Methods of treating renal cancer with anti-PSMA/CD 3 antibodies

文档序号：347724 发布日期：2021-12-03 浏览：37次中文

阅读说明：本技术 用抗psma/cd3抗体治疗肾癌的方法 (Methods of treating renal cancer with anti-PSMA/CD 3 antibodies ) 是由 T·麦克德维特 S·谢蒂 H·谢于 2020-04-17 设计创作，主要内容包括：本文示出了用于治疗癌症的双特异性单克隆抗体和方法。(Bispecific monoclonal antibodies and methods for treating cancer are shown herein.)

1. A method of treating a renal cancer in a patient, the method comprising administering to the patient a safe amount of an anti-PSMA xcd3 antibody fragment, wherein the anti-PSMA x CD3 antibody comprises a first binding domain that specifically binds PSMA and a second binding domain that specifically binds CD3, wherein the first binding domain comprises the Heavy Chain (HC) of SEQ ID NO:7 and the Light Chain (LC) of SEQ ID NO:8, and the second binding domain comprises the Heavy Chain (HC) of SEQ ID NO:17 and the Light Chain (LC) of SEQ ID NO: 18.

2. The method of claim 1, wherein the patient has metastatic renal cancer (mRCC).

3. The method of claim 2, wherein the anti-PSMA x CD3 antibody is administered to the patient Intravenously (IV) at a dose of about 0.1 μ g/kg at week 1.

4. The method of claim 3, wherein the anti-PSMA x CD3 antibody is administered to the patient Intravenously (IV) once weekly beginning at a dose of about 0.1 μ g/kg.

5. The method of claim 3, wherein the anti-PSMA x CD3 antibody is administered to the patient Intravenously (IV) twice weekly beginning at a dose of about 0.1 μ g/kg.

6. A pharmaceutical composition for use in treating a kidney cancer in a patient, the pharmaceutical composition comprising the antigen binding proteins of SEQ ID NOs 7, 8, 17 and 18, wherein the composition is administered to the patient at an initial dose of about 0.1 μ g/kg at week 1.

7. The composition of claim 6, wherein the patient has metastatic renal cancer (mRCC).

Technical Field

The present invention relates to methods of providing treatment of kidney cancer, including metastatic kidney cancer, by administering an anti-PSMA/CD 3 antibody.

Background

Renal cancer is one of the 10 most common cancers, with about 1 in 63 people affected during life, most adults between 50 and 80 years of age. The incidence of renal cancer is highest worldwide in north america, but has steadily increased over the past three decades in developing countries. Metastatic renal cell carcinoma (mRCC) is a disease that predicts a poor prognosis, despite the increasing number of new systemic treatment options, including new targeted therapies and immunotherapy. PSMA is a transmembrane glycoprotein consisting of 750 amino acids and 3 protein domains (a small intracellular domain, a single-pass transmembrane domain, and a large extracellular domain). PSMA has been reported to be expressed in neovasculature of other solid tumors, including lung, bladder and kidney cancers (Chang SS et al, Cancer Res.1999; 59(13): 3192-3198). In a recent study to study PSMA expression in Renal Cell Carcinoma (RCC), immunohistochemical results showed that endothelial PSMA protein was detected in 80% clear cell carcinoma, 14% papillary carcinoma and 72% chromophobe carcinoma (Spatz S, Tolkach et al, J Urol.2018; 199(2): 370-. Further analysis from the same study showed that PSMA expression was significantly associated with lower overall survival in patients in both clear cell and papillary renal cell carcinoma. In another clinical study, the use⁶⁸The PSMA-based radiotracers of Ga are capable of detecting PSMA in metastatic lesions found in patients with clear cell carcinomas (Sawicki LM et al, Eur J Nucl Med Mol imaging.2017; 44(1): 102-. Thus, in addition to prostate cancer, the PSMAxCD3 method may also have therapeutic benefits in patients with histology, such as clear cell renal cell carcinoma.

Disclosure of Invention

General and preferred embodiments are defined by the independent and dependent claims appended hereto, respectively, which are incorporated by reference herein for the sake of brevity. Other preferred embodiments, features and advantages of the various aspects of the present invention will become apparent from the following detailed description when taken in conjunction with the accompanying drawings.

The present invention relates to methods of treating renal cancer, including metastatic Renal Cell Carcinoma (RCC), by administering a safe and effective amount of an anti-PSMAxCD 3 antibody to a subject with metastatic renal cell carcinoma.

In certain embodiments, the invention provides a method of treating a kidney cancer in a patient suffering from kidney cancer, the method comprising, consisting of, and/or consisting essentially of the steps of: administering to the patient an anti-PSMA xccd 3 antibody fragment in a safe amount, wherein the anti-PSMA x CD3 antibody comprises, consists of, and/or consists essentially of: a first binding domain that specifically binds PSMA and a second binding domain that specifically binds CD3, wherein the first binding domain comprises the Heavy Chain (HC) of SEQ ID NO:7 and the Light Chain (LC) of SEQ ID NO:8, and the second binding domain comprises the Heavy Chain (HC) of SEQ ID NO:17 and the Light Chain (LC) of SEQ ID NO: 18.

In another embodiment, the invention provides a method of treating a kidney cancer in a patient suffering from kidney cancer, the method comprising, consisting of and/or consisting essentially of the steps of: administering to the patient an anti-PSMA xcd3 antibody fragment in a safe amount, wherein the anti-PSMA x CD3 antibody comprises a first binding domain that specifically binds PSMA and a second binding domain that specifically binds CD3, wherein the first binding domain comprises the Heavy Chain (HC) of SEQ ID No. 7 and the Light Chain (LC) of SEQ ID No. 8, and the second binding domain comprises the Heavy Chain (HC) of SEQ ID No. 17 and the Light Chain (LC) of SEQ ID No. 18, wherein the patient has metastatic renal cell carcinoma.

In another embodiment, the invention provides a method of treating a kidney cancer in a patient suffering from kidney cancer, the method comprising, consisting of and/or consisting essentially of the steps of: administering to the patient an anti-PSMA xcd3 antibody fragment, wherein the anti-PSMA x CD3 antibody comprises a first binding domain that specifically binds PSMA and a second binding domain that specifically binds CD3, wherein the first binding domain comprises the Heavy Chain (HC) of SEQ ID NO:7 and the Light Chain (LC) of SEQ ID NO:8, and the second binding domain comprises the Heavy Chain (HC) of SEQ ID NO:17 and the Light Chain (LC) of SEQ ID NO:18, wherein the patient has metastatic renal cancer, and wherein the anti-PSMA χ CD3 antibody is administered Intravenously (IV) to the patient at a dose of about 0.1 ug/kg.

In some embodiments, the present invention provides a pharmaceutical composition for use in treating a kidney cancer in a patient, the pharmaceutical composition comprising, consisting of, and/or consisting essentially of the antigen binding proteins of SEQ ID NOs 7, 8, 17, and 18, wherein the composition is administered to the patient at an initial dose of about 0.1 ug/kg.

Drawings

Fig. 1 shows the binding of CD3B146 to primary human T cells.

Figure 2 shows the binding of CD3B146 to cynomolgus monkey primary T cells.

Fig. 3 shows that CD3B146 activates primary human T cells in vitro. Negative controls are shown in white and positive controls in black.

Fig. 4A shows a slow-escalation protocol used in toxicology studies.

Fig. 4B shows a rapid escalation protocol used in toxicology studies.

Figure 5 shows a graph of potential exploration of dose escalation and dose escalation plans and priming dose schedules-part 1 dose escalation protocol and part 2 dose escalation cohort.

Figure 6 shows a schematic overview of the study design-part 1 dose escalation phase. (CRS-cytokine Release syndrome; PK/PD-pharmacokinetics/pharmacodynamics)

Detailed Description

All publications, including patents and patent applications, cited in this specification are herein incorporated by reference as if fully set forth herein.

Definition of

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, exemplary materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells, and the like.

Throughout the specification and claims, the words "comprise", "comprising", and the like, are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense, unless the context clearly requires otherwise; that is, the meaning of "including but not limited to".

By "specifically binds," "specifically binds," or "binds" is meant that the antibody binds to an antigen or epitope within an antigen with a higher affinity than to other antigens. Typically, the antibody is administered at about 5X 10^-8M or less (e.g., about 1X 10)^-9M or less, about 1X 10^-10M or less, about 1X 10^-11M or less or about 1X 10^-12M or less) equilibrium dissociation constant (K)_D) Binding to or an epitope within an antigen, typically the K_DK for binding of the antibody to a non-specific antigen (e.g. BSA, casein)_DAt most one hundredth. Dissociation constants can be measured using the protocols described herein. However, an antibody that binds to an antigen or an epitope within an antigen may have cross-reactivity to other related antigens, e.g. to the same antigen from other species (homologous), such as humans or monkeys, e.g. cynomolgus Macaca fascicularis (cynomolgus, cyno) or chimpanzee (chimpanzee, chimpThe epitope of (1).

"antibody" refers broadly to and includes immunoglobulin molecules, including in particular monoclonal antibodies (including murine monoclonal antibodies, human monoclonal antibodies, humanized monoclonal antibodies, and chimeric monoclonal antibodies), antigen binding fragments, multispecific antibodies (such as bispecific antibodies, trispecific antibodies, tetraspecific antibodies, and the like), dimeric, tetrameric, or multimeric antibodies, single chain antibodies, domain antibodies, and any other modified configuration of an immunoglobulin molecule comprising an antigen binding site with the desired specificity. A "full-length antibody" comprises two Heavy Chains (HC) and two Light Chains (LC) interconnected 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 (consisting of domains CH1, hinge, CH2, and CH 3). 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, termed Complementarity Determining Regions (CDRs) with intervening Framework Regions (FRs). Each VH and VL is composed of three CDRs and four FR segments, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR 4.

"Complementarity Determining Regions (CDRs)" are regions of an antibody that bind antigen. CDRs may be defined using various delineations, such as Kabat (Wu et al, (1970) J Exp Med 132:211-50) (Kabat et al, "Sequences of Proteins of Immunological Interest", 5 th edition, 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, (1996) J Mol Biol 263: 800-15). The correspondence between the various delineations and the variable region numbering is described (see, e.g., Lefranc et al, (2003) Dev Comp Immunol 27: 55-77; Honegger and Pluckthun, (2001) J Mol Biol 309: 657-70; International Immunogenetics (IMGT) database; Web resources, http:// www _ IMGT _ org). Available programs (such as abYsis of UCL Business PLC) can be used to delineate CDRs. As used herein, the terms "CDR," "HCDR 1," "HCDR 2," "HCDR 3," "LCDR 1," "LCDR 2," and "LCDR 3" include CDRs defined by any of the above methods (Kabat, Chothia, IMGT, or AbM), unless the specification expressly indicates otherwise.

Immunoglobulins can be assigned to five major classes, namely IgA, IgD, IgE, IgG and IgM, based on the heavy chain constant domain amino acid sequence. IgA and IgG are further sub-classified into isotypes IgA1, IgA2, IgG1, IgG2, IgG3 and IgG 4. The light chain of an antibody of any vertebrate species can be assigned to one of two completely different types, κ and λ, based on the amino acid sequence of its constant domains.

An "antigen-binding fragment" refers to the antigen-binding portion of an immunoglobulin molecule. The antigen-binding fragment may be a synthetic, enzymatically obtainable or genetically engineered polypeptide and comprises VH, VL, VH and VL, Fab, F (ab')2, Fd and Fv fragments, domain antibodies (dabs) consisting of one VH domain or one VL domain, shark variable IgNAR domains, camelized VH domains, minimal recognition units consisting of amino acid residues that mimic the CDRs of an antibody, such as FR3-CDR3-FR4 portions, HCDR1, HCDR2 and/or HCDR3, and LCDR1, LCDR2 and/or LCDR 3. The VH and VL domains may be linked together via a synthetic linker to form various types of single chain antibody designs, wherein in the case where the VH and VL domains are expressed from separate single chain antibody constructs, the VH/VL domains may pair intramolecularly or intermolecularly to form a monovalent antigen binding site, such as single chain fv (scfv) or diabodies; for example, as described in International patent publications WO1998/44001, WO1988/01649, WO1994/13804 and WO 1992/01047.

"monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibody molecules, i.e., the individual antibodies comprising the population are identical except for possible well-known changes (such as removal of the C-terminal lysine from the heavy chain of the antibody) or post-translational modifications (such as amino acid isomerization or deamidation, methionine oxidation or asparagine or glutamine deamidation). Monoclonal antibodies typically bind to an epitope. Bispecific monoclonal antibodies bind to two different epitopes. Monoclonal antibodies can have heterogeneous glycosylation within the antibody population. Monoclonal antibodies can be monospecific or multispecific, such as bispecific, monovalent, bivalent, or multivalent.

"isolated" refers to a homogeneous population of molecules (such as synthetic polynucleotides or proteins, e.g., antibodies) that have been substantially separated from and/or purified from other components of the system in which the molecules are produced (e.g., recombinant cells), as well as proteins that have been subjected to at least one purification or isolation step. An "isolated antibody" refers to an antibody that is substantially free of other cellular material and/or chemicals, and encompasses antibodies isolated to a higher purity, such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% purity.

"humanized antibody" refers to antibodies in which at least one CDR is derived from a non-human species and at least one framework is derived from a human immunoglobulin sequence. Humanized antibodies may comprise substitutions in the framework such that the framework may not be an exact copy of the expressed human immunoglobulin or human immunoglobulin germline gene sequence.

"human antibody" refers to an antibody that is optimized to have a minimal immune response when administered to a patient. The variable regions of human antibodies are derived from human immunoglobulin sequences. If the human antibody comprises a constant region or a portion of a constant region, the constant region is also derived from a human immunoglobulin sequence. If the variable regions of a human antibody are obtained from a system using human germline immunoglobulins or rearranged immunoglobulin genes, the human antibody comprises heavy and light chain variable regions that are "derived" from sequences of human origin. Exemplary systems of this type are human immunoglobulin gene libraries displayed on phage, and transgenic non-human animals, such as mice or rats carrying human immunoglobulin loci. Due to differences between the systems used to obtain human antibodies and human immunoglobulin loci, the introduction of somatic mutations or deliberate introduction of substitutions into the framework or CDRs, or both, "human antibodies" typically comprise amino acid differences compared to immunoglobulins expressed in humans. Typically, the amino acid sequence of a "human antibody" has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence encoded by a human germline or rearranged immunoglobulin gene. In some cases, a "human antibody" may comprise a consensus framework sequence derived from human framework sequence analysis (Knappik et al, (2000) J Mol Biol 296: 57-86); or binding to synthetic HCDR3 in a human immunoglobulin gene library displayed on phage (Shi et al, (2010) J Mol Biol 397:385-96, and international patent publication WO 2009/085462).

An antibody in which at least one CDR is derived from a non-human species is not included in the definition of "human antibody".

"recombinant" refers to DNA, antibodies and other proteins that are prepared, expressed, formed or isolated by recombinant means when segments from different sources are joined to produce recombinant DNA, antibodies or proteins.

An "epitope" refers to the portion of an antigen that specifically binds to an antibody. Epitopes are typically composed of chemically active (such as polar, non-polar or hydrophobic) surface groups of moieties such as amino acids or polysaccharide side chains, and can have specific three-dimensional structural characteristics as well as specific charge characteristics. Epitopes can be composed of contiguous and/or noncontiguous amino acids that form conformational space units. For discontinuous epitopes, amino acids from different parts of the linear sequence of the antigen are close in three dimensions due to the folding of the protein molecule.

"bispecific" refers to an antibody that specifically binds to two different antigens or two different epitopes within the same antigen. Bispecific antibodies may be cross-reactive to other relevant antigens, e.g. to the same antigen from other species (homologues), such as humans or monkeys, e.g. cynomolgus macaque (cynomolgus, cyno) or chimpanzee (Pan troglodytes), or may bind to an epitope shared between two or more different antigens.

"multispecific" refers to an antibody that specifically binds to two or more different antigens or two or more different epitopes within the same antigen. Multispecific antibodies may be cross-reactive to other related antigens, e.g. to the same antigen from other species (homologous), such as a human or monkey, e.g. macaque or chimpanzee, or may bind an epitope shared between two or more different antigens.

A "variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or reference polynucleotide by one or more modifications (e.g., one or more substitutions, insertions, or deletions).

"vector" refers to a polynucleotide capable of replication within a biological system or of movement between such systems. Vector polynucleotides typically contain elements such as origins of replication, polyadenylation signals, or selectable markers that function to facilitate replication or maintenance of these polynucleotides in biological systems, e.g., cells, viruses, animals, plants, and recombinant biological systems that utilize biological components capable of replicating the vector. The vector polynucleotide may be a single-or double-stranded DNA or RNA molecule or a hybrid of such molecules.

An "expression vector" refers to a vector that can be used in a biological system or a reconstituted biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.

"Polynucleotide" refers to a synthetic molecule comprising a chain of nucleotides or other equivalent covalent chemical covalently linked to a backbone of phosphoroglycoses. cDNA is an exemplary synthetic polynucleotide.

By "polypeptide" or "protein" is meant a molecule comprising at least two amino acid residues joined by peptide bonds to form a polypeptide. Small polypeptides of less than 50 amino acids may be referred to as "peptides".

PSMA refers to prostate specific membrane antigen. The amino acid sequence of full-length human PSMA is shown in SEQ ID NO 1. The extracellular domain spans residues 44-750 of full-length PSMA. All references herein to proteins, polypeptides and protein fragments are intended to refer to the human version of the corresponding protein, polypeptide or protein fragment, unless explicitly indicated as being from a non-human species. Thus, "PSMA" means human PSMA unless specified to be from a non-human species, e.g., "mouse PSMA" or "monkey PSMA" and the like.

1 (all) of SEQ ID NOLong PSMA)

MWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEATNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVHEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDYAVVLRKYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFTVQAAAETLSEVA

"CD 3" refers to an antigen expressed on T cells as part of a multi-molecular T Cell Receptor (TCR) complex and consisting of a homodimer or heterodimer consisting of two or four receptor chains: association of CD3 epsilon, CD3 delta, CD3 zeta and CD3 gamma. Human CD3 epsilon comprises the amino acid sequence of SEQ ID NO. 4. The ectodomain spans residues 23-126 of full-length CD 3. All references herein to proteins, polypeptides and protein fragments are intended to refer to the human version of the corresponding protein, polypeptide or protein fragment, unless explicitly indicated as being from a non-human species. Thus, "CD 3" means human CD3 unless indicated as being from a non-human species, e.g., "mouse CD 3" or "monkey CD 3" and the like.

SEQ ID NO 4 (human CD3 ε)

MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI

"bispecific anti-PSMA/anti-CD 3 antibody", PSMA/CD3 antibody, PSMAxCD3 antibody and the like refer to an antibody that binds PSMA and CD 3.

"in combination with … …" means that two or more therapeutic agents are administered to a patient sequentially in any order, together in admixture, simultaneously as a single agent, or as a single agent.

"PSMA-positive cancer" refers to a cancer tissue or cancer cell that exhibits measurable levels of PSMA protein. The level of PSMA protein can be measured on live or lysed cells using well known assays using, for example, ELISA, immunofluorescence, flow cytometry, or radioimmunoassay.

"sample" refers to collections of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present in a subject. Exemplary samples are biological fluids such as blood, serum and serosal fluids, plasma, lymph, urine, saliva, cystic fluid, tears, excretions, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites such as those associated with non-solid tumors, fluids of the pleura, pericardium, peritoneum, abdominal cavity, and other body cavities, fluids collected by bronchial lavage, liquid solutions in contact with a subject or biological source such as cell and organ culture media (including cell or organ conditioned media), lavage fluids, and the like, tissue biopsy samples, fine needle punctures, or surgically excised tumor tissue.

"cancer cell" or "tumor cell" refers to a cancerous or transformed cell in vivo, ex vivo, or in tissue culture, that has spontaneous or induced phenotypic changes. These changes do not necessarily involve the uptake of new genetic material. Although transformation can occur by infection with a transforming virus and binding to new genomic nucleic acid, or uptake of exogenous nucleic acid, it can also occur spontaneously or following exposure to a carcinogen, thereby mutating an endogenous gene. Transformation/cancer is exemplified by morphological changes in vitro, in vivo and ex vivo, cell immortalization, abnormal growth control, lesion formation, proliferation, malignancy, modulation of tumor-specific marker levels, invasion, tumor growth in a suitable Animal host (such as a nude mouse, etc.) (Freshney, Culture of Animal Cells: A Manual of Basic Technique (3 rd edition, 1994)).

"about" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. In the context of a particular assay, result, or embodiment, "about" means within one standard deviation, or up to a range of 5% (whichever is greater), according to common practice in the art, unless otherwise explicitly stated in the examples or elsewhere in the specification.

"treatment" (or treatment) refers to the treatment of a patient suffering from a pathological disorder and refers to the effect of alleviating the disorder by killing cancer cells and also to the effect of directing the progression of the disease state to be inhibited and includes slowing of the rate of progression, termination of the rate of progression, amelioration of the disorder, and cure of the disorder. Treatment as a prophylactic measure (i.e., prophylaxis) is also included.

"therapeutically effective amount" means an amount effective to treat cancer at a desired dose and for a desired period of time. The therapeutically effective amount may vary depending on the following factors: such as the disease state, age, sex, and weight of the individual, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual. Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, improved patient health as a result of treatment.

According to the invention as defined herein, the term "safe amount" when it relates to administration or treatment with an anti-PSMA xcd3 antigen-binding fragment having a first binding domain specifically binding to PSMA and a second binding domain specifically binding to CD3, wherein the first binding domain comprises the Heavy Chain (HC) of SEQ ID NO:7 and the Light Chain (LC) of SEQ ID NO:8, and the second binding domain comprises the Heavy Chain (HC) of SEQ ID NO:17 and the Light Chain (LC) of SEQ ID NO:18, refers to a favorable risk to benefit ratio of adverse events having a relatively low or reduced frequency and/or low or reduced severity, including adverse vital signs (heart rate, systolic and diastolic pressures, body temperature), adverse standard clinical laboratory tests (hematology, clinical chemistry, urinalysis, lipids, coagulation), anaphylaxis/hypersensitivity reactions, Poor local injection site reactions, or poor EKG.

As used herein, unless otherwise indicated, the term "clinical validation" (used alone or to modify the terms "safe" and/or "effective") means that a clinical trial has proven effective, wherein the clinical trial has met the standards of the U.S. food and drug administration, EMEA, or corresponding national regulatory agency. For example, a clinical study may be a full-scale, randomized, double-blind study for clinical confirmation of the effect of a drug. In some embodiments, "clinical validation" indicates that it has been validated by a clinical trial that has met the standards of the U.S. food and drug administration, EMEA, or corresponding national regulatory agency for phase I clinical trials.

anti-PSMAxCD 3 antibodies

The invention provides a composition comprising a PSMA x CD3 antigen-binding fragment having a first binding domain that specifically binds PSMA and a second binding domain that specifically binds CD3, wherein the first binding domain comprises the Heavy Chain (HC) of SEQ ID No. 7 and the Light Chain (LC) of SEQ ID No. 8, and the second binding domain comprises the Heavy Chain (HC) of SEQ ID No. 17 and the Light Chain (LC) of SEQ ID No. 18. The present invention also relates to a method of treating kidney cancer, comprising, consisting or consisting essentially of the steps of: a safe amount of the above anti-PSMAxCD 3 antibody was administered to human males with kidney cancer.

Unless otherwise specifically indicated, throughout the specification, amino acid residues in the constant region of an antibody are according to the EU index⁵And (6) numbering.

Conventional one-letter and three-letter amino acid codes as shown in table 1 are used herein.

Table 1.

Amino acids	Three letter code	Single letter codeCode
			Alanine	Ala	A
Arginine	Arg	R
			Asparagine	Asn	N
Aspartic acid	Asp	D
			Cysteine	Cys	C
Glutamic acid	Glu	E
			Glutamine	Gln	Q
Glycine	Gly	G
			Histidine	His	H
IsoleucineamideAcid(s)	Ile	I
			Leucine	Leu	L
Lysine	Lys	K
			Methionine	Met	M
Phenylalanine	Phe	F
			Proline	Pro	P
Serine	Ser	S
			Threonine	Thr	T
Tryptophan	Trp	W
			Tyrosine	Tyr	Y
Valine	Val	V

Therapeutic applications

The invention also provides methods of using at least one dual integrin antibody of the invention for modulating or treating at least one PSMA-associated disease in a cell, tissue, organ, animal, or patient as known in the art or as described herein.

The invention also provides methods for modulating or treating at least one kidney cancer-associated disease in a cell, tissue, organ, animal or patient, including but not limited to at least one of advanced solid tumors or metastatic kidney cancer (mRCC).

As used herein, the term "cancer" refers to abnormal growth of cells that are prone to proliferate in an uncontrolled manner, and in some cases refers to metastasis (spread).

As used herein, the term "RCC" refers to metastatic renal cell carcinoma. In some embodiments, the RCC is assessed using a bone scan and a Computed Tomography (CT) or Magnetic Resonance Imaging (MRI) scan.

As used herein, the term "co-administration" or the like encompasses administration of selected therapeutic agents to a patient and is intended to encompass treatment regimens in which these agents are administered by the same or different routes of administration or at the same or different times.

The term "metastasis-free survival" or "MFS" refers to the percentage of patients in a study who have survived for a prescribed period of time or have not died without cancer spread. MFS is typically reported as the time from the start of enrollment, randomization, or treatment in a study. MFS is a report for an individual or study population. In the case of CRPC treatment with antiandrogens, prolongation of metastasis-free survival is an additional time observed in the absence of cancer spread or death (whichever occurs first) compared to treatment with placebo. In some embodiments, the extension of metastasis-free survival is about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, or greater than 20 months. In some embodiments, administration of a safe and effective amount of an antiandrogen provides an extension of metastasis-free survival in a male human, optionally wherein the extension of metastasis-free survival is relative to the average survival of a population of male humans with non-metastatic castration-resistant prostate cancer, said population being treated with a placebo. In some embodiments, metastasis-free survival refers to the time of first evidence of distant metastasis or death (whichever occurs first) from randomization to BICR confirmation of bone or soft tissue.

The term "transfer time" is the time from randomization to the scan time showing the first evidence of a radiographically detectable distant transfer of bone or soft tissue confirmed by BICR. In some embodiments, administration of an antiandrogen provides the patient with improved anti-tumor activity, as measured by Time To Metastasis (TTM).

The term "time to progression of symptoms" is defined as the time from randomization to recording in CRF (whichever occurs earlier): (1) development of Skeletal Related Events (SRE): pathological fractures, spinal cord compression, or the need for surgical intervention or radiation therapy of the bone; (2) the progression or worsening of pain in disease-related symptoms requiring the initiation of new systemic anti-cancer treatments; or (3) development of clinically significant symptoms due to local tumor progression requiring surgical intervention or radiotherapy. In some embodiments, administration of an antiandrogen to a patient provides improved antitumor activity as measured by time to progression of symptoms.

The term "overall survival" is defined as the time from randomization to the death date for any reason. Survival data for patients who survived the analysis were reviewed at the last known date of their survival. In addition, for patients who survived no post-baseline information, data was reviewed on randomized dates; for patients with missed visits or withdrawn consent, the data was reviewed on the last known date of their survival. In some embodiments, administration of an antiandrogen to a patient provides improved anti-tumor activity as measured by overall survival.

As used herein, the term "delay of symptoms associated with disease progression" refers to an increase in the time to progression of symptoms such as pain, urinary obstruction, and quality of life considerations, from the randomized time of administration of the drug trial.

The term "randomization" when referring to a clinical trial refers to the time when a patient is identified as appropriate for the clinical trial and assigned to a treatment group.

The terms "kit" and "article of manufacture" are used synonymously.

Examples

Example 1. materials

Production of PSMA cell lines. Expression vectors exhibiting full-length chimpanzee PSMA (SEQ ID NO:2) or full-length cynomolgus PSMA (SEQ ID NO:3) were generated for use as screening tools for the evaluation of anti-PSMA leads. The vector was transiently transfected into HEK293F cells. Transfected 293F suspension cells were seeded in growth medium plus serum, allowed to become adherent and selected for stable plasmid integration. Single cell populations were selected by serial dilution and PSMA surface receptor expression was quantified by FACS using (PSMA antibody (Center) affinity purified rabbit polyclonal antibody (cat # OAAB02483, Aviva Systems Biology) as the primary antibody, while using R-PE anti-rabbit secondary antibody (cat # 111-116-144, Jackson ImmunoResearch Laboratories, Inc.) and rabbit polyclonal IgG (cat # SC-532, Santa Cruz Biotechnology) as isotype controls).

2 (full length chimpanzee PSMA)

SEQ ID NO. 3 (full length cynomolgus monkey PSMA)

MWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEATNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVLDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRHGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVHEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVYNLTKELKSPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDYAVVLRKYADKIYNISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFTERLQDFDKSNPILLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGDVKRQISVAAFTVQAAAETLSEVA

A lentivirus (Genencopoeia, catalog number EX-G0050-Lv105-10) containing full-length HUMAN PSMA (FOLH1_ HUMAN, SEQ ID NO:1) and puromycin was used to generate a cell line expressing HUMAN PSMA for selection of PSMA-positive cells. HEK293F cells (ATCC) negative for PSMA were transduced with lentiviral particles to overexpress human PSMA. After transduction, cells positively expressing PSMA and resistance marker were selected by treating pooled cells, grown in DMEM + 10% HI FBS (Life Technologies) and supplemented with puromycin at various concentrations (Life Technologies).

In addition to HEK-producing cell lines, several commercial cell lines were used for phage panning and binding and cytotoxicity assays. LNCaP clone FGC cells (ATCC, catalog number CRL-1740) are a commercially available human prostate cancer cell line. C4-2B cells initially developed in MD Anderson and were derived from LNCaP FGC grown in vivo and transferred to bone marrow (Thalmann et al, 1994, Cancer Research 54,2577-81).

Production of soluble PSMA ECD protein. Production of recombinant chimpanzee PSMA extracellular domain (ECD) protein (amino acids 44-750 of ECD, SEQ ID NO:2), recombinant cynomolgus monkey PSMA extracellular domain (ECD) protein (amino acids 44-750 of SEQ ID NO:3) and recombinant human PSMA extracellular domain (ECD) protein (amino acids 44-750 of SEQ ID NO:1) for panning and evaluation of anti-PSMA leads

Example 2 production of anti-chimpanzee and anti-human PSMA antibodies

Panning with recombinant protein. First solution panning of a de novo human Fab-pIX library consisting of VH1-69, 3-23 and 5-51 heavy chain libraries paired with four human VL germline gene (A27, B3, L6, O12) libraries (Shi, L et al, J Mol Biol,2010.397 (2): page 385 and 396, WO2009/085462) was performed using an alternating panning method, wherein a round of phage captures was performed according to the manufacturer's protocol on streptavidin magnetic beads (Invitrogen, catalog No. 112.05D, 62992920) coated with cynomolgus monkey PSMA-Fc, followed by phage captures on ProtG beads (Invitrogen, catalog No. 10003D) coated with cynomolgus monkey PSMA-Fc, followed by Thermo-phage capture on Serra-Double magnetic beads (neutral), catalog number 7815-.

Whole cell panning of anti-PSMA Fab. Additional panning experiments were performed on whole cells using round 1 output from the chimpanzee ECD panning experiment described above or a fresh de novo phage library as input. Briefly, phage were generated by helper phage infection and concentrated by PEG/NaCl precipitation according to standard protocols known in the art. The phage library was previously cleared on untransfected parental HEK293F cells by gentle shaking overnight at 4 ℃. After PEG/NaCl precipitation, the pre-cleared library was incubated with HEK293 cells or LNCAP cells expressing chimpanzee PSMA while gently shaking for 2 hours at 4 ℃. Removal of unbound phage and recovery of phage-bound cells was performed by Ficoll gradient, and after several washing steps, phage-bearing cells were incubated with 1mL of TG-1 E.coli (E.coli) culture for 30min at 37 ℃ without stirring. The resulting mixture was seeded on LB-carbenicillin-1% glucose plates and grown overnight at 37 ℃. The process is then repeated for subsequent panning rounds.

The phage Fab-pIX was converted to Fab-His to generate E.coli supernatants. The resulting phage Fab-pIX hits were converted to Fab-His using standard procedures. Plasmid DNA was isolated from phage-panned E.coli (Plasmid Plus Maxi kit, Qiagen Cat. No. 12963) and subjected to NheI/SpeI restriction digestion. The resulting 5400bp and 100bp fragments were separated on a 0.8% agarose gel and the 5400bp fragment was gel purified (MinElute PCR purification kit, Qiagen Cat. No. 28006). The purified 5400bp band was self-ligated using T4 ligase and the resulting product (encoding the Fab-his fusion) was transformed back into the TG-1 E.coli strain and clonally isolated. Fab-His supernatants were generated from clones by overnight induction of cultures with 1mM IPTG. After centrifugation of the overnight culture, the clarified supernatant was prepared for use in downstream assays. To determine the relative expression levels of the different Fab-his supernatants, the serial dilutions of the supernatants were subjected to an anti-k (Southern Biotech, Cat. No. 2061-05) ELISA. All clones tested showed similar expression of Fab-his (data not shown).

Cell binding of Fab-His fusions from E.coli. Cell-based binding assays were designed to assess the binding ability of individual Fab-his fusions from e.coli supernatants to PSMA-expressing cells. After pIX excision, individual Fab clones were isolated from the 3 rd round output of all panning experiments. Fab clones were tested for binding to HEK cells expressing chimpanzee and cynomolgus PSMA, and binding to human PSMA on LNCaP cells. Briefly, PSMA-expressing cells were aliquoted at a density of 200,000/well into V-shaped plates (CoStar 3357) and incubated with Fab fragment-expressing (100 μ Ι) supernatant for 1 hour on ice. Cells were washed twice with PBS containing 2% FBS and stained with mouse anti-human κ -RPE antibody (Life Technologies, cat # MH10514) on ice for 1 hour. Cells were washed twice with PBS containing 2% FBS and resuspended in 100 μ L of the same wash buffer. Plates were read on a BD FACS Array flow cytometer. FACS data were analyzed in FlowJo software by real-time gating of healthy cell populations using forward and side scatter, and then analyzing PE staining of cells within the gate. The Mean Fluorescence Intensity (MFI) was calculated and derived into Microsoft Excel. Fab clones that showed binding > 3-fold background for all three PSMA species (cynomolgus monkey, chimpanzee and human) and did not show binding to the HEK293 cell line were marked as "primary positive". Fab were sequenced and moved forward to clone into mammalian expression vectors for rescreening. True positives were selected from the binding of Fab supernatants expressed by mammalian cells to PSMA-expressing cell lines.

Mammalian Fab is prepared. To convert E.coli Fab into mammalian expressed Fab, In-Fusion HD clone (ClonTech Cat No. 638918) was used according to the manufacturer's protocol. Briefly, the cloned nucleotide sequences that had passed the preliminary screening and were transferred into the mammalian Fab form were loaded into the "InFu Primer Finder v1.2.3" program (software developed in-house) which generated a list of isotype specific PCR primers used to generate PCR fragments for seamless cloning into the huKappa _ muIgGSP and huG1 Fab expression vectors. These vectors are internal vectors with a CMV promoter based on pcdna3.1. Following the seamless cloning procedure, E.coli clones were isolated, sequence verified and transfected into HEK293 cells using standard protocols. Mammalian PSMA fabs for confirmation of binding to PSMA-expressing cell lines were prepared by harvesting 20ml of supernatant from the transfection after 5 days.

Hits from whole cell panning were rescreened as mammalian supernatants. Confirmation of mammalian expressed Fab supernatants was performed using a whole cell binding assay. Fab binding to chimpanzee, cynomolgus monkey and human PSMA (LNCaP cells) was tested and screened against non-binding to the parental HEK cell line.

Dose response curve of mammal-expressed Fab. Once positive binding of the mammalian-expressed Fab clones to PSMA-expressing cell lines as pure Fab supernatants was confirmed, the supernatants were normalized to protein concentration by Octet or protein gel, and dose response curves were completed using the protocol previously described to confirm PSMA binding.

Preparation of anti-PSMA mAb. Clones that displayed binding to all three PSMA-expressing cells were finally converted by restriction cloning to mAb IgG4 with Fc substitutions S228P, F234A, and L235A (PAA) isotype. Briefly, constructs corresponding to Fab clones that had passed the initial screen were digested with HindIII and ApaI. The gel-purified fragments were ligated into an internal expression vector with CMV promoter for generating human IgG4-PAA expression. The heavy and light chains of each PSMA mAb were expressed using the previously described internal expression vectors, both of which were transiently co-transfected into 293Expi or CHO cell lines for mAb expression.

A monospecific anti-PSMA antibody PSMB127 was generated comprising VH and VL regions having the VH of SEQ ID No. 5 and VL of SEQ ID No. 6, and IgG4 constant regions having substitutions S228P, F234A, and L235A, as described in tables 2 and 3 below.

TABLE 2 VH and VL of PSMB127

TABLE 3 HC and LC of PSMB127

The interaction of parent PSMA mAb PSMB127 with human, chimpanzee, and cynomolgus monkey PSMA ECD was measured by Surface Plasmon Resonance (SPR) using the ProteOn XPR36 system (BioRad). A summary of binding affinities for human, chimpanzee and cynomolgus PSMA ECD is shown below.

TABLE 4 Kd data for PSMB127 anti-human, chimpanzee and cynomolgus PSMA

Example 3 Generation and characterization of anti-CD 3 antibodies

Production of anti-CD 3 antibodies. Commercial anti-CD 3 antibody SP34 (mouse IgG1 isotype anti-human CD3IgG1 antibody) was humanized by human frame adaptation method (Fransson et al, JMB,2010398(2): 214-31). To maintain the conformation of CDR-H3, mouse residues of Val38, Gly48, Gly51 and V59 of VL and Ala at position 48 in VH were retained. These "back mutations" were added to the humanization plan. The resulting anti-CD 3 variant was designated CD3B 146.

The humanized anti-CD 3 antibody binds to cells endogenous to primary T cells. Testing of CD3B146 with Primary human T cells and Primary cynomolgus monkey CD4⁺Binding of cell surface CD3 epsilon on T cells to assess retention of cross-reactivity. CD4 purified from peripheral blood of cynomolgus monkey is used⁺T cells (Zen Bio, Triangle Research Park, USA). In brief introductionInstead, the binding of anti-CD 3 antibodies to cell surface CD3 epsilon was assessed by flow cytometry using primary human T lymphocytes purified by negative selection (Biological Specialty, colomar, USA). Expression supernatants or purified antibodies in culture medium or FACS buffer (BD BioSciences) were normalized to 10 μ g/ml, respectively. 2X 105 cells were aliquoted into each well of a 96-well round bottom plate (CoStar) for labeling. The antibody in the expression supernatant was added to the cells and incubated at 4 ℃ for 45 min. After centrifugation at 1300rpm for 3min and removal of supernatant, 50 μ L of anti-human IgG (H + L) Alexa Fluor 647 secondary antibody (Life technologies Inc.) was incubated with cells at a final concentration of 10 μ g/mL for 30min at 4 ℃ away from direct light, then washed and resuspended in 30 μ L FAC buffer (BD BioSciences). Sample collection was performed on the intellictyt HTFC system using the ForeCyt software.

Two internal phage-derived antibodies with the same Fc region as the therapeutic antibody were used as controls: non-cynomolgus monkey cross-reactive agonistic antibody G11 was used as a positive control and non-binding/non-agonistic antibody CD3B124 was used to assess non-specific binding. The commercially available SP34 antibody was not used as a control in this assay because it is a mouse antibody and the use of a different secondary detection reagent would prevent direct comparison with the variants tested. Although a titration series was run, for clarity, the median concentration is shown in fig. 1 using the mean fluorescence intensity value (FIM). CD3B146 showed strong binding to both human and cynomolgus T cells, indicating that CD3B146 retained species cross-reactivity between human and cynomolgus CD3 epsilon (fig. 1 and 2).

Functional analysis of humanized anti-CD 3 hits in primary T cells. To investigate the ability of the CD3B146 variant to induce human T cell activation by CD3 epsilon cross-linking, primary human T cells were cultured overnight in the presence of bead conjugated antibodies. The next day, cells were harvested and labeled with anti-CD 69 antibody to measure activation. Humanized anti-CD 3 antibody was conjugated to protein a coated magnetic beads (spheerech, Lake forest, USA). The next day, 2 × 10⁵Primary human T cells were seeded in round bottom cell culture plates in triplicate and 2X 10 added⁵Coated beads. After overnight culture at 37 ℃, cells were harvested and used with anti-CD 69AlexaThe 488 antibody (clone FN 50; Biolegend) was labeled to assess the upregulation of the activation marker. Sample collection and analysis was performed as described above for the binding. Several negative controls were run, including T cells alone, T cells containing uncoated beads, and T cells containing isotype control (CD3B94) coated beads. A positive control was run for comparison, including a commercially available SP34-2 antibody (fig. 3).

FN50 anti-CD 69 antibodies have been described as cross-reactive with non-human proteins and were therefore used to test cynomolgus monkey CD4+ T cell activation. CD3B146 showed the ability to activate both human and cynomolgus monkey (fig. 3).

Preparation of anti-CD 3 mAb. CD3B146IgG1 was converted to mAb IgG4PAA GenMab form (Labrijn et al, 2013) with Fc substitutions S228P, F234A and L235A (PAA) and substitutions F405L and R409K (numbering according to the EU index). S233P, F234A and L235A were Fc silent mutations, whereas F405L and R409K mutations would allow heterodimerization with PSMA antibodies containing native IgG4F405 and R409 residues. Briefly, the Heavy Chain (HC) variable region was subcloned onto human IgG4-PAAFc containing the S228P, F234A, L235A, F405L and R409K mutations using an internal expression vector with CMV promoter using standard molecular biology techniques. Light Chain (LC) variable regions were subcloned into human lambda constant regions using an internal expression vector with a CMV promoter using standard molecular biology techniques. The resulting plasmids were transfected into Expi293F cells (Invitrogen) and mAb expressed. anti-CD 3 antibody was purified using standard purification methods: a protein a column with an elution buffer of 100mM NaAc pH3.5 and a neutralization buffer of 2M Tris pH 7.5 and 150mM NaCl. Mab desalting was performed using PD10(Sephadex G25M) column and pool

The resulting monospecific anti-CD 3 antibody was renamed CD3B219 and comprised VH and VL regions having the VH of SEQ ID NO. 15 and the VL of SEQ ID NO. 16, and an IgG4 constant region having S228P, F234A, L235A, F405L and R409K substitutions. CD3B219 comprises the heavy chain of SEQ ID NO 17 and the light chain of SEQ ID NO 18. As a control, a monospecific anti-RSV antibody derived from B21M paired as a blank arm with the CD3 or PSMA arm of a bispecific antibody. The VH and VL sequences of CD3B219 are shown in table 5.

TABLE 5 VH, VL, HC and LC of CD3B219

Example 4 preparation of a PSMAXCD3 bispecific antibody

The formation of the PSMAxCD3 bispecific antibody was performed by combining the PSMA mAb PSMB127(VH SEQ ID NO:5, VL SEQ ID NO:6) with the high affinity CD3B219(VH SEQ ID NO:15, VL SEQ ID NO:16) CD3 arm. The targeting Parent (PSMA) contained the natural IgG4 amino acids F405 and R409, while the killing parent (CD3) contained the F405L GenMab mutation and the R409K mutation.

Parental PSMA and CD3 antibodies were purified using a protein a column with an elution buffer of 100mM NaAc pH3.5 and a neutralization buffer of 2M Tris pH 7.5 and 150mM NaCl. The mAb was desalted using a PD10(Sephadex G25M) column and dialyzed into D-PBS buffer at pH 7.2.

After purification, the parent PSMA antibody was mixed with the desired parent CD3 antibody in 75mM cysteamine-HCl under reducing conditions and incubated at 31 ℃ for 4 h. The recombination reaction is based on a molar ratio, wherein a set amount of PSMA (e.g., 10mg or about 67.8 nanomolar) is combined with a CD3 antibody (e.g., about 71.8 nanomolar), wherein the amount of CD3 antibody added is 6% greater than the PSMA antibody. The concentration of the PSMA antibody stock varied from 0.8mg/mL to 6mg/mL, and the volume of the recombination reaction varied from pair to pair. Recombinants were then dialyzed against PBS to remove reducing agents. The bispecific antibody reaction was performed with an excess of CD3 antibody (ratio) to minimize the amount of unreacted PSMA parent antibody remaining after recombination. After partial reduction of the parent mAb, the reducing agent was removed by overnight dialysis into PBS. The final PSMAxCD3 antibody was named PS3B 27.

Selected PSMA hits were also paired with non-killer arms (blanks) to generate negative controls for test purposes. For the control bispecific antibody B2M1, RSV antibody in the form of IgG4PAA was produced, purified, and combined with CD3 arm CD3B219-F405L, R409K to produce CD3B288(CD 3X blank), or with PSMA arm PSMB162, PSMB126, PSMB130 to produce PS3B37, PS3B39, and PS3B40, respectively (PSMA X blank).

TABLE 6 HC and LC cDNA SEQ ID NO

Table 7 VH, VL, HC and LC proteins SEQ ID NO.

TABLE 8 HC/LC sequences of PSMA X CD3 bispecific antibody (PS3B27) and corresponding SEQ ID NO

TABLE 9 PSMA x The VH and VL chain sequences of the CD3 bispecific antibody (PS3B27) correspond to SEQ ID NOs.

TABLE 10 CDR sequences of the PSMA xCD3 bispecific antibody (PS3B27) and corresponding SEQ ID ID NO.

Example 5 bispecific binding of PSMAxCD3 to PSMA Positive cell lines

The PSMAxCD3 bispecific antibody was tested for binding to the PSMA positive cell line LNCAP, human PSMA-HEK, chimpanzee-PSMA-HEK, and cynomolgus monkey PSMA-HEK. Bound antibody was detected by anti-human kappa light chain PE conjugated detection reagent (Invitrogen). Mean Fluorescence Intensity (MFI) is a measure of binding to bispecific antibodies. Conversion of MFI to relative EC₅₀。EC₅₀Is a commonly used dose response curve in which the half maximal effective concentration or EC₅₀The point is defined as the inflection point of the curve. EC (EC)₅₀Values were determined by measuring cell-bound bispecific and known concentrations. High concentrations result in maximum target antigen binding, i.e. complete binding saturation. The dose response curve is then diluted to background or a dose response curve without bispecific binding. The inflection point of the curve reflects EC₅₀And (4) point. By applying at EC₅₀The ug/ml amount of bispecific antibody was taken at the spot and converted to a molar concentration value based on the Mw of the bispecific antibody to determine the calculated EC₅₀. Bispecific antibodies were normalized to protein concentration and then incubated with the same number of cells expressing human or cynomolgus PSMA. MFI at each concentration was collected by flow cytometry and plotted as a function of concentration. Data were transformed by log10 and then plotted. Non-linear regression of binding curves to determine EC₅₀The value is obtained. Cell-based binding of EC to PS3B127 in whole cells using LNCaP, cynomolgus monkey and chimpanzee PSMA expressing cell lines₅₀Value and calculated EC₅₀The values are shown in Table 11.

₅₀Table 11: cell-based binding EC values。

Example 6 affinity of the PSMA xCD3 bispecific antibody for recombinant PSMA protein

To further evaluate the antibodies, the rate of association and dissociation of chimpanzee PSMA ECD was measured and these hits were inherited from the cell binding assay. The interaction of the PSMAxCD3 bispecific mAb with the target (recombinant chimpanzee, PSMA) was studied by Surface Plasmon Resonance (SPR) using the ProteOn XPR36 system (BioRad). Biosensor surfaces were prepared by coupling anti-human IgG Fc (Jackson ImmunoResearch Laboratory, catalog number 109-005-098) to the modified alginate polymer layer surface of the GLC chip (BioRad, catalog number 176-5011) according to the manufacturer's instructions for amine-based coupling chemistry. Approximately 4400RU (response unit) anti-human IgG Fc antibody was immobilized. Kinetic experiments were performed in running buffer (DPBS + 0.03% P20+ 100. mu.g/ml BSA) at 25 ℃. For kinetic experiments, 100RU of bispecific antibody was captured, followed by injection of analyte (recombinant chimpanzee PSMA ECD) at a concentration of 3.7nM to 300nM (in 3-fold serial dilutions). The bound phase was monitored at 50. mu.L/min for 3 minutes, followed by 15 minutes of buffer flow (dissociation phase). At 100. mu.L/min, with 100mM phosphoric acid (H)₃PO₄Sigma, catalog number 7961) to regenerate the chip surface.

Results for each bispecific antibody are given in k_a(association ratio), k_a(dissociation Rate) and K_D(equilibrium dissociation constant) was recorded. The results are shown in Table 12.

Table 12: PS3B27(PSMB127 x CD3B219) for recombinant human PSMA, recombinant chimpanzee PSMA and recombinant cynomolgus monkey Summary of kinetics and affinity of PSMA (3.7-300 nM). The parameters reported in the table were obtained from the 1:1 langmuir binding model. _D _d _aAffinity, K ═ K/K。

n is 3 independent experiments and repeated 2 times. Results are presented as mean ± standard deviation.

Example 7: toxicology studies

Study toxicology assessment of drugs in studies conducted by IV administration.

The IV administration tolerance of study drugs was evaluated in a single/repeated dose non-GLP exploratory toxicology study. The dosage range is 0.03mg/kg to 3 mg/kg. Different dosage regimens were used for SA males and SM males and females. The most significant and dose limiting toxicity is cytokine release, which is primarily the first dose effect. Plasma cytokines appear to be directly associated with mortality. The increase of Interferon (IFN) -gamma, Interleukin (IL) -2, IL-6, IL-10 and Tumor Necrosis Factor (TNF) -alpha is observed mainly at 0.06mg/kg (Q3D or Q1W). At non-tolerated doses (. gtoreq.0.1 mg/kg), the animals were found to die or euthanize due to side effects, mainly between day1 and day 2 of the first dose. The cause of death could not be determined histologically in all early deceased individuals and was presumably due to severe cytokine release. Microscopic findings on the planned day of necropsy (day 30) for monkeys in the 0.06mg/kg (Q3D and Q1W) and 0.3mg/kg (Q1W) groups included mononuclear infiltrates in the liver, kidney, and gallbladder; minimal to mild tubular degeneration/regeneration; minimal multifocal tubular mineralization; tubular findings or mononuclear interstitial infiltrates around large blood vessels; and mild myelorrhagia. The maximum tolerated dose (most sensitive to study drug-induced cytokine release) for SM men was 0.06mg/kg (Q3D or Q1W). There was a loss of exposure (apparently due to ADA) in most animals dosed for more than 2 weeks, so the duration of the subsequent study was limited to 2 weeks.

In a key GLP study in SM cynomolgus monkeys, study drug was expected to be administered by IV slow bolus injection of Q1W (3 total doses) or Q3D (6 total doses) for 2 weeks. The dose of Q3D administered to males is 0mg/kg, 0.03mg/kg or 0.06 mg/kg. Females received 0mg/kg, 0.06mg/kg or 0.2 mg/kg. The dose of Q1W was 0.06mg/kg for males and 0.2mg/kg for females. Generally, a dose-related increase in cytokine plasma concentration was observed in both male and female monkeys at dose levels ≧ 0.03 mg/kg. Emesis (0.06mg/kg Q3D and 0.2mg/kg Q3D/Q1W) and humpback posture (0.03mg/kg and 0.06mg/kg Q3D) were primarily associated with administration of the first dose. Clinical signs are thought to be associated with cytokine release. One of the 5 females (0.2mg/kg Q1W) was euthanized at day 3 due to a decline in clinical status and possibly due to severe cytokine release. In animals that successfully completed dosing, no macroscopic changes associated with the study drug were observed, but microscopic findings were observed at ≧ 0.03mg/kg (from the planned necropsy at day 16/17). These findings were limited to lymphocyte infiltrates noted in the perivascular regions of the kidney (minimal to mild), liver (minimal to moderate) and gall bladder (mild), which were reversed at the end of the 57 day recovery period, except for a mild perivascular infiltration in the kidneys of 1 female (0.2 mg/kg; Q3D). The highest non-severe toxicity dose (HNSTD) in the critical study was 0.06 mg/kg/dose. After dosing on day1, monkeys administered Q3D (male and female) or Q1W (male) had corresponding mean Cmax of 1.85 μ g/mL or 1.99 μ g/mL, and AUCDay1-4 or AUCDay1-8 were 1.72 μ g-day/mL or 2.37 μ g-day/mL, respectively.

A non-GLP investigational toxicology study was conducted to determine if the dose-limiting cytokine release observed in previous studies could be mitigated. Two methods were tested, which included dose escalation in animals following either priming with low dose (0.01mg/kg) or prophylactic treatment with tocilizumab (IL-6 receptor antagonist). The study drug was administered by IV slow bolus injection of Q3D during the low dose priming study phase, either in a slow intra-animal dose escalation regimen (0.01 → 0.02 → 0.04 → 0.12 → 0.6mg/kg) (fig. 4A) or a fast intra-animal dose escalation regimen (0.01 → 0.03 → 0.1 → 0.4 → 1.5mg/kg) (fig. 4B).

Cross-study clinicopathological changes by IV administration

Cross-study analysis was performed in male and female cynomolgus monkeys comparing the clinical pathology associated with IV administration of study drug in single/repeat dose non-GLP exploratory studies, key GLP toxicology studies (T-2015-.

The changes in the clinical pathological parameters were generally similar in all 3 studies and were not related to the presence or severity of clinical signs in individual animals, including animals that were euthanized early due to a degenerative condition. These findings suggest that the clinical pathological changes themselves are not generally sensitive or specific biomarkers for the clinical signs or overall tolerance associated with the study drug under these study conditions.

Many clinical pathological changes were most pronounced after the first dose, with minor or no consistent changes observed after subsequent doses. These changes include decreased platelets, red cell mass, reticulocytes, lymphocytes and monocytes (except after the escalating dose discussed below), eosinophils, clotting time (except after the escalating dose), Blood Urea Nitrogen (BUN), creatinine, most liver enzymes and bilirubin, as well as changes in phosphorus and electrolytes. Several clinical pathological changes are believed to be likely associated with study drug-related cytokine release and proinflammatory states including acute phase response (proinflammatory states associated with decreased albumin and cholesterol and increased C-reactive protein, triglycerides and globulins) and possibly neutrophil, eosinophil and basophil changes, prolonged clotting time, increased bilirubin and increased BUN and creatinine. The reduction of lymphocytes in all studies is believed to be likely a result of the expected pharmacological activity associated with the involvement of CD 3. Other clinical pathological changes include increased liver enzymes and decreased minerals and electrolytes.

Among these changes, lymphopenia and monocytosis, as well as mild prolongation of Activated Partial Thromboplastin Time (APTT), generally persist longer in animals undergoing dose escalation than in animals repeatedly dosed at the same dose level; the longer duration of these changes is associated with dose escalation in animals and not necessarily with administration of low priming doses. In most studies, other changes usually persist throughout (or begin later in) the dosing phase, including acute phase response, increased alkaline phosphatase, increased some leukocyte parameters (eosinophils, basophils and large unstained cells), and calcium reduction.

Although improved dose level tolerability was noted at low dose priming, the effect was limited by the clinical pathology parameters chosen. The most significant differences in animals undergoing low dose priming were no changes in renal parameters (BUN, creatinine and phosphorus increase) and a persistent presence of lymphopenia and monocytic depletion and mildly prolonged APTT. These differences indicate a prime-related effect, but the lack of a change in renal parameters contributes indefinitely to the improvement in tolerance. In addition, the extended clotting time (most notably APTT) in animals undergoing a low dose priming at all doses (either by 0.6mg/kg or 1.5mg/kg) is generally less than the clotting time in animals at similar doses in the absence of priming.

Local tolerance study when subcutaneous administration of study drug

Local tolerance of SC (subcutaneous) administration of study drug was evaluated in sexually mature male cynomolgus monkeys. Animals received 2 weekly doses of study drug, 0.9% saline or formulation buffer (aqueous solution containing 10mM sodium acetate, 8% sucrose, 0.04% polysorbate 20 and 0.02mg/mL disodium EDTA, ph 5.2). After the two doses, injection sites were assessed up to 96 hours after the dose and animals were necropsied on day 15. No drug-related changes were studied in clinical observations, body weight, qualitative food assessment, general or microscopic findings in injection sites or draining lymph nodes. An increase in the concentration of study drug-associated plasma cytokines (MCP-1, IL-10, IL-6, TNF- α, IFN-) was observed, although significantly lower than that observed when the same dose was administered IV. Study of clinical pathological parameters drug-related changes included lymphocytes, monocytes, eosinophils, large unstained cells, reticulocytes, and thrombocytopenia, as well as acute phase responses (increased C-reactive protein and decreased albumin). These changes are transient after the first dose. After the second dose, the clinical pathology was limited to a mild reduction of lymphocytes. The mean Cmax at day1 and day 8 was 0.28ug/mL and 0.33ug/mL, respectively, and AUCDay0-7 or AUCDay7-14 was 1.35. mu.g/day/mL and 1.58. mu.g/day/mL, respectively

Example 8: phase 1, first human dose escalation study of study drugs in patients with advanced solid tumors

Abbreviations

TABLE 13 abbreviations used throughout the examples are as follows

Definition of terms

TABLE 14 terminology used throughout this example。

AUC	Area under serum concentration versus time curve
		AUC(_t1-t2)	Area under concentration-time curve from time t1 to time t2
CL
		C_max	Maximum serum concentration observed
C_min	Observed minimum serum concentration
		EC_20、50、90	Drug concentrations required to produce 20%, 50% or 90% of maximum effect
RA	Cumulative ratio
		t_1/2	End slope of the curve with semilogarithmic drug concentration versus time (lambda.)_z) Associated apparent elimination half-life
T_max	Time corresponding to last quantifiable serum concentration
		VSS	Volume of distribution

1. Summary of the solution

1.1. Summary of the invention

The study drug was developed to develop bispecific antibodies for evaluating the therapeutic potential of targeting Prostate Specific Membrane Antigen (PSMA) for CD 3-mediated T cell redirection. The study drug was human IgG4 antibody. By antibodies from 2 species: the controlled fragment antigen binding arms of PSMB127 and CD3B219 were exchanged to generate bispecific antibodies. PSMB127 is a whole cell panning anti-PSMA antibody derived from a phage library on a PSMA overexpressing cell line. CD3B219 is an anti-CD 3 epsilon antibody derived from the common domain antibody SP34, which is further humanized and affinity matured.

PSMA is a transmembrane protein expressed in normal prostate and its expression is increased during malignant transformation, including expression on bone metastases. In addition, PSMA is overexpressed in neovasculature of other malignancies. It is hypothesized that the study drug, a bispecific antibody targeting both PSMA and CD3, will direct the body's immune cells to kill these PSMA-overexpressing malignant cells. The mechanism of action of the study drug achieved T cell-mediated cytotoxicity by recruiting CD 3-expressing T cells to PSMA-expressing target cells. This mechanism for cell killing is unique, providing an opportunity to treat patients whose disease has been demonstrated to be resistant to current therapies.

Goal, endpoint and hypothesis

TABLE 15 targets, endpoints and assumptions

Hypothesis

No formal statistical hypothesis testing will be performed in this study. The study will be evaluated as follows:

dose escalation (part 1): RP2D of the study drug can be identified such that < 33% of participants experience dose-limiting toxicity (DLT).

Dose extension (part 2): study drug was safe and showed preliminary clinical activity at RP 2D.

Figures 5 and 6 provide a graph of potential exploration of dose escalation and dose extension plans and priming dose schedules.

Overall design

This is a FIH, open label, multicenter, phase 1 study used to evaluate the safety, pharmacokinetics, pharmacodynamics and preliminary clinical activity of study drug monotherapy in late stage cancer participants. The study will be performed in 2 sections: dose escalation (part 1) and dose extension (part 2). In part 1, adult males with metastatic castration resistant prostate cancer (mCRPC) with recurrent disease following Androgen Receptor (AR) targeted therapy will be enrolled. Dose escalation will be supported by an improved continuous re-assessment method (mCRM) based on statistical model Bayesian Logistic Regression Model (BLRM) using dose Escalation (EWOC) principles governing overdose. The study will start with an accelerated titration followed by a standard titration phase. The goal of part 1 was to determine the MTD of the study drug and select the dose and regimen to be used in part 2 dose escalation (i.e., RP 2D). The purpose of section 2 was to further evaluate safety, pharmacokinetics, pharmacodynamics and biomarkers (blood and tissue), as well as to evaluate the preliminary clinical activity of study drugs in mCRPC and Renal Cell Carcinoma (RCC).

After the first 2 study drug administrations (and any priming doses, if administered), the participants will be hospitalized for 48 hours to facilitate safety monitoring and pharmacokinetic assessments. Subsequent hospitalizations for study drug administration will be necessary for participants who meet certain safety criteria (previous grade 2 neurological toxicity, dose escalation in patients for the priming schedule, or previous grade 2 CRS that did not resolve to grade 1 within 72 hours). To minimize the risk associated with the expected infusion-related reactions (IRR), corticosteroid prodrugs were required prior to the first administration of the study drug and subsequent doses for participants who experienced neither grade ≦ 1 IRR nor CRS after the first dose would be reduced or eliminated.

During the study, the study evaluation group (SET) will monitor safety, especially at each dose escalation step of part 1. The study will begin with a weekly dosing schedule. Alternate schedules (e.g., twice weekly or prime schedules) may be explored based on the emerging data determined by the SET.

Participants will continue to receive study medication until radiographic disease progression, clear clinical progression, unacceptable toxicity, withdrawal of consent, investigator or sponsor decision, or study termination. The end of the study (study completion) is defined as the last safety assessment for the last participant in the study.

Number of participants

Approximately 70 participants will receive treatment in this study. However, the amount of samples will depend on the number of queues explored.

Study of drugs and duration

TABLE 16 study drug duration

Efficacy assessment

Clinical activity will be assessed using the following assessments: computed Tomography (CT) scans with neck, chest, abdomen and pelvis contrast; magnetic Resonance Imaging (MRI) (i.e., for sites that are not adequately imaged using CT). Additional assessments of participants with mCRPC included serum Prostate Specific Antigen (PSA) and a whole-body bone scan: (^99mTc). Assessment of prostate treatment response will be performed according to prostate cancer working group 3(PCWG3) criteria and response assessment criteria for solid tumors (RECIST), version 1.1, to assess the progression of soft tissue lesions (CT or MRI). RECIST version 1.1 will evaluate the therapeutic response of RCC.

Pharmacokinetic, biomarker and immunogenicity assessment

Blood samples were collected to characterize the serum pharmacokinetics and anti-drug antibodies of the study drug. Blood samples will also be collected to assess pharmacodynamics, safety, and biomarkers predictive of response or resistance to study drug treatment. Forced fresh tumor biopsies from accessible sites of metastatic disease will be collected from selected PK/PD cohorts of parts 1 and 2 before and during the study to assess PSMA expression and pharmacodynamic markers in tumor tissues.

Security assessment

The safety of study drugs will be assessed by physical examination (including assessment of the basal nervous system), ECOG physical performance status, clinical laboratory tests, vital signs, electrocardiogram, adverse event monitoring. The concomitant medication use will be recorded. The severity of adverse events will be assessed using the "national cancer institute adverse event general term criteria" (version 5.0). Cytokine release syndrome has been identified as an adverse event of particular concern and will require enhanced reporting and data collection.

Statistical method

No formal statistical hypothesis testing will be performed in this study. Dose escalation will be supported by mCRM using EWOC principle based on statistical model BLRM.

1.2. Scheme(s)

Figures 5 and 6 provide a graph of potential exploration of dose escalation and dose extension plans and priming dose schedules.

1.3. Activity schedule

TABLE 17 Activity Schedule-weekly administration Schedule parts 1 and 2

a. The study center visit for each plan may be ± 2 days from the date of the plan. Assessments and procedures (including laboratory testing) can be performed up to 48 hours prior to the planned study drug administration. Based on emerging data, the planned assessment schedule can be adjusted by the sponsor to preserve patient safety or fully characterize the PK or PK/PD characteristics of the study drug. Additional (i.e., unintended) blood samples for cytokine profile, PK, or PD assessment can be collected up to 8 times during the first 4 cycles of treatment with the study drug.

b. Must be signed prior to the first study-related activity.

c. Disease characteristics include tumor type and histology, time of diagnosis, tumor stage at diagnosis and screening, available pathology and molecular data, previous anti-cancer therapies, and date of recent disease progression.

d. See section 8.2.

e. Physical examination was completed at screening. Examination for symptoms and disease will be performed prior to administration of all study drugs. During physical examination at screening, a baseline neurological examination was performed at least every 12 hours prior to the first treatment dose and any priming dose, and during hospitalization. For drug administration as an outpatient, a neurological examination can be performed as clinically indicated.

g. Laboratory evaluation description:

the inclusion and non-exclusion criteria presented in section 5.1 and section 5.2, respectively, must be met before the study drug is first administered.

On study drug administration days, there was no need to repeat laboratory assessments performed within 48 hours prior to infusion.

Additional samples may be collected and analyzed as clinically indicated.

Laboratory evaluations will be performed at the local laboratory.

Pregnancy tests performed at screening and before the first administration of the study drug must be highly sensitive serum (β human chorionic gonadotropin [ β hCG ]).

h. Vital signs were assessed for the first dose of study drug immediately prior to initiation of infusion, every 30 minutes during infusion, at the end of IV flush, and 1, 2, and 3 hours after the end of IV flush. All other infusions: immediately before infusion begins, every 30 minutes during infusion, end of IV flush, and as clinically indicated. Oxygen saturation and body temperature were monitored on the same schedule as vital signs. Vital signs and O2 saturation were monitored until normalized after CRS event.

i. See section 6.5.3 for instructions on the drug to be administered prior to study drug administration.

j. For a weekly dosing schedule, drug administration must be separated by at least 5 days for each study. The actual dose administered (μ g) will be calculated based on the participant's body weight (kg) at baseline on study day1 (see Table 24)

k. Efficacy evaluation is seen in section 8.1. Baseline assessments are acceptable if performed within 6 weeks (42 days) prior to the first administration of the study drug.

Objective response according to RECIST v1.1 must be confirmatory scanned after 4 weeks.

If the study drug is discontinued before disease progression begins, disease assessment should continue according to local standard of care (see section 8).

The same method used to assess disease state at baseline should be used throughout the study.

-if there is a delay in the study treatment schedule, the disease assessment should not be delayed.

Information may be obtained by telephone contact every 12 weeks after discontinuation of study medication until one of the discontinuation criteria in section 7.2 is met.

Treatment end visit was completed no more than 30(+7) days after the last study drug administration and before the start of new anti-cancer therapy (whichever occurred first) (see section 8 treatment end visit).

TABLE 18 Activity Schedule-weekly dosing Schedule for biomarkers, pharmacokinetic and immunogenic samples Part 1

a. All reasonable attempts should be made to collect samples within ± 10% of the planned sampling time (i.e. calculated from the end of IV flush) and the collection time must be recorded.

b. The sample will be shipped to the laboratory designated by the sponsor; the analysis will be performed by the sponsor. Repetitive or unplanned samples (i.e., pharmacokinetic, pharmacodynamic, biomarker) may be taken for safety reasons or for technical issues with the sample.

c. Unless the collection of biopsies presents a safety risk, participants with accessible lesions enrolled in the selected PK/PD cohort in sections 1 and 2 must agree to perform a forced fresh tumor biopsy.

Fresh biopsies at screening can be collected within 6 weeks (42 days) before the first administration of study drug, provided that active anticancer therapy is not started during this period.

Post-treatment tumor biopsy sample collection time (i.e. between 4 and 8 weeks after completion of DLT assessment period and after treatment initiation) can be changed by SET based on emerging data.

The samples will be sent to a central laboratory designated by the sponsor (see "laboratory manual" for details).

d. Samples will be collected in two different tubes (see "laboratory manual" for details).

e. If a suspected level 2 IRR or level 2 CRS event is observed or reported, then the following unplanned samples should be collected:

pharmacokinetic/immunogenicity samples: as close as possible to the time of occurrence of the event, 24 hours and 72 hours after the start of the event.

-cytokine sample: within 4 hours after the start of the event.

f. Receptor occupancy samples will be collected for the dose escalation cohort treated with doses of 1 μ g/kg or higher.

g. If the 72 hour sampling time point occurs on a weekend, a sample may be collected at 96 hours.

h. For all subsequent doses, blood samples before dosing and immediately after EOI (+ -15 minutes) should be collected for PK.

TABLE 19 Activity Schedule-weekly dosing Schedule for biomarkers, pharmacokinetic and immunogenic samples Section 2

Abbreviations: CRS ═ cytokine release syndrome; CTC ═ circulating tumor cells; ctDNA ═ circulating tumor DNA; CyTOF ═ time-of-flight cytometry; ending the EOF (IV) flushing; EOT-end of treatment; IRR ═ infusion-related reactions; IV is intravenous; sequencing; PK ═ pharmacokinetics; SET — research evaluation group; TCR ═ T cell receptor; TBNK ═ T cells, B cells, natural killer cells.

a. All reasonable attempts should be made to collect samples within ± 10% of the planned sampling time (i.e. calculated from the end of IV flush) and the collection time must be recorded.

Fresh biopsies at screening can be collected within 6 weeks (42 days) before the first administration of study drug, provided that active anticancer therapy is not started during this period.

Post-treatment tumor biopsy sample collection time (i.e. between 4 and 8 weeks after completion of DLT assessment period and after treatment initiation) can be changed by SET based on emerging data.

The samples will be sent to a central laboratory designated by the sponsor (see "laboratory manual" for details).

d. If a suspected level 2 IRR or level 2 CRS event is observed or reported, then the following unplanned samples should be collected:

pharmacokinetic/immunogenicity samples: as close as possible to the time of occurrence of the event, 24 hours and 72 hours after the start of the event.

-cytokine sample: within 4 hours after the start of the event.

e. For all subsequent doses, blood samples before dosing and immediately after EOI (+ -15 minutes) should be collected for PK.

f. Samples will be collected in two different tubes (see "laboratory Manual" for details)

g. If the 72 hour sampling time point occurs on a weekend, a sample may be collected at 96 hours.

2. Introduction to the design reside in

The study drug was a humanized immunoglobulin G4 proline, alanine (IgG4PAA) bispecific antibody targeting the CD3 receptor complex on T lymphocytes (T cells) and the Prostate Specific Membrane Antigen (PSMA) expressed on tumor cells and tumor associated neovasculature. The study drug was designed to promote T cell activation in close proximity to PSMA-expressing target cells, followed by lysis of tumor cells by cytotoxic T cells (Buhler P, Wolf P, Gierschner D et al; Cancer Immunol Immunother.2008; 57(1): 43-52).

A summary of in vitro and in vivo pharmacology, safety pharmacology, and toxicology is presented in this section. The term "research drug" throughout this document refers to a research drug, and the term "sponsor" refers to an entity listed in a contact information page to be provided as a separate document.

2.1. Study the basic principle

2.1.1. Prostate specific membrane antigen

PSMA is a transmembrane glycoprotein consisting of 750 amino acids and 3 protein domains (a small intracellular domain, a single-pass transmembrane domain, and a large extracellular domain).

PSMA is highly expressed in prostate cancer and is reported to also express 0 in neovasculature of other solid tumors including lung, bladder and renal cancers. In a recent study investigating PSMA expression in Renal Cell Carcinoma (RCC), immunohistochemical results showed that endothelial PSMA protein 0 was detected in 80% of clear cell renal cell carcinomas, 14% of papillary carcinomas and 72% of chromophobe carcinomas. Further analysis from the same study showed that PSMA expression was significantly associated with lower overall survival in patients in both clear cell and papillary renal cell carcinoma. In another clinical study, the use⁶⁸The PSMA-based radiotracer of Ga enables detection of PSMA in metastatic lesions found in patients with clear cell carcinoma⁰. Thus, the PSMAxCD3 method may have therapeutic benefit for patients with histology, such as clear cell renal cell carcinoma.

2.1.2.CD3 redirection method

Recently, several approaches have been developed to redirect T cells to tumor surface antigens. These methods include drugs that disrupt tumor tolerance by T cell checkpoint blockade (McDermott DF, Atkins MB. cancer Med.2013; 2(5):662-673) and bispecific T cell adaptors (BiTEs) (CD3xCD19) targeting CD19,(Borateuzumab) ((B))[ US FDA product Label]. Thousand Oaks, USA, Amgen Inc.; 12 months in 2018).

The lack of sufficient immune presence in the tumor microenvironment in PSMA-positive tumors such as mCRPC may explain the lack of efficacy of checkpoint inhibitor monotherapy in prostate cancer. T cell redirection is an important approach to enhance the immunogenicity of such tumors.

The mechanism by which PSMA is targeted is currently being evaluated in clinical studies for the treatment of prostate cancer is expected to be two other CD3 redirection approaches similar to the study drug. First, an Fc-competent bivalent bispecific CD3-PSMA molecule (Hernandez-Hoyos G, Sewell T, Bader R et al, Mol Cancer ther.2016; 15(9): 2155-. Second, the Fc-free CD3-PSMA bispecific T cell adaptor (BITE) molecule (Klinger M, Benjamin J, Kischel R, Stienen S, Zugmaier G.Harnesing Immunol Rev.2016; 270(1): 193-208). Preliminary clinical data from this phase 1 study indicate that up to 80 μ g/day of dose is tolerated and a radiographic response is induced in CRPC patients. Another study of trispecific T cell activating construct (TriTAC) compounds was also evaluated in mCRPC (Lemon B, Aaron W, Austin R et al Cancer research.2018, abstract 1773).

The study drug contained a mutated IgG4Fc with significantly reduced binding to Fc γ R, but uninterrupted binding to FcRn to ensure extended half-life (t)_1/2). Compared to Fc-competent bivalent bispecific CD3-PSMA molecules and TriTAC compounds, the study drug was more similar to endogenous human IgG antibodies, which could lead to a reduced production of anti-drug antibodies (ADA) and ultimately to an improved pharmacokinetic exposure and efficacy profile.

2.1.3. Basic principle of initial dose

The first human FIH starting dose of 0.1 μ g/kg was selected using the minimum expected biological Effect level (MABEL) method. The results of in vitro cytotoxicity experiments and Good Laboratory Practice (GLP) toxicology studies in cynomolgus monkeys are considered to be compatible with those based on european medicines administration and FDA industry guidelines: non-clinical evaluation of S9 anticancer drugs determined that the recommendations for the starting dose were consistent (FDA u.s.department of health and public service, the industry guidelines for non-clinical evaluation of S9 anticancer drugs, 3 months 2010).

Performing in vitro cytotoxicity assaysTo characterize study drug-induced T cell activation, PSMA + tumor cell killing, and cytokine release. These assays were performed using purified human T cells from 6 healthy human donors and C4-2B, a human prostate cancer cell line that expresses PSMA and exhibits sensitivity to T cell-mediated killing. Purified T cells from healthy donors, but not cancer patients, were used to obtain a more conservative estimate of the initial dose of MABEL. Among the readings evaluated (T cell activation, cytotoxicity and cytokine release), T cell activation was shown to be the most sensitive reading (20). Median Effective Concentration (EC) of activated T cells from 6 normal donors₂₀Values determine the MABEL concentration at 0.023nM (3.45 ng/mL).

The human pharmacokinetics of the study drug was predicted from cynomolgus pharmacokinetic data using the allometric growth law. A clinical starting dose of 0.1. mu.g/kg after the first dose is predicted to yield about 0.020nM C_maxIt was slightly lower than the MABEL concentration of 0.023nM as determined above.

The following considerations are also critical in determining the starting dose:

selecting a purified T cell system (instead of whole blood) as the effector cell population, since PSMA-expressing target cells are reported not to be present in peripheral circulation in any significant amount.

The C4-2B cell line was physiologically associated with PSMA target expression, similar to that observed in prostate cancer. Of the several prostate cancer cell lines evaluated (22-RV, C4-2/C4-2B and LNCAP/LNCAP-AR), C4-2B is the one most sensitive to T cell-mediated killing of target cells.

The effector to target (E: T) ratios of 3:1, 5:1, 10:1 and 20:1 were evaluated in an in vitro cytotoxicity assay, and the E: T ratio of 3:1 was selected to provide a conservative estimate of the starting dose.

Based on the highest non-severe toxic dose of 0.06mg/kg from key GLP toxicology studies (HNSTD), using body surface area transformation methods, the equivalent dose of human HNSTD is 20 μ g/kg, and the maximum recommended starting dose based on HNSTD is 3.3 μ g/kg, which is 33 times higher than the proposed MABEL-based starting dose.

The lowest dose of study drug tested in cynomolgus monkeys was 0.01 mg/kg. At this dose level, minimal levels of cytokine release and minimal clinical signs and symptoms were observed.

TABLE 20T cell mediated cytotoxicity, cytokine release and T cells with study drugs using C4-2B cells Summary of exposure-response analysis of cell activation assays

Abbreviations: EC (EC)₂₀The concentration of drug required to produce 20% of the maximal effect.

Based on the overall evaluation of in vitro and in vivo data, and the choice of FIH starting dose based on MABEL, a weekly dose of 0.1 μ g/kg of study drug should result in drug exposure with minimal biological activity in the participants treated in this study.

According to prediction, t of the study drug_1/2In humans, this supports the decision to start the study with a weekly dosing schedule, about 4.9 days (at doses with non-linear clearance saturation). An alternative dosing schedule for twice weekly treatments can be explored. Monoclonal antibodies may exhibit faster clearance at lower doses due to target-mediated drug treatment. The decision to change from once weekly to twice weekly schedule will be determined by the research evaluation group (SET) based on emerging pharmacokinetic, pharmacodynamic and safety data.

2.2. Background of the invention

2.2.1. Summary of non-clinical studies

PSMA tumor and normal tissue expression profiles

PSMA protein was detected in 26 out of 30 patient samples from patients with prostate adenocarcinoma tumor samples, most of which showed a heterogeneous staining pattern for PSMA. To assess PSMA expression on human normal tissues, PSMA protein staining was performed on human tissue-microarrays by immunohistochemistry. Of all the different tissues tested, only prostate, brain, kidney, liver, breast, small intestine and salivary glands were positive for PSMA. Overall, PSMA expression in extraprostatic normal tissues appears to be highly restricted, mainly in the cytoplasm, and expressed at much lower levels than in prostatic tumor tissues. These results are generally consistent with those reported in the literature (Kinoshita Y, Kurastukuri K, Landas S et al, World J Surg.2006; 30: 628-.

Study of drug binding to prostate tumor cell lines

The study drug specifically bound endogenous PSMA-expressing prostate tumor cell lines in a concentration-dependent manner as measured by flow cytometry on all PSMA-expressing tumor cell lines tested (C4-2B, LNCaPAR, 22RV 1). In contrast, the study drug did not bind to PC-3 cells, the PSMA negative cell line.

Study of drug-mediated T cell activation

To measure study drug-mediated T cell activation, PSMA-positive tumor cell lines were co-cultured with donor T cells from 6 normal donors for 48 hours in the presence of study drug. Study drug induced a dose-dependent increase in CD25 expression, a marker of T cell activation in PSMA-positive cell line (C4-2B), but not in PSMA-negative cells (PC-3). For the PSMA positive cell line C4-2B, the median EC (EC) was determined for all donors from 3 separate experiments_20/50/90) And report (EC)₂₀：0.02nM，EC₅₀：0.06nM，EC₉₀: 0.40 nM). The 2 blank antibodies did not produce T cell activation in either the C4-2B or PC-3 cell lines.

Study of drug-mediated in vitro T cell-dependent cytotoxicity of prostate tumor cell line

To measure the ability of study drugs to induce cytotoxicity of PSMA-expressing tumor cells, donor T cells were co-cultured with tumor target cells at a 3:1 ratio for 72 hours and conjugated with increasing amounts of study drug or lacking CD3 or PSMA fragment antigenBlank antibodies from the arms were incubated together. The study drug caused dose-dependent cytotoxicity only in the PSMA-positive C4-2B cell line and not in the PSMA-negative PC-3 cell line. For the C4-2B cell line, the median EC values for all 6 donors from 3 separate experiments were calculated and reported (EC)₂₀：0.04nM，EC₅₀：0.08nM，EC₉₀: 0.31 nM). The 2 blank antibodies did not produce T-cell dependent cytotoxicity in either the C4-2B or PC-3 cell lines.

Study of the Effect of drugs in vivo prostate tumor xenograft models

The efficacy of the study drug was evaluated in a human PSMA-positive prostate tumor xenograft model LNCaP Androgen Receptor (AR) tumor. Established tumors were implanted into non-obese diabetic (NOD) Severe Combined Immunodeficiency (SCID) gamma (NSG) mice transplanted with human T cells. Statistically significant anti-tumor efficacy was observed at 2.5, 5.0 and 10mg/kg dose levels of study drug, achieving 51%, 72% and 74% Tumor Growth Inhibition (TGI) (p <0.0001) compared to vehicle-treated control mice, respectively.

Study of the in vivo pharmacodynamic Effect of drugs on CD8+ T cell tumor infiltration

To determine whether the anti-tumor activity of the study drug correlates with immune cell infiltration into the tumor, LNCaP AR tumor-bearing mice were injected with human T cells and sera and tumors were collected from phosphate buffered saline control-treated mice or from mice treated with 2.5mg/kg, 5.0mg/kg, and 10mg/kg study drug. At all dose levels of study drug, a time-dependent increase in tumor CD8+ T cell infiltration was observed by immunohistochemical staining.

Conclusion

In vitro and in vivo results indicate that study drugs specifically bind PSMA-expressing tumor cells, induce T cell activation, and effectively redirect T cells to induce cytotoxicity of PSMA-expressing tumor cells.

2Summary of non-clinical toxicology, pharmacokinetics and safety pharmacology

2.2.2.1. Toxicology

Cynomolgus monkeys were selected as the pharmacologically relevant toxicology species because the study drug had similar binding affinity to cynomolgus PSMA and CD3 (compared to humans) and similar functional activity (cytotoxicity) on cells expressing cynomolgus and human PSMA. Rodents are pharmacologically irrelevant.

As outlined herein, the potential toxicity of the study drug was characterized in 3 studies in cynomolgus monkeys.

non-GLP exploratory toxicology studies

In non-GLP exploratory studies (n ═ 1 to 6), tolerance of the Intravenous (IV) study drug was assessed in cynomolgus monkeys (0.03mg/kg to 3mg/kg) using several dose regimens in standard, Sexual Maturity (SM) males and SM females. The most significant dose-limiting toxicity (DLT) is cytokine release, which is primarily the first dose effect. Plasma cytokines appear to be directly associated with mortality. Increases in IFN-. gamma.IL-2, IL-6, IL-10 and TNF-. alpha.were observed. It was noted that the sexually mature male cynomolgus monkeys were most sensitive to the effects of the study drug and had higher cytokine release than the standard male and sexually mature females. After days 10 to 15, a significant loss of exposure was observed in most monkeys (due to the anti-drug antibody [ ADA ]), and thus the duration of the subsequent study was limited to 2 weeks. At the Maximum Tolerated Dose (MTD) of 0.06mg/kg, the dosing frequency was well tolerated once every 3 days (Q3D; 8 doses total) and once weekly (Q1W; 4 doses total), and the majority (and highest) of cytokine release was observed at the first dose.

At the non-tolerated dose, monkeys were moribund or euthanized between day1 (. gtoreq.6 hours) and day 2 of the first dose, except for one female (0.6mg/kg) euthanized on day 8 (after the first dose). Mortality in this study was generally correlated with plasma cytokine levels. The cause of death could not be determined histologically in all early deceased individuals and was presumably due to severe cytokine release. Microscopic findings on the planned date of necropsy (day 30) included mononuclear infiltration in the liver, kidney, gall bladder, minimal to mild tubulointerstitial degeneration/regeneration, mineralization (0.06mg/kg, Q3D; 8 doses), tubular findings or mononuclear interstitial infiltration around large vessels, and mild bone marrow hypercellularity. In addition, minimal multifocal tubular mineralization was noted in the kidneys of a single female receiving a 0.3mg/kg dose. No histological association associated with mortality was identified in early necessaries. The MTD of SM males (most sensitive) was 0.06mg/kg (Q3D or Q1W).

Glp toxicology study

In a key GLP study in SM cynomolgus monkeys, study drug was administered by IV bolus injection Q1W (3 total doses) or Q3D (6 total doses) for 2 weeks, followed by a 6-week recovery period. The dose of Q3D administered to males is 0mg/kg, 0.03mg/kg or 0.06 mg/kg; females received 0mg/kg, 0.06mg/kg or 0.2 mg/kg. The dose of Q1W was 0.06mg/kg for males and 0.2mg/kg for females. Clinical signs (vomiting, humpback posture) are mainly associated with the administration of the first dose and are usually not observed during the latter dosing phase (consistent with cytokine release). Generally, a dose-related increase in cytokine plasma concentration was observed in both male and female monkeys at dose levels ≧ 0.03 mg/kg.

One of the 5 females (0.2mg/kg Q1W) was euthanized on day 3 due to a decline in clinical condition. The cause of death of the monkey could not be determined and could be due to severe cytokine release. In monkeys that successfully completed dosing, no changes in drug-related body weight, food consumption, physical examination measurements, and ocular effects were studied, and there were no abnormalities in Electrocardiogram (ECG) or changes in blood pressure, heart rate, respiration rate, body temperature, urinalysis, gross autopsy findings, or absolute or relative organ weight. Study drug-related microscopic findings of > 0.03mg/kg (from planned autopsy on day 16/17) were limited to lymphocyte infiltrates noted in perivascular regions of kidney (minimal to mild), liver (minimal to moderate), and gall bladder (mild). All microscopic findings resolved after a six-cycle recovery period of 57 days, except for a mild perivascular infiltrate remaining in the kidneys of 6 females receiving 0.2 mg/kg. HNSTD in the key study was 0.06 mg/kg/dose.

non-GLP research study (role of cytokine release managed using low dose prime or prophylactic toluzumab)

non-GLP studies were performed to determine if the dose-limiting cytokine release observed in previous studies could be mitigated. Two methods were tested, which included dose escalation in animals following either a priming dose or prophylactic treatment with tollizumab.

In the low dose prime portion of the study period, study drug was administered by IV slow bolus injection on days 1, 4, 7, 10 and 13 in slow dose escalation (0.01 → 0.02 → 0.04 → 0.12 → 0.6mg/kg) and fast intra-animal escalation (0.01 → 0.03 → 0.1 → 0.4 → 1.5 mg/kg). Both increasing cohorts successfully completed dosing, with no mortality and significant improvement in clinical signs, and no study drug-related effects on changes in apparent food consumption or physical examination measurements. Improvement in clinical signs (sporadic mild to moderate vomiting, liquid feces, transient and minimal changes in body temperature on day 1) may be associated with low levels of cytokine release at the 0.01mg/kg prime dose and significantly reduced cytokine release at subsequent escalating doses. At the planned necropsy on day 19, mixed cell infiltration into multiple organs and degeneration/regeneration of tubular (minimal) and acinar cells (minimal to mild) in kidney and prostate were observed in two dose-escalation cohorts, respectively. Additional changes believed to be consistent with a systemic inflammatory response include hematopoietic aggregates in the heart (in the fast ascending cohort) and mononuclear cell infiltration in the two dose ascending dose cohorts, as well as fibrin accumulation within the femoral tibial synovial joint. None was found to be considered disadvantageous.

During the tollizumab prophylactic treatment study period, study drug was administered by IV slow bolus injection at 0, 0.1, 0.3, or 0.9mg/kg on days 1 and 8 after the single dose of tollizumab given the previous day (approximately 8 to 24 hours prior to study drug administration). Tolizumab appears to have some protective effect (0.1mg/kg) or delayed mortality (0.3mg/kg) when compared to observations in studies that did not have previous pretreatment with trulizumab. Touzumab did not improve tolerability in monkeys receiving 0.9mg/kg, and monkeys were euthanized approximately 7 hours after dosing on day 1. Prophylactic tollizumab appeared to have no discernible effect on study drug-mediated cytokine release (or related clinical signs), and microscopic and clinical pathology findings were similar to those noted in studies without pre-treatment with tollizumab.

Summary of clinical pathological changes noted in the Cross-Studies

Cross-study analysis of male SM monkeys was performed to compare the clinical pathological changes associated with study drug administration in non-GLP exploratory studies, 2-week key GLP toxicity studies, and non-GLP low dose priming studies. Changes in the clinicopathological parameters were generally similar in all 3 studies and represent a systemic inflammatory response. These findings were not correlated with the presence or severity of clinical signs in individual monkeys, including monkeys that were euthanized early due to a degenerative condition. The clinical pathological changes themselves are generally not sensitive or specific biomarkers for studying drug-related clinical signs or overall tolerance. Observed changes include a decrease in leukocyte counts (neutrophil, lymphocyte, monocyte and eosinophil counts), an increase in neutrophil, eosinophil and basophil counts in some studies, a decrease in erythrocyte pellets, a decrease in platelet counts, an increase in acute phase reactants, an increase in alkaline phosphatase, an increase in renal parameters such as urea nitrogen and creatinine, a decrease in serum calcium, an increase in clotting time, an increase in enzymatic activity and an increase in bilirubin. The above findings do not indicate a discernible dose-dependent relationship.

2.2.2.1.1. Tissue cross reaction

GLP cross-reactivity studies were performed in frozen sections of normal human tissues with study drug and its anti-PSMA parental (bivalent) antibody (positive control). No unexpected tissue cross-reactivity of the study drug was observed. Due to PSMA expression in these tissues, membrane staining of epithelial cells in the prostate and staining of extracellular material using the study drug and the anti-PSMA parent antibody are expected. Based on the expression of CD3 epsilon on T cells, it was expected that monocytes were stained with the study drug alone.

2.2.2.1.2. Assays in human serum or whole blood

The study drug did not cause hemolysis in human whole blood and was compatible with human serum at in vitro concentrations of 0.010mg/mL and 10mg/mL

2.2.2.1.3. Cytokine release

In an in vitro assay, study drugs induced statistically significant and concentration-dependent cytokine release in 6 of 10 cytokines measured in whole blood from healthy donors (IL-1 β, IL-2, IL-8, IL-10, IFN- γ, and TNF- α).

2.2.2.2. Safety pharmacology

No drug-related changes were studied in clinical observations of body temperature, blood pressure, heart rate, respiratory rate or neurobehavioral. No abnormalities in the rhythm or ECG waveform morphology associated with the study drug were found at any dose level based on comparison of the pre-and post-dose ECGs. Hypotension and tachycardia, possibly associated with cytokine release, were observed in monkeys after treatment with other CD3 shift antibodies.

2.2.2.3. Non-clinical pharmacokinetics and immunogenicity

Pharmacokinetics/toxicology (PK/TK) of study drugs were characterized after single IV administration in cynomolgus monkeys at the expected doses of 0.3mg/kg, 0.6mg/kg and 3mg/kg as part of a non-GLP exploratory toxicology study in standard age (adolescents-2.5 to 4 years) or SM male monkeys. Based on limited data from surviving monkeys, study drug exposure increased with dose in a manner approximately proportional to dose over the range of doses tested. Similar Clearance (CL), distribution volume (Vss) and t were estimated for each dose group_1/2. Compared to typical IgG-based therapeutic monoclonal antibodies, study drugs showed relatively high CL (18.69 mL/day/kg to 26.17 mL/day/kg) and shorter t_1/2(2.48 days to 3.12 days).

Multiple post-IV-administration studies characterized in SM cynomolgus GLP toxicology studiesPK/TK of the drug. Monkeys received IV bolus injections of study drug Q3D (6 doses) or Q1W (3 doses) for 2 weeks, followed by a 6-week recovery period. Due to expected differences in tolerance and sex, male monkeys received doses of 0.03mg/kg and 0.06mg/kg of Q3D, and 0.06mg/kg of Q1W, respectively; female monkeys received 0.06mg/kg and 0.2mg/kg doses of Q3D, and 0.2mg/kg dose of Q1W, respectively. Average C_maxAnd AUC increases in a manner approximately proportional to dose over the range of doses tested. The mean drug cumulative ratio after Q3D administration was in the range of 1.30 to 1.57 in the 0.03mg/kg and 0.06mg/kg dose groups and 0.95 for the 0.2mg/kg dose group. Following Q1W administration, there was no systemic accumulation of study drug. A reduction in drug exposure following the fifth Q3D dose or the second Q1W dose was observed in multiple monkeys compared to PK/TK following the first dose on day1, which may be associated with the development of ADA. There was no significant PK/TK difference between male and female monkeys.

As part of the non-GLP exploratory toxicology study and the non-GLP exploratory toxicity study in cynomolgus monkeys, the PK/TK of study drugs after multiple (i.e., Q3D or Q1W) IV administrations were also examined, and the results were similar. In a non-GLP investigational toxicity study of SM cynomolgus monkeys, study drug was administered by IV injection on days 1, 4, 7, 10, and 13 in slow dose escalation (0.01 → 0.02 → 0.04 → 0.12 → 0.6mg/kg) and fast escalation (0.01 → 0.03 → 0.1 → 0.4 → 1.5mg/kg), respectively, study drug exposure increasing with dose in a manner approximately proportional to dose. Average C after the highest dose of 1.5mg/kg in GLP toxicology studies_maxAnd AUC higher than after 0.06mg/kg Q3D IV dose>10 times.

The immunogenicity of the study drug in cynomolgus monkeys was evaluated in a non-GLP exploratory toxicity study and a GLP toxicity study. Forty out of 56 monkeys treated with IV doses of study drug tested positive for ADA. Of the other 16 monkeys, 13 had no suitable samples for immunogenicity determination (i.e., no samples on or after day 13), and therefore their ADA status was not evaluable; the remaining 3 monkeys tested ADA negative. Overall, the incidence of ADA was high for the study drug. The immunogenicity of animals is not expected to predict human immunogenic responses.

2.3. Benefit/risk assessment

This was the first clinical study to study drugs. The potential risk and mitigation strategies are based on safety data obtained from non-clinical studies, known mechanisms of action (i.e., T cell activation and tumor cell lysis), and routes of administration. Although PSMA expression in normal tissues is highest in prostate tissues, relatively low levels of membrane expression are also detected in brain, kidney, liver, breast, small intestine and salivary glands (see section 2.2.1). Thus, there is potential to study drug-induced toxicity in these organs. Safety monitoring will include frequent laboratory assessments (hematochemistry and hematology) and physical examinations including neurological assessments to monitor potential toxicity in these organs.

Potential risks are indicated below. Prevention of immune effects and PSMA expression patterns is discussed in section 6.1.2. A dose modification guide is provided in section 6.6.

Immune effects: guidelines for managing these potential safety risks of pre-treatment drugs are provided in section 6.1.2.

Infusion-related reactions (IRR) (section 6.1.2.1)

Immune-related adverse events (section 6.1.2.2)

-Cytokine Release Syndrome (CRS) (section 6.1.2.3)

Potential toxicity caused by PSMA expression pattern:

tumor lysis syndrome-monitoring adverse events and chemical parameters after first study drug administration

Nephrotoxicity-monitoring adverse events and chemical parameters

Hepatotoxicity-monitoring adverse events and chemical parameters

Nervous system toxicity (section 6.1.2.4)

Mumps/salivary gland toxicity-monitoring adverse events

Gastrointestinal toxicity-monitoring adverse events

Clinical laboratory abnormalities: consistent with the expected pharmacological function of CD3 involvement, the most notable changes in laboratory parameters observed in cynomolgus monkey toxicology studies include changes in leukocytes (primarily lymphopenia, monocyte and eosinophil depletion, sometimes followed by increases in these and other leukocytes), increases or decreases in neutrophils, thrombocytopenia, red cell mass depletion, acute phase reactions, increases in renal parameters, prolongation of clotting time, and increases in liver enzyme activity and bilirubin.

It is unknown whether there are clinical benefits associated with study drug treatment. Study drugs have the potential to result in effective killing of PSMA-expressing target cells, such as tumors or tumor-associated neovasculature cells, and may result in increased overall survival in patients with advanced disease and limited treatment options.

3. Target and endpoint

TABLE 21 target and endpoint。

Suppose that

No formal statistical hypothesis testing will be performed in this study. The study will be evaluated as follows:

dose escalation (part 1): RP2D for the study drug can be identified such that < 33% of participants experience DLT.

Dose extension (part 2): study drug was safe and showed preliminary clinical activity at RP 2D.

3.1.1. Research medicine

The study drug was developed to evaluate bispecific antibodies targeting PSMA for the therapeutic potential of CD 3-mediated T cell redirection. The study drug was an engineered human IgG4 antibody. By the antibodies from 2 parent antibodies: the controlled fragment antigen binding arms of PSMB127 and CD3B219 were exchanged to generate bispecific antibodies. PSMB127 is a whole cell panning anti-PSMA antibody derived from a phage library on a PSMA overexpressing cell line. CD3B219 is an anti-CD 3 epsilon antibody derived from the common domain antibody SP34, which is further humanized and affinity matured. It was hypothesized that the study drug would induce enhanced T cell-mediated cytotoxicity by recruiting CD 3-expressing T cells to PSMA-expressing cells. This will result in activation of T cells and induce subsequent PSMA-positive cell lysis mediated by cytotoxic T cells.

4. Design of research

4.1. Overall design

This is a FIH, open label, multicenter, phase 1 study used to evaluate the safety, pharmacokinetics, pharmacodynamics and preliminary clinical activity of study drug monotherapy in late stage cancer participants. Approximately 70 participants will receive treatment in this 2-part study. Additional participants may be recruited if a priming dose schedule is explored. Once participants are determined to be eligible (i.e., inclusion/exclusion criteria) for the study and informed consent has been provided for study participation, study medication will be administered as an IV infusion. The overall safety of study treatment was continuously assessed by SET throughout the study (see section 4.1.4). Preliminary clinical activity will be assessed according to the assessment outlined in section 8.1. The pharmacodynamics of the study drug will be characterized by pre-treatment and in-treatment biopsies in the selected cohort as determined by the sponsor (see table 19).

Part 1 (dose escalation)

Part 1 of the study was designed to determine the MTD of study drug in participants with metastatic castration resistant prostate cancer (mCRPC) and select RP2D and protocol. Dose escalation will begin with the MABEL based starting dose and proceed as shown in figure 5. Dose escalation will be supported using an adaptive design dose escalation strategy guided by an improved continuous re-evaluation method (mCRM) based on statistical model Bayesian Logistic Regression Model (BLRM) with dose Escalation (EWOC) principles governing overdose. Dose escalation will be performed in 2 phases: an accelerated titration phase and a standard titration phase.

The decision making by the research panel will be based on an examination of all available data, including but not limited to pharmacokinetics, pharmacodynamics, safety and efficacy. Dose escalation will be performed according to the dose escalation strategy outlined in section 4.1.1.

In section 1a, a single participant cohort was recruited with a SET-specified dose during accelerated dose escalation. Up to 12 additional participants in the pharmacokinetic/pharmacodynamic (PK/PD) cohort may be treated with doses determined by SET to be safe to better understand safety, pharmacokinetics, pharmacodynamics and primary clinical activity. Once grade 2 or 3 non-hematological toxicity or grade 3 hematological toxicity of anemia, neutropenia or thrombocytopenia has occurred, the study will transition from the accelerated titration phase to the standard titration phase and begin the recruitment of 3 to 6 participants per cohort. Standard titration can occur without priming (part 1 b), or at the priming dose (part 1 c) if toxicity ≧ 2 CRS. Additional participants may be enrolled in the PK/PD cohort during standard dose escalation to obtain additional data.

Part 2 (dose extension)

Once RP2D was identified, participants with mCRPC and RCC (20 per cohort) were treated to confirm the safety, pharmacokinetics, pharmacodynamics and preliminary clinical activity of study drugs at RP 2D.

Overall treatment plan

The treatment and priming dose schedules are described in table 24. Priming a priming dose may be considered to reduce toxicity.

Treatment dose schedule: predicted t at 4.9 days based on saturation dose scaled from cynomolgus monkey model_1/2The study will be started with a once weekly therapeutic dose. The starting dose will be 0.1 μ g/kg administered by IV infusion once a week. An alternate schedule of twice weekly treatment doses may be explored. The decision to switch from once weekly to twice weekly therapy will be based on the emerging data and will be made after SET approval. Dose escalation decisions and subsequent dose levels will be determined based on statistical models using all available safety, pharmacokinetic, pharmacodynamic and clinical activity data to identify safe and tolerable RP 2D. After the study drug RP2D has been identified in section 1,enrollment of part 2 will begin.

Prior to the first dose of study drug, corticosteroid prodrugs will be administered to minimize the risk associated with IRR (see table 30). For subsequent doses, corticosteroid prodrugs can be reduced or omitted. For participants who experience grade 2 or higher IRR, a pre-infusion corticosteroid will be required for at least 1 subsequent dose administered to the participant.

Priming dose schedule: since bispecific T cell adaptor antibodies such as bornauzumab may cause acute cytokine-mediated toxicity associated with first dose administration, a priming dose strategy has been effectively used for these antibodies. In this study, the priming dose schedule will be initiated after the first occurrence of CRS levels greater than or equal to 2. One or more initial lower doses may be administered prior to a subsequent higher therapeutic dose to mitigate acute toxicity that may be associated with T cell activation and cytokine release. Selection of priming doses is described in section 4.1.1.

Required standard of hospitalization and discharge

Part 1: after IV wash, participants will be hospitalized for at least 48 hours for the first 2 treatment doses and any relevant priming doses of study drug. Hospitalization will be optional for subsequent doses unless certain safety criteria are met: previous grade 2 neurological toxicity, dose escalation in patients to the priming schedule, or previous grade 2 CRS that did not resolve to grade 1 within 72 hours. If any of these toxicities occurred during study drug administration, participants will be hospitalized for at least 48 hours after the next study drug administration (post IV flush) to monitor signs and symptoms associated with CRS or nervous system toxicity.

Section 2: based on experience from section 1, hospitalization may not be required. However, if the participants had previous grade 2 neurological toxicity or previous grade 2 CRS that did not resolve to grade 1 within 72 hours, hospitalization would be required for at least 48 hours after the next study drug administration.

Discharge standard

The following criteria must be met before the participant is discharged from the hospital: vital signs and oxygen saturation within the normal range, including the absence of fever (defined as body temperature ≦ 100.4 ° f (38 ℃) for at least 24 hours), and the absence of any significant grade ≧ 2 adverse events not attributed to the underlying disease.

Treatment discontinuation/follow-up

Participants will receive study medication until radiographic disease progression, clear clinical progression, unacceptable toxicity, or any other standard of discontinuation of treatment is met (see section 7). However, treatment beyond disease progression is contemplated (see section 8.1.2). For participants who discontinue study treatment for reasons other than disease progression (e.g., adverse events), disease assessment will continue according to local standard of care until disease progression or a new anti-cancer therapy is initiated (or another study exit criterion is met). After discontinuation of treatment, participants will have an end of treatment (EOT) visit within 30(+7) days after the last dose of study drug and will continue to follow-up in the study as outlined in section 8.

Data cutoff and end of study

The sponsor will establish a clinical data expiration date for Clinical Study Report (CSR) analysis reports, which may occur before the end of the study. The data cutoff will be communicated to the research center. Participants who continued to receive study medication or who were in a follow-up state after data expiration would continue to monitor according to table 7 until the end of the study. These data will be reported in the final CSR to the appropriate health authorities. The final data from the research center will be sent to the sponsor (or designee) after completion of the final participant visit to the research center, within the time frame specified in the clinical trial protocol. Study end (study completion) is defined in section 4.4.

4.1.1. Rule of dose escalation

Part 1: dose escalation decisions will be made by SET based on safety, pharmacodynamic, pharmacokinetic and other biomarker data for the mCRM with all DLT data and all previous dose levels. Preliminary clinical activity (if available) will also be reviewed by SET at each dose escalation step.

In part 1, the mCRM will proceed in 2 stages: (1) an accelerated titration phase and (2) a standard titration phase (with and without priming). Dose escalation will begin with the weekly administered therapeutic dose; based on emerging data, twice weekly dosing can begin. As described later in this section, the priming schedule may be explored. The mCRM will proceed as follows:

part 1 a-accelerated titration

The following rules apply during accelerated titration using mCRM.

Dose escalation will start from a single (at least 1) participant cohort.

If more than 1 participant is treated at a certain dosage level, the first participant treated at that given dosage level must be observed for 48 hours before subsequent participants are treated.

At least 1 participant who has completed the DLT assessment period needs to be assessed before SET determines that the dose is safe and before the next cohort enrollment (see section 4.1.3).

Dose escalation will be performed as directed by BLRM using EWOC guidelines (i.e., providing the highest recommended dose), except that the next dose level cannot exceed the 3.5-fold increment of the previous dose.

The study may switch from accelerated titration to standard titration if one of the following occurs during the DLT assessment period:

grade 2 non-hematologic toxicity of anemia, neutropenia or thrombocytopenia or more

Grade 3 or more of hematological toxicity: part 1 b-standard titration without priming.

-one or more ≧ 2-level CRS events: part 1 c-with priming standard titration.

Up to 12 additional participants may be enrolled into the PK/PD cohort at doses determined to be safe by SET to obtain additional pharmacokinetic, pharmacodynamic or biomarker data. Once the criteria for stopping accelerated dose titration have been met, the dose escalation is converted to a standard titration as described below.

Part 1 b-Standard titration (without priming)

The following rules apply during standard titration using mCRM.

At least 3 participants need to be evaluated for dose levels to complete the DLT evaluation period before determining the next cohort of doses (section 4.1.1).

The first participant treated at a given dosage level must be observed for 48 hours before subsequent participants are treated.

Primary model by DLT (see section 9.1.1)

If no participant in the cohort experiences a DLT, dose escalation of the therapeutic dose may be performed as directed by BLRM using EWOC guidelines (i.e., providing the highest recommended dose), except that the next dose level may not exceed the 3.5-fold increment of the previous dose.

If one participant in the queue experiences a DLT during a DLT period, the SET (as directed by the BLRM using the EWOC principle) can do either;

consent to recruit additional participants before determining the next dosage level or

Reevaluating the cohort based on all available data and updated DLT probabilities, and determining the next dose cohort that is guided by BLRM using EWOC principles (i.e. providing the highest recommended dose)

If 2 participants in a particular dose cohort experience a DLT, further recruitment to that dose cohort will cease and the SET will re-evaluate the cohort based on all available data and updated DLT probabilities. Based on re-evaluation of the dose cohort, additional participants may be recruited to the current or lower dose cohort only if the dose level still complies with EWOC guidelines and is agreed to by SET.

Up to 12 additional participants may be enrolled into the PK/PD cohort at doses determined to be safe by SET to obtain additional pharmacokinetic, pharmacodynamic or biomarker data.

If a ≧ 2-level CRS event is observed, the study may initiate priming (section 1 c).

Part 1 c-Standard titration (with priming)

The priming dose was administered on day1, followed by the therapeutic dose on day 8. However, based on review of available data and after review by SET, more than one priming dose may be administered.

The priming dose will be determined as follows：

If the first CRS event is grade 2 or 3, the dose level at which the first event occurs will extend to at least 6 participants.

-if no additional ≧ 2-rank CRS is observed, this dose level will be considered as the priming dose.

-if additional participants have ≧ 2 levels of CRS, no observation that the previous dose level for CRS will extend to at least 6 participants.

If no more than 1 of 6 participants experienced grade 2 or grade 3 CRS, then the dose level would be considered a day1 priming dose.

If the first CRS event is ≧ 4-level CRS, no previous dose level for CRS observed will extend to at least 6 participants.

-if no more than 1 of 6 participants experienced grade 2 or grade 3 CRS at this lower dose level, that dose level would be considered a day1 priming dose.

Initial priming queue

In the first priming cohort, the treatment dose will be determined as follows:

the first therapeutic dose will be determined by mCRM.

-if the first CRS event is > 2-rank, the therapeutic dose may be reduced to a dose below that at which > 2-rank CRS is observed.

At least 3 participants need to be evaluated on a priming schedule to complete the DLT evaluation period before determining the dose for the next cohort (section 4.1.3).

The first participant treated at a given dosage level must be observed for 48 hours before treating subsequent participants.

Primary model determined by DLT

If no participant in the cohort experiences a DLT, dose escalation may be conducted as directed by BLRM using the EWOC principle (i.e., providing the highest recommended dose), except that the next dose level may not exceed the 100% increment of the previous dose.

If one participant in the queue experiences a DLT during a DLT period, the SET (as directed by the BLRM using the EWOC principle) can do either;

consent to recruit additional participants before determining the next dosage level or

Up to 12 additional participants may be enrolled into the PK/PD cohort at doses determined to be safe by SET to obtain additional pharmacokinetic, pharmacodynamic or biomarker data.

Multiple dose levels and dose schedule queues can be recruited in parallel, provided that all the above criteria are met, and that each of the new dose queues/schedules is recommended by the SET and supported by a statistical model that utilizes EWOC principles.

Temporary administration table

A sample temporary dosing schedule is provided at 22. Dose levels will be discussed in the SET meeting (see section 4.1.4) and will be changed based on the emerging data. Intermediate dose level increments ensure safety for study participants. The actual ascending dose level will be guided by mCRM based on BLRM. The maximum dose level for this study has not been identified.

TABLE 22 temporary dosing table

4.1.2. determination of RP2D

RP2D will be determined after reviewing all available pharmacokinetic, pharmacodynamic, safety and efficacy data from at least 6 participants treated with RP2D and at least 12 participants with pharmacokinetic data in all cohorts, and will take into account the recommended dose of BLRM. One or more RP2D may be selected.

Once RP2D was identified, an extended cohort of 2 participants with mCRPC and RCC (approximately 20 per cohort) was treated to confirm the safety, pharmacokinetics, pharmacodynamics and preliminary clinical activity of study drugs at RP 2D.

4.1.3. Definition of dose limiting toxicity

The DLT assessment period was defined as the first 21 days of treatment. If one or more priming doses are explored, the priming period will be included in the DLT assessment period. Participants who do not complete the DLT period for reasons other than DLT may be replaced. If a participant receives less than 75% of each of the prescribed doses for reasons other than toxicity (e.g., disease progression, missed appointments, non-compliance, participant withdrawal) during the time period, the participant can be replaced with a new participant as judged by SET. The SET will consider all available security data from non-evaluable participants. The criteria for DLT are summarized in the table below. Dose-limiting toxicity leading to discontinuation of treatment is described in section 7. These events were evaluated according to the "national cancer institute adverse event general terminology standard" (NCI CTCAE version 5.0).

^aTABLE 23 dose limiting toxicity criteria

Abbreviations: ALP ═ alkaline phosphatase; ALT ═ alanine aminotransferase; AST ═ aspartate aminotransferase; CRS ═ cytokine release syndrome; DLT-dose-limiting toxicity; GGT ═ γ -glutamyl transferase; IRR ═ infusion-related reactions; ULN is the upper normal limit.

a. Unless toxicity is clearly due to a potential malignancy or an extrinsic cause.

b. Optimal supportive care (including electrolyte and hormone supplementation, where clinically applicable) according to institutional standards.

c. The Hai's law standard is defined as ALT or AST value ≥ 3 × ULN, total bilirubin ≥ 2 × ULN, and ALP ≤ 2 × ULN; there is no alternative cause.

d. Grade 3 chemical abnormalities that occur during the DLT phase need to be repeated within 72 hours to confirm the grade or resolve.

4.1.4. Research evaluation group

Participant security and study progress SET monitoring will be established by the sponsor throughout the study. The committee will continuously monitor all treatment emergent data (e.g., pharmacokinetics, pharmacodynamics, safety) throughout the study to ensure continued safety of the participants enrolled in the study. Cumulative data for late toxicity will be monitored.

The SET will be hosted by the research responsible physician of the sponsor. Members will include primary researchers, application clinical scientists, safety physicians (application safety management team chairman), statisticians, clinical pharmacologists, and additional application workers (as the case may be). The team will be in regular meetings throughout the study and it should be applied that the requirements of the sponsor or researcher are made at any time during the study to evaluate the emerging security signals. The documentation of the meeting results will be maintained by the sponsor. Decisions are communicated to researchers, and decisions that may affect participant safety (e.g., adverse changes in risk/benefit assessments) will also be communicated to regulatory agencies in time as needed.

Dose escalation decisions and changes to treatment and procedure schedules will be made by the SET. The schedule for the dose escalation meeting will depend on the frequency of DLTs and whether/when to determine the MTD or Maximum Administered Dose (MAD) or when to determine RP 2D.

SET may also decide to modify the study in progress or stop further enrollment into one or more cohorts if it is determined that sudden toxicity of treatment results in adverse changes in participant risk/benefit. The recruitment may be temporarily suspended for the SET to evaluate the newly emerging data, if desired. The SET chapter will outline the communication plan regarding the decisions or recommendations made by the SET.

4.2. Scientific rationale for research and design

Recently introduced T cell redirecting bispecific agents represent a particularly promising form of immunotherapy. Bispecific agents use hetero-bivalent binding through 2 separate antigen recognition domains; one to recognize tumor antigens and the other to target CD3 on T cells to achieve tumor clearance and circumvent many resistance mechanisms (Ramados NS, Schulman AD, Choi SH et al, J Am Chem Soc. 2015; 137(16): 5288-.

PSMA is a transmembrane protein expressed in normal prostate and its expression is increased during malignant transformation, including expression on bone metastases (Chang SS et al, urology.2001; 57(4): 801-. Furthermore, PSMA is overexpressed in neovasculature of other malignancies (Baccala A et al, urology.2007; 70(2):385- & 390; Chang SS. Rev Urol.2004; 6 (appendix 10): S13-S18; Chang SS et al, Cancer Res.1999; 59(13):3192- & 3198). It is hypothesized that the study drug will direct the immune cells of the body to kill these malignant cells that overexpress PSMA. The mechanism of action of the study drug achieved T cell-mediated cytotoxicity by recruiting CD 3-expressing T cells to PSMA-expressing target cells. This mechanism for cell killing is unique, providing an opportunity to treat patients whose disease has been demonstrated to be resistant to current therapies.

4.2.1. Investigating specific ethical design considerations

The study is ongoing to evaluate safety, pharmacokinetics, pharmacodynamics and potential clinical benefit of study drugs upon repeated dosing to participants with mCRPC or RCC. The results of this study will provide useful information for further development of this compound. A major ethical concern is that the risks and benefits associated with administering study drugs in this FIH study are unknown. To assess drug-related risks in humans in studies, tumor cell lines were used for in vitro and in vivo assessments. Preclinical toxicology and PK/PD studies were performed in cynomolgus monkeys as this is the only relevant species that demonstrated binding of both the PSMA and CD3 arms of the study drug. Although non-clinical studies suggest the potential for antitumor activity in the dose range proposed for evaluation in this study, the therapeutic benefit of the study drug in humans has not been determined. The main findings of the study drugs identified in the studies performed in cynomolgus monkeys were related to cytokine release (dose limiting) and a generalized systemic inflammatory response.

The disease of the participants may not respond to the study drug, or the participants may receive sub-therapeutic doses, especially in the lower dose cohort. Furthermore, toxicity not observed in preclinical studies may occur. Based on preclinical assessments, there is reason to believe positive risk-benefit characteristics based on preclinical data. To ensure the health status of the participants of the treatment in this study, safety and clinical benefit will be closely monitored, as discussed throughout the protocol.

As with all FIH dose-finding PK/PD studies, there are risks associated with venipuncture and multiple blood sample collections. To avoid multiple venipuncture causing additional discomfort and other potential toxic effects, IV indwelling catheters were allowed to be used in this study (see investigator product prep [ IPPI ] for further details). The blood sample collection protocol is designed to collect a minimum number of blood samples that accurately and completely describe the PK/PD characteristics of the study drug. This minimizes the number of venipuncture and total volume of blood collected from each participant during the study. Most blood samples will be collected during the first 8 cycles of treatment. The total blood volume to be collected is considered an acceptable amount of blood collected from the population in this study over the period of time based on the criteria of the american red cross.

The timing of the imaging is designed to acquire progression events and allow clinical researchers to make treatment decisions in a timely manner, while balancing this with preventing overexposure of the participants to radiation. Efficacy assessments will be made as recommended by the internationally recognized solid tumor response assessment criteria (RECIST) v1.1 or the PCWG3 criteria.

Participants with tumor biopsies may be at risk for toxicity associated with the biopsy procedure, including pain, bleeding, and infection, as well as any risk of local or general anesthesia provided according to local standards of care.

The potential participants will be fully informed of the risk and requirements of the study and during the study, the participants will be provided with any new information that may influence their decision to continue participation. They will tell that they agreed that participation in the study was voluntary and could be withdrawn at any time without giving any reason and without harming or losing the benefit they were entitled to. Only participants who are fully able to understand the risk, benefit and potential adverse events of the study and voluntarily provided their consent will be recruited.

4.3. Rationalization of the dosage

See section 2.1.3 for initial dose rationale.

4.4. Definition of study termination

If a participant has died or does not meet the criteria for withdrawal from the study (see section 7), he or she will be considered to have completed the study. The end of the study (study completion) was considered the final safety assessment for the last participant in the study.

5. Study population

Screening of eligible participants will be performed within 30 days prior to administration of study medication. "failure to screen" under conditions that allow any screening procedure to be repeated refers to section 5.4.

Inclusion and exclusion criteria for recruiting participants in this study are described below. If there is a question about these criteria, the researcher must negotiate with an appropriate sponsor representative and resolve any issues before the research recruits participants. Abstinence is not allowed.

5.1. Inclusion criteria

Each potential participant must meet all criteria for the following study enrollment:

1. is more than or equal to 18 years old.

2. Revised criteria according to revision 1.

2.1 histology:

part 1: metastatic crpc (mcrpc) with histological confirmation of adenocarcinoma. Permitting adenocarcinomas with small cell or neuroendocrine features.

mCRPC is defined as: total serum testosterone ≦ 50ng/dL or 1.7nmol/L with evidence of progressive disease, defined as 1 or more PCWG3 criteria (Scher HI et al, J Clin Oncol.2016; 34(12):1402-1418) increased on at least 2 consecutive occasions separated by at least 1 week with PSA levels ≧ 1ng/mL, lymph node or visceral progression as defined by RECIST 1.1 with PCGW3 modifications, and/or 2 or more new lesions appearing in bone scans.

Section 2:

1. mCRPC as defined above.

2. Pathologically confirmed metastatic RCC, defined by WHO 2016 classification.

3. Modified criteria according to revision 1.

3.1 previous treatments were as follows:

parts 1 and 2: mCRPC-at least 1 prior series of novel AR targeted therapies for mCRPC (i.e., abiraterone acetate, apalutamide, enzalutamide). Patients who received prior chemotherapy also qualify for at least 1 prior series of novel Androgen Receptor (AR) targeted therapies.

Section 2: RCC-at least two existing approaches for systemic treatment of metastatic or locally advanced disease (e.g., mammalian targets of anti-vascular endothelial growth factor VEGFR, checkpoint inhibitors, or rapamycin (mTOR) inhibitors).

4. Measurable or evaluable disease:

part 1: measurable or evaluable disease of prostate cancer.

Section 2: at least one measurable lesion that can be accurately assessed at baseline by CT (or MRI with CT disabled) and that is suitable for repeated assessment according to RECIST v 1.1. If the only site where the measurable disease has been previously irradiated, the disease progression and 4 week interval since completion of radiotherapy need to be recorded. In addition, lesions selected at baseline or at treatment for biopsy cannot be selected as target lesions for disease assessment.

5. Evidence of disease progression for previous therapies requiring new treatment lines.

mCRPC: if a participant is receiving treatment with a gonadotropin releasing hormone agonist analogue (GnRH), i.e. a participant who has not undergone bilateral orchiectomy, the treatment must begin before the study drug is first administered and must continue throughout the study.

7. Unless the collection of biopsies presents a safety risk, participants with accessible lesions enrolled in the selected PK/PD cohort and section 2 must agree to perform a forced fresh tumor biopsy.

8. Eastern Cooperative Oncology Group (ECOG) fitness status level is 0 or 1.

9. Within 3 weeks prior to the first administration of the study drug, the hematological laboratory parameters were in the following ranges, independent of transfusion or growth factors. Participants had to be independent of transfusion:

a. hemoglobin is greater than or equal to 9g/dL

b. Absolute neutrophil count ≥ 1.5X 10⁹/L

c. Platelet count is not less than 100X 10⁹/L

10. The chemical laboratory parameters were in the following ranges:

a. serum albumin is more than or equal to 3.0g/dL

b. Calculated or measured creatinine clearance>50mL/min/1.73m²

c. Total bilirubin content is less than or equal to 1.5 multiplied by the Upper Limit of Normal (ULN); in participants with Gilbert syndrome, direct and indirect bilirubin is measured if total bilirubin is greater than or equal to 1.5 × ULN, and if direct bilirubin is within normal limits, the participants may qualify

d. Aspartate Aminotransferase (AST) and alanine Aminotransferase (ALT) of less than or equal to 2.5 × ULN

11. The cardiac parameters were in the following ranges:

a. left ventricular ejection fraction within tissue normal limits

b. Corrected QT intervals (QTcF or QTcB) were ≦ 480 milliseconds based on the mean of three replicate assessments performed at 5 minute intervals (+ -3 minutes). This standard does not apply to participants with pacemakers.

12. Women with fertility potential must have negative, highly sensitive serum (β -human chorionic gonadotropin [ β -hCG ]) at screening and prior to first dose of study drug. Urine pregnancy tests were required every 4 weeks during the treatment period.

Women must be a control guide and collection of purples information):

has no fertility potential

Has fertility potential and

to carry out a highly effective, preferably user-independent, contraceptive method (failure rate < 1%/year when used consistently and correctly), and agree to maintain a highly effective method at the time of receiving the study drug and until 30 days after the last administration. Pregnancy tests (serum or urine) within 30 days after the last study drug administration.

13. In addition to high-efficiency methods of contraception that are not dependent on the user, there is a need for male or female condoms with or without spermicidal agents, such as condoms containing spermicidal foam/gel/film/cream/suppository. Male and female condoms should not be used together (due to the risk of frictional failure).

14. A male participant must wear a condom while engaging in any activity that allows ejaculation to pass to another person. The benefit of using an efficient contraceptive method by a female partner should also be advised to the male participant, as the condom may burst or leak.

15. The use of contraceptive (birth control) measures as described above by men or women should comply with local regulations regarding acceptable methods of contraception for those participating in clinical studies. Typical failure rates of use may be different from those when used consistently and correctly. The use should be in accordance with local regulations regarding the use of contraceptive methods by participants participating in clinical studies.

16. Women must agree not to donate eggs (ova, oocytes) for assisted reproduction during the study and for at least 30 days after the last study drug administration.

17. The male participants must agree not to donate sperm for reproductive purposes during the study and within a minimum of 90 days after the last dose of study drug.

18. The proscribed items and restrictions specified in the scheme are willing and able to be followed.

19. An informed Consent (CF) must be signed, indicating that he or she knows the purpose of the study and the required procedures and is willing to participate in the study.

5.2. Exclusion criteria

Any potential participants who meet any of the following criteria will be excluded from participating in the study:

1. history of brain metastases or known brain metastases.

2. Adenomas, eosinophilic tumors, and mesenchymal renal cell tumors.

3. Modified criteria according to revision 1.

3.1-mCRPC, with primary histology of prostate neuroendocrine or small cell carcinoma tumors.

Non-metastatic CRPC.

4. At least 2 weeks between discontinuation of prior anti-cancer treatments (including radiotherapy) and first dose of study drug, and toxicity has returned to grade ≦ 1 or baseline.

5. Prior treatments with PSMA-targeted therapies include, but are not limited to, chimeric antigen T cell receptors, PSMA T cell redirection therapies, PSMA-targeted monoclonal antibodies, including antibody drug conjugates. Allowing previous treatment with PSMA-targeted vaccines.

6. Solid organ or bone marrow transplantation.

7. Seizures or known conditions that may be prone to seizures or intracranial tumors such as schwannomas and meningiomas that cause edema or tumor effects.

8. Other active malignancies requiring systemic treatment less than or equal to 12 months prior to enrollment.

9. Any of the following was present within 6 months prior to screening:

a. myocardial infarction

b. Severe or unstable angina pectoris

c. Clinically significant ventricular arrhythmias

d. Congestive heart failure (New York Heart Association type II to IV)

e. Cerebrovascular accident or transient ischemic attack

f. Arterial events of any grade

10. Venous thromboembolic events (i.e., pulmonary embolism) within 1 month prior to first administration of study drug; no complications (< 2 grade) deep vein thrombosis are considered to be excluded.

11. Uncontrolled hypertension (grade 2 or more); participants who received antihypertensive treatment were allowed.

12. Known allergic reactions, hypersensitivity reactions or intolerance to the investigational drugs or their excipients (see "investigator manual").

13. Any other anti-cancer treatment or research agent for the treatment of advanced disease is used simultaneously.

14. Active infections or conditions requiring treatment with systemic antibiotics within 7 days prior to the first administration of the study drug.

15. Immunosuppressive doses of systemic drugs, such as corticosteroids (dose >10 mg/day prednisone or equivalent) were received within 2 weeks prior to the first administration of the study drug. A single course of treatment of corticosteroids is allowed as prophylaxis for imaging contrast (i.e., for participants allergic to contrast).

16. Active autoimmune diseases that require systemic immunosuppressive drugs (i.e., chronic corticosteroids, methotrexate, or tacrolimus) over the past 2 years.

17. Major surgery (e.g., requiring general anesthesia). Participants must recover adequately without sequelae for at least 3 weeks prior to initiation of study drug. Central venous catheters were allowed to be inserted under general anesthesia 1 week prior to the start of study medication. Note that: participants who plan to perform surgery under local anesthesia may participate.

18. Active or chronic hepatitis b or c infection. Hepatitis b infection is defined as a positive detection of both the hepatitis b surface antigen (HBsAg) and an antibody to either the hepatitis b surface antigen or the core antigen (anti-HBs and anti-HBc, respectively). Hepatitis c infection was defined as positive for hepatitis c antibodies.

Participants who tested positive for anti-HBs or anti-HBc had to have hepatitis b DNA by performing the polymerase chain reaction and confirmed negative before study drug administration. Participants who tested positive for hepatitis c antibody are eligible if previously treated and achieved a sustained viral response, which is defined as a negative viral load of hepatitis c after completion of the hepatitis treatment.

19. History of Human Immunodeficiency Virus (HIV) antibody positivity, or testing HIV positive at screening.

20. Live vaccines were administered 28 days before the first administration of study drug; allowing vaccination with inactivated vaccines such as annual influenza vaccines.

21. Pregnancy, lactation or pregnancy scheduled at the time of enrollment of the study or within 30 days after the last study drug administration.

22. The study was scheduled to become the father of the child at the time of enrollment or within 90 days of the last study drug administration.

23. The investigators considered participation in the study to be inconsistent with the best interests of the participants (e.g., impaired health) or any condition that might prevent, limit or confound the assessment specified by the protocol.

Note that: the investigator should ensure that all study recruitment (inclusion/exclusion) criteria have been met at the time of screening and prior to the first administration of study drug. A participant should be excluded from participating in the study if his or her clinical status changes after screening but before the first administration of study drug (including any available laboratory results or receiving additional medical records) such that he or she no longer meets all eligibility criteria. Section 5.4, "screening failure" describes options for retesting.

5.3. Lifestyle considerations

Potential participants must be willing and able to comply with the following lifestyle restrictions during the course of the study to be eligible for participation:

1. therapies that must be discontinued or replaced at least 4 weeks before the study drug is first administered include drugs known to lower seizure thresholds and products that lower PSA levels. Details of therapies that are forbidden and limited during the study are referenced in section 6.5.2.

2. Agreement to follow all requirements (e.g. contraceptive requirements) indicated in the eligibility (inclusion and exclusion) criteria that must be met during the study.

3. The dose escalated participants had to be willing to stay at least 48 hours after the first and second therapeutic doses and any priming dose (if administered) from the end of study drug infusion (IV flush) and hospitalization as described in section 4.1.

4. The participants must agree to avoid driving and engaging in dangerous occupations or activities during the time periods described in section 6.1.2.4.

5.4. Failure of the screen

Participant identification, recruitment, and screening logs

Participants meeting the screening failure criteria may be rescreened. During the screening phase, only one retest (to re-assess eligibility) of the exception screening value that resulted in rejection is allowed. The final results obtained before the first administration of the study drug will be used to determine eligibility. The measurements collected at a time closest to but before the start of study drug administration will be defined as baseline values for safety assessment and treatment decisions.

If a participant's clinical status changes after screening but before the first administration of study drug (including any available laboratory results or receiving additional medical records) such that he or she no longer meets all eligibility criteria, the participant should be excluded from participating in the study.

Researchers agreed to complete a log of participant identification and recruitment to allow each participant to be easily identified during and after the study. This document will be reviewed by the sponsor research center contacts to ensure their integrity. The participant identification and recruitment logs will be considered confidential and will be submitted by the researcher in the study documentation. To ensure the confidentiality of the participants, no copying must be done. All reports and communications related to the study will identify the participants by their identification and age when initially agreed with informed consent (as allowed by local regulations). In the case where participants were not recruited into the study, the date and age seen when initial informed consent (as allowed by local regulations) would be used.

6. Research medicine

6.1. Study drug administration

Description of study drugs and diluents

The study drug was a fully humanized IgG 4-based bispecific antibody against CD3 and PSMA receptors, produced by culturing recombinant chinese hamster ovary cells, followed by isolation, chromatographic purification, and formulation.

The manufacture and provision of study drugs and diluents will be responsible for the sponsor. Study drug administration will be collected in source documents and electronic case report forms (eCRF). For details on emergency medications, refer to section 6.5.4. For the definition of study drug overdose, refer to section 8.4.

For the purposes of this study, "study drug" refers to study drug and its diluent. All administration information must be recorded in the eCRF. The enrollment staggered interval for participants in dose escalation is provided in section 4.1.1. Infusion times and recommendations can be adjusted by the sponsor based on emerging safety information negotiated with the researcher. Such changes will be recorded in the study file, SET meeting record, or IPPI revision. Infusion durations that exceed the planned length of time due to IV bag overfill, secondary device calibration factors, or participant factors not controlled by the administrator will not be considered protocol deviations. The actual infusion time should be accurately recorded. The table provides details of drug administration.

TABLE 24 study drug administration

6.1.1. Standard of retreatment

Prior to each dose, participants will be assessed for possible toxicity that may have occurred. Laboratory results and general physical condition must be reviewed. Toxicities and complications must return to grade 1 or baseline (except for hair loss). Participants must not develop a fever for at least 72 hours. Treatment with study drug can be resumed as long as the clinical status of the participants meets all re-treatment criteria outlined in 25 and does not meet all treatment discontinuation criteria presented in section 7.1.

TABLE 25 criteria for retreatment before each dosing

a. Blood transfusions and growth factors may be used to manage hematological toxicity.

b. It must have recovered sufficiently from toxicity and stopped transfusion or growth factors at least 5 days before the next study drug administration.

In all cases of clinically significantly impaired wound healing or impending surgery or potential bleeding complications, it is recommended to discontinue dosing, carefully monitor appropriate clinical laboratory data (e.g., blood clotting), and, where applicable, administer supportive therapy. When deemed safe according to the investigator's assessment, dosage administration can be resumed with the appropriate dosage determined by negotiation with the sponsor.

6.1.2. Management guidelines for potential toxicity

Where applicable, optimal supportive care should be applied. The management of specific potential toxicities indicated in section 2.3 is outlined in this section. Appropriate personnel and appropriate resuscitation equipment should be available at any time in or near the infusion room and trained physicians should be available at any time during infusion of study medication. Resources required for resuscitation include medications such as epinephrine and nebulized bronchodilators; medical devices such as oxygen, tracheostomy devices and defibrillators. Vital signs and laboratory parameters must be monitored periodically until toxicity normalizes. In the case of IRR or CRS events, unplanned pharmacokinetic, immunogenicity, cytokine and pharmacodynamic samples should be collected.

6.1.2.1. Management of infusion-related reactions

Participants experiencing IRR manifested as asthma, flushing, hypoxemia, fever, chills, shiver, bronchospasm, headache, rash, itching, arthralgia, hypotension or hypertension or other symptoms should have symptoms managed according to the recommendations provided in 26.

All level 3 or level 4 IRRs should be reported to the sponsor medical inspector within 24 hours. If the event meets the criteria for a serious adverse event, the criteria for a serious adverse event report in section 8.3 is followed. After the initial IRR event, prophylactic drugs must be administered as described in section 6.5.3 before the next study drug infusion.

TABLE 26 infusion correlationDose modification and guidance for response management

6.1.2.2. Management and prevention of immune-related adverse events

Study drugs may lead to specific immune related adverse events (irAE). Continuous, careful monitoring and timely management of irAE may help mitigate more severe toxicity. Symptomatic and best supportive care measures for a particular potential irAE should be performed immediately upon clinical indication and should comply with institutional standards. These treatments may include corticosteroids and other immunosuppressive agents required for the particular irAE.

6.1.2.3. Prevention and management of cytokine release syndrome

Since the specific mode of action of the study drug is based on T cell binding and activation and cytokine release in the tumor environment, adverse events with CRS should be expected. The limited clinical experience with T-cell activating bispecific antibodies appears to indicate that CRS occurs most frequently within minutes to hours after the start of infusion, Klinger M et al, blood.2012,119(26): 6226-; lee DW et al, blood.2014,124(2): 188-; zimmerman Z et al, Int Immunol.2015,27(1): 31-37.

Clinical symptoms indicative of CRS may include, but are not limited to, fever, shortness of breath, headache, tachycardia, hypotension, rash, and hypoxia caused by cytokine release. Effects on other organs such as hallucinations, confusion, headaches, epilepsy, dysphasia, tremors, or other nervous system toxicities are also contemplated. Potential life-threatening complications of CRS may include cardiac dysfunction, adult respiratory distress syndrome, kidney and liver failure, and disseminated intravascular coagulation. Participants should be closely monitored for early signs and symptoms indicative of CRS, and study drug infusion should be immediately discontinued. Laboratory tests for coagulation and inflammatory markers may be performed as clinically indicated to monitor disseminated intravascular coagulation and inflammation, which may serve as markers for coagulation and inflammationThe manifestation of CRS occurs. Cytokine release syndrome will be collected as an adverse event of particular interest (see section 8.3.5) and will be evaluated according to NCI CTCAE version 5.0. Clinical management recommendations for CRS are provided in table 27 below, and include treatment with toslizumab.(Tulizumab). Prescription information. South San Francisco, CA, Genentech, Inc; 2017. Administration of toslizumab should be considered ≧ grade 2 CRS (according to CTCAE v 5.0); additionally, toclizumab may be administered according to institutional standards of care guidelines. Thus, it was ensured that toslizumab was available at the study center prior to infusion of study drug (see section 6.5.4). See section 4.1 for hospitalization requirements for CRS events.

TABLE 27 cytokine Release syndrome management guidelines

The source is as follows: according to Kymriah^TM(tisagenleceucel) United states packaging Specification Kymriah^TM[ US FDA Package Specification]And (5) modifying. East Hanover, USA Novartis Pharmaceutical Corporation; year 2018, month 5.

Dose modification/discontinuation guidelines for participants experiencing CRS are provided in table 28. Post-treatment drugs should be administered as needed. Participants had to be hospitalized as described in section 4.1.

TABLE 28 guidance for dose modification for cytokine release syndrome management

a. See section 6.6.2 for dose reduction schedules.

6.1.2.4. Adverse events of the nervous system

It is unknown to study whether drugs cause toxicity to the nervous system; however, this is a potential risk due to PSMA expression (cytoplasm) in glial cells of the cerebellum and spinal cord. In addition, neurotoxicity has been observed using CD3 redirecting agents such as CD19xCD3 bornaemezumab⁰. The etiology of these toxicities is unclear and can be associated with CD19 expression, T cell redirection, or cytokine release in general. In clinical trials with bornauzumab (CD19xCD3 BiTE), neurological toxicity occurred in about 50% of patients and included encephalopathy, convulsions, language disorders, disturbance of consciousness, confusion and disorientation, and disturbance of coordination and balance. Most events resolved after bornaemezumab discontinuation, but some resulted in discontinuation of treatment. Monitoring of signs and symptoms associated with nervous system effects will occur throughout the study.

Based on the particular mode of action of the study drug, severe or severe neurological toxicity may occur. Early recognition of neurological adverse events is crucial for management. Participants should be monitored for neurological toxicity including, but not limited to, language disorders, convulsions and disturbances of consciousness, confusion, disorientation or disturbance of coordination and balance. If participants notice impaired motor function (e.g., weakness), changes in sensation (e.g., numbness), or symptoms suggestive of a possible central nervous system abnormality (such as a new headache episode or a change in mental state), they should be advised to seek medical assessment.

Participants should also be advised to avoid driving and engaging in dangerous occupations or activities, such as operating heavy or potentially dangerous machinery, during the first 72 hours post-treatment, and to extend to the first 4 weeks of treatment for participants experiencing grade 2 or greater nervous system toxicity that would impair such activities. These limits should be enforced again if the participant's status deteriorates at any time.

The study center personnel will perform a basic nervous system examination to assess the nervous system status as shown at 29. If these or other nervous system toxicities are observed, the sponsor medical inspector must be consulted. Dose modification/discontinuation guidelines for participants who experienced neurological toxicity are provided in table 29. Post-treatment drugs should be administered as needed. Participants who experienced nervous system toxicity had to be hospitalized as described in section 4.1.

TABLE 29 guidance for dose modification for nervous system toxicity management

a. See section 6.6.2 for dose reduction schedules.

6.2. Preparation/processing/storage/accountability

Storage device

Study drugs must be stored at controlled temperatures. A detailed description of the storage conditions and handling of study drugs will accompany the clinical drug supply to the clinical study center. The research drug label will contain information that meets applicable regulatory requirements

Responsibility of

The investigator was responsible for ensuring that all study drugs and diluents received at the study center were inventoried throughout the study and accounted for. Study medication and diluents administered to participants must be recorded on the study medication liability chart. All study medications and diluents will be stored and disposed of according to the instructions of the applicant. The study center personnel must not mix the contents of the study drug container.

Study medications must be handled strictly according to protocol and container labels and must be stored in limited access areas at the study center or in lock cabinets under appropriate environmental conditions. During a field monitoring visit, unused study medication must be available for validation by the applicant's study site reviewer. Return of unused study medication to the sponsor will be recorded on the study medication return table. When the research center is an authorized destruction unit and the research drug supply is destroyed on site, this must also be recorded on the research drug return table.

Potentially hazardous materials, such as used ampoules, needles, syringes and vials containing hazardous liquids, should be immediately handled in a safe manner and therefore will not be retained for the purpose of studying drug liability.

Study medication should be distributed under the supervision of qualified members of the investigator or study center personnel or by hospital/clinic pharmacists. Study medication and diluents will only be provided to the participants of the study. The study drug or diluent may not be relabeled or redistributed for use by other participants. Researchers agree neither to distribute nor store study medications from any location other than the research location agreed with the sponsor.

6.3. Measures to minimize the deviation: randomization and blinding

Not applicable.

6.4. Study of drug compliance

Study medication was administered as an intravenous infusion in the desired form by a qualified physician who was the primary researcher or listed as a sub-researcher. Drug supply for each participant will be inventoried and accounted for throughout the study. Administration of the study drug must also be recorded in the participant's source document.

An interactive network response system would be used to assign each participant enrolled in the study a centrally supplied study treatment kit. Study medication cannot be used for any purpose other than that outlined in the present protocol, including other human studies, animal surveys, or in vitro tests.

The intravenous study drug will be administered under direct observation by qualified study center personnel in the controlled environment of the clinical study center. Details of each administration will be recorded in the eCRF (including the date of IV infusion, start and stop times, and volume of infusion). Participants will review the precautions associated with the use of study medication and the concomitant medications banned.

At the termination of the study, or at the request of the sponsor or its designee, the pharmacist must return the study medication to the sponsor or its designee after all of the medication supplies have been accounted for, unless it is destroyed at a site where both the sponsor and the site agree.

6.5. Concomitant therapy

During screening, previous series of therapies should be recorded on the eCRF. Throughout the study, in addition to those listed in section 6.5.2, the investigator may prescribe any concomitant medications or treatments deemed necessary to provide adequate supportive care. All medications other than study medication (including prescription and over-the-counter products, and blood transfusions of blood products) must be recorded throughout the study, from the time the ICF is signed until 30 days after the last administration of the study medication or, if earlier, until the start of subsequent anti-cancer treatment. This includes any concomitant therapy and any drug used to treat or support an adverse event or severe adverse event. The information recorded will include a description of the type of drug, the dosing regimen, the route of administration, the duration of treatment and its indications.

Effective pre-existing therapies should not be modified for the express purpose of bringing participants into the study. mCRPC participants who did not undergo orchiectomy will remain on androgen deprivation therapy or investigator-selected GnRH analogs throughout the study treatment. All medications should be recorded in the appropriate section of the eCRF.

6.5.1. Allowed therapy

Participants will receive full supportive care during the study. The following are examples of supportive therapies that may be used during the study:

standard supportive care regimens (antiemetics, antidiarrheals, anticholinergics, spasmolytics, antipyretics, antihistamines, analgesics, antibiotics and other antimicrobials, histamine receptor [ H2] antagonists or proton pump inhibitors, and other drugs intended to treat disease symptoms or signs or adverse events) according to institutional standards and as deemed necessary by the investigator, as indicated clinically.

According to standard institutional practice, the recorded complications of infection are treated with oral or IV antibiotics or other anti-infective agents deemed appropriate by the treatment investigator for a given infectious condition.

Allowing growth factor support, erythropoietin stimulating agents and blood transfusions such as red blood cells and platelets to treat symptoms or signs of neutropenia, anemia or thrombocytopenia according to local standard of care; these agents are not allowed as a prophylactic treatment during the DLT phase.

As shown in the table, corticosteroids used as a pre-treatment drug for study medication are allowed and if the daily dose is less than 10mg prednisone or equivalent, they are allowed for treatment of pre-existing diseases. Corticosteroids may be used as a prophylaxis for imaging contrast.

Best supportive care to prevent or manage potential toxicity as indicated in section 6.1.2.

Palliative radiotherapy on bone lesions.

GnRH agonists and antagonists

Drugs that allow for a reduction in PSA levels (e.g., megestrol acetate, estrogen, progesterone, 5 α -reductase inhibitors [ e.g., finasteride, dutasteride ]) if started before the study drug is first administered.

6.5.2. Forbidden or limited therapy

The following drugs were banned during the study. The sponsor must be informed in advance (or as soon as possible thereafter) of any instances of administering the forbidden therapy.

Any chemotherapy, anti-cancer immunotherapy (except for research drugs), experimental therapy or radiotherapy of visceral lesions.

Drugs known to lower seizure threshold.

To minimize the potential effects of CRS on CYP450 enzyme activity, which in turn may affect the blood concentration of CYP450 substrates, concomitant administration of CYP450 substrates, especially those with a narrow therapeutic index (e.g., warfarin), should be suspended for 48 hours during the first dose administration of study drug. The potential toxicity from all CYP450 substrates of the participants should be monitored and the dosage of concomitant drugs can be adjusted as needed.

Prohibiting the administration of a chronic dose of corticosteroid exceeding 10mg prednisone or equivalent per day for >10 days, in addition to managing adverse events.

Other immunosuppressive agents, unless used as a regimen-specific pretreatment drug or to treat adverse events (e.g., CRS).

No routine blood transfusions should be performed on the day of study drug administration.

Herbal products.

6.5.3. Pre-infusion drug

Prior to each study drug infusion, participants in this study had to receive a prodrug as shown in table 30 below. If study drug infusion is discontinued for 4 hours or more due to acute toxicity, the antihistamine and antipyretic treatments in Table 30 should be administered again. The pre-infusion drugs may change based on emerging safety and other data as determined by the SET.

TABLE 30 drugs administered before study drug infusion

Abbreviations: CRS ═ cytokine release syndrome; IRR ═ infusion-related reactions; IV is intravenous.

a. The pre-infusion drug is only required prior to the first therapeutic dose and the priming dose (if administered).

6.5.4. First-aid medicine

Clinical management recommendations for CRS include treatment with toslizumab.⁰Therefore, the site must be assured in administering the study drugTolizumab was previously available at this site. The research site will provide tositumumab rescue medications, which will be sourced locally and reimbursed by the sponsors. The date and time of the rescue medication administration, as well as the name and dosage regimen of the rescue medication, must be recorded.

6.5.5. Subsequent anti-cancer therapy

Subsequent anti-cancer therapies administered after the last dose of study drug (including start and end dates and optimal response, if available) should be recorded in the eCRF.

6.6. Dose modification

Any dose/dose adjustment should be supervised by medically qualified research center personnel (primary or secondary investigators, unless immediate safety risks arise). Dose delay and dose reduction are the primary methods used to manage toxicity. A priming dose schedule may be implemented for the particular toxicity described in section 6.6.3. If toxicity meets the criteria for discontinuation of treatment in section 7.1, treatment will be discontinued.

6.6.1. Dose delay

If the dose is delayed by more than 72 hours, subsequent doses will be delayed, ensuring a minimum 5-day interval between weekly doses and a 3-day interval between twice weekly doses. For the events outlined in section 6.1.2, the dose escalation schedule shown in table 31 should be followed to negotiate with the sponsor.

In case DLT occurs during treatment (table), the treatment must be temporarily suspended and supportive therapy administered, as indicated clinically. For other grade 3 clinically significant toxicities during treatment, supportive therapy should be administered and treatment may be suspended as indicated clinically.

If toxicity subsides to grade ≦ 1 or baseline within 28 days, treatment can be re-initiated with the sponsor negotiation except that the criteria for the cause of discontinuation are met (see section 7).

6.6.2. Dose reduction

If determined to be of greatest concern to the participants, the study medication may be restarted at the same or a lower dose after consultation with the sponsor medical inspector, provided that the criteria for discontinuing study therapy in section 7 are not met. The lower dose levels shown in the table represent the claimed safe dose levels previously evaluated.

TABLE 31 dosage reduction Schedule

Dose reduction	Dosage level
		Current dose	Current dose
First dose reduction	Less than 1 dosage level or less^a
		The second dose is reduced	Lower than 2 dosage levels or less^a

a if deemed clinically appropriate and after discussion between the sponsor medical inspector and researcher, optionally

Lower doses were selected. Lower dosage levels are those evaluated and claimed to be safe.

6.6.3. Dose modification during priming dose

If toxicity occurs during priming dose administration:

all retreatment criteria in section 6.1.1 must be met before the next priming or therapeutic dose of study drug is administered.

If grade 2 toxicity subsides to baseline or grade ≦ 1 within 72 hours, the participants may continue study treatment at the final priming dose level.

If CRS grade 3 occurs during or after the priming dose but resolves to grade 1 within 72 hours, the dose will be reduced as described in the table. Dose re-escalation may be considered after negotiation with the sponsor.

Study treatment was discontinued permanently if grade 4 CRS occurred during or after the priming dose.

For other class 3 toxicities, retreatment is allowed under negotiation with the sponsor.

6.7. Study drug after completion of study

The sponsor will ensure that participants who continue to benefit from treatment with the study medication will be able to continue treatment after the data for CSR expires. Participants will also be instructed that they will not get the study medication after they complete/discontinue it, and they should return to their attending physician to determine the standard of care.

7. Discontinuation of study drug and discontinuation/withdrawal of participants

7.1. Discontinuation of study drug

If a participant had to discontinue the study medication, he or she would not automatically withdraw from the study. The study medication of the participants must be discontinued if:

participants received concurrent (non-scheduled) anti-cancer therapy.

Confirm disease progression unless the investigator judges that continued treatment with the study drug is in line with the best benefit of the participants after obtaining written approval by the sponsor medical inspector.

Prevention of complications from further administration of study drug

Participants refused to further treatment with study drug

Pregnancy of the participant

Adverse events did not resolve to grade ≦ 1 within 4 weeks of the last dose of study drug, so that study drug was discontinued continuously for more than 28 days unless the sponsor medical inspector and investigator agreed otherwise based on evidence of clinical benefit.

Grade 3 or 4 non-hematologic toxicity occurs despite 2 dose reductions and best supportive care unless otherwise agreed by the sponsor medical inspector and investigator based on evidence of clinical benefit.

Recurrence of grade 3 IRR after 2 consecutive doses of study drug

Level 4 IRR (section 6.1.2.1).

·CRS：

Level 2 or level 3 CRS which is not improved to level 1 or less within 7 days

Level 3 CRS not improved to level 2 or less within 5 days

Omicron two independent level 3 CRS events (recurrence)

Omicron 4 grade CRS

Relapse of grade 3 or any grade 4 nervous system toxicity (section 6.1.2.4)

Grade 4 hematological toxicity recurs despite 2 dose reductions and optimal supportive care unless the sponsor medical inspector and investigator agree otherwise based on evidence of clinical benefit

After discontinuation of treatment, participants should complete the EOT visit. The main reason for discontinuation of treatment will be documented in the eCRF. Participants who quit for reasons other than toxicity will be replaced at the discretion of the sponsor (see section 4.1.1).

7.2. Participant suspension/withdrawal from study

Participants will exit the study for any of the following reasons:

lost follow-up

Consent to withdraw

Application discontinuation study

When a participant exits before completing a study, the reason for the exit should be recorded in the eCRF and source documents. The study medication assigned to the exiting participant is not assigned to another participant.

EOT and post-treatment follow-up assessments should be obtained if the participants discontinue study medication. If the reason for exiting the study is to revoke consent, no additional evaluation is allowed.

7.2.1. Revocation of researchUse of the sample

Participants who exited the study will have the following options with respect to the study sample:

the collected sample will be retained and used according to the participants' original informed consent to study the sample.

The participant can revoke consent to the study sample, in which case the sample will be destroyed and no further testing will be performed. To initiate the sample destruction process, the researcher must inform the sponsor research center contact about revoking consent to the research sample and request that sample destruction be performed. The sponsor research center contact will then contact the biomarker representative to perform sample destruction. If requested, the researcher will receive written confirmation from the sponsor that the sample has been destroyed.

Withdrawal of study samples while remaining in the main study

Participants may revoke consent from the study sample while remaining in the study. In such cases, the study sample will be destroyed. The sample destruction process will proceed as described above.

Use of withdrawn samples in future studies

Participants may revoke consent for study sample use. In such cases, the sample will be destroyed after clinical studies are no longer needed. Details of the sample retention used for the study are given in the ICF.

7.3. Loss of visit

If a participant is out of visit, the research center personnel must make all reasonable effort to contact the participant and determine the reason for the abort/exit. Follow-up measures must be recorded. Reference is made to section 7.2 for "participant suspension/withdrawal from study".

8. Research evaluation and procedure

SUMMARY

The study was divided into 3 phases: a screening phase, a treatment phase and a post-treatment follow-up phase. The "activity schedule" summarizes the frequency and timing of the study process and the assessments applicable to the study.

All planned assessments, including clinical laboratory tests, must be completed and the results reviewed at each clinical visit. If multiple evaluations of the same point in time are planned, it is recommended to execute the procedure in the following order: ECG, vital signs, blood draw. Treatment decisions will be based on safety and disease assessments performed at the center. If clinically indicated, more frequent study visits may be made and clinical assessments may be repeated more frequently.

Blood collection for pharmacokinetic and pharmacodynamic assessments should be kept as close to the time specified as possible. Other measurements may be taken at an earlier time than the specified point in time, if desired. The actual date and time of the evaluation will be recorded in the source document and the eCRF or laboratory application form. Repetitive or unplanned samples (i.e., pharmacokinetic, pharmacodynamic, biomarker) may be taken for safety reasons or for technical issues with the sample. Additional serum or urine pregnancy tests may be performed as determined by the needs of the investigator or as required by local regulations to determine the absence of pregnancy at any time during the participation of the participants in the study. For each participant, approximately 23mL of blood will be drawn during the screening phase. During the treatment phase, most samples will be collected during the first 8 cycles of treatment. During this period, about 450mL (weekly schedule) to 490mL (twice weekly schedule) of blood will be drawn. If a priming schedule is implemented, an additional 25mL may be required. The samples will be or be evaluated for safety, pharmacokinetic and pharmacodynamic parameters.

If the study drug is peripherally infused, a blood sample must be drawn from a vein or through the centerline contralateral to the arm where the study drug is infused. If the study drug is infused through the centerline, a blood sample must be drawn from a vein in either arm.

Screening stage

All participants must sign the ICF before proceeding with any study-related procedures. The screening phase begins at the time of the first screening evaluation and within 30 days before the first administration of the study drug. During screening, if the assessment is made as part of a participant's routine clinical assessment and is not specific to the study, the assessment need not be repeated after signed informed consent has been obtained, provided that the assessment meets the study requirements and is made within a specified time frame prior to the study drug being first administered. Test results such as radiological tests (e.g., MRI and CT scans) are acceptable for screening if performed within 6 weeks (42 days) before the first administration of the study drug. Fresh tumor biopsy samples (from accessible sites of metastatic disease) were required for screening. However, samples obtained within 6 weeks (42 days) of the first administration of the study drug were acceptable provided that the participants did not receive active anti-cancer therapy during this time frame. These samples will be sent to a central laboratory designated by the sponsor (see "laboratory manual" for details).

Stage of treatment

The treatment phase began on day1 with administration of study drug and continued until the EOT visit was completed. During the treatment phase, biopsy samples will be collected from the selected queue. To facilitate safety monitoring, participants will be hospitalized as outlined in section 4.1. During the study drug infusion, vital signs, body temperature and oxygen saturation measurements will be monitored at regular intervals. At each visit of the study center, participants will be assessed for possible toxicity. Participants may continue to receive study medication until any of the treatment discontinuation criteria outlined in section 7 are met. For participants who discontinue treatment due to disease progression, the disease progression table must be completed and sent to the sponsor medical inspector before treatment is discontinued. Upon discontinuation of study medication, participants will complete the EOT visit.

End of treatment

All participants required an EOT visit, including those who discontinued study medication for any reason, except for missed visits, death, or consent to withdrawal of study participation. EOT visits will be completed 30(+7) days after the last dose of study drug or before (whichever occurs first) the start of a new anti-cancer therapy. If the participants are unable to return to the study center for the EOT visit, or if the EOT visit occurs before 30 days after the last administration of the study drug, the participants should be contacted to collect adverse events and concomitant drugs until 30 days after the last administration of the study drug or until the initiation of a subsequent anti-cancer therapy.

Post-treatment phase (follow-up visit)

The post-treatment follow-up phase begins after the EOT visit and will continue until one of the withdrawal criteria in section 7.2 is met. If the study drug is discontinued before the onset of disease progression, as defined by disease specific response criteria, the results of the disease assessment, performed according to local standard of care, should be recorded on the eCRF. Once disease progression is confirmed, no subsequent disease assessment is required.

Survival status and subsequent anti-cancer therapy will be obtained every 12 weeks after the EOT visit until the end of the study, unless the participants die, lose visits, or withdraw consent. Adverse events were collected up to 30 days after the last dose of study drug. The researcher may re-contact the participant or designated representative to obtain long-term follow-up information regarding the security or survival status of the participant as indicated in the informed consent form. If information about survival is obtained through a telephone contact, a paper file of the communication must be provided in the source file for review. If the participant died, the date and reason for the death would be collected and recorded on the eCRF (if or when available). Where local laws permit, public records may be used to record deaths and to obtain survival status.

Sample collection and processing

The actual date and time of sample collection must be recorded in the eCRF or laboratory application form. Instructions regarding the collection, handling, storage and transport of samples can be found in the "laboratory manual"/research center test drug and procedure manual (SIPPM) to be provided. The collection, handling, storage and transport of the samples must be carried out under the specified and, where applicable, controlled temperature conditions indicated in the laboratory manual/SIPPM. The timing and frequency of all sample collections are referenced to the "activity schedule".

Study specificityMaterial

The following supplies were provided to the investigator:

study protocol

Investigator manual

Research center SIPM

Laboratory Manual

IPPI and ancillary supplies

ECG manual

ECG machine

Interactive network response System Manual

Electronic data acquisition Manual

Sample ICF

8.1. Efficacy assessment

Disease assessments include the assessments listed below. The frequency timing of these evaluations is provided in the "activity schedule".

The same method should be used at baseline and throughout the study (CT scan or MRI or^99mTc bone scan) for disease assessment to characterize each identified and reported lesion to record disease status. Ultrasound, fluorine¹⁸F-fluorodeoxyglucose Positron Emission Tomography (PET) and normal X-ray are unacceptable methods for assessing disease response. Imaging should not be delayed due to delays in study drug administration.

The response to treatment will be assessed by the investigator at the study center and the results will be recorded in the eCRF. If clinically indicated, the unplanned assessment should be considered and the results collected in the eCRF. If requested by the sponsor, the images should be retained until the study is complete to facilitate central review.

Efficacy evaluations included the following：

mCRPC cancer alone: PSA and whole body bone Scan: (^99mTc)

mCRPC and RCC:

CT scan

·MRI

Assessment of the therapeutic response of prostate cancer will be performed according to the PCWG3 standard (Sawicki LM et al, Eur J Nucl Med Mol imaging.2017; 44(1): 102-. 107). Response to treatment of RCC participants with baseline CT or MRI measurable disease was assessed by RECIST version 1.1, Eisenhauer EA, therase P, bogairts J, et al. New response evaluation criteria in solid tumors: revised RECIST guidelines (version 1.1). (Eur J cancer. 2009; 45(2): 228-.

Participants with objective responses according to RECIST v1.1 must undergo confirmatory scans after 4 weeks. If a participant was assessed as having a Partial Response (PR) or Complete Response (CR) at any time during study drug treatment, but not confirmed after 4 weeks or more, the best response of the participant would be classified as stable disease/progressive disease/unevaluable based on the next immediate assessment of the participant. During the study, CT or MRI scans of known lesion locations will be used to assess disease response.

If symptomatic exacerbations occur without recording radiographic progression, the clinical findings used to make this determination must be designated as "clinical disease progression" in the eCRF and recorded in the source document. Various efforts should be made to document objective progress through radiographic confirmation, even after discontinuing treatment symptomatic deterioration. Clinical activity will be reported by researchers in eCRF.

After recording disease progression, participants will have an EOT visit and enter the post study treatment follow-up phase (section 8). For participants who discontinued study treatment prior to disease progression, efficacy assessments according to the standard of care at the study center will continue after EOT visit until disease progression is documented, new anti-cancer therapy is initiated, up to 52 weeks or study end, whichever comes first; the results should be recorded in the CRF.

8.1.1. Assessment of disease response and progressive disease

8.1.1.1. Soft tissue lesion assessment (CT or MRI, physical examination)

The baseline disease burden will be assessed using IV imaging using CT scans of the neck, chest, abdomen and pelvis plus other regions as appropriate. Participants who are intolerant of IV contrast agents may be CT scanned with oral contrast agents and the reason for not using IV contrast agents will be recorded in the source document. Subsequent efficacy assessments during the study will include radiographic imaging of all disease sites recorded at baseline.

Magnetic resonance imaging can be used to assess sites of disease that cannot be adequately imaged using CT (in any case where MRI is required, it must be an imaging technique used to assess disease at baseline and at all subsequent response assessments). For all other disease sites, MRI assessment does not replace the required neck, chest, abdomen and pelvis CT scans unless CT scans are contraindicated. Brain MRI is only required at the time of clinical indication. If MRI is contraindicated, CT scanning of the head may be used.

For participants with palpable/superficial lesions, clinical disease assessments should be made by physical examination at baseline and throughout study drug treatment, as indicated clinically. The irradiated or excised lesions will be considered unmeasurable and only disease progression monitored.

8.1.1.2. Bone lesion assessment in prostate cancer

Bone disease in participants with prostate cancer will be assessed according to PCWG3 (i.e., assessing the duration of response) as follows:

progression of soft tissue lesions as defined in RECIST v1.1 as measured by CT or MRI.

Progression of bone lesions observed by bone scanning and based on PCWG 3. Under these criteria, any bone progression must be confirmed by a follow-up scan after ≧ 6 weeks. Week 8 scans (scans after the first treatment) were applied as reference scans against which all subsequent scans were compared to determine progression. A bone scan is defined as one of the following:

1. compared to the baseline scan, participants who observed a 8 week scan with ≧ 2 new bone lesions would need to be confirmatory scans after ≧ 6 weeks and would fall into one of the following classes 2:

a. participants who confirmed scans (which were taken after ≧ 6 weeks) showed ≧ 2 new lesions compared to the week 8 scan (i.e., a total of ≧ 4 new lesions compared to the baseline scan) would be considered to have bone scan progression at week 8.

b. Participants who did not show ≧ 2 new lesions compared to the week 8 scan would not be considered to have bone scan progression at that time. The week 8 scan will be considered the reference scan to which the subsequent scans are compared.

2. For participants who did not have ≧ 2 new bone lesions from the baseline scan at week 8, if these new lesions were confirmed by a follow-up scan at ≧ 6 weeks later, the first scan time point showing ≧ 2 new lesions from the week 8 scan would be considered the bone scan progression time point.

8.1.1.3. Assessment of immune response or soft tissue pathology

Researchers can assess response to treatment based on immune-RECIST v1.1 (irrecist) (Seymour l. et al, Lancet oncol.2017; 18(3), e143-e 152).

8.1.2. Treatment after initial disease progression

In the event that progressive disease exists according to RECIST v1.1 or PCWG3 prostate criteria, but the treating physician strongly believes that continued study treatment will be in line with the best benefit of the participants, the participants may then be allowed to continue using the study medication through written approval by the sponsor medical inspector. In this case, after recording the progressive disease, local therapy such as radiation may be performed according to the standard of care.

Once the specific criteria for disease progression as defined by RECIST v1.1 or the PCWG3 prostate criteria are met, if clinically necessary (but not earlier than 4 weeks of previous assessments), repeated efficacy assessments should be made at the next assessment time point according to the protocol plan or earlier to confirm disease progression. This allows for continued treatment in spite of initial radiological advances, taking into account the observation that some participants may have transient tumor-burning responses several months before immunotherapy began, but develop subsequent disease responses (Zimmerman Z et al, Int Immunol.2015; 27(1): 31-37). If the participants are clinically stable as defined by the following criteria, they should continue study treatment at the discretion of the treating physician while awaiting confirmation of disease progression:

absence of clinical signs and symptoms indicative of disease progression

Clinical disease progression without immediate therapeutic intervention

Without a decline in ECOG physical Performance status

Absence of progressive tumors at critical anatomical sites (e.g., spinal cord compression) requiring urgent alternative medical intervention

If, after evaluation, the participant is deemed clinically unstable, he or she may withdraw from study treatment without repeated imaging to confirm progressive disease.

Participants will be required to provide written informed consent (e.g., in accordance with local regulations or requirements) before proceeding with study treatment. All procedures indicated in the "activity schedule" will continue according to the scheme.

8.2. Security assessment

Security will be monitored by the SET. Details regarding the research panel are provided in section 4.1.4. Safety will be measured by adverse events, clinical laboratory test results, ECG, vital sign measurements, physical examination findings including basic nervous system examinations. Safety monitoring can be done more frequently if clinically indicated, and researchers should evaluate adverse events according to standard practice.

Adverse events

Adverse events will be reported and followed by the investigator. Adverse events will be ranked according to NCI CTCAE version 5.0. Any clinically relevant changes that occur during the study must be recorded in the "adverse events" section of the eCRF. Researchers will follow any clinically significant toxicity that persists at the end of the study until resolution or a clinically stable condition is reached.

The study will include the following assessments of safety and tolerability according to the time points provided in the "activity schedule".

8.2.1. Physical examination

General physical examination

The screening physical examination includes at least the examination of the participant's height, weight, general appearance, skin, ears, nose, throat, lungs, heart, abdomen, limbs, musculoskeletal system, lymphatic system, and nervous system. Thereafter, physical examinations for symptoms and for diseases will be performed at subsequent time points. The exception will be recorded in the appropriate section of the eCRF. Body weight will also be measured. Clinically significant post-baseline abnormalities should be recorded as adverse events.

Examination of the nervous system

The study centre personnel will perform a basic nervous system examination. Will be evaluated during the screening and treatment phases with physical examination to assess central nervous system related toxicity in the participants. Any clinically significant change from baseline will be recorded as an adverse event.

ECOG physical fitness status

The ECOG fitness status scale will be used to grade the changes in the participants' activities of daily living.

8.2.2. Vital signs

Body temperature, pulse/heart rate, respiration rate, blood pressure and oxygen saturation will be evaluated. Blood pressure and pulse/heart rate measurements will be evaluated using a fully automated device. Manual techniques will only be used when the automated device is not available. Blood pressure and pulse/heart rate measurements should be taken after an undisturbed (e.g., television, cell phone) rest for at least 5 minutes in a quiet environment.

8.2.3. Electrocardiogram

Triplicate 12 lead ECGs will be performed by qualified study center personnel using an ECG machine provided by the applicant that automatically calculates heart rate and measures pulse rate and RR, QRS, QT and QTc intervals. The 3 individual ECG traces should be acquired in succession as close as possible at intervals of about 5 minutes (+ -3 minutes). During the collection of the ECG, the participants should be in a quiet environment, undisturbed (e.g., television, cell phone). Participants should rest in a supine position for at least 5 minutes prior to ECG collection and should avoid speaking or moving arms or legs for at least 10 minutes prior to ECG collection. It is important to note that for both screening and in-study ECGs, the actual test time should be consistent for each time point to minimize variability in the results obtained.

Additional cardiovascular assessments should be performed clinically appropriate to ensure participant safety. Clinical researchers will review the results, including ECG morphology, for immediate management. Abnormalities noted at screening should be included in the medical history. The ECG data will be submitted to a central laboratory and reviewed by a cardiologist for interval measurements and general interpretation.

8.2.4. Echocardiography or multi-gated acquisition scan

Echocardiography (ECHO) or multi-gated acquisition (MUGA) scans (if ECHO is not available) will be performed at screening to establish baseline cardiac status. If clinically indicated, further evaluation will be performed.

8.2.5. Clinical safety laboratory assessment

Clinical laboratory samples will be collected. Researchers must review laboratory reports, record the review, and record any clinically relevant changes that occur during the study in the adverse events section of the eCRF. Laboratory reports must be submitted with the source document. Laboratory certificates or certifications and normal scopes of the research center laboratory facility must be submitted to the sponsor before the research center recruits any participants. If a participant performs a laboratory evaluation at a laboratory facility other than the laboratory facility associated with the research site, the researcher must also submit the facility's laboratory certificate or certification and normal scope to the sponsor. Laboratory reports must be submitted with the source document.

8.3. Adverse events and serious adverse events

Timely, accurate, and complete reporting and analysis of safety information from clinical studies is crucial to protecting participants, researchers, and sponsors, and is enforced by global regulatory bodies. The sponsor has established a Standard Operating program (Standard Operating Procedures) that meets global regulatory requirements to ensure proper reporting of security information; all clinical studies conducted by the sponsor or affiliates thereof will be conducted according to those procedures.

Adverse events will be reported by the participants (or, where appropriate, by the caregiver, surrogate, or legally acceptable representative of the participants) from the time of obtaining informed consent on the signature and date of note until up to 30 days after the last dose of study drug or, if earlier, until the start of subsequent anti-cancer therapy (see section 8.3.1, time period for reporting adverse events). The expected events will not be recorded and reported as this is a FIH study where all serious adverse events are important to understand the safety of the product.

8.3.1. Time period and frequency for collecting adverse event and severe adverse event information

All adverse events

All adverse events and special reporting events, whether severe or not, will be reported from the time the signed and dated ICF was obtained up to 30 days after the last dose of study drug, or if earlier until the start of subsequent anti-cancer therapy, and may include a link for safety follow-up. Researchers will follow up with adverse events and grade according to NCI CTCAE version 5.0. Participants with grade 3 or higher toxicity or non-resolved adverse events leading to discontinuation of study drug will continue to be evaluated until grade 1 or baseline is restored, the event is considered irreversible, the study is complete, or up to 6 months, whichever occurs first.

Severe adverse events must be reported using a severe adverse event table, including those adverse events reported spontaneously to the investigator within 30 days after the last dose of study drug. The sponsor will evaluate any safety information that the researcher spontaneously reports beyond the time frame specified in the project.

Serious adverse events

All serious adverse events that occurred during the study must be reported by the study site personnel to the appropriate sponsor contact within 24 hours of the event. Information about the serious adverse events will be transmitted to the sponsor using a "serious adverse event table" that must be completed and signed by the physician at the research center and transmitted to the sponsor within 24 hours. Initial and follow-up reports of serious adverse events should be made by facsimile (fax).

8.3.2. Follow-up of adverse events and severe adverse events

Researchers will follow up with adverse events, including pregnancy.

8.3.3. Regulatory reporting requirements for severe adverse events

The sponsors assume responsibility for appropriate reporting of adverse events to the regulatory body. The sponsor will also report to the investigator (and the leader of the research institution, if needed) all Suspected Unexpected Severe Adverse Reactions (SUSAR). Unless otherwise required and documented by the independent ethics committee/institutional review board (IEC/IRB), the researcher (or sponsor, if required) must report SUSAR to the appropriate IEC/IRB that approved the protocol.

8.3.4. Pregnancy

Initial reports of companion pregnancies for all female participants or male participants must be reported to the sponsor by the study centre personnel using an appropriate pregnancy notification form within 24 hours after they know about the event. Abnormal pregnancy outcomes (e.g., spontaneous abortion, fetal death, dead fetus, congenital abnormalities, ectopic pregnancy) are considered serious adverse events and must be reported using a "severe adverse event table". Any participant who was pregnant during the study had to discontinue treatment with study medication. Follow-up information will be required regarding the outcome of the pregnancy and any postpartum sequelae of the infant.

8.3.5. Adverse events of particular interest

Any level of cytokine release syndrome will be followed as part of the sponsor's standard safety monitoring activities. Regardless of the severity (i.e., severe and non-severe adverse events), these events will be reported to the sponsor within 24 hours of recognition of the event, and enhanced data collection will be required. CRS events (any level) must be followed up until recovery or until no further improvement.

8.4. Treatment of overdose

Since this is the first experience in studying drugs in humans, the MTD is unknown; therefore, an excess cannot be defined. In the case of a dosage error > 25% of the expected dose, the investigator or treating physician should:

contact the sponsor medical inspector immediately.

Closely monitor the participants for AE/SAE and laboratory abnormalities until the study drug can no longer be detected systemically (at least 5 days).

Serum samples for pharmacokinetic analysis were obtained as soon as possible and repeated for 5 consecutive days in sequence from the date of the last administration of the study drug.

Record the prescribed dose in the eCRF.

Record the actual dose administered in eCRF.

8.5. Pharmacokinetics and immunogenicity

8.5.1. Evaluation of

A venous blood sample was taken to measure the following serum concentrations:

study drug and anti-study drug antibodies. Each serum sample was divided into 3 aliquots (1 each for pharmacokinetic, anti-study drug antibodies and back-up). The collection of samples for analysis of study drug serum concentrations and antibodies to study drugs can additionally be used to assess safety or efficacy aspects addressing issues arising during or after the study period, to further characterize immunogenicity, or to assess relevant biomarkers (e.g., the possible presence of soluble PSMA). These serum samples were not subjected to genetic analysis. The confidentiality of the participants will be maintained. Additional information regarding the collection, handling and transportation of biological samples can be found in "laboratory manuals".

8.5.2. Analysis program

Pharmacokinetics

Serum samples are analyzed by or under the supervision of the sponsor using validated, specific and sensitive immunoassays to determine the concentration of the study drug.

Immunogenicity

The detection and characterization of anti-study drug antibodies will be performed by the sponsor or under the supervision of the sponsor using validated assay methods. All samples collected for detection of antibodies against the study drug will also be evaluated against study drug serum concentrations to enable interpretation of antibody data.

8.5.3. Pharmacokinetic parameters and evaluation

Blood samples were collected during the study for measuring the pharmacokinetics of the study drug at the time points summarized in table 19. Samples will also be collected at the end of treatment visit after study drug discontinuation.

The exact date and time of blood sampling of all samples collected on the laboratory application form must be recorded. Sample collection requires reference to a "laboratory manual". The collected samples must be stored under specific controlled conditions of temperature indicated in the "laboratory manual".

The collected samples may additionally be used to assess safety or efficacy aspects of addressing issues that arise during or after the study period, or addressing issues with drug properties that may arise later, if desired. The confidentiality of the participants will be maintained. Additional information regarding the collection, handling and transportation of biological samples can be found in "laboratory manuals".

Pharmacokinetic parameters

Pharmacokinetic parameters of the individual will be estimated and descriptive statistics for each dose level will be calculated. Can also explore C_maxAnd dose dependence of AUC. Pharmacokinetic parameters may include, but are not limited to, C_max、T_max、AUC_(t1-t2)、AUC_tau、C_minAnd an accumulation Ratio (RA); if enough data is available for estimation, the parameters will be calculated. In addition, exploratory population pharmacokinetics-based methods are also applicable to pharmacokinetic analysis.

8.5.4. Assessment of immunogenicity (anti-study drug antibody)

Anti-study drug antibodies will be evaluated according to table 19 in serum samples collected from all participants during both part 1 and part 2. In addition, serum samples will also be collected at the final visit from participants who discontinued study medication or exited the study.

Serum samples will be used to assess the immunogenicity of antibodies against the study drug. The collection of samples for immunogenicity analysis may additionally be used to assess the safety or efficacy aspects of addressing issues arising during or after the study period.

8.6. Pharmacodynamics of medicine

Cytokine production from peripheral blood was analyzed before and after study drug treatment. The assay will monitor the levels of cytokines that can signal activation of immune cells, which may include, but are not limited to, IL-1 β, IL-2, IL-6, IL-8, IL-10, IFN- γ, and TNF- α δ.

To determine whether treatment with study drugs results in increased anti-tumor activity through redirected T cell mediated killing of PSMA positive tumor cells and increased activation of cytotoxic T cells, whole blood samples and metastatic tissue samples can be analyzed by methods such as flow cytometry or time of flight cytometry (cytef) to assess tumor and immune cell populations. Fresh tissue tumor biopsies from accessible sites of metastatic disease will be collected and tested for PSMA expression and pharmacodynamic markers in the tumor.

Whole blood samples can be analyzed using flow cytometry to assess peripheral immune cell populations. Venous blood samples will be collected for exploratory assessment of CD3 Receptor Occupancy (RO) on T cells by flow cytometry. For more details on tumor tissue sample requirements, preparation and transport reference is made to the "laboratory manual".

8.7. Genetics and science

Pharmacogenomics or pharmacogenetics will not be evaluated in this study.

8.8. Biomarkers

Biomarker assessment in this study will focus on the following goals: 1) assessing the potential contribution of immune responses indicative of T cell responses in tumors and blood as research drugs; 2) assessing cytokine production in response to study drug administration; and 3) assessing other markers predictive of response to treatment, including PSMA expression.

PSMA is often expressed at high levels on certain tumors compared to normal human prostate. Previous studies showed variable expression of PSMA expression in patients with mCRPC. In addition, neuroendocrine tumors of the prostate gland showed resistance to PSMA-targeted therapy. Thus, expression of PSMA and neuroendocrine markers will be assessed from tumors by IHC. Pre-and post-treatment expression of PSMA and neuroendocrine markers in tumors can be assessed to assess treatment efficacy. Tumor samples will be collected from the selected cohort.

The baseline tumor immune status predicts the response and therefore T cell activation, depletion and other immune cells affecting T cell response will be assessed from the baseline tumor and post-treatment. Immune cell responses in tumors and peripheral blood will be assessed before and after treatment. Cytokines released due to T cell activation will be assessed from serum samples collected before and after infusion. In addition, PBMCs will be collected and stored. Potential future uses may include the identification of immunophenotypic subpopulations that respond differently to study drugs.

During part 2, in addition to the biomarkers described above, circulating tumor DNA and CTCs will be collected and used to explore changes in T cell clonality, identify markers that predict response/resistance, and assess immune characteristics in peripheral blood and tumors.

Biomarkers will be assessed in tumor tissue samples, whole blood and serum. Biomarker samples can be used to help solve emerging problems and enable the development of safer, more effective and ultimately individualized therapies. These samples will only be collected at research centers where local regulations and transportation logistics allow and the analysis will be performed at a central laboratory.

To understand the changes in tumor microenvironment before and after treatment with study drugs, RNA samples from metastatic tumor sources were subjected to next generation RNA sequencing. Genes and genomes will correlate with treatment outcomes.

Stop analysis

Biomarker analysis depends on the availability and clinical response rate of appropriate biomarker assays. If during or at the end of the study, the analysis will not have sufficient scientific value for biomarker assessment, or if there are not enough samples or responders to allow sufficient biomarker assessment to be performed, biomarker analysis may be postponed or not performed. In cases where the study terminates early or shows poor clinical efficacy, biomarker assessment is accomplished based on the reasonableness and expected utility of the data.

Additional collection

If it is determined at any time prior to completion of the study that additional material from the formalin fixed paraffin embedded tumor sample is needed to successfully complete the protocol specified analysis, the sponsor may request that additional material be retrieved from the existing sample. In addition, based on emerging scientific evidence, the sponsor may request additional material from previously collected tumor samples for retrospective analysis during or after completion of the study. In this case, such analysis is specific to the study associated with the study drug or the disease being studied.

8.9. Health economics or medical resource utilization and health economics

Not applicable.

9. Statistical considerations

No formal hypothesis testing will be performed. Descriptive statistics will be used to summarize the data. Continuous variables will be summarized as desired using the number, mean, standard deviation, coefficient of variation, median and range of observations. The classification values will be summarized using the number and percentage of observations as needed.

9.1. Statistical assumptions

Not applicable. Dose escalation will be guided by the statistical model described below.

9.1.1. Statistical model supporting dose escalation

The DLT probability derived by the two-parameter BLRM using EWOC principle will be the main guide to help with dose escalation and RP2D recommendation, which is equal to or lower than the estimated MTD.

The incidence of DLT, e.g., whether DLT occurred during the DLT assessment period (section 4.1.3), is the primary variable for dose escalation. These cumulative DLT data from the qualified participants of the DLT evaluable analysis group will be used to model the relationship between dose of study drug and DLT. The two-parameter BLRM will be used to calculate the probability of DLT at dose d.

logit(π(d))＝log(α)+β·log(d/d*)，α>0，β>0

Where pi (d) is the probability of DLT when the study drug is administered as a single agent at dose d, d is the planned dose during the DLT assessment period, and logit (pi (d)) -log [ pi (d)/{ 1-pi (d) } ], and d is the reference dose.

DLT probability by BLRM

The probability of true DLT rate for each dose level will be summarized as follows:

[ 0%, 20%) underdose interval

[ 20%, 33%) target toxicity interval

[ 33%, 100% ] excessive toxicity interval

As described above, when all participants in the dose cohort complete the DLT assessment period, the probability of DLT will be calculated by the BLRM. The DLT probability at all dose levels of the study drug will be used to recommend the highest dose level for the next dose cohort. The highest dose will need to meet the EWOC principle, i.e. the probability of estimating the DLT rate within the excessive toxicity interval is less than 25% and the probability of estimating the DLT rate within the target toxicity interval is highest. In addition, the dose selection and decision of MTD or RP2D for the next cohort will follow the rules described in section 4.1.1.

9.2. Sample size determination

During dose escalation, 1 or more participants will be enrolled at a dose level during the accelerated titration phase and 3 or more participants will be enrolled at a dose level during the standard titration phase, with at least 6 of the participants being enrolled with safe and tolerable RP 2D. The total number of enrolled participants will depend on the frequency of DLTs and when RP2D is determined. The maximum sample size was about 70 participants.

Since section 2 is aimed at assessing the safety and preliminary clinical activity of study drugs at RP2D, a sample size of about 20(mCRPC and RCC) was chosen to provide a point estimate with reasonable accuracy. The table describes the point estimates at the selected frequency for the event type of interest (e.g., objective response or adverse event of particular interest) and their 90% accurate confidence intervals (two-sided).

TABLE 32 Point estimates and 90% accurate confidence intervals

Specifically, if the true probability of an event of interest is 15% or higher, the probability of not observing a participant experiencing the event is less than 5%.

9.3. Populations for analysis

The analytical population of this study is defined as follows:

all treatment analysis groups: this group consisted of participants who received at least 1 dose of study drug. This analysis group will be considered primary and will be used for all safety and efficacy summaries.

DLT evaluable analysis group: this group is a subset of the "all treatments analysis" group. Participants who received at least 75% of the planned dose of study drug during the DLT observation period defined in section 4.1.3 will be included in the analysis.

Biomarker analysis panel: this group consisted of all participants who received at least 1 dose of study drug and had at least 1 pre-treatment or post-treatment biomarker measurement.

Pharmacokinetic analysis group: this group consisted of all participants who received at least 1 dose of study drug and had at least 1 evaluable concentration measurement of study drug.

9.4. Statistical analysis

9.4.1. Efficacy analysis

Endpoint definition

Overall Response Rate (ORR) is defined as the proportion of participants with PR or better according to disease specific response criteria. The investigator will assess the response to treatment.

The duration of response (DOR) will be calculated from the date of initial recording of the response (PR or better) to the date of first recording of evidence of progressive disease as defined in the disease specific response criteria or death due to any cause, whichever occurs first. For non-progressing and surviving participants who responded to disease treatment (CR or PR), data will be censored at the final disease assessment before starting any subsequent anti-cancer therapy.

Time To Response (TTR) is defined as the time from the date the study drug was first administered to the date the response was first recorded.

Analytical method

The overall response rate will be tabulated along with 90% accurate confidence intervals on either side. In addition, the number and percentage of participants in each response category will be tabulated. For response time, the results will be summarized using descriptive statistics, including mean, median, standard deviation, and range of participants with responses. For DOR, a descriptive summary will be made using the Kaplan-Meier method.

9.4.2. Security analysis

All safety analyses will be performed on data from the "all treatment analysis group". The baseline value for safety assessment is defined as the value collected at the time closest to but prior to the start of the first study drug administration. Safety parameters to be assessed are the incidence, severity and type of adverse events, clinically significant changes found by physical examination of participants, vital sign measurements, clinical laboratory and other clinical test results (e.g., ECG). The reasons for exposure to study drug and discontinuation of study drug will be tabulated. Adverse events will be summarized by system organ categories, preferred terminology, worst-case grade experienced by participants and dose level.

Adverse events

The verbatim terms used by researchers in eCRF to identify adverse events will be encoded with the supervised active medical dictionary (MedDRA). A study drug emergent adverse event is an adverse event that occurs during the study drug phase, or is the result of a pre-existing condition that worsens from baseline. All reported adverse events will be included in the analysis. For each adverse event, the percentage of participants who experienced at least 1 occurrence of a given event will be summarized by dose level/dose cohort.

For participants who die, discontinue study medication due to adverse events, or experience severe or severe adverse events, a summary, list, dataset, or participant narrative may be provided as appropriate. The list of DLTs will use DLT evaluable analysis groups. DLTs will be listed and the incidence is summarized by major system organ categories, preferred terms, worst grade and type of adverse event, and dose level.

Clinical laboratory testing

Laboratory data will be summarized by the type of laboratory test. Reference ranges will be used in the summary of laboratory data. Descriptive statistics for each laboratory analyte were calculated at baseline, and the values observed at each scheduled time point and the change from baseline were calculated. The worst toxicity rating during treatment will be given according to NCI CTCAE version 5.0. The change from baseline to worst toxicity level experienced by the participants during the study will be provided as a shift table. A list of participants for which any laboratory results are outside the reference range will be provided.

Electrocardiogram (ECG)

The effect of study drugs on QTc will be evaluated by means of descriptive statistics and frequency lists. Pharmacokinetic/pharmacodynamic models will be explored to understand and characterize the exposure-response relationship.

Vital signs

Descriptive statistics of body temperature, pulse/heart rate and blood pressure (systolic and diastolic) values and changes from baseline at each planned time point were summarized. The percentage of participants whose summary value exceeded the clinically significant limit.

9.4.3. Other analysis

Pharmacokinetic analysis

The data from the "pharmacokinetic analysis group" will be subjected to pharmacokinetic analysis. All serum concentrations or missing data below the lowest quantifiable concentration will be so labeled in the concentration database. In summary statistics, concentrations below the quantifiable lower limit will be considered zero. If the participant's data does not allow for adequate assessment of the parameters, it is excluded from pharmacokinetic parameter analysis. All participants and samples excluded from the analysis will be clearly recorded in the CSR.

Data will be presented for all participants with available serum concentrations per dose level. If the participant's data does not allow for accurate assessment of pharmacokinetics (e.g., incomplete administration of the study drug; missing information on dosing and sampling times; insufficient concentration data for pharmacokinetic parameter calculations), the participant will be excluded from pharmacokinetic analysis.

Descriptive statistics the dose cohort for the pharmacokinetic parameters of the study drug was summarized at each sampling time point for the study drug serum concentration. The mean serum concentration time curve will be plotted, and the individual serum concentration time curve may also be plotted.

If appropriate data is available, population pharmacokinetic analysis of serum concentration-time data for study drugs can be performed using non-linear mixed effect modeling. Details will be given in a separate population pharmacokinetic analysis plan, and the results of the population pharmacokinetic analysis will be presented in a separate report.

Biomarker analysis

Biomarker analysis will be stratified by clinical covariates or molecular subgroups using appropriate statistical methods (e.g., parametric or non-parametric, univariate or multivariate, analysis of variance, or survival analysis, depending on the endpoint). Correlation of baseline expression levels or changes in expression levels with responses to event time endpoints will identify a subset of responsiveness (or resistance) in addition to genes and pathways that are attenuated following treatment with study drug.

Any pharmacokinetic measurements will be listed, tabulated, and plotted where appropriate. Participants may be grouped by queue, dose schedule, or clinical response. Since this is an open label study without a control group, statistical analysis will be performed to help understand the results.

The results of the biomarker analysis may be presented in a separate report. Planned analysis is based on the availability of clinically valid assays and can be postponed if emerging research data shows that there is no possibility to provide useful scientific information.

Receptor occupancy analysis

Descriptive statistics will be used to summarize the study drug CD3RO results. The relationship between the serum concentration of the drug and RO and the relationship between RO and downstream pharmacodynamic effects will be explored. The results of any such analysis may be presented in a separate report.

Immunogenicity assays

All participants who received at least 1 dose of study drug and had the appropriate sample for detecting the antibodies to the study drug (i.e., participants who obtained at least 1 sample after the first administration of the study drug) will be pooled for incidence of antibodies to the study drug. A list of participants who were positive for antibodies to the study drug will be provided. For participants who were positive for antibodies to the study drug, the maximum titers of antibodies to the study drug will be summarized. Other immunogenicity assays may be performed to further characterize the resulting immune response.

Pharmacodynamic analysis

Pharmacodynamic samples received by the contract supplier or sponsor after the expiration date will not be analyzed and are therefore excluded from the pharmacodynamic analysis. Correlations between baseline levels of the selected markers and changes from baseline and clinical response will be explored. The results of this analysis will be presented in a separate report.

Pharmacokinetic/pharmacodynamic analysis

Pharmacokinetic/pharmacodynamic models will be explored to understand and characterize the exposure-response relationships of key efficacy, safety, and pharmacodynamic/biomarker endpoints. The details will be provided in a separate analysis plan and the analysis results may be aggregated in a separate report.

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