes. Exemplary such systems are phage-displayed human immunoglobulin gene libraries and transgenic non-human animals, such as mice or rats, carrying human immunoglobulin loci. A "human antibody" typically contains amino acid differences compared to immunoglobulins expressed in humans due to differences in the systems used to obtain human antibodies and human immunoglobulin loci, the introduction of somatic mutations or intentional substitutions into frameworks or CDRs, or both.

[0094] Typically, 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 in amino acid sequence to the amino acid sequence encoded by a human germline immunoglobulin or rearranged immunoglobulin gene. Optionally, a "human antibody" may contain consensus framework sequences 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 incorporated into a phage-displayed human immunoglobulin gene library, e.g., as described in Shi et al., (2010) J Mol Biol 397:385-96 and WO 2009 / 085462. Antibodies in which at least one CDR is derived from a non-human species are not included in the definition of "human antibody."

[0095] Transgenic animals, such as mice, rats, or chickens, carrying human immunoglobulin (Ig) loci in their genomes can be used to generate antibodies for use in the methods of the disclosure, as described, for example, in U.S. Patent No. 6,150,584, WO 1999 / 45962, WO 2002 / 066630, WO 2002 / 43478, WO 2002 / 043478, and WO 1990 / 04036. The endogenous immunoglobulin loci of such animals can be disrupted or deleted, and at least one complete or partial human immunoglobulin locus can be inserted into the animal's genome using homologous or non-homologous recombination, using transchromosomes, or using minigenes. 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 engaged to provide human antibodies against a selected antigen.

[0096] Antibodies generated by immunizing non-human animals can be humanized using methods well known in the art. Generally, humanized or engineered antibodies have one or more amino acid residues derived from a non-human source, such as, but not limited to, a mouse, rat, rabbit, non-human primate, or other mammalian source. Exemplary humanization techniques involving the selection of a human acceptor framework include CDR-grafting (U.S. Pat. No. 5,225,539), SDR-grafting (U.S. Pat. No. 6,818,749), resurfacing (Padlan, (1991) Mol Immunol 28:489-499), specificity-determining residue resurfacing (U.S. Pat. App. Pub. No. 2010 / 0261620), human framework adaptation (U.S. Pat. No. 8,748,356), or superhumanization (U.S. Pat. No. 7,709,226). These methods involve grafting the CDRs or a subset of CDR residues of a parent antibody onto a human framework that can be selected based on overall homology to the parent framework, based on similarity in CDR length or identity of canonical structure, or a combination thereof.

[0097] The humanized antigen-binding domain may be further optimized to improve its selectivity or affinity for the desired antigen by incorporating altered framework support residues to retain binding affinity (backmutation), or by introducing diversity into any of the CDRs, e.g., to improve the affinity of the antigen-binding domain, by techniques such as those described in WO 1090 / 007861 and WO 1992 / 22653.

[0098] The anti-TMEFF2xCD3 bispecific antibodies used in accordance with the present disclosure can be produced by recombinant means, including production from mammalian cells or transgenic preparations, as described herein or known in the art, or purified from other biological sources. The antibodies used in the methods of the present disclosure can be produced by cell lines, mixed cell lines, immortalized cells, or clonal populations of immortalized cells, as known in the art. Cell lines can be engineered to express the antibodies of the present disclosure, and the antibodies can be produced intracellularly, in the periplasmic space, or directly secreted into the medium.

[0099] The cell lysate or supernatant containing the anti-TMEFF2xCD3 bispecific antibody can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography. Other techniques for protein purification are also available, such as fractionation on ion exchange columns, ethanol precipitation, reverse-phase HPLC, chromatography on silica, and chromatography on anion or cation exchange resins.

[0100] Treatment method In some embodiments, the methods of the disclosure are carried out by administering a TMEFF2xCD3 bispecific antibody comprising a first variable domain that specifically binds TMEFF2 and a second variable domain that specifically binds CD3, wherein the first variable domain that specifically binds TMEFF2 comprises a VH of SEQ ID NO: 13 and a VL of SEQ ID NO: 14, and the second variable domain that specifically binds CD3 comprises a VH of SEQ ID NO: 17 and a VL of SEQ ID NO: 18.

[0101] In some embodiments, the methods of the disclosure are carried out 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.

[0102] In some embodiments, the methods of the disclosure are carried out by administering a TMEFF2xCD3 bispecific antibody comprising a first variable domain that specifically binds TMEFF2 and a second variable domain that specifically binds CD3, wherein the first variable domain that specifically binds 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 CD3 comprises a heavy chain (HC) of SEQ ID NO: 19 and a light chain (LC) of SEQ ID NO: 20.

[0103] In some embodiments, the methods of the disclosure are carried out 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 an HCDR of a VH having the amino acid sequence of SEQ ID NO: 13 and an LCDR of a VL having the amino acid sequence of SEQ ID NO: 14, and the second binding domain that binds to CD3 comprises an HCDR of a VH having the amino acid sequence of SEQ ID NO: 17 and an LCDR of a VL having the amino acid sequence of SEQ ID NO: 18, and (b) the first binding domain that binds to TMEFF2 comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 1, 2, 3, 4, 5, and 6, 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 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.

[0104] In some embodiments, the methods of the present disclosure induce T cell activation and proliferation in a target-specific manner without significant T cell activation or proliferation in the periphery.

[0105] In some embodiments, the methods of the present disclosure induce T cell activation and proliferation in a target-specific manner.

[0106] In some embodiments, the methods of the present disclosure induce infiltration of activated and proliferative T cells and inflammatory cells in the prostate.

[0107] In some embodiments, the methods of the present disclosure increase the number of intraprostatic T cells.

[0108] In some embodiments, the methods of the present disclosure do not induce significant numbers of suppressor T cell markers (PD1+Ki-67- and CD25+FoxP3).

[0109] In some embodiments, the methods of the present disclosure induce myeloid cell infiltration.

[0110] composition The methods of the disclosure are also practiced by administering a composition comprising an anti-TMEFF2xCD3 bispecific antibody described herein.

[0111] In the methods of the disclosure, the antibody may also be administered as a pharmaceutical composition comprising a therapeutically effective amount of an anti-TMEFF2xCD3 bispecific antibody and, optionally, a pharmaceutically acceptable carrier. [Example]

[0112] The following examples are provided to further illustrate some of the embodiments disclosed herein and are intended to illustrate, but not limit, embodiments of the present disclosure.

[0113] Example 1. Preparation and Description of Test Agents An exemplary TMEFF2xCD3 bispecific antibody Ab1 is used as a test agent in the Examples.

[0114] Ab1 is an immunoglobulin (Ig) G4-proline, alanine, alanine (IgG4 PAA) duobody® bispecific antibody that simultaneously binds to the cluster of differentiation (CD3) receptor complex on T lymphocytes (T cells) and to a transmembrane protein with an epidermal growth factor-like domain and two follistatin-like domains (TMEFF2) on tumor cells. Through this binding activity, the bispecific antibody is hypothesized to mediate 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. Ab1 exhibits reduced binding to the crystallizable fragment (Fc) gamma receptor due to leucine-to-alanine mutations at positions 234 and 235, and reduced fragment-antigen-binding arm exchange due to a serine-to-proline mutation at position 228.

[0115] Ab1 was developed to evaluate the therapeutic potential of targeting TMEFF2 for CD3-mediated T cell redirection. This bispecific antibody was generated by controlled fragment antigen binding (Fab) arm exchange from two antibodies, TMEB762 and CD3B376. TMEB762 is an anti-TMEFF2 antibody generated by immunizing Omni rats (OMT™). CD3B376 is an anti-CD3ε antibody generated by immunizing Omni rats (OMT™).

[0116] The generation of Ab1 is described in U.S. Patent Application Publication No. 16,417,889, entitled "Monospecific and multispecific anti-TMEFF2 antibodies and their uses," published November 28, 2019, which is incorporated herein by reference in its entirety. Assays for assessing the functional activity and structural properties, e.g., amino acid sequence, of Ab1 are also described in detail in U.S. Patent Application Publication No. 16,417,889, which is incorporated herein by reference in its entirety.

[0117] Using the Kabat description, the TMEFF2 binding domain of Ab1 (TMCB150) comprises an HCDR1 of the amino acid sequence SYSMS (SEQ ID NO: 1), an HCDR2 of the amino acid sequence VISGSGSGTDYADSVKG (SEQ ID NO: 2), and an HCDR3 of the amino acid sequence MPLNSPHDY (SEQ ID NO: 3), as well as an LCDR1 of the amino acid sequence RASQGIRNDLG (SEQ ID NO: 4), an LCDR2 of the amino acid sequence AASSLQS (SEQ ID NO: 5), and an LCDR3 of the amino acid sequence LQDYNYPLT (SEQ ID NO: 6).

[0118] Using the Kabat description, the CD3 binding domain of Ab1 (CD3B376) comprises an HCDR1 of the amino acid sequence NNNAAWS (SEQ ID NO: 7), an HCDR2 of the amino acid sequence RTYYRSKWLYDYAVSVKS (SEQ ID NO: 8), and an HCDR3 of the amino acid sequence GYSSSFDY (SEQ ID NO: 9), as well as an LCDR1 of the amino acid sequence TGTSSNIGTYKFVS (SEQ ID NO: 10), an LCDR2 of the amino acid sequence EVSKRPS (SEQ ID NO: 11), and an LCDR3 of the amino acid sequence VSYAGSGTLL (SEQ ID NO: 12).

[0119] The VH VL, HC, and LC sequences of Ab1 (TMCB150) TMEFF2 and CD3 binding domains are listed below.

[0120] Ab1 TMEFF2-binding domain VH, VL, HC, and LC VH amino acid sequence of TMCB150 TMEFF2 binding domain, SEQ ID NO: 13 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYSMSWVRQAPGKGLEWVSVISGSGGFTDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARMPLNSPHDYWGQGTLVTVSS

[0121] TMCB150 TMEFF2 binding domain VL amino acid sequence, SEQ ID NO: 14 DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDYNYPLTFGGGTKVEIK

[0122] TMCB150 TMEFF2 binding domain HC amino acid sequence, SEQ ID NO: 15 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYSMSWVRQAPGKGLEWVSVISGSGGFTDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARMPLNSPHDYWGQG TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGP PCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0123] TMCB150 TMEFF2 binding domain LC amino acid sequence, SEQ ID NO: 16 DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDYNYPLTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0124] TMCB150 TMEFF2 binding domain HC nucleic acid sequence, SEQ ID NO: 23

[0125] TMCB150 TMEFF2 binding domain LC nucleotide sequence, SEQ ID NO: 24 GACATCCAGATGACCCAGAGCCCTAGCAGCCTGAGCGCTAGCGTGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGGCATCAGAAACGACCTGGGCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCCT GCAGAGCGGAGTGCCTAGCAGGTTCAGCGGAAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCTGCAGGACTACAACTACCCCCTGACATTCGGCGGCGGCACCAAGGTGGAGATCAAGC GTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

[0126] VH, VL, HC and LC of Ab1 CD3 binding domain VH amino acid sequence of TMCB150 CD3 binding domain, SEQ ID NO: 17 QVQLQQSGPRLVRPSQTLSLTCAISGDSVFNNNAAWSWIRQSPSRGLEWLGRTYYRSKWLYDYAVSVKSRITVNPDTSRNQFTLQLNSVTPEDTALYYCARGYSSSFDYWGQGTLVTVSS

[0127] VL amino acid sequence of TMCB150 CD3 binding domain, SEQ ID NO: 18 QSALTQPASVSGSPGQSITISCTGTSSNIGTYKFVSWYQQHPDKAPKVLLYEVSKRPSGVSSRFSGSKSGNTASLTISGLQAEDQADYHCVSYAGSGTLLFGGGTKLTVL

[0128] TMCB150 CD3 binding domain HC amino acid sequence, SEQ ID NO: 19 QVQLQQSGPRLVRPSQTLSLTCAISGDSVFNNNAAWSWIRQSPSRGLEWLGRTYYRSKWLYDYAVSVKSRITVNPDTSRNQFTLQLNSVTPEDTALYYCARGYSSSFDYWG QGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG PPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0129] TMCB150 CD3 binding domain LC amino acid sequence, SEQ ID NO: 20 QSALTQPASVSGSPGQSITISCTGTSSNIGTYKFVSWYQQHPDKAPKVLLYEVSKRPSGVSSRFSGSKSGNTASLTISGLQAEDQADYHCVSYAGSGTLLFGGGTKLT VLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0130] TMCB150 CD3 binding domain HC nucleic acid sequence, SEQ ID NO: 25

[0131] TMCB150 CD3 binding domain LC nucleic acid sequence, SEQ ID NO: 26 CAGTCTGCTCTGACCCAGCCTGCCTCCGTGTCTGGCTCCCGGCCAGTCCATCACCATCAGCTGTACCGGCACCTCCTCCAACATCGGCACCTACAAGTTCGTGTCCTGGTATCAGCAGCACCCCGACAAGGCCCCCAAAGTGCTGCTGTACGAGGTGTCC AAGCGGCCCTCTGGCGTGTCCTCCAGATTCTCCGGCTCCAAGTCTGGCAACACCGCCTCCTGACCATCAGCGGACTGCAGGCTGAGGACCAGGCCGACTACCACTGTGTGTCCTACGCTGGCTCTGGCACCCTGCTGTTTGGCGGAGGCACCAAGCTGACC GTGCTGGGTCAGCCCAAGGCTGCACCCAGTGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGAGCCGTGACAGTGGCCTGGAAGGCCGATAGCAGCCCCGTCAAGGCGGGA GTGGAGACCACCACACCCTCCAAACAAAGCAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCA

[0132] The term "test drug" refers to Ab1 as described herein and to the test drug used in the pharmacodynamic study described in Example 2 and the clinical trial protocol described in Example 3.

[0133] Example 2. Preclinical Pharmacodynamic Study in Cynomolgus Monkeys The pharmacodynamics of the test drug were evaluated in cynomolgus monkeys to understand the dosing and on-target effects of the test drug in the prostate.

[0134] Test Plan Two dosing regimens of intravenous test drug were evaluated in male cynomolgus monkeys (3-8 years old): a single fixed dose (0.075 mg / kg) and an ascending dose (0.075 mg / kg, followed by 0.3 mg / kg one week later). Each dosing regimen included 12 male cynomolgus monkeys, which were equally divided into four groups and received either the test drug or a control. Prostate tissue, blood, and serum were collected for further analysis 24, 72, and 168 hours after administration of the 0.075 mg / kg fixed dose and 24, 72, and 168 hours after administration of the 0.3 mg / kg dose of the ascending dose regimen.

[0135] Sample collection For fixed doses of study drug, whole blood was collected pre-dose, 6 hours post-dose, and immediately prior to necropsy at all additional time points (24, 72, and 168 hours). Prostate tissue was collected at three necropsy time points.

[0136] For the test drug escalation dosing, samples were collected at the same time points as for the fixed dosing, with some slight adjustments. Whole blood was collected before and 6 hours after the priming dose, and before and 6 hours after the 1-week escalation dose. Whole blood was collected immediately before autopsy at 24, 72, and 168 hours after the escalation dose. Prostate tissue was collected as described for the fixed dosing.

[0137] All samples were processed by flow cytometry.

[0138] Cell surface staining 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 minced into smaller pieces and processed according to standard procedures for further analysis. Data analysis was performed using FlowJo version 10 to determine frequency and counts. Cell counting was facilitated by the addition of a fixed amount of Precision Counting Beads. Conversion of acquired cell events to cells / mL was calculated as follows:

[0139]

number

[0140] Test drug concentration A validated and purpose-built ECLIA (electrochemiluminescence immunoassay) method on the MSD® platform was used to quantify test drug concentrations in serum and prostate tissue, respectively. The minimum quantifiable concentrations in serum and prostate tissue lysate samples were 0.01 μg / mL and 1.25 ng / mL, respectively. After escalating dosing, an approximately dose-proportional increase in test drug was observed ( FIG. 10 ). The median peak test drug exposure in the prostate was 11.0% of serum for the first dose and 8.7% after the second dose.

[0141] T cell infiltration Test drug administration increased prostate infiltration of CD8+ T cells and CD4+ T cells and reduced peripheral T cells (Figures 11A-11B, 12A-12F). The reduction in peripheral CD8+ T cells was more pronounced with fixed doses of test drug.

[0142] T cell activation and proliferation Both CD4+ and CD8+ prostate T cells expressed markers of activation and proliferation upon test drug dosing (Figures 13A-F). The magnitude of T cell activation appeared to attenuate slightly with increasing doses of test drug.

[0143] Suppressive T cell markers Test drugs had minimal effects on suppressor T cell markers (PD-1+Ki67-, CD4+CD25+Foxp3) (Figures 14A-14C and 15A-15B).

[0144] Myeloid cell infiltration Test drug dosing resulted in an influx of pro-inflammatory cells (dendritic cells, myeloid cells, B cells) (Figures 16A-16B, 17A-17C). Escalating test drug dosing showed a decrease in immature and pro-inflammatory myeloid subpopulations in prostate tissue compared with fixed dosing. Total B cell numbers were lower with escalating test drug dosing compared with fixed dosing.

[0145] conclusion Study drug administration increased the number of intraprostatic T cells. T cell activation and proliferation was observed in a target-specific manner, and no significant T cell activation or proliferation was detected in the periphery. Regulatory T cell markers (PD1+Ki-67- and CD25+FoxP3) were not observed in significant numbers after study drug administration. Myeloid cell infiltrates support an active immune microenvironment in the prostate after study drug administration.

[0146] Example 3. Phase 1 Clinical Trial Protocol Summary This is a first-in-human (FIH), open-label, multicenter, Phase 1 dose-escalation study to evaluate the safety, pharmacokinetics (PK), pharmacodynamics (PD), and preliminary clinical activity of the investigational drug monotherapy in participants with metastatic castration-resistant prostate cancer (mCRPC). A diagram of the study design is provided in Figure 1.

[0147] Dose escalation was supported by the modified continual reassessment method (mCRM). The goal was to determine the maximum tolerated dose (MTD) of study drug.

[0148] Safety was monitored throughout the study by the Study Evaluation Team (SET), particularly at each dose escalation step. The study began with a once-weekly (QW) dosing schedule by subcutaneous (SC) injection. An alternative dosing schedule of once every two weeks (Q2W) was investigated based on emerging data determined by the SET.

[0149] Approximately 73 participants were treated in this study.

[0150] [Table 2]

[0151] Purpose, endpoint The objectives and endpoints of this study are listed in Table 33.

[0152] [Table 3]

[0153] Efficacy evaluation Clinical activity will be assessed using the following assessments: computed tomography (CT) scan of the chest, abdomen, and pelvis with contrast as clinically indicated; magnetic resonance imaging (MRI) may be substituted as clinically indicated (i.e., for areas not adequately imaged using CT). Additional assessments for participants with mCRPC include serum prostate-specific antigen (PSA) and whole-body bone scan ( 99m Response to treatment will be assessed according to the Prostate Cancer Working Group 3 (PCWG3) criteria and Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 to assess progression of soft tissue lesions (CT or MRI).

[0154] Pharmacokinetic, biomarker, and immunogenicity assessment Blood samples will be collected to characterize serum pharmacokinetics of the study drug and anti-drug antibodies. Blood samples will also be collected to assess pharmacodynamics, safety, and biomarkers predictive of response or resistance to study drug treatment. Archival tissue samples from metastatic (non-prostate) tumor lesions (collected at any time prior to enrollment) and archival prostate samples collected within 15 months of providing informed consent will be requested, if available, to assess TMEFF2 expression and T cell infiltration. In addition, selected participants in the PK / PD cohort in Part 1 and all participants in Part 2 will undergo fresh tumor biopsies to evaluate pharmacodynamic markers in tumor tissue. Selected PK / PD cohorts may undergo additional blood sample collection.

[0155] Safety evaluation Safety in drug trials will be assessed by physical examination (including neurological examination), vital signs, Eastern Cooperative Oncology Group (ECOG) performance status, laboratory tests, electrocardiograms (ECG), ophthalmologic examination, and adverse event (AE) monitoring (including dose-limiting toxicities [DLTs], serious adverse events [SAEs], and adverse events of special interest [AESIs]). Concomitant medication use will be recorded. The severity of adverse events will be assessed using the National Cancer Institute Common Terminology Criteria for 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-onset retinal abnormalities or non-infectious inflammatory ophthalmopathy (grade ≥ 2) have been identified as adverse events of particular interest, requiring enhanced reporting and data collection.

[0156] Rationale for Starting Dose A first-in-human (FIH) starting dose of 300 μg administered SC approximates a 4 μg / kg dose (with an estimated median body weight of 75 kg and 100% bioavailability).

[0157] In vitro cytotoxicity assays were performed to characterize test drug-induced T cell activation, TMEFF2-positive tumor cell killing, and cytokine release. These assays were performed using purified human T cells from healthy human donors and LNCaP-AR, a human prostate cancer cell line that expresses TMEFF2 and exhibits sensitivity to T cell-mediated killing. The median cytotoxicity was shown to be the most sensitive and considered most predictive of T cell-mediated killing (Table 4). A MABEL concentration of 1.0 nM (0.15 μg / mL) was used to determine the median cytotoxicity EC 20 was determined from the value.

[0158] [Table 4]

[0159] The PK model MABEL predicted mean test drug serum concentrations in humans after a single dose of 300 μg, assuming a median body weight of 75 kg, are shown in Figure 2.

[0160] Based on the overall evaluation of the in vitro and in vivo data, and the MABEL-based FIH starting dose selection, a 300 μg once-weekly SC dose of study drug should result in minimally bioactive drug exposure in participants treated in this study.

[0161] Test Plan Overall Design This is an 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 were treated in this study. Once participants are determined to be eligible for the study (i.e., meet the inclusion / exclusion criteria) and provide informed consent to participate in the study, the study drug will be administered as a SC injection.

[0162] The pharmacodynamics of the study drug were further characterized in selected PK / PD cohorts as determined by the Study Evaluation Team (SET).

[0163] A diagram of the study design is shown in Figure 1.

[0164] Dose escalation The study was designed to select the RP2D and regimen and determine the MTD (if possible) of the study drug in participants with mCRPC. Dose escalation will begin with a MABEL-based starting dose of 300 μg (0.3 mg) and progress as shown in Figure 1. Only the starting dose was prespecified. After the starting dose, subsequent doses were selected based on consideration of all available data, including, but not limited to, pharmacokinetics, pharmacodynamics, safety, and preliminary clinical activity. Dose escalation decision-making was guided by a modified continuous reassessment method (mCRM).

[0165] Multiple dose levels were enrolled in parallel with each new dose level / schedule recommended by SET and supported by a statistical model using the Escalation with Overdose Control (EWOC) principle. To better understand safety, tolerability, pharmacokinetics, pharmacodynamics, or preliminary antitumor activity, additional participants were enrolled in one or more dose cohorts (referred to as PK / PD cohorts) at or below the dose deemed safe by SET.

[0166] Dose escalation was performed in single or multiple consecutive cohorts of participants at the SET-assigned dose. The following guidelines were applied during dose escalation:

[0167] If more than one participant was treated at a dose level, the first participant treated at that given dose level was observed for a minimum of 2 days before treating any subsequent participants.

[0168] Dose escalation proceeded in half-log increments or less.

[0169] Dose escalation decisions were made by SET based on mCRM, utilizing all available safety, pharmacokinetic, and other biomarker data from all previous dose levels. Preliminary clinical activity, if available, was investigated by SET during dose escalation.

[0170] Part 2 (dose expansion) In Part 2, the RP2D / regimen of study drug determined in Part 1 will be administered to additional participants with mCRPC to confirm the safety, pharmacokinetics, pharmacodynamics, and preliminary clinical activity of the study drug. Additional histologies (in addition to mCRPC) will be considered for dose expansion based on new data from Part 1.

[0171] Treatment Dose Schedule The study began with a Q1W therapeutic dose. Alternative dosing schedules of Q2W were evaluated based on safety and pharmacokinetic data emerging after SET approval.

[0172] To minimize the risks associated with cytokine release syndrome (CRS) and infusion-related reactions, corticosteroid, antihistamine, and antipyretic premedication were administered prior to the first dose of study medication. Premedication doses or schedules were reduced or omitted for subsequent doses based on SET review of available data, and on a case-by-case basis, corticosteroid premedication was reduced or omitted for subsequent doses. For participants experiencing Grade 2 or higher CRS or IRR, pretreatment corticosteroids were required for at least one subsequent dose administered to that participant.

[0173] Determination of the recommended phase 2 dose (RP2D) The RP2D is determined after review of all available data, including safety, pharmacokinetics, pharmacodynamics, and clinical activity, from at least six participants treated at the dose level being considered for the RP2D declaration, taking into account pharmacokinetic data from at least 12 participants across all dose levels, and dose recommendations from a Bayesian Logistic Regression Model (BLRM).

[0174] Definition of Dose-Limiting Toxicity (DLT) The DLT assessment period is defined as the first 21 days of treatment.

[0175] [Table 5]

[0176] Administration route This study investigated the SC route of administration.

[0177] Treatment Discontinuation / Follow-up Participants received study drug until radiographic disease progression, overt clinical progression, unacceptable toxicity, or any other treatment discontinuation criteria were met. Participants who were able to return clinically for evaluation after treatment discontinuation had an End-of-Treatment (EOT) visit within 30 (±7) days after the last dose of study drug.

[0178] Study population The inclusion and exclusion criteria for enrolling participants in this study are described below.

[0179] Inclusion criteria Each potential participant met all of the following criteria to be enrolled in the study: 18 years of age or older, Histology: metastatic CRPC (mCRPC) with histological confirmation of adenocarcinoma. Small cell or adenocarcinoma with neuroendocrine features is permitted; ·Measurable or assessable disease; · Prior treatment with at least one prior novel AR-targeted therapy (i.e., abiraterone acetate, apalutamide, enzalutamide, dultamide) or chemotherapy (e.g., docetaxel); Eastern Cooperative Oncology Group (ECO) performance status grade of 0 or 1; Hematology parameters within the following ranges, independent of blood transfusions within 7 days or growth factors within 3 weeks prior to the first dose of study drug: Participants must not be transfusion-dependent (hemoglobin ≥ 9 g / dL, absolute neutrophil count ≥ 1.5 x 10 9 / L, platelet count ≥75×10 9 / L); 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 syndrome, if total bilirubin is ≥ 1.5 × ULN, direct and indirect bilirubin will be measured, and participants will be eligible if direct bilirubin is within the normal range, and aspartate aminotransferase (AST) and alanine aminotransferase (ALT) ≤ 2.5 × ULN (or ≤ 4 × ULN for participants with tumorous liver lesions).

[0180] Exclusion criteria Any potential participant who met any of the following criteria was excluded from participation in the study. Known brain metastases. Concomitant use of any other anti-cancer treatment (including non-palliative radiation therapy) or investigational drug; preceding anti-cancer treatment (including non-palliative radiation therapy) must be discontinued for at least 2 weeks before the first administration of the investigational drug. Toxicity related to previous anticancer therapy, excluding alopecia and vitiligo, is grade ≤1 or has not returned to baseline. Symptomatic diabetic retinopathy with macular edema or active exudative age-related macular degeneration (AMD) within the past 12 months, and any symptomatic ophthalmitis or optic neuropathy (e.g., uveitis, optic neuritis). ·Solid organ or bone marrow transplantation. Seizures or known conditions that may predispose to seizures, or intracranial masses such as schwannomas and meningiomas causing edema or a mass effect. Any of the following within six months prior to signing the informed consent: Myocardial infarction b. Severe or unstable angina C. Clinically significant ventricular arrhythmia d. Congestive heart failure (New York Heart Association Class III-IV) e. Transient ischemic attack f. Cerebrovascular accident. Venous thromboembolic events (i.e., pulmonary embolism) and uncomplicated (grade ≤2) deep vein thrombosis within 1 month prior to the first dose of study drug are not considered exclusions. Grade ≥2 peripheral neuropathy or neuropathic pain. Clinically significant pulmonary impairment, particularly the need for supplemental oxygen use (>2Λ by nasal cannula) to maintain adequate oxygenation. Known allergy, hypersensitivity, or intolerance to the study drug or its excipients Concurrent use of any other anti-cancer therapy or investigational drug for the treatment of advanced disease. Active infection or condition requiring treatment with a systemic anti-infective within 7 days prior to the first dose of study drug. - Received an immunosuppressive dose of systemic medication, such as a corticosteroid (dose >10 mg / day of prednisone or equivalent), within 3 days prior to the first dose of study medication. A single dose of corticosteroid is permitted as prophylaxis for imaging with contrast (i.e., for participants with an allergy to contrast). If corticosteroids were used to treat an immune-mediated adverse event related to previous therapy, 7 or more days must have elapsed since the last dose of corticosteroids. Active autoimmune disease within 12 months prior to signing consent that requires systemic immunosuppressive medication (e.g., chronic corticosteroids, methotrexate, or tacrolimus). Underwent major surgery (e.g., requiring general anesthesia). Participants must have recovered satisfactorily without sequelae for at least 3 weeks before the first dose of study medication. Active or chronic hepatitis B or C infection. Hepatitis B infection defined by a positive test for hepatitis B surface antigen (HBsAg). Hepatitis C infection defined by a positive hepatitis C antibody. -History of positive antibody test for Human Immunodeficiency Virus (HIV). - Planning to father a child while enrolled in this study or within 90 days after the last dose of study drug. Any condition where, in the investigator's opinion, participation would not be in the participant's best interest (e.g., would compromise their health) or could interfere with, limit, or confound protocol-specified evaluations. Received 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 licensed for emergency use (e.g., COVID-19) and non-live vaccines (e.g., influenza) are acceptable.

[0181] Administration of test drug Administration of test drug The study began using SC injection as the route of administration, with a once-weekly dosing schedule. An alternative study drug dosing schedule of Q2W was also investigated. Dose escalation began with a starting dose of 300 μg administered SC.

[0182] Test Evaluation and Procedures Efficacy evaluation The following assessments were used to evaluate clinical activity:

[0183] The same methodology (CT scan or MRI or MRI) was used at baseline and throughout the study period to characterize each identified and reported lesion and record disease status. 99m Tc bone scan) was used to assess the disease. Ultrasound, 18 [F]-fluorodeoxyglucose positron emission tomography (PET) and plain x-ray were not acceptable methods of assessing disease response. Imaging should not be delayed due to delays in study drug administration. Efficacy assessments included: PSA, whole body bone scan ( 99m Tc), CT scan, or MRI. Evaluation of treatment response for prostate cancer was performed according to the Prostate Cancer Working Group 3 (PCWG3) criteria.

[0184] Participants with an objective response per RECIST v1.1 must have had a confirmatory scan performed after 4 weeks. If a participant was assessed for a partial response (PR) or complete response (CR) at any time during study treatment but did not have a confirmatory response after 4 weeks or more, the participant's best response was classified as stable disease / progressing disease / inevaluable disease, depending on the participant's next most recent assessment. During the study, disease response was assessed using CT or MRI scans of known lesion locations.

[0185] Assessment of disease response and disease progression Soft tissue lesion evaluation (CT or MRI, physical examination) Baseline disease burden was assessed using CT scans with IV contrast of the chest, abdomen, and pelvis, and other areas as needed. Participants who could not tolerate IV contrast could have a CT scan with oral contrast, and the reason for not using IV contrast was recorded in the original documentation. Subsequent efficacy assessments during the study included radiographic imaging of all disease sites documented at baseline.

[0186] Magnetic resonance imaging was used to evaluate areas of disease that could not be adequately imaged using CT (if MRI was preferred, it should be the imaging technique used to evaluate disease at baseline and all subsequent response assessments). For all other areas of disease, MRI evaluation did not replace CT scans of the chest, abdomen, and pelvis, which were required unless CT scans were contraindicated. Brain MRI was required only if clinically indicated. Head CT scans were used when MRI was contraindicated.

[0187] For participants with palpable / superficial lesions, clinical disease assessment by physical examination was performed at baseline and throughout study drug treatment, as clinically indicated. Irradiated or excised lesions were considered non-measurable and were monitored only for disease progression.

[0188] Evaluation of bone lesions in prostate cancer Bone disease in participants with prostate cancer was assessed according to PCWG3 (i.e., duration of response was assessed) as follows: Progression of soft tissue disease as measured by CT or MRI as defined by RECIST v1.1. Progression of bone lesions as observed by bone scan and based on PCWG3. Under these criteria, bone progression must be confirmed by a subsequent scan at least 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 progression is defined as one of the following: 1. Participants whose week 8 scan is observed to have two or more new bone lesions compared to the baseline scan must have a confirmatory scan performed 6 weeks or more later and may fall into one of the following two categories: a. Participants whose confirmatory scan (performed 6 weeks or later) shows 2 or more new lesions compared to the week 8 scan (i.e., a total of 4 or more new lesions compared to the baseline scan) are considered to have bone scan progression at week 8. b. Participants whose confirmatory scan did not show two or more new lesions compared to the week 8 scan were not considered to have bone scan progression at that time point. The week 8 scan was considered the reference scan to which subsequent scans were compared. 2. For participants whose week 8 scan does not have two or more new bone lesions compared to the baseline scan, the first scan time point showing two or more new lesions compared to week 8 will be considered the bone scan progression time point if these new lesions are confirmed by a subsequent scan 6 weeks or more later.

[0189] Pharmacokinetic and immunogenicity evaluation Venous blood samples were collected for measurement of serum concentrations of test drug and anti-test drug antibodies. Blood samples were obtained from the arm opposite the arm into which the test drug was injected when the test drug was administered via a peripheral vein. At time points where both serum concentrations and immunogenicity were assessed, a single blood draw was collected and serum samples were divided into separate aliquots. Samples collected for analysis of test drug serum concentrations and antibodies to the test drug can additionally be used to assess safety or efficacy aspects that address concerns arising during or after the study period, for further characterization of immunogenicity or evaluation of relevant biomarkers (e.g., the potential presence of soluble TMEFF2).

[0190] Population for analysis For the purposes of analysis, the following populations are defined: All-Treatment Analysis Set: This set consists of participants who received at least one dose of study drug. This analysis set is considered primary and will be used for all safety and efficacy summaries. DLT-Evaluable Analysis Set: This set is a subset of the "All Treatments Analysis" set. Participants who receive at least 75% of the planned doses of study drug during the DLT observation period, as defined, will be included in this analysis. Biomarker analysis set: This set consists of all participants who received at least one dose of study drug and had at least one pre- or post-treatment biomarker measurement. Pharmacokinetic analysis set: This set consists of all participants who received at least one dose of study drug and had at least one evaluable concentration measurement of study drug.

[0191] statistical analysis Efficacy analysis Endpoint definition The PSA response rate (RR) was defined as the proportion of participants whose PSA decreased by at least 30% or more from baseline. The maximum change at any time during the study was reported for each participant using a waterfall plot. Whenever possible, participants should remain in the study until radiography or symptom progression to reflect changes in clinical status. Analysis of the primary endpoint was performed on the entire treatment population, and PSA RRs were presented with 90% two-sided exact CIs.

[0192] Overall response rate (ORR) was defined as the proportion of participants with PR or better by RECIST v1.1 response criteria without evidence of bone progression by PCWG3. Response to treatment was assessed by investigators.

[0193] Duration of response (DOR) was calculated from the first documented date of response (PR or better), as defined by PCWG3 or RECIST v1.1 response criteria, to the first documented date of evidence of disease progression or death from any cause (whichever occurred first). For participants who responded to treatment without disease progression (CR or PR) and were surviving, data were censored at the last disease assessment before the start of any subsequent anticancer therapy.

[0194] Time to response (TTR) is defined as the time from the date of first administration of study drug to the date of the first documented response.

[0195] Analysis method Overall response rates were tabulated along with their two-sided 90% exact confidence intervals. Additionally, the number and percentage of participants in each response category were tabulated. For TTR, results were summarized using descriptive statistics, including the mean, median, standard deviation, and range of responding participants. For DOR, the Kaplan-Meier method was used for descriptive summaries.

[0196] Safety analysis All safety analyses were performed on data from the "all-treatment analysis set." Baseline safety assessments were defined as values ​​collected closest to but prior to the start of the first study drug administration. Safety parameters evaluated included incidence, severity, and type of adverse events, clinically significant changes in participants' physical examination findings, vital sign measurements, laboratory results, and other laboratory results (e.g., ECG). Exposure to study drug and reasons for study drug discontinuation 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.

[0197] Adverse events Terms used by the investigator to identify adverse events on the CRF were coded verbatim using the Medical Dictionary for Regulatory Activities (MedDRA). Study drug-emergent adverse events were those occurring during the study drug phase or those that were the result of a pre-existing condition that worsened from baseline. All reported treatment-emergent adverse events were included in the analysis. For each treatment-emergent adverse event, the percentage of participants with at least one occurrence of the specific event was summarized by dose level / dose cohort.

[0198] Summaries, lists, datasets, or participant narratives of participants who died due to an adverse event, discontinued study drug, or had a severe or serious adverse event were provided, as appropriate. DLT listings used the DLT-evaluable analysis set. DLTs were listed and incidence summarized by primary system organ class, preferred term, worst grade and type of adverse event, and dose level.

[0199] Example 4. Safety and Preliminary Clinical Activity of Investigational Agents for the Treatment of Metastatic Castration-Resistant Prostate Cancer (mCRPC) The study drug was administered subcutaneously (SC) at doses ranging from 0.3 mg to 6 mg once weekly (Q1W) and 2 to 6 mg once every two weeks (Q2W). A total of nine dose levels were tested (Q1W: 300 μg, 1 mg, 1.5 mg, 3 mg, and 6 mg; Q2W: 2 mg, 3 mg, 4 mg, and 6 mg), administered to a total of 82 patients.

[0200] Pharmacokinetics Preliminary Pharmacokinetics Preliminary PK of the study drug was evaluated in 71 subjects from the ongoing FIH study. Preliminary data after SC injection of study drug are available at doses ranging from 0.3 to 6.0 mg Q1W (Cohorts 1-4 and 9) and at doses ranging from 2.0 to 6.0 mg every 2 weeks (Q2W; Cohorts 5-8). Baseline characteristics are shown in Table 6.

[0201] [Table 6] Abbreviations: AR, antigen receptor; ECOG, Eastern Cooperative Oncology Group; PSA, prostate-specific antigen; SD, standard deviation. a includes the lungs, liver, adrenal glands, and central nervous system, b includes the pelvis and extrapelvis, c PSA was not entered into the database for one patient at the time of data cut.

[0202] After the first SC injection of study drug ranging from 0.3 to 6.0 mg, C max and AUC 0~168時間 Both increased approximately dose-proportionally. Moderate increases in mean serum concentrations of the study drug were observed at all dose levels (Figure 3). Median T max generally occurred between 72 and 168 hours (Table 7).

[0203] [Table 7] Abbreviations: AUC(168h) = area under the serum concentration-time curve from 0 to 168 h, AUC(336h) = area under the serum concentration-time curve from 0 to 336 h, 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 reach maximum concentration. For evaluable subjects ≥ 3, the mean ± SD is shown; for evaluable subjects < 3, only the mean is shown. Subjects lacking concentrations around the expected Cmax were excluded from descriptive statistics. a For AUC(168 hours) and AUC(168 hours, dn), n=4. b n=9 for AUC(168h), AUC(336h), AUC(168h, dn), and AUC(336h, dn). c For AUC(336 h) and AUC(336 h, dn), n=9. d n=4 for AUC(168 hr), AUC(336 hr), AUC(168 hr, dn), and AUC(336 hr, dn).

[0204] After multiple SC injections of the study drug, steady state was achieved after the seventh SC injection with Q1W dosing and the fourth SC injection with Q2W dosing (Figures 4 and 5, respectively). At steady state, serum trough concentrations (C トラフ ) increased approximately dose-proportionally after Q1W dosing, whereas C トラフ Values ​​appeared comparable for the 2.0-4.0 mg Q2W cohorts (Table 8). The mean accumulation ratios for AUC were approximately 4.9 and 1.6 for Q1W and Q2W dosing, respectively.

[0205] [Table 8] Abbreviation:AR AUC = accumulation ratio, C トラフ = serum trough concentration, d n = dose normalized to 1 mg, n = number of subjects, QXW = every X weeks, SC = subcutaneous, SD = standard deviation. C トラフ is the concentration observed immediately before the start of the dosing interval. For evaluable subjects ≥3, the mean ± SD is shown; for evaluable subjects <3, only the mean is shown. a AR AUC For, n=1. b AR AUC For, n=2. c AR AUC For, n=1. d AR AUC About n=2

[0206] Effect of body weight on pharmacokinetics The effect of body weight on pharmacokinetics is shown in Figures 6 and 7A and 7B. Subjects with low body weight are likely to have a lower volume of distribution and clearance, resulting in higher PK exposure when compared to subjects with high body weight.

[0207] Effectiveness Efficacy / Pharmacodynamics Preliminary PD data for 73 subjects were analyzed: 38 in the Q1W SC dosing cohort and 35 in the Q2W SC dosing cohort. Maximum prostate-specific antigen (PSA) reductions of at least 50% were reported for eight subjects: six in the Q1W SC dosing cohort and two in the Q2W SC dosing cohort (Figure 8). An additional seven subjects achieved a maximum PSA reduction of at least 30%, four of whom were dosed Q1W and three of whom were dosed Q2W.

[0208] Of the 38 subjects with available data, the maximum reduction in the sum of target lesion diameters by RECIST 1.1 was at least 30% (partial response or better) for seven subjects (Figure 9): five subjects from the Q1W SC dosing cohort and two subjects from the Q2W SC dosing cohort. Confirmed partial responses were observed in five patients.

[0209] Summary of preliminary efficacy data Preliminary efficacy data (PSA and RECIST response) are summarized in Table 9.

[0210] [Table 9] * Both patients had intrapatient dose escalation to 3 mg QW, one before confirmed PR and one after PR. ** One patient had a uPR (Day 127) before dose reduction to 3 mg Q2W due to an AE. PR was confirmed during a subsequent scan (Day 175). The same patient had a PSA50 before dose reduction. Another patient had a PSA50 after dose reduction to 3 mg QW due to a G3 decline (DLT) on Day 16.

[0211] Safety and Tolerability Nature and frequency of adverse events Treatment-emergent AEs were experienced by at least 10% of subjects. Overall, 71 of 73 subjects (97.3%) reported at least one TEAE (Tables 10 and 11). The most frequently 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 frequent TEAEs did not differ significantly between doses.

[0212] [Table 10] AE, adverse event; AST, aspartate aminotransferase; COVID, coronavirus disease; Q1W, once every week; Q2W, once every 2 weeks; SC, subcutaneous; TEAE, treatment-emergent adverse event.

[0213] [Table 11] AE, adverse event; AST, aspartate aminotransferase; COVID, coronavirus disease; Q1W, once every week; Q2W, once every 2 weeks; SC, subcutaneous; TEAE, treatment-emergent adverse event.

[0214] Treatment-emergent serious adverse events (SAEs) Overall, treatment-emergent serious adverse events (SAEs) were reported for 31 (42.5%) subjects in the clinical trial study (17 subjects in the Q1W SC dosing cohort and 14 subjects in the Q2W SC dosing cohort) (Table 12). SAEs considered related to study drug by the investigator are described as follows: In the 1.0 mg and 3.0 mg study drug Q1W SC dosing cohorts, one subject each experienced a grade 2 SAE of vomiting. In the 1.5 mg study drug Q1W SC dosing cohort, one subject experienced a grade 2 SAE of CRS. In the 6.0 mg study drug Q1W SC cohort, one subject experienced a grade 3 SAE of asthenia, and one subject experienced a grade 2 SAE of imbalance, grade 2 confusion, and grade 3 fall (the fall TEAE occurred after the study drug dose was reduced to 3.0 mg). While study drug treatment was discontinued, the subject also experienced a grade 3 SAE of orthostatic hypotension. In the 4.0 mg Q2W SC cohort, one subject experienced a grade 3 SAE of increased aspartate aminotransferase (AST), and in the 6.0 mg study drug Q2W SC cohort, one subject experienced a grade 2 SAE of fever and grade 2 confusion, and one subject experienced a grade 1 SAE of fever.

[0215] Dose-limiting toxicity Two subjects experienced a total of three DLTs in the study. One subject in the 6.0 mg study drug Q1W SC cohort experienced a DLT of a 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 not recovered / resolved, and study treatment had been discontinued. Ten days later, the same subject experienced a DLT of grade 3 syncope, which recovered / resolved.

[0216] Grade 3 or higher treatment-emergent adverse events Grade 3 or higher TEAEs were reported in 40 (54.8%) subjects in this study. Grade 3 or higher TEAEs reported in two or more subjects (incidence ≥ 5%) were anemia (13 subjects, 18%), fatigue (8 subjects, 11%), lymphopenia (5 subjects, 7%), asthenia (4 subjects, 6%), 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).

[0217] Treatment discontinuation occurred in seven patients who experienced at least one TEAE (increased blood creatinine [n=1], weight loss [n=1], arthralgia [n=1], back pain [n=1], orthostatic hypotension [n=2], hypotension [n=1], pulmonary edema [n=1]). Of the seven patients, two patients discontinued the study due to treatment-related AES (both with orthostatic hypertension).

[0218] Cytokine release syndrome occurred in 4 of 73 patients (6%) and was considered grade 1 or 2.

[0219] Six deaths occurred during the study (disease progression [n=4]; pulmonary edema [n=1]; and COVID-19 [n=1]), all considered unrelated to the study drug. Dose-limiting cytotoxicity was reported in two patients (all grade 3; fall hospitalization [n=1]; orthostatic hypotension and syncope [n=1]).

[0220] In conclusion, the study drug shows a tolerable safety profile at the specified dose in mCRPC patients with PSA50 and RECIST responses.

Claims

1. A pharmaceutical composition used to treat cancer in a subject or to slow its progression, wherein the pharmaceutical composition comprises an anti-TMEF2 × CD3 bispecific antibody and is administered subcutaneously to the subject.

2. The pharmaceutical composition according to claim 1, wherein the subcutaneous dose is approximately 0.3 mg to approximately 6 mg of the bispecific antibody.

3. The pharmaceutical composition according to claim 2, wherein the subcutaneous dose is administered once a week or once every two weeks at a dose of approximately 1.5 mg.

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

5. The anti-TMEFF2 × CD3 bispecific antibody comprises a first binding domain that binds to TMEFF2 and a second binding domain that binds to CD3. a. The first binding domain that binds to TMEFF2 includes a VH HCDR having the amino acid sequence of SEQ ID NO: 13 and a VL LCDR having the amino acid sequence of SEQ ID NO: 14, and the second binding domain that binds to CD3 includes a VH HCDR having the amino acid sequence of SEQ ID NO: 17 and a VL LCDR having the amino acid sequence of SEQ ID NO:

18. b. The first binding domain that binds to TMEFF2 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 1, 2, 3, 4, 5, and 6, respectively, and the second binding domain that binds to CD3 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 7, 8, 9, 10, 11, and 12, respectively. c. The first binding domain that binds to TMEFF2 includes VH and VL of SEQ ID NOs. 13 and 14, respectively, and the second binding domain that binds to CD3 includes VH and VL of SEQ ID NOs. 17 and 18, respectively, and / or d. The pharmaceutical composition according to claim 4, wherein 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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