Methods for using multispecific binding proteins

Abstract

This specification provides anti-CD19 antibodies and multispecific binding proteins that bind to CD19, CD3, and serum albumin. Also provided are pharmaceutical compositions comprising these antibodies or multispecific binding proteins, expression vectors and host cells for producing these antibodies or multispecific binding proteins, and methods for using these antibodies or multispecific binding proteins in the treatment of cancers, including relapsed and / or refractory non-Hodgkin lymphoma and cancers that express CD19 at low levels.

Description

[Technical Field]

[0001] Related applications This patent application is a U.S. Provisional Patent Application No. 63 / 494970 filed on April 7, 2023, a U.S. Provisional Patent Application No. 63 / 494973 filed on April 7, 2023, a U.S. Provisional Patent Application No. 63 / 464258 filed on May 5, 2023, a U.S. Provisional Patent Application No. 63 / 464259 filed on May 5, 2023, and a U.S. Provisional Patent Application No. 63 / 464259 filed on June 29, 2023. We claim priority rights to U.S. Provisional Patent Application No. 63 / 511125, U.S. Provisional Patent Application No. 63 / 581767 filed on 11 September 2023, U.S. Provisional Patent Application No. 63 / 591594 filed on 19 October 2023, and U.S. Provisional Patent Application No. 63 / 605804 filed on 4 December 2023, the contents of which are incorporated in their entirety by reference.

[0002] Reference to electronic sequence listings This application includes a sequence listing submitted electronically in a computer-readable file format, which is incorporated herein by reference in its entirety. The computer-readable file, created on March 16, 2024, is named 67300WO01_SequenceListing.xml and has a size of 45,643 bytes.

[0003] This disclosure relates to anti-CD19 antibodies and multispecific binding proteins that bind to CD19, CD3, and optionally serum albumin for the treatment of cancer. [Background technology]

[0004] Bispecific molecules, such as BiTE® (registered trademark) (bispecific T cell engager) constructs, are recombinant protein constructs made from two flexibly linked antibody-derived binding domains. One binding domain of a BiTE® construct is specific for a selected tumor-associated surface antigen on target cells, and the second binding domain is specific for CD3, a subunit of the T cell receptor complex on T cells. This design allows the BiTE® construct to transiently connect T cells to target cells while potently activating the intrinsic cytolytic ability of T cells against target cells.

[0005] The CD3 receptor complex is a protein complex composed of four polypeptide chains. In mammals, the complex includes the CD3γ (gamma) chain, the CD3δ (delta) chain, and two CD3ε (epsilon) chains. The CD3γ (gamma) chain, CD3δ (delta) chain, and CD3ε (epsilon) chain are cell surface proteins highly related to the immunoglobulin superfamily that contain a single extracellular immunoglobulin domain. These chains associate with the T cell receptor (TCR) to form the TCR-CD3 complex and generate activation signals in T lymphocytes upon antigen binding. Approximately 95% of T cells express the αβ TCR, which includes an α (alpha) chain and a β (beta) chain. Two TCRζ (zeta) chains are also present in the TCR-CD3 complex. The αβ TCR plays a role in recognizing antigens presented by the major histocompatibility complex (MHC). When the TCR binds to the antigenic peptide and MHC complex, T lymphocytes are activated through a series of biochemical events mediated by associated enzymes, coreceptors, special adapter molecules, and activated or released transcription factors.

[0006] CD19, also known as B cell surface antigen B4 or Leu-12, is a transmembrane protein expressed in B lymphocytes and follicular dendritic cells. CD19 is a co-receptor of the B cell antigen receptor complex on B lymphocytes (see Carter et al. (2002) Science, 256:105-7, van Zelm et al. (2006) N. Eng. J. Med., 354:1901-12). Along with the B cell receptor (BCR), CD19 modulates endogenous and antigen receptor-induced signaling thresholds crucial for B cell clonal proliferation and humoral immunity. CD19 is a human B cell surface marker expressed from the early stages of pre-B cell development to terminal differentiation into plasma cells. It is also expressed in many non-Hodgkin lymphoma (NHL) cells and certain leukemias. Antibodies that bind to CD19 have been developed and are being tested in clinical trials for lymphoid cancers such as B-cell malignancies (see, for example, Hekman et al. (1991) Cancer Immunol. Immunother., 32:364-72; Vlasfeld et al. (1995) Cancer Immunol. Immunother., 40:37-47; Corny et al. (1995) J. Immunother. Emphasis Tumor Immunol., 18:231-41; and Manzke et al. (2001) Int. J. Cancer, 91:516-22). Furthermore, a BiTE® construct called blinatumomab is being developed for clinical use.

[0007] BiTE® constructs are thought to undergo rapid clearance from the body. Therefore, they can rapidly penetrate many areas of the body, are rapidly produced, and are easier to handle; however, their in vivo application may be limited by their short in vivo persistence. Blinatumomab and solitomab have short in vivo half-lives, and therefore, long-term administration via continuous intravenous infusion may be required to achieve their therapeutic effects. However, such continuous intravenous infusion can be inconvenient for patients and may increase treatment costs.

[0008] Important developments have been made in the construction of anti-CD19 antibodies and bispecific binding proteins, but there remains a need for new and useful proteins for cancer treatment that have sufficient therapeutic effects, formats that are easy to manufacture, and favorable pharmacokinetic properties such as an extended half-life.

Summary of the Invention

[0009] Described herein, in certain embodiments, is a method of treating an individual having CD19-low expressing cancer and needing treatment thereof, the method comprising administering to the individual a bispecific binding protein comprising: a) a first antigen-binding site comprising a heavy chain variable domain (VH) that binds to human CD19 and comprises complementarity determining regions HCDR1, HCDR2, and HCDR3 and a light chain variable domain (VL) that comprises complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, 7, 8, 9, and 10, respectively; and b) a second antigen-binding domain comprising a VH that binds to CD3 and comprises complementarity determining regions HCDR1, HCDR2, and HCDR3 and a VL that comprises complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 15, 16, 18, 19, 20, and 21, respectively.

[0010] In some embodiments, CD19-low expressing cancer is classified as low expressing by flow cytometry. In some embodiments, CD19-low expressing cancer is classified by having from about 325 to about 17,000 CD19 molecules per cell. In some embodiments, CD19-low expressing cancer is classified by having less than about 3,000 CD19 molecules per cell.

[0011] In some embodiments, the VH of the first antigen-binding domain contains an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to that of SEQ ID NO: 1, and the VL of the first antigen-binding domain contains an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to that of SEQ ID NO: 2. In some embodiments, the VH of the first antigen-binding domain contains the amino acid sequence of SEQ ID NO: 1, and the VL of the first antigen-binding domain contains the amino acid sequence of SEQ ID NO: 2. In some embodiments, the first antigen-binding site contains an amino acid sequence that is at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the first antigen-binding site contains the amino acid sequence of SEQ ID NO: 11.

[0012] In some embodiments, the VH of the second antigen-binding site contains an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to that of SEQ ID NO: 12, and the VL of the second antigen-binding site contains an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to that of SEQ ID NO: 13. In some embodiments, the VH of the second antigen-binding site contains the amino acid sequence of SEQ ID NO: 12, and the VL of the second antigen-binding site contains the amino acid sequence of SEQ ID NO: 13. In some embodiments, the second antigen-binding site contains an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to that of SEQ ID NO: 22. In some embodiments, the second antigen-binding site contains the amino acid sequence of SEQ ID NO: 22.

[0013] In some embodiments, the polyspecific binding protein further includes a half-life extension domain. In some embodiments, the half-life extension domain includes a third antigen-binding site that binds to human serum albumin. In some embodiments, the half-life extension domain is not located between the first and second antigen-binding sites in the polypeptide chain. In some embodiments, the third antigen-binding site includes a VH comprising complementarity-determining regions HCDR1, HCDR2, and HCDR3, where HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs. 26, 27, and 29, respectively.

[0014] In some embodiments, the VH of the third antigen-binding site contains an amino acid sequence that is at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 24. In some embodiments, the VH of the third antigen-binding site contains the amino acid sequence of SEQ ID NO: 24. In some embodiments, the polyspecific binding protein contains, from N-terminus to C-terminus, a) a third antigen-binding site that binds to human serum albumin and contains an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 24; b) a first antigen-binding site that binds to CD19 and contains an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 11; and c) a second antigen-binding site that binds to human CD3 and contains an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 22. In some embodiments, the polyspecific binding protein includes, from N-terminus to C-terminus, a) a third antigen-binding site that binds to human serum albumin and contains the amino acid sequence of SEQ ID NO: 24, b) a first antigen-binding site that binds to CD19 and contains the amino acid sequence of SEQ ID NO: 11, and c) a second antigen-binding site that binds to human CD3 and contains the amino acids of SEQ ID NO: 22. In some embodiments, the polyspecific binding protein contains an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 41. In some embodiments, the polyspecific binding protein contains the amino acid sequence of SEQ ID NO: 41.

[0015] Also described herein is a method for treating relapsed and / or refractory non-Hodgkin lymphoma (NHL) in an individual in need thereof, the method comprising: a) a first antigen-binding site comprising a heavy chain variable domain (VH) that binds to human CD19 and includes complementarity-determining regions HCDR1, HCDR2, and HCDR3, and a light chain variable domain (VL) that includes complementarity-determining regions LCDR1, LCDR2, and LCDR3, respectively, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, SEQ ID NO: 4 A method comprising administering a multispecific binding protein comprising a first antigen-binding site comprising amino acid sequences 5, 7, 8, 9, and 10 and a second antigen-binding domain comprising VH, which binds to CD3 and comprises complementarity-determining regions HCDR1, HCDR2, and HCDR3, and VL, which comprises complementarity-determining regions LCDR1, LCDR2, and LCDR3, respectively. In some embodiments, the individual has relapsed after treatment with CD19-targeted therapy or is resistant to CD19-targeted therapy. In some embodiments, the CD19-targeted therapy comprises blinatumomab. In some embodiments, the CD19-targeted therapy comprises CD19 CAR-T cell therapy. In some embodiments, the VH of the first antigen-binding domain contains an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to that of SEQ ID NO: 1, and the VL of the first antigen-binding domain contains an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to that of SEQ ID NO: 2. In some embodiments, the VH of the first antigen-binding domain contains the amino acid sequence of SEQ ID NO: 1, and the VL of the first antigen-binding domain contains the amino acid sequence of SEQ ID NO: 2. In some embodiments, the first antigen-binding site contains an amino acid sequence that is at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 11.In some embodiments, the first antigen-binding site includes the amino acid sequence of SEQ ID NO: 11. In some embodiments, the VH of the second antigen-binding site includes an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to that of SEQ ID NO: 12, and the VL of the second antigen-binding site includes an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to that of SEQ ID NO: 13. In some embodiments, the VH of the second antigen-binding site includes the amino acid sequence of SEQ ID NO: 12, and the VL of the second antigen-binding site includes the amino acid sequence of SEQ ID NO: 13. In some embodiments, the second antigen-binding site includes an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to that of SEQ ID NO: 22. In some embodiments, the second antigen-binding site includes the amino acid sequence of SEQ ID NO: 22. In some embodiments, the polyspecific binding protein further includes a half-life extension domain. In some embodiments, the half-life extension domain includes a third antigen-binding site that binds to human serum albumin. In some embodiments, the half-life extension domain is not located between the first and second antigen-binding sites in the polypeptide chain. In some embodiments, the third antigen-binding site includes a VH containing complementarity-determining regions HCDR1, HCDR2, and HCDR3, where HCDR1, HCDR2, and HCDR3 contain the amino acid sequences of SEQ ID NOs. 26, 27, and 29, respectively. In some embodiments, the VH of the third antigen-binding site contains an amino acid sequence that is at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO. 24. In some embodiments, the VH of the third antigen-binding site contains the amino acid sequence of SEQ ID NO. 24.In some embodiments, the polyspecific binding protein includes, from N-terminus to C-terminus, a) a third antigen-binding site that binds to human serum albumin and includes an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 24; b) a first antigen-binding site that binds to CD19 and includes an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 11; and c) a second antigen-binding site that binds to human CD3 and includes an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 22. In some embodiments, the polyspecific binding protein contains an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 41. In some embodiments, the polyspecific binding protein contains the amino acid sequence of SEQ ID NO: 41.

[0016] In some embodiments, administration results in B cell depletion in the subject within 96 hours of administration of the polyspecific binding protein. In some embodiments, administration results in persistent B cell depletion lasting for at least 90 days after administration of the polyspecific binding protein. In some embodiments, administration results in persistent B cell depletion lasting for at least 90 days after the last administration of the polyspecific binding protein.

[0017] In some embodiments, subjects are administered a dose of at least about 30 μg of the polyspecific binding protein. In some embodiments, subjects are administered a dose of at least about 40 μg of the polyspecific binding protein. In some embodiments, subjects are administered a dose of at least about 50 μg of the polyspecific binding protein. In some embodiments, subjects are administered a dose of at least about 60 μg of the polyspecific binding protein.

[0018] In some embodiments, the polyspecific binding protein is administered once a week. In some embodiments, the polyspecific binding protein is administered once every two weeks. [Brief explanation of the drawing]

[0019] [Figure 1A] This is a schematic diagram of the six-domain arrangement of a single-chain polyspecific binding protein. The CD19-binding domain in the form of scFv, the CD3-binding domain in the form of scFv, and the HSA-binding domain in the form of sdAb are linked in different directions. The top of each construct represents the N-terminus, and the bottom of each construct represents the C-terminus of a given polypeptide chain. [Figure 1B] This disclosure shows the design of a multispecific binding protein according to an aspect of this disclosure.

[0020] [Figure 2A] In vitro data comparing tAB0050 and blinatumomab in their ability to redirect lysis are illustrated. CD19 expression in the EMT6 cell line is shown. The parental, low-CD19 strain (clone 6), and high-CD19 strain (clone 5) were characterized by flow cytometry. [Figure 2B] In vitro data comparing tAB0050 and blinatumomab in their ability to redirect lysis are illustrated. Cytotoxicity data in the parental strain are also shown. [Figure 2C] In vitro data comparing tAB0050 and blinatumomab in their ability to redirect lysis are illustrated. Cytotoxicity data in low CD19 cell lines are shown. [Figure 2D]In vitro data comparing tAB0050 and blinatumomab in their ability to redirect lysis are illustrated. Cytotoxicity data in high CD19 cell lines are shown.

[0021] [Figure 3A] This shows the activity of tAB0050 in co-culture of human T cells with low CD19 expression. In vitro data of tAB0050 when redirecting the lysis of low-CD19-expressing cell lines are illustrated. [Figure 3B] This shows the activity of tAB0050 in co-culture of human T cells with low CD19 expression. It also shows the expression of CD69 and CD25. [Figure 3C] This shows the activity of tAB0050 in co-culture of human T cells with low CD19 expression. It also shows TNFα expression. [Figure 3D] This shows the activity of tAB0050 in co-culture of human T cells with low CD19 expression. It also shows IFNγ expression.

[0022] [Figure 4A] tAB0050 induces T cells and T cell-dependent cytotoxicity (TDCC). Chemically induced CD19-expressing CHO cells were co-cultured for 68 hours with isolated T cells at a 10:1 E:T ratio in the presence or absence of the indicated concentration of tAB0050 (n=4). The lysis curves evaluated by flow cytometry based on 7-AAD uptake in CD19-expressing CHO cells are shown. [Figure 4B] tAB0050 induces T cell induction and T cell-dependent cytotoxicity (TDCC). Chemically induced CD19-expressing CHO cells were co-cultured for 68 hours with isolated T cells at a 10:1 E:T ratio in the presence or absence of the indicated concentrations of tAB0050 (n=4). The CD25 expression profile as a marker of CD8+ T cell activation is illustrated. [Figure 4C]tAB0050 induces T cell induction and T cell-dependent cytotoxicity (TDCC). Chemically induced CD19-expressing CHO cells were co-cultured for 68 hours with isolated T cells at a 10:1 E:T ratio in the presence or absence of the indicated concentrations of tAB0050 (n=4). Ki67 expression profiles as a marker of CD8+ T cell activation are illustrated. [Figure 4D] tAB0050 induces T cell induction and T cell-dependent cytotoxicity (TDCC). Chemically induced CD19-expressing CHO cells were co-cultured for 68 hours with isolated T cells at a 10:1 E:T ratio in the presence or absence of the indicated concentration of tAB0050 (n=4). The supernatant from the co-culture of CD19-expressing CHO cells and PBMCs was analyzed for INFγ and is shown in the figure. [Figure 4E] tAB0050 induces T cells and T cell-dependent cytotoxicity (TDCC). Chemically induced CD19-expressing CHO cells were co-cultured for 68 hours with isolated T cells at a 10:1 E:T ratio in the presence or absence of the indicated concentrations of tAB0050 (n=4). Cytotoxicity or INFγ production was observed at 72 hours with tAB0050 or blinatumomab at a concentration of 225 pM (n=10). [Figure 4F] tAB0050 induces T cell induction and T cell-dependent cytotoxicity (TDCC). Chemically induced CD19-expressing CHO cells were co-cultured for 68 hours with isolated T cells at a 10:1 E:T ratio in the presence or absence of the indicated concentrations of tAB0050 (n=4). Wilcoxon signed-rank test is shown.

[0023] [Figure 5A] This disclosure describes the design and characterization of tAB0050 according to one aspect of this disclosure. It also includes a summary of Biacore binding data for tAB0050 to target proteins. [Figure 5B] This disclosure describes the design and characterization of tAB0050 according to one aspect of this disclosure. The binding of tAB0050 to human, cynomolgus monkey, and mouse peripheral blood mononuclear cells is shown (n=3 donors).

[0024] [Figure 6] This shows the binding of tAB0050 in the presence or absence of serum albumin.

[0025] [Figure 7A] This shows T cell activation and potential induction of TDCC after administration of tAB0050. RAMOS cells co-cultured for 48 hours with tAB0050 at concentrations shown in the absence of albumin, with an E:T ratio of 10:1 and PBMCs derived from six healthy donors. The lysis curve, evaluated by flow cytometry based on 7-AAD uptake in RAMOS cells, is also shown. [Figure 7B] This shows T cell activation and potential induction of TDCC after administration of tAB0050. It also shows RAMOS cells co-cultured for 48 hours with 6 healthy donor-derived PBMCs at the concentrations of tAB0050 shown in the absence of albumin, with an E:T ratio of 10:1. CD25 and CD69 expression in CD4+ and CD8+ T cells is also shown. [Figure 7C] This shows the potential induction of T cell activation and TDCC after administration of tAB0050. RAMOS cells co-cultured with PBMCs for 48 hours at the concentrations of tAB0050 shown in and out of human serum albumin, with an E:T ratio of 10:1 (n=10). [Figure 7D] This shows the potential induction of T cell activation and TDCC after administration of tAB0050. The supernatant from co-cultures of RAMOS cells and PBMCs in 2C in the presence of albumin was analyzed by Luminex for the indicated cytokines. [Figure 7E] This shows the potential induction of T cell activation and TDCC after administration of tAB0050. The supernatant from co-cultures of RAMOS cells and PBMCs in 2C in the presence of albumin was analyzed by Luminex for the indicated cytokines.

[0026] [Figure 8A]Raji cells co-cultured with PBMCs for 48 hours in the presence or absence of tAB0050 and human albumin at the indicated concentrations, with or without, at an E:T ratio of 10:1 (n=8). The lysis curves evaluated by flow cytometry upon 7-AAD uptake in RAMOS cells and the CD69 expression profile as a marker of CD4+ and CD8+ T cell activation are shown. [Figure 8B] This shows tAB0050 redirection lysis of endogenous B cells in the presence or absence of human albumin. [Figure 8C] This shows T cell activation by tAB0050 in the presence of lymphoma target cell lines Ramos and Raji.

[0027] [Figure 9A] This demonstrates the in vivo efficacy of tAB0050. It shows that hCD3ε-expressing BALB / c mice were inoculated with hCD19-expressing A20 cells. Mice were treated once with IV when the tumor volume reached approximately 100 mm3. [Figure 9B] This study demonstrates the in vivo efficacy of tAB0050. It shows that 2 × 10⁵ Raji cells were IV-engrafted into immunodeficient NCG mice, followed by IP transplantation of 2 × 10⁷ PBMCs the following day. Mice were treated weekly with IV therapy starting on day 1. Statistics were calculated against vehicle or PBS using ANOVA with multiple comparison tests at d10 for group A and d15 for group B. [Figure 9C] This study demonstrates the in vivo efficacy of tAB0050. Immunodeficient NCG mice were IV-transplanted with 2 × 10⁵ Raji cells. The following day (day 1), 2 × 10⁷ PBMCs from a healthy donor were intravenously transplanted. tAB0050 was administered weekly, either IV-transplanted or via spongiform fusion, starting on day 1. All treatment conditions using tAB0050 resulted in a statistically significant response compared to the control group, as measured by ANOVA at day 14 (p<0.0001). [Figure 9D] This demonstrates the in vivo efficacy of tAB0050. A representative emission image of C is shown. [Figure 9E]This demonstrates the in vivo efficacy of tAB0050. A graph shows that hCD3ε-expressing BALB / c mice were inoculated with A20 cells expressing varying levels of hCD19. Mice were treated IV once weekly with 0.1 mg / kg of tAB0050 or control when their tumor volume reached approximately 100 mm3.

[0028] [Figure 10A] The quantification of flow cytometry is illustrated. It shows the decrease in normal B cells and tumor cells in peripheral blood after treatment with tAB0050 in the huPBMC Raji B.luc mouse model. [Figure 10B] The quantification of flow cytometry is illustrated. The total number of T cells and the frequency of T cell activation in peripheral blood after treatment with tAB0050 in the huPBMC Raji B.luc mouse model are shown. [Figure 10C] The quantification of flow cytometry is illustrated. The pharmacodynamic changes in the bone marrow after a single administration of tAB0050 in the huPBMC Raji B.luc mouse model are shown.

[0029] [Figure 11A] This shows cytokine induction in peripheral blood after treatment with tAB0050 in the huPBMC Raji B.luc mouse model. [Figure 11B] This shows cytokine induction in peripheral blood after treatment with tAB0050 in the huPBMC Raji B.luc mouse model.

[0030] [Figure 12A] This is the PK profile of tAB0050 after a single dose in the huPBMC Raji B.luc mouse model. [Figure 12B] This table shows the PK parameters of tAB0050 after a single treatment with tAB0050 in the huPBMC Raji B.luc mouse model.

[0031] [Figure 13]The relative biological activity of tAB0050 after incubation in human serum, as measured by a T cell activation reporter assay in the presence of Raji target cells, is illustrated.

[0032] [Figure 14A] The study design, pharmacokinetics, B cell depletion, T cell redistribution, and cytokine release in cynomolgus monkeys in response to a single IV or SC administration of tAB0050 are illustrated. Female monkeys (n=2) were administered 0.1 or 1 mg / kg via either IV (blue bar) or SC (red bar). The study design is shown. Blood samples were collected at predetermined time points. [Figure 14B] This diagram illustrates the study design, pharmacokinetics, B cell depletion, T cell redistribution, and cytokine release in cynomolgus monkeys in response to a single IV or SC administration of tAB0050. Female monkeys (n=2) were administered 0.1 or 1 mg / kg via either IV (blue bar) or SC (red bar). The diagram shows the results after a single IV or SC administration. [Figure 14C] This diagram illustrates the study design, pharmacokinetics, B cell depletion, T cell redistribution, and cytokine release in cynomolgus monkeys in response to a single IV or SC administration of tAB0050. Female monkeys (n=2) were administered 0.1 or 1 mg / kg via either IV (blue bar) or SC (red bar). Absolute B cell count. [Figure 14D] This diagram illustrates the study design, pharmacokinetics, B cell depletion, T cell redistribution, and cytokine release in cynomolgus monkeys in response to a single IV or SC administration of tAB0050. Female monkeys (n=2) were administered 0.1 or 1 mg / kg via either IV (blue bar) or SC (red bar). Absolute T cell count. [Figure 14E] This diagram illustrates the study design, pharmacokinetics, B cell depletion, T cell redistribution, and cytokine release in cynomolgus monkeys in response to a single IV or SC administration of tAB0050. Female monkeys (n=2) were administered 0.1 or 1 mg / kg via either IV (blue bar) or SC (red bar). Cytokines were measured by Luminex.

[0033] [Figure 15A] The serum concentration-time profile of CLN-978 (tAB0050) in female cynomolgus monkeys via the IV route (0.1 mg / kg) is illustrated. [Figure 15B] The serum concentration-time profile of CLN-978 (tAB0050) in female cynomolgus monkeys via the IV route (1 mg / kg) is illustrated. [Figure 15C] The serum concentration-time profile of CLN-978 (tAB0050) in female cynomolgus monkeys via the SC pathway (0.1 mg / kg) is illustrated. [Figure 15D] The serum concentration-time profile of CLN-978 (tAB0050) in female cynomolgus monkeys via the SC route (1 mg / kg) is illustrated.

[0034] [Figure 16A] The table and key parameters show the toxicological parameters of CLN-978(tAB0050) in the serum of female cynomolgus monkeys administered via the IV route. [Figure 16B] The table and key parameters show the toxicological parameters of CLN-978(tAB0050) in the serum of female cynomolgus monkeys administered via the SC route.

[0035] [Figure 17] This is an explanatory diagram of a human model. T cell engagers (TCEs) are administered via SC and absorbed into the central compartment, where they can be distributed to the peripheral or tumor compartment, eliminated, or crosslinked to CD3 or CD19 on T cells and normal or malignant B cells, respectively. CD3 and CD19 are synthesized and internalized in each compartment.

[0036] [Figure 18]Figures A and B illustrate dose range simulations to illustrate predictions of the initiation and effective doses. Figure A illustrates the mean TpT in tumor projection. The black dashed lines represent 60 TpT (underline, initiation dose criterion) and 472 TpT (overline, effective dose criterion). Note that 240 μg (blue) crosses the lower black line on day 28, and 1850 μg (cyan) crosses the upper black line on day 28, achieving the initiation and effective dose criteria, respectively. Figure B illustrates the estimated PK profile.

[0037] [Figure 19] This is a diagram illustrating the first phase 1, open-label, multicenter, human dose-escalation and dose-expansion trial of tAB0050 in patients with relapsed or refractory (R / R) B-cell non-Hodgkin lymphoma (B-NHL).

[0038] [Figure 20] This graph shows the preliminary clinical pharmacokinetics (PK) of CLN-978 overlaid with simulated PK from a preclinical PK model.

[0039] [Figure 21A] This graph shows B cell depletion, T cell activation, and cytokine analysis in subjects treated with CLN-978. [Figure 21B] This graph shows B cell depletion, T cell activation, and cytokine analysis in subjects treated with CLN-978. [Figure 21C] This graph shows B cell depletion, T cell activation, and cytokine analysis in subjects treated with CLN-978. [Figure 21D] This graph shows B cell depletion, T cell activation, and cytokine analysis in subjects treated with CLN-978. [Figure 21E] This graph shows B cell depletion, T cell activation, and cytokine analysis in subjects treated with CLN-978. [Figure 21F] This graph shows B cell depletion, T cell activation, and cytokine analysis in subjects treated with CLN-978.

[0040] [Figure 22] Graphs A-C show the depletion of deep B cells in the bone marrow (A), spleen (B), and lymphoid tissue (C) after SC administration of CLN-97 in cynomolgus monkeys.

[0041] [Figure 23] This table summarizes clinical observations from three patients treated once weekly with 30 μg of CLN-978 administered subcutaneously.

[0042] [Figure 24] These images show the response to CLN-978 treatment in patients who received 30 μg of CLN-978 subcutaneously once a week. [Modes for carrying out the invention]

[0043] This specification provides a polyspecific binding protein comprising a first domain that binds to CD19 (e.g., human CD19), a second domain that binds to CD3 (e.g., human CD3), and optionally a half-life extension domain, the half-life extension domain being a third domain that binds to serum albumin (e.g., human serum albumin) for treating diseases and disorders associated with abnormal cells expressing CD19, such as certain B-cell malignant hematological diseases. Figure 1A is a schematic diagram of the arrangement of six domains of a single-chain polyspecific binding protein. Figure 1B shows a design of a polyspecific binding protein according to an aspect of this disclosure. In one embodiment, the polyspecific binding protein is tAB0050.

[0044] To facilitate understanding of this disclosure, several terms and phrases are defined below.

[0045] The term “multispecific binding protein” refers to a protein or protein conjugate that can bind to two or more different targets (e.g., two or more different antigens or two or more different epitopes of the same antigen). For example, a multispecific binding protein can bind to two or more different targets via two or more different binding domains. The structure and / or function of a multispecific binding protein can be based on the structure and / or function of an antibody, e.g., a full-length or whole immunoglobulin molecule, an antibody heavy chain variable domain (VH) and / or light chain variable domain (VL), and / or a single-chain antibody. In one example, each of the binding domains of a multispecific binding protein relating to this disclosure contains the minimum structural requirements of an antibody that enable target binding. These minimum requirements may be defined, for example, by the presence of at least three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3 of the VH domain) and / or three light chain CDRs (i.e., CDR1, CDR2, and CDR3 of the VL domain). Alternative approaches to defining the minimum structural requirements of an antibody include defining a specific target epitope to which the antibody binds, or referring to known antibodies that the antibody competes with to bind to the same epitope to which a known antibody binds. Antibodies on which the constructs according to this disclosure are based include, for example, monoclonal antibodies, recombinant antibodies, chimeric antibodies, deimmunized antibodies, humanized antibodies, and human antibodies.

[0046] Any of the binding domains of the polyspecific binding proteins described herein may include the group of CDRs mentioned above. These CDRs may be contained within a VH and / or VL framework. For example, an Fd fragment has two VH domains and often retains the antigen-binding function of several intact antigen-binding domains. Additional examples of antibody fragment, antibody variant, or binding domain formats include: (1) Fab fragments, i.e., monovalent fragments having VL, VH, CL, and CH1 domains; (2) F(ab')2 fragments, i.e., bivalent fragments having two Fab fragments linked by disulfide crosslinks in the hinge region; (3) Fd fragments having two VH and CH1 domains; (4) Fv fragments having VL and VH domains in a single arm of the antibody; (5) dAb fragments having a VH domain (Ward et al., (1989) Nature 341:544-546); (6) isolated complementarity-determining regions (CDRs); and (7) single-chain Fv (scFv), which may, for example, be derived from an scFv library.

[0047] The multispecific binding proteins relating to this disclosure may also include “multibodies” such as di-scFv or bi(s)-scFv, scFv-Fc, scFv-zipper, scFab, Fab2, Fab3, diabody, single-chain diabody, tandem diabody (tandab), tandem di-scFv, tandem tri-scFv, triabody, or tetrabody, or antibody-modified fragments also called antibody variants, such as nanobodies containing a single variable domain (which may be VH (also called VHH in relation to sdAb) or VL that specifically binds to an antigen or epitope independently of other V regions or domains) or single-domain antibodies such as single-variable-domain antibodies.

[0048] As used herein, the terms “single-chain Fv,” “single-chain antibody,” and “scFv” refer to a single polypeptide chain antibody fragment that contains variable regions from both the heavy and light chains but lacks a constant region. Generally, single-chain antibodies further include a peptide linker connecting the VH and VL domains, which enables the single-chain antibody to form a desired structure for binding to an antigen. In certain embodiments, single-chain antibodies may also be bispecific, multispecific, human, humanized, and / or synthetic.

[0049] Furthermore, the “polyspecific binding proteins” described herein may be monovalent, divalent, or polyvalent / multivalent constructs. Additionally, the “polyspecific binding proteins” described herein may include molecules consisting of only one polypeptide chain or molecules consisting of multiple polypeptide chains, where the chains may be identical (homodimer, homotrimer, or homooligomer) or different (heterodimer, heterotrimer, or heterooligomer).

[0050] The domains of the polyspecific binding proteins of this disclosure may be linked via one or more peptide bonds and / or peptide linkers. The term “peptide linker” according to this disclosure includes an amino acid sequence linking two domains. A peptide linker may also be used to fuse a third domain to another domain of the polyspecific binding protein of this disclosure. An essential technical feature of such peptide linkers is that they do not contain polymerization activity.

[0051] The terms “binding domain” or “(antigen) binding domain” are, in relation to this disclosure, used to characterize a domain that (specifically) binds to or interacts with a given target epitope or target molecule (antigen), e.g., CD19, serum albumin, and CD3, respectively. The structure and function of the first, second, and / or third binding domains may be based on the structure and / or function of an antibody, e.g., a full-length or whole immunoglobulin molecule. The binding domains may be derived from the VH and / or VL or VHH domains of the antibody or its fragments. For example, a binding domain may comprise three light chain CDRs (i.e., CDR1, CDR2, and CDR3 of the VL domain) and / or three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3 of the VH domain). A binding domain may also comprise VHH CDRs (i.e., CDR1, CDR2, and CDR3 of the VHH region).

[0052] The terms “variable domain” and “variable region” are used interchangeably and refer to the portion of an antibody or immunoglobulin domain that exhibits variability in its sequence and is involved in determining the specificity and binding affinity of a particular antibody. Variability is not uniformly distributed throughout the antibody’s variable domain; it is concentrated in the respective subdomains of the heavy and light chain variable regions. These subdomains are called “hypervariable regions” or “complementarity-determining regions” (CDRs). The more conserved (i.e., non-hypervariable) portions of the variable domain are called “framework” regions (FRMs or FRs), which provide a scaffold for six CDRs in three-dimensional space to form the antigen-binding surface.

[0053] In this disclosure, any one of the binding domains of a polyspecific binding protein may include a single-domain antibody (sdAb). A single-domain antibody contains a single monomeric antibody variable domain that can selectively bind to a specific antigen independently of other variable regions or domains. The first single-domain antibodies were engineered from heavy chain antibodies found in camelids, and these are called VHH fragments. Cartilaginous fish also have heavy chain antibodies (IgNARs), from which VHH fragments were developed. NAR Single-domain antibodies, known as fragments, can be obtained. An alternative approach involves splitting a dimeric variable domain from a common immunoglobulin, e.g., human or rodent, into monomers to obtain VH or VL single-domain antibodies. While most research on single-domain antibodies currently relies on heavy-chain variable domains, nanobodies derived from light chains have also been shown to specifically bind to target epitopes. Examples of single-domain antibodies include nanobody and single-variable-domain antibodies.

[0054] As used herein, the term “antigen-binding site” refers to the portion of an immunoglobulin molecule or its derivatives or variants that is involved in antigen binding. In human antibodies, the antigen-binding site is formed by amino acid residues in the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly variable regions within the V regions of the heavy and light chains, called “hypervariable regions,” are inserted between more conserved adjacent regions known as “framework regions” or “FR.” Thus, the term “FR” refers to the amino acid sequences found naturally between and adjacent to the hypervariable regions within the immunoglobulin. Within a human antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to this three-dimensional surface of the bound antigen, and the three hypervariable regions of the heavy and light chains are called “complementarity-determining regions” or “CDR.” In certain animals, such as camels and cartilaginous fish, the antigen-binding site is formed by a single antibody chain that provides a “single-domain antibody.” The antigen-binding site can be present in an intact antibody, in an antigen-binding fragment of an antibody that retains an antigen-binding surface, or in a recombinant polypeptide such as scFv, or by linking a heavy-chain variable domain to a light-chain variable domain within a single polypeptide using a peptide linker.

[0055] As used herein, the term “antibody” refers to a protein or protein conjugate containing an antigen-binding site. Antibodies may be monospecific or polyspecific (e.g., bispecific).

[0056] As used herein, the terms "a" and "an" mean "one or more" and include the plural form unless otherwise appropriate from the context.

[0057] The terms “recipient,” “individual,” “subject,” “host,” and “patient” are used interchangeably herein and, in some embodiments, refer to any mammalian subject, in particular human, to whom diagnosis, treatment, or therapy is desired. For therapeutic purposes, “mammal” refers to any animal classified as a mammal, including humans, domesticated and livestock, as well as laboratory, zoo, athletic, or companion animals, such as dogs, horses, cats, cattle, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, and monkeys. In some embodiments, the mammal is human. None of these terms require medical supervision.

[0058] As used herein, the term "CD19 low expression" refers to low CD19 expression in cancer. Cancer can be classified as "CD19 low expression" if the number of CD19 molecules per cell is approximately 17,000 or less, more specifically approximately 3,000 or less. The number of CD19 molecules can be determined by flow cytometry.

[0059] As used herein, the term “effective dose” means an amount of a compound (e.g., a compound of this disclosure) sufficient to produce a beneficial or desired result. An effective dose may be administered in one or more doses, applications, or prescriptions and is not intended to be limited to a particular formulation or route of administration. As used herein, the term “treat” includes any effect resulting in improvement of a condition, disease, disorder, etc., such as reduction, reduction, regulation, improvement, or elimination, or improvement of their symptoms.

[0060] As used herein, the term “pharmaceutical composition” refers to a combination of an active agent and an inactive or active carrier that makes the composition essentially suitable for in vivo or ex vivo diagnostic or therapeutic use.

[0061] As used herein, the term “pharmaceutically acceptable carrier” refers to any standard pharmaceutical carrier, such as phosphate-buffered saline, water, emulsion (e.g., oil / water or water / oil emulsion), and various types of wetting agents. The composition may also contain stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA (1975).

[0062] Throughout the description, where a composition is described as having, including, or comprising certain components, or where a process and method is described as having, including, or comprising certain steps, it is intended that, in addition, there exist compositions of the disclosure that are essentially composed of or comprise the components described, and processes and methods of the disclosure that are essentially composed of or comprise the processing steps described.

[0063] As a general rule, compositions with specified percentages are based on weight unless otherwise specified. Furthermore, if a definition is not provided for a variable element, the previous definition for that variable element takes precedence.

[0064] I. Polyspecific binding proteins In some embodiments, the disclosure provides a multispecific binding protein comprising a first domain (e.g., a first antigen-binding site) that binds to CD19 (e.g., human CD19), a second domain (e.g., a second antigen-binding site) that binds to CD3 (e.g., human and / or Macaca CD3) such as CD3ε (epsilon), CD3δ (delta), and / or CD3γ (gamma), and optionally a half-life extension domain. The multispecific binding protein is configured to spatially bring CD19-expressing cells, such as B cells, into close proximity with CD3-expressing cells, such as T cells, thereby enhancing the cytotoxicity of CD3-expressing cells against CD19-expressing cells. The optionally half-life extension domain may be a third domain (e.g., a third antigen-binding site) that binds to serum albumin (e.g., HSA).

[0065] Each antigen-binding site of a polyspecific binding protein can take various forms, such as a single-chain variable fragment (scFv), a Fab fragment, or a single-domain antibody (sdAb). In some embodiments, the first antigen-binding site comprises an scFv. In some embodiments, the second antigen-binding site comprises an scFv. In some embodiments, the third antigen-binding site comprises an sdAb.

[0066] In some embodiments, the polyspecific binding protein further comprises an antibody Fc region. The presence of the Fc region may extend the serum half-life of the polyspecific binding protein. Depending on the specific Fc subtype and variant used, the Fc region may also alter the activity (e.g., cytotoxic activity) of the polyspecific binding protein.

[0067] In some embodiments, the polyspecific binding protein does not contain an antibody Fc region. The absence of Fc contributes to a smaller size of the polyspecific binding protein, which may result in improved tissue permeability and pharmacokinetic properties. In some embodiments, the polyspecific binding protein consists of, or is essentially, first, second, and third antigen-binding sites and linkers between them. In some embodiments, the polyspecific binding protein is essentially composed of first, second, and third antigen-binding sites.

[0068] In some embodiments, the polyspecific binding protein monovalently binds to CD19, CD3, and / or serum albumin. Excluding additional binding domains reduces the risk of nonspecific immune cell activation and decreases the size of the polyspecific binding protein.

[0069] A. Antigen binding site for CD19 This disclosure provides, in some embodiments, antigen-binding sites that bind to CD19 (e.g., human CD19). This disclosure also provides antibodies containing antigen-binding sites. CDR sequences are identified by the Kabat numbering scheme unless indicated by an asterisk (*). [Table 1]

[0070] In some embodiments, the antigen-binding site that binds to CD19 is in the form of an scFv. In certain embodiments, VH is located on the C-terminal side of VL. In some embodiments, VH is located on the N-terminal side of VL. In some embodiments, VH and VL are linked by a peptide linker, e.g., a linker disclosed in the following subsection E titled "Linker". To stabilize the scFv, the amino acid residues at position 44 of VH and position 100 (below Kabat numbering) of VL can be substituted with Cys, thereby promoting the formation of a disulfide bond between VH and VL. Thus, in some embodiments, VH and VL contain Cys at positions 100 and 44, respectively.

[0071] In some embodiments, the antigen-binding sites that bind to CD19 of the present disclosure include a VH having at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) the same amino acid sequence as the VH of the antibody disclosed in Table 1, and a VL having at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) the same amino acid sequence as the VL of the same antibody disclosed in Table 1. In some embodiments, the antigen-binding site includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the VH and VL sequences of the antibodies disclosed in Table 1, determined under Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (see, for example, Chothia C & Lesk AM, (1987) J Mol Biol 196:901-917), MacCallum (see MacCallum RM et al., (1996) J Mol Biol 262:732-745), IMGT (see Lefranc, (1999) The Immunologist, 7, 132-136), or any other CDR determination method known in the art.

[0072] In some embodiments, the antigen-binding site that binds to CD19 is derived from CNG-CD19-701. In some embodiments, the antigen-binding site includes a VH containing the HCDR1, HCDR2, and HCDR3 sequences represented by SEQ ID NOs: 3, 5, and 6, respectively, and a VL containing the LCDR1, LCDR2, and LCDR3 sequences represented by SEQ ID NOs: 8, 9, and 10, respectively. In some embodiments, the antigen-binding site includes a VH containing the HCDR1, HCDR2, and HCDR3 sequences represented by SEQ ID NOs: 4, 5, and 7, respectively, and a VL containing the LCDR1, LCDR2, and LCDR3 sequences represented by SEQ ID NOs: 8, 9, and 10, respectively. In some embodiments, the antigen-binding site includes VH containing at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) the same amino acid sequence as SEQ ID NO: 1, and VL containing at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) the same amino acids as SEQ ID NO: 2. In some embodiments, VH and VL contain the amino acid sequences of SEQ ID NO: 1 and 2, respectively.

[0073] In some embodiments, the antigen-binding site is present in human CD19 or its extracellular fragments as a monomer, measured by surface plasmon resonance (SPR) with a K content of 2 nM, 1 nM, or less than 0.5 nM. D It binds via [a specific molecule]. In some embodiments, the antigen-binding site is present on human CD19 or its extracellular fragments with K in the range of 0.5–2 nM, 0.5–1 nM, or 0.5–0.5 nM, as measured by SPR when the antigen-binding site is present as a monomer. D They are joined together.

[0074] In some embodiments, the antigen-binding site derived from CNG-CD19-701 binds to human CD19 or its extracellular fragment with a K of 2 nM, 1 nM, or 0.5 nM or less, as measured by surface plasmon resonance (SPR) when the antigen-binding site exists as a monomer. D In some embodiments, the antigen-binding site derived from CNG-CD19-701 binds to human CD19 or its extracellular fragment with a K in the range of 0.5-2 nM, 0.5-1 nM, or 0.5-0.5 nM, as measured by SPR when the antigen-binding site exists as a monomer. D

[0075] In some embodiments, the antigen-binding site derived from CNG-CD19-701 binds to human CD19 or its extracellular fragment with a K of 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM or less, as measured by SPR when the antigen-binding site exists as a monomer. D In some embodiments, the antigen-binding site derived from CNG-CD19-701 binds to human CD19 or its extracellular fragment with a K in the range of 0.5-0.4 nM, 0.5-0.3 nM, 0.5-0.2 nM, or 0.5-0.1 nM, as measured by SPR when the antigen-binding site exists as a monomer. D

[0076] In some embodiments, the antigen-binding site derived from CNG-CD19-701 binds to cynomolgus CD19 with a K of 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, or 3 nM or less, as measured by SPR when the antigen-binding site exists as a monomer. D In some embodiments, the antigen-binding site derived from CNG-CD19-701 binds to cynomolgus CD19 with a K in the range of 1-8 nM, 1-7 nM, 1-6 nM, 1-5 nM, 1-4 nM, or 1-3 nM, as measured by SPR when the antigen-binding site exists as a monomer. D

[0077] ​​​ This disclosure also provides antigen-binding sites that compete for binding to CD19 (e.g., human CD19) with antibody or antigen-binding sites including the VH, VL, and / or scFv sequences presented in Table 1.

[0078] antigen-binding site that binds to B-CD3 The second antigen-binding site of the polyspecific binding protein binds to CD3 (e.g., human CD3 and / or Macaca CD3). In some embodiments, the second antigen-binding site binds to CD3ε (epsilon). In some embodiments, the second antigen-binding site binds to CD3δ (delta). In some embodiments, the second antigen-binding site binds to CD3γ (gamma).

[0079] In some embodiments, the second antigen-binding site of the polyspecific binding protein binds to the N-terminal epitope of the CD3ε chain. In some embodiments, the second antigen-binding site binds to an epitope localized to amino acid residues 1-27 of the human CD3ε extracellular domain. This epitope or its homologous variant is also present in certain non-human primates. Therefore, in certain embodiments, the second antigen-binding site binds to CD3 in different primates, such as humans, New World primates (e.g., Callitrichidae, Saguinus Oedipus, or Saimiri sciureus), Old World primates (e.g., baboons and macaques), gibbons, and non-homininae. Callitrichidae and Saguinus Oedipus are New World primates belonging to the family Callitrichidae, while Saimiri sciureus is a New World primate belonging to the family Cebidae. In some embodiments, the second antigen-binding site binds to human CD3ε and / or Macaca CD3ε. In some embodiments, the second antigen-binding site further binds to Callitrix jacchus, Saguinus Oedipus, and / or Saimiri sciureus CD3ε. [Table 2]

[0080] In some embodiments, the second antigen-binding site comprises a VH having at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) the same amino acid sequence as the VH of the antibody disclosed in Table 2, and a VL having at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) the same amino acid sequence as the VL of the same antibody disclosed in Table 2. In some embodiments, the antigen-binding site includes Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (see, for example, Chothia C & Lesk AM, (1987), J Mol Biol 196:901-917), MacCallum (see MacCallum RM et al., (1996) J Mol Biol 262:732-745), IMGT (see Lefranc, (1999) The Immunologist, 7, 132-136), or HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 determined under any other CDR determination method known in the art with respect to the VH and / or VL sequences of the antibodies disclosed in Table 2. In some embodiments, the antigen-binding site includes the VH and VL sequences of the antibody disclosed in Table 2.

[0081] In some embodiments, the second antigen-binding site that binds to CD3 is derived from CNG-CD3-1. In some embodiments, the second antigen-binding site includes a VH containing the HCDR1, HCDR2, and HCDR3 sequences represented by SEQ ID NOs. 14, 16, and 17, respectively, and a VL containing the LCDR1, LCDR2, and LCDR3 sequences represented by SEQ ID NOs. 19, 20, and 21, respectively. In some embodiments, the second antigen-binding site includes a VH containing the HCDR1, HCDR2, and HCDR3 sequences represented by SEQ ID NOs. 15, 16, and 18, respectively, and a VL containing the LCDR1, LCDR2, and LCDR3 sequences represented by SEQ ID NOs. 19, 20, and 21, respectively. In some embodiments, the second antigen-binding site includes VH containing at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) the same amino acid sequence as SEQ ID NO: 12, and VL containing at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) the same amino acids as SEQ ID NO: 13. In some embodiments, VH and VL of the second antigen-binding site contain the amino acid sequences of SEQ ID NO: 12 and SEQ ID NO: 13, respectively.

[0082] Such antigen-binding sites can take the form of scFv. In certain embodiments, VH is located on the C-terminal side of VL. In some embodiments, VH is located on the N-terminal side of VL. In some embodiments, VH and VL are linked by a peptide linker, for example, a linker disclosed in the following subsection E titled "Linker". In some embodiments, the second antigen-binding site includes the amino acid sequence of SEQ ID NO: 22. To stabilize the scFv, the amino acid residues at position 44 of VH and position 100 (below Kabat numbering) of VL can be substituted with Cys, thereby promoting the formation of a disulfide bond between VH and VL. Thus, in some embodiments, VH and VL contain Cys at positions 100 and 44, respectively.

[0083] In some embodiments, the second antigen-binding site comprises an sdAb containing a VH including complementarity-determining regions HCDR1, HCDR2, and HCDR3. In some embodiments, the VH comprises an amino acid sequence that is at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the VH of the sdAb antibody shown in Table 2. In some embodiments, the VH comprises the HCDR1, HCDR2, and HCDR3 sequences of the antibody presented in Table 2. In some embodiments, the VH comprises the amino acid sequence of the VH of the sdAb presented in Table 2.

[0084] In some embodiments, the second antigen-binding site competes for binding to CD3 (e.g., human CD3 and / or Macaca CD3) with an antibody or its antigen-binding fragment containing the VH, VL, and / or scFv sequences presented in Table 2.

[0085] In some embodiments, the second antigen-binding site of the polyspecific binding protein is a dissociation constant (K) of CD3 (e.g., human CD3 and / or Macaca CD3) of about 0.1 nM to about 1 μM.D ) is used to join. K D This can be measured by methods known in the art. In some embodiments, K D This is measured by SPR against CD3 or its extracellular fragments immobilized on a chip. In some embodiments, K D This is measured by flow cytometry for CD3 expressed on the cell surface.

[0086] In some embodiments, the second antigen-binding site is measured by SPR and has a K of 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, or less than 10 pM. DIt binds to CD3. For example, in a particular embodiment, the first antigen-binding site is measured by SPR in the following ranges: approximately 10 pM to approximately 1 nM, approximately 10 pM to approximately 0.9 nM, approximately 10 pM to approximately 0.8 pM, approximately 10 pM to approximately 0.7 nM, approximately 10 pM to approximately 0.6 nM, approximately 10 pM to approximately 0.5 nM, approximately 10 pM to approximately 0.4 nM, approximately 10 pM to approximately 0.3 nM, and approximately 10 pM to approximately 0.2 nM. M, about 10 pM to about 0.1 nM, about 10 pM to about 50 pM, 0.1 nM to about 10 nM, about 0.1 nM to about 9 nM, about 0.1 nM to about 8 nM, about 0.1 nM to about 7 nM, about 0. 1nM to about 6nM, about 0.1nM to about 5nM, about 0.1nM to about 4nM, about 0.1nM to about 3nM, about 0.1nM to about 2nM, about 0.1nM to about 1nM, about 0.1nM to about 0 .5nM, about 0.5nM to about 10nM, about 0.5nM to about 9nM, about 0.5nM to about 8nM, about 0.5nM to about 7nM, about 0.5nM to about 6nM, about 0.5nM to about 5nM, Approximately 0.5nM to approximately 4nM, approximately 0.5nM to approximately 3nM, approximately 0.5nM to approximately 2nM, approximately 0.5nM to approximately 1nM, approximately 1nM to approximately 10nM, approximately 1nM to approximately 9nM, approximately 1nM to approximately 8nM , about 1nM to about 7nM, about 1nM to about 6nM, about 1nM to about 5nM, about 1nM to about 4nM, about 1nM to about 3nM, about 1nM to about 2nM, about 2nM to about 10nM, about 3nM to about K in the range of 10nM, about 4nM to about 10nM, about 5nM to about 10nM, about 6nM to about 10nM, about 7nM to about 10nM, about 8nM to about 10nM, or about 9nM to about 10nM D Then combine it with CD3.

[0087] In some embodiments, the second antigen-binding site is measured by BLI and has a K of 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or less than 0.1 nM. DIt binds to CD3. For example, in a particular embodiment, the first antigen-binding site is measured by BLI in the following ranges: approximately 0.1 nM to approximately 20 nM, approximately 0.1 nM to approximately 19 nM, approximately 0.1 nM to approximately 18 nM, approximately 0.1 nM to approximately 17 nM, approximately 0.1 nM to approximately 16 nM, approximately 0.1 nM to approximately 15 nM, approximately 0.1 nM to approximately 14 nM, approximately 0.1 nM to approximately 13 nM, approximately 0.1 nM to approximately 12 nM, approximately 0.1 nM to approximately 11 nM, approximately 0.1 nM to approximately 10 nM, approximately 0.1 nM to approximately 9 nM, approximately 0.1 nM to approximately 8 nM, approximately 0.1 nM to approximately 7 nM, approximately 0.1 nM to approximately 6 nM, and approximately 0.1 nM. ~about 5nM, about 0.1nM to about 4nM, about 0.1nM to about 3nM, about 0.1nM to about 2nM, about 0.1nM to about 1nM, about 0.1nM ~about 0.5nM, about 1nM to about 50nM, about 1nM to about 40nM, about 1nM to about 30nM, about 1nM to about 20nM, about 1nM to about 19 nM, about 1nM to about 18nM, about 1nM to about 17nM, about 1nM to about 16nM, about 1nM to about 15nM, about 1nM to about 14nM, about 1 K in the range of nM to about 13 nM, about 1 nM to about 12 nM, about 1 nM to about 11 nM, about 1 nM to about 10 nM, or about 1 nM to about 5 nM D Then combine it with CD3.

[0088] In some embodiments, the second antigen-binding site is determined by SPR to have a K content of 1nM, 2nM, 3nM, 4nM, 5nM, 6nM, 7nM, 8nM, 9nM, 10nM, 20nM, 30nM, 40nM, 50nM, 60nM, 70nM, 80nM, 90nM, or 100nM or higher, as measured by SPR. D It then binds to CD3 (for example, human CD3, for example, human CD3ε). In some embodiments, the second antigen-binding site is K in the range of approximately 1 nM to approximately 100 nM, approximately 1 nM to approximately 90 nM, approximately 1 nM to approximately 80 nM, approximately 1 nM to approximately 70 nM, approximately 1 nM to approximately 60 nM, approximately 1 nM to approximately 50 nM, approximately 1 nM to approximately 40 nM, approximately 1 nM to approximately 30 nM, approximately 1 nM to approximately 20 nM, approximately 1 nM to approximately 10 nM, approximately 10 nM to approximately 100 nM, approximately 10 nM to approximately 90 nM, approximately 10 nM to approximately 80 nM, approximately 10 nM to approximately 70 nM, approximately 10 nM to approximately 60 nM, approximately 10 nM to approximately 50 nM, approximately 10 nM to approximately 40 nM, approximately 10 nM to approximately 30 nM, or approximately 10 nM to approximately 20 nM, as measured by SPR.D It binds to CD3. In some embodiments, the second antigen-binding site is 10nM, 20nM, 30nM, 40nM, 50nM, 60nM, 70nM, 80nM, 90nM, 100nM, 200nM, 300nM, 400nM, 500nM, 600nM, 700nM, 800nM, 900nM, or 1 μM or more of K as measured by BLI. D The second antigen-binding site binds to CD3 (e.g., human CD3, e.g., human CD3ε). In some embodiments, the second antigen-binding site is measured by BLI in the following ranges: about 10 nM to about 1 μM, about 10 nM to about 900 nM, about 10 nM to about 800 nM, about 10 nM to about 700 nM, about 10 nM to about 600 nM, about 10 nM to about 500 nM, about 10 nM to about 400 nM, about 10 nM to about 300 nM, and about 10 nM to about 2 K in the range of 00nM, approximately 10nM to approximately 100nM, approximately 100nM to approximately 1μM, approximately 100nM to approximately 900nM, approximately 100nM to approximately 800nM, approximately 100nM to approximately 700nM, approximately 100nM to approximately 600nM, approximately 100nM to approximately 500nM, approximately 100nM to approximately 400nM, approximately 100nM to approximately 300nM, or approximately 100nM to approximately 200nM D Then combine it with CD3.

[0089] In some embodiments, the second antigen-binding site, when present in the form of a Fab, has a melting temperature of at least 60°C, at least 65°C, at least 70°C, at least 75°C, or at least 80°C. In some embodiments, the second antigen-binding site, when present in the form of a Fab, has a melting temperature in the range of 60-85°C, 60-80°C, 60-75°C, 60-70°C, 60-65°C, 65-85°C, 65-80°C, 65-75°C, 65-70°C, 70-85°C, 70-80°C, 70-75°C, 75-85°C, 75-80°C, or 80-85°C.

[0090] C. Half-life extension domain In some embodiments, the multispecific binding protein includes a half-life extension domain. As used herein, the term “half-life extension domain” refers to a protein domain that, within a subject (e.g., the subject’s blood), extends the half-life of the protein to which it is fused. Exemplary half-life extension domains include Fc domains, serum albumin domains, and protein domains that bind to serum albumin. In some embodiments, the half-life extension domain of the multispecific binding protein includes a third antigen-binding site that binds to serum albumin (e.g., HSA). The serum albumin-binding domain is thought to facilitate the recycling of the multispecific binding protein by binding to the fetal Fc receptor (FcRn), thereby potentially extending the serum half-life of the multispecific binding protein. Therefore, in certain embodiments, the third antigen-binding site does not bind to the D-III domain of HSA (the domain that mediates the interaction between HSA and FcRn). In some embodiments, the third antigen-binding site extends the serum half-life of the multispecific binding protein.

[0091] In some embodiments, the third antigen-binding site is an antigen-binding site that binds to serum albumin derived from a single-domain antibody listed in Table 3 (e.g., human serum albumin (HSA)). CDR sequences are identified by the Kabat numbering scheme unless indicated by an asterisk (*). [Table 3]

[0092] In some embodiments, the antigen-binding site that binds to serum albumin includes a VH having at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) the same amino acid sequence as the VH of the antibody disclosed in Table 3. In some embodiments, the antigen-binding site includes HCDR1, HCDR2, and HCDR3 of the VH sequence of the antibody disclosed in Table 3, determined under Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (see, for example, Chothia C & Lesk AM, (1987), J Mol Biol 196:901-917), MacCallum (see MacCallum RM et al., (1996) J Mol Biol 262:732-745), IMGT (see Lefranc, (1999) The Immunologist, 7, 132-136), or any other CDR determination method known in the art. In some embodiments, the antigen-binding site includes the HCDR1, HCDR2, and HCDR3 sequences of the antibody disclosed in Table 3. In some embodiments, the antigen-binding site includes the VH sequence of the antibody disclosed in Table 3.

[0093] In some embodiments, the antigen-binding site that binds to serum albumin is derived from CNG-HSA-101. In some embodiments, the antigen-binding site includes a VH containing the HCDR1, HCDR2, and HCDR3 sequences represented by SEQ ID NOs. 25, 27, and 28, respectively. In some embodiments, the antigen-binding site includes a VH containing the HCDR1, HCDR2, and HCDR3 sequences represented by SEQ ID NOs. 26, 27, and 29, respectively. In some embodiments, the antigen-binding site includes a VH containing at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequence to SEQ ID NO. 24. In some embodiments, the VH contains the amino acid sequence of SEQ ID NO. 24.

[0094] In some embodiments, the antigen-binding site has a K content of 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, or less than 3 nM, as measured by SPR when the antigen-binding site is present as a monomer. D It binds to human serum albumin. In some embodiments, the antigen-binding site has a K content in the range of 1-10 nM, 1-9 nM, 1-8 nM, 1-7 nM, 1-6 nM, 1-5 nM, 1-4 nM, or 1-3 nM, as measured by SPR when the antigen-binding site is present as a monomer. D It then binds to human serum albumin.

[0095] In some embodiments, the antigen-binding site derived from CNG-HSA-101 has a K content of 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, or less than 3nM, as measured by SPR when the antigen-binding site is present as a monomer. DIt binds to cynomolgus monkey serum albumin. In some embodiments, the antigen-binding site derived from CNG-HSA-101 has a K content in the range of 1-9 nM, 1-8 nM, 1-7 nM, 1-6 nM, 1-5 nM, 1-4 nM, or 1-3 nM, as measured by SPR when the antigen-binding site is present as a monomer. D It then binds to cynomolgus monkey serum albumin.

[0096] In some embodiments, the antigen-binding site derived from CNG-HSA-101 has a K content of 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, or less than 10 nM, as measured by SPR when the antigen-binding site is present as a monomer. D It binds to mouse serum albumin. In some embodiments, the antigen-binding site derived from CNG-HSA-101 has a K in the range of 1-100 nM, 1-90 nM, 1-80 nM, 1-70 nM, 1-60 nM, 1-50 nM, 1-40 nM, 1-30 nM, 1-20 nM, or 1-10 nM, as measured by SPR when the antigen-binding site is present as a monomer. D It then binds to mouse serum albumin.

[0097] In some embodiments, the antigen-binding site derived from CNG-HSA-101 is the first K D It binds to human serum albumin, and the second K D It then binds to mouse serum albumin. Here, the second K D The first K DThe ratios to 1 are in the range of 0.5–10, 0.5–9, 0.5–8, 0.5–7, 0.5–6, 0.5–5, 0.5–4, 0.5–3, 0.5–2, 0.9–10, 0.9–9, 0.9–8, 0.9–7, 0.9–6, 0.9–5, 0.9–4, 0.9–3, 0.9–2, 1–10, 1–9, 1–8, 1–7, 1–6, 1–5, 1–4, 1–3, or 1–2. Antigen-binding sites with ratios close to 1 are understood to have a more similar affinity to mouse serum albumin compared to human serum albumin, thereby allowing for more accurate evaluation of the pharmacokinetics of antigen-binding sites or proteins containing them using mouse models.

[0098] The melting temperature represents the thermal stability of the antigen-binding site and can be measured by differential scanning fluorescence assay, for example, as described in Durowoju et al. (2017) J.Vis.Exp. (121):55262. The thermal stability of the antibody or its fragments may be enhanced by incorporating CDRs into a stable backbone, introducing non-standard disulfide bonds, or introducing other mutations, as described in McConnell et al. (2014) MAbs, 6(5):1274-82 and Goldman et al. (2017) Front.Immunol., 8:865. In some embodiments, the antigen-binding site derived from CNG-HSA-101 has a melting temperature of 60°C or higher, as measured by differential scanning fluorescence assay. In some embodiments, the antigen-binding site derived from CNG-HSA-101 has a melting temperature of 65°C or higher, as measured by differential scanning fluorescence assay.

[0099] In some embodiments, a third antigen-binding site competes for binding to serum (e.g., human serum albumin) and / or for binding to protein A, with antibody or antigen-binding sites containing VH sequences as presented in Table 3.

[0100] In some embodiments, the third antigen-binding site has a melting temperature of at least 50°C, at least 55°C, at least 56°C, at least 57°C, at least 58°C, at least 59°C, at least 60°C, at least 61°C, at least 62°C, at least 63°C, at least 64°C, at least 65°C, at least 70°C, at least 75°C, or at least 80°C. In some embodiments, the third antigen-binding site has a melting temperature in the range of 50-80°C, 50-70°C, 50-65°C, 50-60°C, 50-55°C, 55-70°C, 55-65°C, 55-60°C, 56-65°C, 56-60°C, 57-65°C, 57-60°C, 58-65°C, 58-60°C, 59-65°C, 59-60°C, 60-80°C, 60-75°C, 60-70°C, 60-65°C, 65-80°C, 65-75°C, 65-70°C, 70-80°C, or 70-75°C.

[0101] D. Construct Format The first, second, and third antigen-binding sites can take various forms. In some embodiments, the first, second, and / or third antigen-binding sites include two antibody-variable domains (e.g., VH and VL). VH and VL can be mutated to introduce a disulfide bond that stabilizes the antigen-binding site (e.g., between H44 and L100) (see Zhao et al. (2010) Int. J. Mol. Sci., 12(1):1-11). In some embodiments, the first, second, and / or third antigen-binding sites include a single antibody-variable domain (e.g., sdAb).

[0102] In antigen-binding sites including VH and VL, VH and VL can be linked to form scFv. VH may be located on the N-terminal or C-terminal side of VL. VH and VL are typically linked via a linker, such as a peptide linker. Examples of peptide linker sequences are provided in the following subsection E, titled “Linkers”. In some embodiments, VH of the antigen-binding domain is linked to VL of the antigen-binding domain via a peptide linker having the amino acid sequences listed in Table 4. In certain embodiments, VH of the antigen-binding domain is linked to VL of the antigen-binding domain via a peptide linker having the amino acid sequence of SEQ ID NO: 36, 37, or 40, with VH located on the N-terminal side of VL. In other specific embodiments, VH of the antigen-binding domain is linked to VL of the antigen-binding domain via a peptide linker having the amino acid sequence of SEQ ID NO: 36, 37, or 40, with VH located on the C-terminal side of VL.

[0103] In some embodiments, VH and VL reside on separate polypeptide chains, and the formation of the VH-VL complex is facilitated by additional domains such as the constant regions CH1 and CL of the antibody. Thus, in certain embodiments, the polyspecific binding protein comprises a Fab containing VH and VL as disclosed herein.

[0104] In some embodiments, the polyspecific binding protein of this disclosure comprises a first antigen-binding site comprising a single antibody-variable domain, a second antigen-binding site comprising a single antibody-variable domain, and a third antigen-binding site comprising a single antibody-variable domain. In some embodiments, the polyspecific binding protein comprises a first antigen-binding site in sdAb format, a second antigen-binding site in sdAb format, and a third antigen-binding site in sdAb format.

[0105] In some embodiments, the polyspecific binding protein of this disclosure comprises a first antigen-binding site comprising a single antibody-variable domain, a second antigen-binding site comprising a single antibody-variable domain, and a third antigen-binding site comprising two antibody-variable domains. In some embodiments, the polyspecific binding protein comprises a first antigen-binding site in sdAb format, a second antigen-binding site in sdAb format, and a third antigen-binding site in scFv format.

[0106] In some embodiments, the polyspecific binding protein of this disclosure includes a first antigen-binding site comprising a single antibody-variable domain, a second antigen-binding site comprising two antibody-variable domains, and a third antigen-binding site comprising a single antibody-variable domain. In some embodiments, the polyspecific binding protein includes a first antigen-binding site in sdAb format, a second antigen-binding site in scFv format, and a third antigen-binding site in sdAb format.

[0107] In some embodiments, the polyspecific binding protein of this disclosure includes a first antigen-binding site comprising a single antibody-variable domain, a second antigen-binding site comprising two antibody-variable domains, and a third antigen-binding site comprising two antibody-variable domains. In some embodiments, the polyspecific binding protein includes a first antigen-binding site in sdAb format, a second antigen-binding site in scFv format, and a third antigen-binding site in scFv format.

[0108] In some embodiments, the polyspecific binding protein of this disclosure comprises a first antigen-binding site comprising two antibody-variable domains, a second antigen-binding site comprising a single antibody-variable domain, and a third antigen-binding site comprising a single antibody-variable domain. In some embodiments, the polyspecific binding protein comprises a first antigen-binding site in scFv format, a second antigen-binding site in sdAb format, and a third antigen-binding site in sdAb format.

[0109] In some embodiments, the polyspecific binding protein of this disclosure includes a first antigen-binding site comprising two antibody-variable domains, a second antigen-binding site comprising a single antibody-variable domain, and a third antigen-binding site comprising two antibody-variable domains. In some embodiments, the polyspecific binding protein includes a first antigen-binding site in scFv format, a second antigen-binding site in sdAb format, and a third antigen-binding site in scFv format.

[0110] In some embodiments, the polyspecific binding protein of this disclosure comprises a first antigen-binding site comprising two antibody-variable domains, a second antigen-binding site comprising two antibody-variable domains, and a third antigen-binding site comprising a single antibody-variable domain. In some embodiments, the polyspecific binding protein comprises a first antigen-binding site in scFv format, a second antigen-binding site in scFv format, and a third antigen-binding site in sdAb format.

[0111] In some embodiments, the polyspecific binding protein of this disclosure includes a first antigen-binding site comprising two antibody-variable domains, a second antigen-binding site comprising two antibody-variable domains, and a third antigen-binding site comprising two antibody-variable domains. In some embodiments, the polyspecific binding protein includes a first antigen-binding site in scFv format, a second antigen-binding site in scFv format, and a third antigen-binding site in scFv format.

[0112] The three antigen-binding sites of the polyspecific binding protein can be linked in one of the following configurations, from amino to carboxyl: (i) First antigen-binding site (CD19 binding domain) - Second antigen-binding site (CD3 binding domain) - Third antigen-binding site (serum albumin binding domain), (ii) First antigen-binding site (CD19 binding domain) - Third antigen-binding site (serum albumin binding domain) - Second antigen-binding site (CD3 binding domain), (iii) Second antigen-binding site (CD3 binding domain) - First antigen-binding site (CD19 binding domain) - Third antigen-binding site (serum albumin binding domain), (iv) Second antigen-binding site (CD3 binding domain) - Third antigen-binding site (serum albumin binding domain) - First antigen-binding site (CD19 binding domain), (v) Third antigen-binding site (serum albumin-binding domain) - First antigen-binding site (CD19-binding domain) - Second antigen-binding site (CD3-binding domain), and (vi) Third antigen-binding site (serum albumin-binding domain) - Second antigen-binding site (CD3-binding domain) - First antigen-binding site (CD19-binding domain), Here, the dash above represents a peptide bond and / or a linker (e.g., a peptide linker).

[0113] In some embodiments, the third antigen-binding site is not located between the first and second antigen-binding sites. Constructs having such a format are intended to have a favorable therapeutic effect and in vivo half-life. In some embodiments, the third antigen-binding site is located either at the N-terminus of both the first and second antigen-binding sites, or at the C-terminus of both the first and second antigen-binding sites. In some embodiments, the third antigen-binding site is located at the N-terminus of both the first and second antigen-binding sites. In some embodiments, the third antigen-binding site is located at the C-terminus of both the first and second antigen-binding sites.

[0114] The position (N-terminus or C-terminus) of one antigen-binding site relative to another antigen is determined under the definitions of "N-terminus" and "C-terminus" known in the art when a single polypeptide chain contains both antigen-binding sites. If an antigen-binding site contains two separate polypeptide chains, its position (N-terminus or C-terminus) relative to the other antigen-binding site (having either one or two polypeptide chains) can similarly be determined when a single polypeptide chain contains at least one of the former polypeptide chains and at least one of the latter. If antigen-binding site A is N-terminal to antigen-binding site B, and antigen-binding site B is N-terminal to antigen-binding site C, then antigen-binding site A is considered to be N-terminal to antigen-binding site C, even if antigen-binding sites A and C do not exist in any single, common polypeptide chain. More complex structures of polyspecific binding proteins are also being considered, some of which may have configurations that are difficult to characterize using the aforementioned terms "N-terminus" and "C-terminus," for example, when the relative positions of two antigen-binding sites on one polypeptide chain and another are different, or when loop structures are present.

[0115] According to this disclosure, the polyspecific binding protein and its constituent binding domains are in the form of one or more polypeptides. Such polypeptides may comprise a proteinaceous portion and a non-proteinaceous portion (e.g., a chemical linker or a chemical crosslinking agent such as glutaraldehyde). In some embodiments, the polyspecific binding protein of this disclosure comprises a first antigen-binding site, a second antigen-binding site, and a third antigen-binding site, all linked together to form a single polypeptide chain. In some embodiments, the first, second, and third antigen-binding sites, for example, in the above combination, take the form of scFv and / or sdAb to form a single polypeptide chain.

[0116] E. Linker As described above, the antigen-binding sites of the polyspecific binding proteins of this disclosure may be linked via peptide bonds or linkers (e.g., peptide linkers). In some embodiments, at least two adjacent antigen-binding sites are connected by a linker (e.g., a peptide linker). In some embodiments, each pair of adjacent antigen-binding sites is connected by a linker (e.g., a peptide linker).

[0117] In some embodiments, the three antigen-binding sites of a polyspecific binding protein may be linked by linkers (e.g., peptide linkers) denoted as L1 and L2 in one of the following configurations, in the direction from amino to carboxyl: (i) First antigen-binding site (CD19 binding domain) - L1 - Second antigen-binding site (CD3 binding domain) - L2 - Third antigen-binding site (serum albumin binding domain), (ii) First antigen-binding site (CD19 binding domain) - L1 - Third antigen-binding site (serum albumin binding domain) - L2 - Second antigen-binding site (CD3 binding domain), (iii) Second antigen-binding site (CD3 binding domain) - L1 - First antigen-binding site (CD19 binding domain) - L2 - Third antigen-binding site (serum albumin binding domain), (iv) Second antigen-binding site (CD3 binding domain) - L1 - Third antigen-binding site (serum albumin binding domain) - L2 - First antigen-binding site (CD19 binding domain), (v) Third antigen-binding site (serum albumin-binding domain) - L1 - First antigen-binding site (CD19-binding domain) - L2 - Second antigen-binding site (CD3-binding domain), and (vi) Third antigen-binding site (serum albumin-binding domain) - L1 - Second antigen-binding site (CD3-binding domain) - L2 - First antigen-binding site (CD19-binding domain). It should be understood that in a given construct, L1, L2, or both L1 and L2 may be replaced by peptide bonds.

[0118] When a single polypeptide chain contains two adjacent antigen-binding sites, it is understood that the peptide linker connecting the two antigen-binding sites represents the amino acid sequence between them. When the antigen-binding sites contain two separate polypeptide chains, one of which exists in a single common polypeptide as an adjacent antigen-binding site or its polypeptide chain, the peptide linker connecting the two antigen-binding sites represents the amino acid sequence between them in the common single polypeptide.

[0119] In some embodiments, linkers L1 and L2 are peptide linkers. The appropriate lengths of L1 and L2 can be selected independently. For example, in certain embodiments, L1 and / or L2 have an amino acid residue length of about 50 or less. In some embodiments, L1 consists of about 50 or fewer amino acid residues. In some embodiments, L1 consists of about 20 or fewer amino acid residues. In some embodiments, L2 consists of about 50 or fewer amino acid residues. In some embodiments, L2 consists of about 20 or fewer amino acid residues. In some embodiments, L1 and L2 independently consist of about 50 or fewer amino acid residues. In some embodiments, L1 and L2 independently consist of about 20 or fewer amino acid residues.

[0120] In some embodiments, peptide linkers L1 and L2 have optimized lengths and / or amino acid compositions. In some embodiments, L1 and L2 are the same length and have the same amino acid composition. In some embodiments, L1 and L2 are different. In some embodiments, L1 and / or L2 are "short," i.e., consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues. Thus, in certain cases, the linker consists of about 12 or fewer amino acid residues. In some embodiments, L1 and / or L2 are "long," e.g., consisting of 15, 20, or 25 amino acid residues. In some embodiments, L1 and / or L2 consist of about 3 to about 15 consecutive amino acid residues, e.g., 8, 9, or 10.

[0121] With respect to the amino acid composition of L1 and L2, the peptides are selected to confer flexibility to the multispecific binding protein of this disclosure, to not interfere with the binding domain, and to withstand cleavage by proteases. For example, glycine and serine residues generally confer protease resistance. Examples of linkers suitable for ligating the domains of the multispecific binding protein include (GS) n (GGS) n (GGGS) n (GGSG) n (GGSGG) n , and (GGGGS) n Examples include, but are not limited to, the following. In the formula, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, L1 and / or L2 are independently selected from the peptide sequences listed in Table 4. In some embodiments, L1 and / or L2 are independently selected from SEQ ID NOs: 30-40. In some embodiments, L1 and / or L2 are independently selected from SEQ ID NOs: 36, 37, or 40. In some embodiments, L1 and / or L2 include the amino acid sequence of SEQ ID NOs: 36, 37, or 40. In some embodiments, L1 and / or L2 consist of the amino acid sequence of SEQ ID NOs: 36, 37, or 40. In some embodiments, each of L1 and L2 includes the amino acid sequence of SEQ ID NOs: 36, 37, or 40. In some embodiments, L1 and L2 each consist of the amino acid sequence of SEQ ID NO: 36, 37, or 40. [Table 4]

[0122] Linkers such as the peptide linkers disclosed herein may also be used to connect the VH and VL of the scFv, as referred to in subsection D above, titled “Construction Format”.

[0123] F. Polyspecific binding protein The polyspecific binding proteins, including scFv that binds to CD19, scFv that binds to CD3, and sdAb that binds to serum albumin, are listed in Table 5 below. In some embodiments, tAB0050 may also be called CLN-978. [Table 5] tAB0050 (Sequence ID 41) KVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMTWVRQAPGKGLEWVSSISGSGSDTLYADSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTIGGSLSPSSQGTLVTVSSGGGGSGGGSEIVLTQSPATLSLSPGERATLSCSCSASSSVGYM HWYQQKPGQAPRLLIYDTSKLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCFQGSVYPFTFGQGTKLEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSQTLSLTCTVSGGSISTSTMGVGWIRQHPGKGLEWIGFIWWDDDKRYNPNLKS RVTMSVDTSKNQFSLKLSSVTAADTAVYYCARMELWSYYFDYWGQGTLVTVSSGGGGSGGGSDIVMTQSPDSLAVSLGERATINCKSSQSLLNARTGKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC KQSYSRRTFGGGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGFNIKDYYMHWVRQAPGQRLEWMGWIDLENANTIYDAKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDAYGRYFYDVWGQGTLVTVSS

[0124] The antigen-binding sites listed in a given row in Table 5 can be linked via peptide linkers as described in subsection E above, in the arrangement specified in the row. In some embodiments, at least two adjacent antigen-binding sites are linked by a peptide linker having the amino acid sequence of SEQ ID NO: 40. In some embodiments, each of two adjacent antigen-binding sites is linked by a peptide linker having the amino acid sequence of SEQ ID NO: 40. This forms a polyspecific binding protein present within a single polypeptide.

[0125] In some embodiments, the polyspecific binding protein includes the amino acid sequence of SEQ ID NOs. 24, 11, and 22 from the N-terminus to the C-terminus. In some embodiments, the polyspecific binding protein includes the amino acid sequence of SEQ ID NO. 41.

[0126] In some embodiments, the polyspecific binding protein includes an antigen-binding site that binds to CD19 as disclosed herein, an antigen-binding site that binds to CD3, and a half-life extension domain that includes an antibody Fc region. The polyspecific binding protein can take various formats to combine the antigen-binding site and the Fc region.

[0127] In some embodiments, the polyspecific binding protein comprises an anti-CD19 antibody in IgG antibody format fused with a CD3-binding scFv at the C-terminus of the IgG Fc region. In some embodiments, the polyspecific binding protein comprises a first polypeptide chain from N-terminus to C-terminus, comprising the VH antigen-binding site that binds to CD19, the CH1 domain, hinge, CH2 domain, and CH3 domain of the IgG antibody (e.g., human IgG1, IgG2, IgG3, or IgG4), and the CD3-binding scFv; and a second polypeptide chain from N-terminus to C-terminus, comprising the VL antigen-binding site that binds to CD19 and the light chain constant (CL) domain of the IgG antibody. In some embodiments, the CD3-binding scFv comprises the VL domain located on the N-terminal side of the VH domain. In some embodiments, the IgG antibody is a human IgG1 antibody. In some embodiments, the polyspecific binding protein comprises two of the first polypeptide chains and two of the second polypeptide chains, thereby forming a dimeric antibody Fc region.

[0128] G. Therapeutic activity The multispecific binding proteins disclosed herein are designed to bind simultaneously to B cells and T cells. T cell recruitment promotes B cell lysis, accompanied by cytolytic synapse formation and delivery of perforin and granzymes. The involved T cells are capable of sequential target cell lysis and are unaffected by immune escape mechanisms that prevent peptide antigen processing and presentation or clonal T cell differentiation. See, for example, WO2007042261A2. Thus, binding of the multispecific binding proteins to target B cells destroys the target cells and / or impairs the progression of B cell-related disease. In certain embodiments of the present invention, the B cell-related disease is relapsed and / or refractory non-Hodgkin lymphoma (NHL).

[0129] The cytotoxicity mediated by the polyspecific binding proteins of this disclosure can be measured in vitro in various ways. Effector cells may be, for example, stimulated enriched (human) CD8-positive T cells or unstimulated (human) peripheral blood mononuclear cells (PBMCs). If the target cells are of macaque origin, or express or are transfected with the macaque target cell surface antigen bound by the first domain, then the effector cells should also be of macaque origin, such as a macaque T cell line, e.g., 4119LnPx. The target cells must express CD19, e.g., human or macaque CD19. The target cells may be cell lines (e.g., CHO) stably or transiently transfected with CD19. Alternatively, the target cells may be cell lines that spontaneously express CD19, such as B lymphocytes. The effector-to-target cell (E:T) ratio is usually about 10:1, but may vary. Killing of target cells is 51 It can be measured by a Cr release assay (incubation time of approximately 18 hours) or a FACS-based cytotoxicity assay (incubation time of approximately 48 hours). Other methods for measuring cell death, such as MTT or MTS assays, ATP-based assays including bioluminescence assays, sulforhodamine B (SRB) assays, WST assays, cloning assays, and ECIS techniques, are well known to those skilled in the art.

[0130] In some embodiments, the cytotoxic activity mediated by the polyspecific binding proteins disclosed herein is measured by the cell-based cytotoxic assay described above. 50 Represented by a value, this EC 50 The value corresponds to the half-effect concentration (the concentration of the multispecific binding protein that induces a cytotoxic response between baseline and maximum). In some embodiments, the EC of the multispecific binding protein 50The values ​​are ≤5000pM, for example, ≤4000pM, ≤3000pM, ≤2000pM, ≤1000pM, ≤500pM, ≤400pM, ≤300pM, ≤200pM, ≤100pM, ≤50pM, ≤20pM, ≤10pM, ≤5pM, ≤4pM, ≤3pM, ≤2pM, or ≤1pM.

[0131] Stimulate / enrich CD8 + When T cells are used as effector cells, compared to unstimulated PBMCs, EC 50 It is understood that the value is generally lower. Furthermore, when target cells express high levels of the target cell surface antigen compared to when the level of the target antigen is low, EC 50 It is understood that the values ​​are generally lower. For example, stimulated / enriched human CD8 + When using T cells as effector cells (or when using either target cell surface antigen transfect cells such as CHO cells or target cell surface antigen-positive human cell lines as target cells), the EC of the polyspecific binding protein 50 The values ​​are ≤1000pM, for example, ≤500pM, ≤250pM, ≤100pM, 50pM, ≤10pM, or ≤5pM. When human PMBCs are used as effector cells, the EC of multispecific binding proteins 50 The values ​​are ≤5000pM, for example, ≤4000pM, ≤2000pM, ≤1000pM, ≤500pM, ≤200pM, ≤150pM, ≤100pM, ≤50pM, ≤10pM, or ≤5pM. When macaque T cell lines such as LnPx4119 are used as effector cells and cell lines transfected with macaque target cell surface antigens such as CHO cells are used as target cell lines, the EC of polyspecific binding proteins 50 The values ​​are ≤2000pM, for example, ≤1500pM, ≤1000pM, ≤500pM, ≤300pM, ≤250pM, ≤100pM, ≤50pM, ≤10pM, or ≤5pM.

[0132] Therefore, in a particular embodiment, EC 50 The value is stimulated / enriched human CD8 +It is measured using T cells as effector cells. In some embodiments, EC 50 The values ​​are measured using human PBMCs as effector cells. In some embodiments, EC 50 The values ​​are measured using macaque T cell lines such as LnPx4119 as effector cells and cells engineered to express macaque CD19 (e.g., CHO cells) as target cells.

[0133] In some embodiments, the polyspecific binding proteins of this disclosure do not induce or mediate the lysis of non-CD19-expressing cells. The terms “do not induce lysis” or “do not mediate lysis,” or their grammatical equivalents, mean that the polyspecific binding proteins do not induce or mediate the lysis of more than 30% (e.g., 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, or 5%) of non-CD19-expressing cells at concentrations up to 500 nM, where the lysis of CD19-expressing cell lines is set to 100%.

[0134] In some embodiments, the polyspecific binding proteins disclosed herein are more effective in killing CD19-expressing cells (e.g., cancer cells) than the corresponding respective anti-CD19 or anti-CD3 monoclonal antibodies at the same molar concentration.

[0135] The cytotoxic activity of multispecific binding proteins can be measured in the presence or absence of serum albumin (e.g., HSA). In some embodiments, the cytotoxic activity disclosed above is measured in the absence of serum albumin (e.g., HSA). In some embodiments, the cytotoxic activity disclosed above is measured in the substantial absence of serum albumin (e.g., HSA). In some embodiments, the cytotoxic activity disclosed above is measured in the presence of serum albumin (e.g., HSA), for example, in the presence of serum albumin (e.g., HSA) at concentrations of approximately 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, or 50 mg / mL.

[0136] In some embodiments, the polyspecific binding protein of this disclosure is used in the presence of serum albumin, in the absence of serum albumin, or substantially in the absence of serum albumin in the presence of EC 50 Similar EC values 50 The value kills CD19-expressing cells. In some embodiments, the EC of a multispecific binding protein kills CD19-expressing cells in the presence of serum albumin. 50 The values ​​increase by 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 times or less compared to the absence or substantial absence of serum albumin. The presence of serum albumin (e.g., serum albumin at approximately 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, or 50 mg / mL) also increases the EC of polyspecific binding proteins. 50 It is understood that the values ​​may change nonspecifically. The nonspecific effect is observed in the presence and absence of serum albumin in the EC of a control protein that does not contain a serum albumin-binding domain. 50 This can be evaluated by comparing the values. In some embodiments, the magnification change is offset by the nonspecific effect of serum albumin on control proteins, such as bispecific proteins that bind to CD19 and CD3.

[0137] H. Size of the structure In some embodiments, the molecular weight of the polyspecific binding protein is approximately 40 kD to approximately 100 kD. In some embodiments, the molecular weight of the polyspecific binding protein is at least 60 kD, at least 65 kD, at least 70 kD, at least 75 kD, at least 80 kD, at least 85 kD, at least 90 kD, or at least 95 kD. Generally, smaller size contributes to faster diffusion and tissue penetration, but it is understood that size reduction may not be as important for indications where target cells (e.g., cancer cells) are substantially present in the blood.

[0138] In some embodiments, the molecular weight of the polyspecific binding protein is approximately 40kD to 90kD, 40kD to 80kD, 40kD to 70kD, 40kD to 60kD, 40kD to 50kD, 50kD to 100kD, 50kD to 90kD, 50kD to 80kD, 50kD to 70kD, 50kD to 60kD, 60kD to 100kD, and These ranges are 60kD to approximately 90kD, approximately 60kD to approximately 80kD, approximately 60kD to approximately 70kD, approximately 65kD to approximately 100kD, approximately 65kD to approximately 90kD, approximately 65kD to approximately 80kD, approximately 65kD to approximately 70kD, approximately 70kD to approximately 100kD, approximately 70kD to approximately 90kD, approximately 70kD to approximately 80kD, approximately 80kD to approximately 100kD, approximately 80kD to approximately 90kD, or approximately 90kD to approximately 100kD. In some embodiments, the polyspecific binding protein is lower than 40kD. In some embodiments, the polyspecific binding proteins are approximately 50kD to 90kD, 50kD to 80kD, 50kD to 70kD, 50kD to 60kD, 60kD to 90kD, 60kD to 80kD, 60kD to 70kD, 65kD to 90kD, 65kD to 80kD, 65kD to 70kD, 70kD to 90kD, or 70kD to 80kD.

[0139] I. Serum half-life Fusion proteins have been developed to increase the in vivo half-life of small proteins, particularly antibody fragments. For example, fusion with heterodimeric antibody Fc regions, such as Fc having one or more mutations that extend the in vivo half-life, is described in U.S. Patent Publications US20140302037A1, US20140308285A1, and PCT Publications WO2014144722A2, WO2014151910A1, and WO2015048272A1. Alternative strategies include fusion with human serum albumin (HSA) or HSA-binding peptides (see, for example, PCT Publications WO2013128027A1 and WO2014140358A1). Fetal Fc receptors (FcRn) appear to be involved in extending the lifespan of circulating albumin (see Chaudhury et al. (2003) J.Exp.Med., 3:315-22). Albumin and IgG bind non-cooperatively to separate sites on FcRn, forming tripolecules (see ibid.). The binding of human FcRn to HSA and human IgG is pH-dependent, stronger at acidic pH and weaker at neutral or physiological pH (see ibid.). This observation suggests that albumin-containing proteins and protein complexes, as well as those containing IgG (especially Fc), are protected from degradation through pH-sensitive interactions with FcRn (see ibid.). By measuring the ability of individual HSA domains to bind to immobilized soluble human FcRn using surface plasmon resonance (SPR), it was shown that FcRn and albumin interact via the D-III domain of albumin in a pH-dependent manner, at different sites from the IgG binding site. (See Chaudhury et al. (2006) Biochemistry 45:4983-90 and PCT Publication No. WO2008068280A1).

[0140] This disclosure provides multispecific binding proteins with extended half-lives. In some embodiments, the multispecific binding proteins have a serum half-life of at least 24, 36, 48, 60, 72, 84, or 96 hours. In some embodiments, the multispecific binding proteins have a serum half-life of at least about 50 hours. In some embodiments, the multispecific binding proteins have a serum half-life of at least about 100 hours. Methods for measuring serum half-life are known in the art. In some embodiments, serum half-life is measured in non-human primates. In some embodiments, serum half-life is measured in humans.

[0141] In some embodiments, 50 hours after intravenous administration to a subject, the serum concentration of the polyspecific binding protein is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the serum concentration of the polyspecific binding protein in the subject 1 hour after administration.

[0142] In some embodiments, the polyspecific binding protein has a serum half-life at least 20% longer than the control polyspecific binding protein, and the control polyspecific binding protein includes a first domain identical to the first antigen site of the polyspecific binding protein and a second domain identical to the second antigen-binding site of the polyspecific binding protein, but does not include a third domain identical or substantially identical to the third antigen-binding site of the polyspecific binding protein. In some embodiments, the control polyspecific binding protein is identical to the polyspecific binding protein except that it lacks a half-life extension domain. In some embodiments, the serum half-life of the polyspecific binding protein is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% longer than the serum half-life of the control polyspecific binding protein. In some embodiments, the serum half-life of the polyspecific binding protein is at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times longer than the serum half-life of the control polyspecific binding protein.

[0143] II. Preparation method The antibodies and polyspecific binding proteins described above can be prepared using recombinant DNA techniques well known to those skilled in the art. For example, one or more isolated polynucleotides encoding an antibody or polyspecific binding protein can be ligated to, for example, a constant region coding sequence and other suitable nucleotide sequences including an expression regulatory sequence to generate a conventional gene expression construct (i.e., an expression vector) encoding the desired antibody or polyspecific binding protein. The preparation of a specified gene construct is within the realm of routine art for those skilled in the art.

[0144] Nucleic acids encoding a desired antibody or polyspecific binding protein can be incorporated into an expression vector (ligated), which can then be introduced into host cells via conventional transfection or transformation techniques. Exemplary host cells include E. coli cells, Chinese hamster ovary (CHO) cells, human fetal kidney 293 (HEK293) cells, HeLa cells, small hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and myeloma cells that do not otherwise produce IgG proteins. Transformed host cells can be grown under conditions that allow the host cells to express the gene encoding the antibody or polyspecific binding protein.

[0145] Specific expression and purification conditions will vary depending on the expression system used. For example, when expressing a gene in E. coli, the gene is first cloned into an expression vector by placing an appropriate bacterial promoter (e.g., Trp or Tac) and the engineered gene downstream of a prokaryotic signaling sequence. The expressed protein may be secreted. The expressed protein may accumulate in refractive bodies or inclusion bodies, which can be recovered after cell disruption by French press or sonication. The refractive bodies can then be solubilized, and the protein may be refolded and / or cleaved by methods known in the art.

[0146] When expressing a manipulated gene in a eukaryotic host cell, such as a CHO cell, it is first inserted into an expression vector containing a suitable eukaryotic promoter, secretory signal, poly(A) sequence, and stop codon. Optionally, the vector or gene construct may include enhancers and introns. In embodiments involving a fusion protein containing an antibody or a portion thereof, the expression vector may optionally include a sequence encoding all or part of the constant region, allowing for the expression of all or part of the heavy or light chain. The gene construct can be introduced into a eukaryotic host cell using conventional techniques.

[0147] The antibodies or polyspecific binding proteins disclosed herein may comprise a single polypeptide chain. In this case, the host cell may be transfected with a single vector expressing the polypeptide (e.g., including an expression regulatory sequence operably linked to the nucleotide sequence encoding the polypeptide). Alternatively, the antibodies or polyspecific binding proteins disclosed herein may comprise two or more polypeptides. In this case, the host cell may be co-transfected with multiple expression vectors, e.g., one expression vector expressing each polypeptide. The host cell may also be transfected with a single expression vector expressing two or more polypeptides. For example, the coding sequences of two or more polypeptides may be operably linked to different expression regulatory sequences (e.g., promoters, enhancers, and / or intra-sequence ribosome entry sites (IRESs)). The coding sequences of two or more polypeptides may also be separated by a ribosome skipping sequence or a self-cleaving sequence, e.g., a 2A peptide.

[0148] In some embodiments, an N-terminal signal sequence is included in the protein construct to express an antibody or a polyspecific binding protein. Exemplary N-terminal signal sequences include those derived from interleukin-2, CD-5, IgG kappa light chain, trypsinogen, serum albumin, and prolactin.

[0149] Following transfection, a single clone can be isolated for cell bank creation using methods known in the art, such as limiting dilution, ELISA, FACS, microscopy, or cloniPIX. The clone can be cultured under conditions suitable for bioreactor scale-up and maintenance expression of antibodies or polyspecific binding proteins.

[0150] Antibodies or polyspecific binding proteins can be separated and purified using methods known in the art, including centrifugation, deep filtration, cell lysis, homogenization, freeze-thaw, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed-mode chromatography.

[0151] Mai. Pharmaceutical composition This disclosure also features pharmaceutical compositions containing a therapeutically effective amount of the antibodies or polyspecific binding proteins described herein. These compositions can be formulated for use in various drug delivery systems. One or more physiologically acceptable excipients or carriers may also be included in the composition for appropriate formulation. Suitable formulations for use in this disclosure can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985. For a brief review of methods for drug delivery, see, for example, Langer (Science 249:1527-1533, 1990).

[0152] In some embodiments, the pharmaceutical composition may contain formulation materials for modifying, maintaining, or preserving the composition's pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution rate or release rate, adsorption, or osmosis. In this embodiment, suitable formulation materials include amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine), antimicrobial agents, antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite), buffers (e.g., boric acid, bicarbonate, Tris-HCl, citric acid, phosphoric acid, or other organic acids), fillers (e.g., mannitol or glycine), chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)), complexing agents (e.g., caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (e.g., glucose, mannose, or dextrin), proteins (e.g., serum albumin, gelatin, or immunoglobulin), colorants, flavoring agents, and diluents, emulsifiers, hydrophilic polymers (e.g., polyvinylpyrrolidone), low molecular weight polypeptides, and salt formation agents. Counterions (e.g., sodium), preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (e.g., glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (e.g., mannitol or sorbitol), suspending agents, surfactants or wetting agents (e.g., Pluronic acid, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, Triton, tromethamine, lecithin, cholesterol, tyroxapal), stability enhancers (e.g., sucrose or sorbitol), tonicity enhancers (e.g., alkali metal halides (preferably sodium chloride or potassium chloride), mannitol sorbitol), delivery medium, diluents, excipients and / or pharmaceutical adjuvants (Remington's This includes, but is not limited to, Pharmaceutical Sciences, 18th ed. (see Mack Publishing Company, 1990).

[0153] In some embodiments, the pharmaceutical composition may contain nanoparticles, such as polymer nanoparticles, liposomes, or micelles (see Anselmo et al. (2016) BIOENG.TRANSL.MED.1:10-29).

[0154] In some embodiments, the pharmaceutical composition may include sustained-release or controlled-release formulations. Techniques for formulating sustained-release or controlled-release means, such as liposome carriers, biodegradable microparticles or porous beads, and depot injections, are also known to those skilled in the art. Examples of sustained-release preparations include porous polymer microparticles or semipermeable polymer matrices in the form of molded articles such as films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gammaethyl-L-glutamic acid, poly(2-hydroxyethyl-inetaacrylate), ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyric acid. Liposomes may also be used as sustained-release compositions, which may be prepared by any of several methods known in the art.

[0155] Pharmaceutical compositions containing antibodies or polyspecific binding proteins disclosed herein can be provided in dosage unit forms and can be prepared by any suitable method. The pharmaceutical compositions are formulated to suit their intended route of administration. Examples of routes of administration include intravenous (IV), intradermal, inhalation, transdermal, topical, transmucosal, intrathecal, and rectal administration. In some embodiments, the antibodies or polyspecific binding proteins disclosed herein are administered by IV infusion. In some embodiments, the antibodies or polyspecific binding proteins disclosed herein are administered by intratumoral infusion. Useful formulations can be prepared by methods known in the pharmaceutical field. See, for example, Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990). Suitable formulation components for parenteral administration include sterile diluents such as sterile water for injection, physiological saline, fixative oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates, or phosphates; and isotonic modifiers such as sodium chloride or dextrose.

[0156] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). The carrier must be stable under manufacturing and storage conditions and must be protected from microorganisms. The carrier may be a solvent or dispersion medium, for example, containing water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), or suitable mixtures thereof.

[0157] Intravenous drug delivery formulations may be contained in syringes, pens, or bags. In some embodiments, the bag is connected to a channel including a tube and / or needle. In some embodiments, the formulation is a lyophilized formulation or a liquid formulation. In some embodiments, the formulation may be freeze-dried and contained in about 12 to 60 vials. In some embodiments, the formulation is lyophilized, with 45 mg of the lyophilized formulation contained in one vial. In some embodiments, about 40 mg to about 100 mg of the lyophilized formulation is contained in one vial. In some embodiments, a therapeutic dose of protein in the intravenous drug formulation is obtained by combining lyophilized formulations from 12, 27, or 45 vials. In some embodiments, the formulation is a liquid formulation and is stored as about 250 mg / vial to about 1,000 mg / vial. In some embodiments, the formulation is a liquid formulation and is stored as about 600 mg / vial. In some embodiments, the formulation is a liquid formulation and is stored as about 250 mg / vial.

[0158] These compositions may be sterilized by conventional sterilization techniques or aseptically filtered. The resulting aqueous solutions may be packaged for immediate use or lyophilized, and the lyophilized preparations may be combined with a sterile aqueous carrier before administration. The pH of the preparations is typically 3 to 11, more preferably 5 to 9 or 6 to 8, most preferably 7 to 8, for example, 7 to 7.5. The resulting compositions in solid form may be packaged in multiple single-dose units, each containing a fixed amount of the above-mentioned drug(s). The solid-form compositions may also be packaged in containers for flexible quantities.

[0159] In some embodiments, the Disclosure provides extended shelf-life formulations containing the proteins of the Disclosure in combination with mannitol, citrate monohydrate, sodium citrate, disodium phosphate dihydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, polysorbate 80, water, and sodium hydroxide.

[0160] In some embodiments, aqueous formulations containing the protein of the Disclosure are prepared in a pH buffer solution. The buffer of the Disclosure may have a pH in the range of about 4 to about 8, for example, about 4.5 to about 6.0, or about 4.8 to about 5.5, or about 5.0 to about 5.2. The intermediate range of pH described above is also intended to be part of the Disclosure. For example, it is intended to include a range of values ​​using any combination of the values ​​listed above as upper and / or lower limits. Examples of buffers that control pH within this range include acetates (e.g., sodium acetate), succinates (such as sodium succinate), glucons, histidine, citrates, and other organic acid buffers.

[0161] In some embodiments, the formulation includes a buffer system containing citrate and phosphate to maintain the pH in the range of about 4 to about 8. In some embodiments, the pH range is about 4.5 to about 6.0, or about pH 4.8 to about 5.5, or about 5.0 to about 5.2. In some embodiments, the buffer system includes citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, and / or sodium dihydrogen phosphate dihydrate. In some embodiments, the buffer system includes about 1.3 mg / ml of citric acid (e.g., 1.305 mg / ml), about 0.3 mg / ml of sodium citrate (e.g., 0.305 mg / ml), about 1.5 mg / ml of disodium phosphate dihydrate (e.g., 1.53 mg / ml), about 0.9 mg / ml of sodium dihydrogen phosphate dihydrate (e.g., 0.86), and about 6.2 mg / ml of sodium chloride (e.g., 6.165 mg / ml). In some embodiments, the buffer system contains 1–1.5 mg / ml of citric acid, 0.25–0.5 mg / ml of sodium citrate, 1.25–1.75 mg / ml of disodium phosphate dihydrate, 0.7–1.1 mg / ml of sodium dihydrogen phosphate dihydrate, and 6.0–6.4 mg / ml of sodium chloride. In some embodiments, the pH of the formulation is adjusted with sodium hydroxide.

[0162] Polyols that act as isotonic agents and can stabilize antibodies or polyspecific binding proteins may also be included in the formulation. The polyol is added to the formulation in amounts that may vary with respect to the desired isotonicity of the formulation. In some embodiments, the aqueous formulation is isotonic. The amount of polyol added may also vary with respect to the molecular weight of the polyol. For example, a smaller amount of monosaccharide (e.g., mannitol) is added compared to a disaccharide (e.g., trehalose). In some embodiments, the polyol used as an isotonic agent in the formulation is mannitol. In some embodiments, the mannitol concentration is about 5 to about 20 mg / ml. In some embodiments, the mannitol concentration is about 7.5 to 15 mg / ml. In some embodiments, the mannitol concentration is about 10 to 14 mg / ml. In some embodiments, the mannitol concentration is about 12 mg / ml. In some embodiments, the polyol sorbitol is included in the formulation.

[0163] Detergents or surfactants may also be added to the formulation. Exemplary detergents include nonionic detergents, such as polysorbates (e.g., polysorbate 20, 80, etc.) or poloxamers (e.g., poloxamer 188). The amount of detergent added is such that it reduces aggregation of the formulated antibody and / or minimizes the formation of particulate matter in the formulation and / or reduces adsorption. In some embodiments, the formulation includes a surfactant that is a polysorbate. In some embodiments, the formulation may contain the detergent polysorbate 80 or Tween 80. Tween 80 is a term used to describe polyoxyethylene (20) sorbitan monooleate (see Fiedler, Lexikon der Hifsstoffe, Editio Cantor Verlag Aulendorf, 4th edi., 1996). In some embodiments, the formulation may contain approximately 0.1 mg / mL to approximately 10 mg / mL of polysorbate 80, or approximately 0.5 mg / mL to approximately 5 mg / mL of polysorbate 80. In some embodiments, approximately 0.1% of polysorbate 80 is added to the formulation.

[0164] In some embodiments, the protein product of this disclosure is formulated as a liquid formulation. The liquid formulation can be supplied at a concentration of 10 mg / mL in any USP / Ph Eur Type I 50R vial, which is closed with a rubber stopper and sealed with an aluminum crimp seal. The stopper may be made of an elastomer compliant with USP and Ph Eur. In some embodiments, the liquid formulation is diluted with 0.9% physiological saline.

[0165] In some embodiments, the liquid formulations of the present disclosure are prepared as a 10 mg / mL concentration solution in combination with sugars at a stabilizing level. In some embodiments, the liquid formulations are prepared on an aqueous carrier. In some embodiments, the stabilizer is added in an amount less than or equal to the amount that would result in a viscosity undesirable or unsuitable for intravenous administration. In some embodiments, the sugars are disaccharides, such as sucrose. In some embodiments, the liquid formulations may also contain one or more of the following: buffers, surfactants, and preservatives.

[0166] In some embodiments, the pH of the liquid formulation is set by the addition of a pharmaceutically acceptable acid and / or base. In some embodiments, the pharmaceutically acceptable acid is hydrochloric acid. In some embodiments, the base is sodium hydroxide.

[0167] The aqueous carriers of interest in this specification are pharmaceutically acceptable (safe and non-toxic for administration to humans) and useful for the preparation of liquid formulations. Examples of carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffer solutions (e.g., phosphate-buffered saline), sterile saline, Ringer's solution, or dextrose solution.

[0168] Preservatives may be optionally added to the formulations herein to reduce bacterial activity. The addition of preservatives can facilitate, for example, the manufacture of multi-use (multi-dose) formulations.

[0169] Antibodies or polyspecific binding proteins may be freeze-dried to produce a freeze-dried formulation containing the protein and a freeze-protecting agent. The freeze-dried protective agent may be a sugar, such as a disaccharide. In some embodiments, the freeze-dried protective agent is sucrose or maltose. The freeze-dried formulation may also contain one or more of the following: buffers, surfactants, fillers, and / or preservatives.

[0170] The amount of sucrose or maltose useful for stabilizing the lyophilized pharmaceutical may be such that the weight ratio of protein to sucrose or maltose is at least 1:2. In some embodiments, the weight ratio of protein to sucrose or maltose is 1:2 to 1:5. In some embodiments, before lyophilization, the pH of the formulation is set by adding a pharmaceutically acceptable acid and / or base. In some embodiments, the pharmaceutically acceptable acid is hydrochloric acid. In some embodiments, the pharmaceutically acceptable base is sodium hydroxide. Before lyophilization, the pH of the solution containing the protein of this disclosure may be adjusted to 6 to 8. In some embodiments, the pH range of the lyophilized pharmaceutical is 7 to 8.

[0171] The actual dose levels of the active ingredient in the pharmaceutical compositions of this disclosure may be varied to obtain an amount of the active ingredient that is effective in achieving a desired therapeutic response for a particular patient, composition, and mode of administration without causing toxicity to the patient.

[0172] A specific dose may be a uniform dose for each patient, for example, 50–5,000 mg of protein. Alternatively, the patient's dose may be adjusted to the patient's approximate body weight or surface area. Other factors in determining an appropriate dose may include the disease or condition to be treated or prevented, the severity of the disease, the route of administration, and the patient's age, sex, and medical condition. Those skilled in the art can routinely make further improvements to the calculations necessary to determine an appropriate dose for treatment, particularly in light of the dose information and assays disclosed herein. Doses can also be determined by using known assays to determine doses to be used in combination with appropriate dose-response data. Individual patient doses can be adjusted while observing the progression of the disease. Blood levels of the targetable construct or complex in the patient can be measured to determine whether the dosage needs to be adjusted to reach or maintain an effective concentration. Pharmacogenic genomics can be used to determine which targetable constructs and / or complexes, and their dosages, are most likely to be effective for a given individual (Schmitz et al., Clinica Chimica Acta 308:43-53, 2001; Steimer et al., Clinica Chimica Acta 308:33-41, 2001).

[0173] Generally, the dosage based on body weight is approximately 0.1 μg to 100 mg per kg of body weight, for example, approximately 0.1 μg to 100 mg / kg body weight, approximately 0.1 μg to 50 mg / kg body weight, approximately 0.1 μg to 10 mg / kg body weight, approximately 0.1 μg to 1 mg / kg body weight, approximately 0.1 μg to 100 μg / kg body weight, approximately 0.1 μg to 50 μg / kg body weight, approximately 0.1 μg to 10 μg / kg body weight, approximately 0.1 μg to 1 μg / kg body weight, and approximately 0.1 μg to 0.1 μg / kg body weight. Weight, about 0.1 μg to about 100 mg / kg body weight, about 0.1 μg to about 50 mg / kg body weight, about 0.1 μg to about 10 mg / kg body weight, about 0.1 μg to about 1 mg / kg body weight, about 0.1 μg to about 100 μg / kg body weight, about 0.1 μg to about 10 μg / kg body weight, about 0.1 μg to about 1 μg / kg body weight, about 1 μg to about 100 mg / kg body weight, about 1 μg to about 50 mg / kg body weight, about 1 μg to about 10 mg / kg body weight, about 1 μg to about 1 mg / kg body weight, about 1 μg to about 100 μ g / kg body weight, about 1 μg to about 50 μg / kg body weight, about 1 μg to about 10 μg / kg body weight, about 10 μg to about 100 mg / kg body weight, about 10 μg to about 50 mg / kg body weight, about 10 μg to about 10 mg / kg body weight, about 10 μg to about 1 mg / kg body weight, about 10μg to about 100μg / kg body weight, about 10μg to about 50μg / kg body weight, about 50μg to about 100mg / kg body weight, about 50μg to about 50mg / kg body weight, about 50μg to about 10mg / kg body weight, about 50μg to about 1 The dosages are mg / kg body weight, approximately 50 μg to 100 μg / kg body weight, approximately 100 μg to 100 mg / kg body weight, approximately 100 μg to 50 mg / kg body weight, approximately 100 μg to 10 mg / kg body weight, approximately 100 μg to 1 mg / kg body weight, approximately 1 mg to 100 mg / kg body weight, approximately 1 mg to 50 mg / kg body weight, approximately 1 mg to 10 mg / kg body weight, approximately 10 mg to 100 mg / kg body weight, approximately 10 mg to 50 mg / kg body weight, and approximately 50 mg to 100 mg / kg body weight.

[0174] In some embodiments, subjects may be administered a dose of at least 20 μg of polyspecific binding protein. In some embodiments, subjects may be administered a dose of at least 30 μg of polyspecific binding protein. In some embodiments, subjects may be administered a dose of at least 40 μg of polyspecific binding protein. In some embodiments, subjects may be administered a dose of at least 50 μg of polyspecific binding protein.

[0175] The dosage may be administered once or more daily, weekly, monthly, or annually, or once every 2 to 20 years. Those skilled in the art can easily estimate the number of administrations based on the measured residence time and concentration of the target construct or complex in body fluids or tissues. The administration of this disclosure may be intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, intrathoracic, intrathecal, or intracavitary by catheter perfusion or direct intrafocal injection. This may be administered once or more daily, once or more weekly, once or more monthly, and once or more annually. In some embodiments, the administration is intravenous. In some embodiments, the administration is subcutaneous.

[0176] IV. Application to Treatment Antibodies or multispecific binding proteins are intended to be used either alone or in combination with other therapeutic agents.

[0177] A. Efficacy This disclosure provides a method for treating or improving proliferative disorders, neoplastic diseases, inflammatory diseases, immunodeficiencies, autoimmune diseases, infectious diseases, viral diseases, allergic reactions, parasitic reactions, graft-versus-host diseases, or host-versus-graft diseases in subjects requiring such treatment, the method comprising the administration of a multispecific binding protein or an antibody bound to CD19 as disclosed herein.

[0178] In some embodiments, the cancer being treated is non-Hodgkin lymphoma, such as B-cell lymphoma. Curative therapies for many patients with relapsed and / or refractory (R / R) NHL remain unmet. Numerous advances have been made in therapies, including CAR-T and bispecific T-cell engagers (TCEs), with response rates of approximately 80–90%, although 45–50% of these relapse. Reduced target expression contributes to resistance after CAR-T or TCE therapy.

[0179] CD19 has been clinically and commercially validated for the treatment of patients with relapsed / refractory B-cell acute lymphoblastic leukemia (ALL) and non-Hodgkin lymphoma (NHL). Since T-cell engagers (TCEs) exhibit clinical activity in many B-cell malignancies, the following modalities have also been well-validated: e.g., CD19-targeted Blincyto® (ALL), several CD20-targeted TCEs (NHL), and BCMA-targeted TCEs (MM). Further rationale for the development of tAB0050 includes its activity against malignant cells expressing very low levels of CD19, the mechanism of resistance after CD19 CAR-T therapy, the convenience of being an off-the-shelf product with the potential for CAR-T-like complete response (CR) rates, and its competitiveness against CD19-targeted ADCs and Fc-enhanced IgG1s that require higher levels of CD19 target expression.

[0180] In some embodiments, non-Hodgkin lymphomas are B-cell lymphomas such as diffuse large B-cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma, follicular lymphoma (FL), small lymphocytic lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma (e.g., nodal, extranodal, or mucosal-associated), extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, chronic lymphocytic leukemia, or primary central nervous system lymphoma. In some embodiments, the cancer to be treated is multiple myeloma. In some embodiments, the cancer to be treated is acute lymphoblastic leukemia (ALL). In some embodiments, ALL is relapsed / refractory adult and pediatric ALL.

[0181] In some embodiments, the cancer to be treated is relapsed and / or refractory non-Hodgkin lymphoma (NHL). In some embodiments, relapsed and / or refractory non-Hodgkin lymphoma (NHL) is a relapsed and / or refractory B-cell lymphoma such as diffuse large B-cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma, follicular lymphoma (FL), small lymphocytic lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma (e.g., (nodal, extranodal, or mucosal-associated), extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, chronic lymphocytic leukemia, or primary central nervous system lymphoma.

[0182] In some embodiments, individuals have relapsed after treatment with CD19-targeted therapy or are resistant to CD19-targeted therapy. In some embodiments, the CD19-targeted therapy includes blinatumomab, coltuximab tansine, MOR208 (XmAb-5574), MEDI-551, denintuzumab mahodotin, DI-B4, tapritumomab paptox, XmAb5871, MDX-1342, AFM11, MDX-1342, roncastuximab tesillin, SAR3419, Combotox, DT2219ARL, SGN-CD19A, AFM11, or GBR401. In some embodiments, the CD19-targeted therapy includes blinatumomab. In some embodiments, the CD19-targeted therapy includes CD19 CAR-T cell therapy. In some embodiments, CD19 CAR-T cell therapy comprises tisagenlecleucel, axicapbutagensiloleucel, or brexcapbutagenautoleucel.

[0183] In some embodiments, the individual relapses after treatment with at least two lines of therapy. In some embodiments, the individual relapses after treatment with at least three lines of therapy. In some embodiments, the individual relapses after treatment with at least four lines of therapy. In some embodiments, the individual relapses after treatment with at least five lines of therapy. In some embodiments, the treatment is CD19-targeted therapy. In some embodiments, the treatment is a CD20 monoclonal antibody. In some embodiments, the treatment is an anthracycline. In some embodiments, the treatment is an alkylating agent. In some embodiments, at least two treatment lines include CD19-targeted therapy, a CD20 monoclonal antibody, an anthracycline, an alkylating agent, or a combination thereof. In some embodiments, at least two treatment lines include a CD20 monoclonal antibody and an anthracycline. In some embodiments, at least two treatment lines include a CD20 monoclonal antibody and an alkylating agent.

[0184] In some embodiments, the cancer to be treated is advanced non-Hodgkin lymphoma. In some embodiments, advanced non-Hodgkin lymphoma is an advanced B-cell lymphoma such as diffuse large B-cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma, follicular lymphoma (FL), small lymphocytic lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma (e.g., nodal, extranodal, or mucosal-associated), extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, chronic lymphocytic leukemia, or primary central nervous system lymphoma. In some embodiments, the cancer to be treated is advanced multiple myeloma. In some embodiments, the cancer to be treated is advanced acute lymphoblastic leukemia (ALL).

[0185] Described herein are methods for treating an individual having a CD19-low-expressing cancer and requiring treatment thereof, comprising administering to the individual a multispecific binding protein described herein. In some embodiments, the individual has low levels of CD19 expression. In some embodiments, the individual has low levels of CD19 expression as a result of downregulation by cancer therapy (e.g., CD19-targeted therapy). In some embodiments, the individual does not respond to cancer therapy. In some embodiments, the individual has received cancer therapy, but the cancer therapy does not improve the cancer, does not prevent cancer regression, does not control the cancer, or a combination thereof. In some embodiments, the individual has a CD19-low-expressing cancer that is progressive, metastatic, unresectable, or a combination thereof. In some embodiments, the individual has a CD19-low-expressing cancer that is progressive, metastatic, unresectable, or a combination thereof after at least one, two, three, or four cancer treatments, or after more than four cancer treatments.

[0186] In some embodiments, the individual has a CD19-low-expressing cancer and has received at least one cancer therapy (e.g., antibody therapy, chemotherapy, or radiation). In some embodiments, the individual has a CD19-low-expressing cancer and has received at least two cancer therapies. Anti-cancer therapies include, but are not limited to, surgery, chemotherapy, radiation therapy, cryotherapy, hormone therapy, immunotherapy, cytokine therapy, and combinations thereof. In some embodiments, the cancer treatment includes CD19-targeted therapy. In some embodiments, the CD19 targeted therapy includes blinatumomab, coltuximab tansine, MOR208 (XmAb-5574), MEDI-551, denintuzumab mahodotin, DI-B4, tapritumomab paptox, XmAb5871, MDX-1342, AFM11, MDX-1342, roncastuximab tesillin, SAR3419, Combotox, DT2219ARL, SGN-CD19A, AFM11, or GBR401. In some embodiments, the CD19 targeted therapy includes blinatumomab. In some embodiments, the CD19 targeted therapy includes CD19 CAR-T cell therapy. In some embodiments, the CD19 CAR-T cell therapy includes tisagenlecleucel, axicapbutagen siroleucel, or brexcapbutagen autoleucel. In some embodiments, the treatment is a CD20 monoclonal antibody. In some embodiments, the treatment is an anthracycline. In some embodiments, the treatment is an alkylating agent. In some embodiments, at least two treatment lines include CD19 targeted therapy, a CD20 monoclonal antibody, an anthracycline, an alkylating agent, or a combination thereof. In some embodiments, at least two treatment lines include a CD20 monoclonal antibody and an anthracycline. In some embodiments, at least two treatment lines include a CD20 monoclonal antibody and an alkylating agent.

[0187] Various CD19-low-expressing cancers for treatment are intended herein. In some embodiments, the CD19-low-expressing cancer is metastatic cancer. In some embodiments, the CD19-low-expressing cancer includes solid tumors. In some embodiments, the CD19-low-expressing cancer is non-Hodgkin lymphoma, such as B-cell lymphoma. In some embodiments, non-Hodgkin lymphoma is B-cell lymphoma such as diffuse large B-cell lymphoma, primary mediastinal B-cell lymphoma, follicular lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, chronic lymphocytic leukemia, or primary central nervous system lymphoma. In some embodiments, the CD19-low-expressing cancer targeted for treatment is multiple myeloma. In some embodiments, the CD19-low-expressing cancer is acute lymphoblastic leukemia (ALL). In some embodiments, ALL is relapsed / refractory adult and pediatric ALL. In some embodiments, the CD19-low-expressing cancer to be treated is relapsed and / or refractory non-Hodgkin lymphoma (NHL). In some embodiments, the CD19-low-expressing cancer is B-cell lymphoma.

[0188] In some embodiments, CD19 low-expression cancers are classified as low-expression by various methods. In some embodiments, the methods include immunohistochemistry, immunocytochemistry (ICC), in situ hybridization (ISH), flow cytometry, enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), blotting methods (e.g., Western, Southern, and Northern blotting), or labeling within, on the surface of, or on an electrophoresis system. In some embodiments, the method includes flow cytometry. In some embodiments, the method includes immunohistochemistry. In some embodiments, the method includes, but is not limited to, fluorescence in situ hybridization (FISH), and immunohistochemical in situ hybridization. In some embodiments, the method includes, but is not limited to, next-generation sequencing (NGS), single-molecule real-time sequencing, Polony sequencing, ligation sequencing, reversible terminator sequencing, proton detection sequencing, ion semiconductor sequencing, nanopore sequencing, electronic sequencing, pyrosequencing, Maxam-Gilbert sequencing, chain termination (e.g., Sanger) sequencing, +S sequencing, and synthesis sequencing.

[0189] Some of the features of tAB0050 include the fact that the highly affinity human anti-CD19 scFv can target NHL cells expressing very low levels of CD19, the humanized anti-human serum albumin-conjugated single-domain antibody (VHH) has an extended half-life, and the human scFv binds to the immutable CD3 epsilon chain of the T cell receptor (TCR). Some of the highlights of the preclinical data include effective B cell depletion with a single dose and potent tumor killing in low levels of CD19 expression.

[0190] B. Combination therapy The methods and compositions described herein may be used alone or in combination with other therapeutic agents and / or modalities. The term “administered in combination,” as used herein, is understood to mean that two (or more) different treatments are delivered to a subject in the course of the subject’s illness, resulting in a overlap in the therapeutic effects on the patient at some point. In some embodiments, the delivery of one treatment is still occurring at the commencement of the second delivery, so as to overlap in terms of administration. This may be referred to herein as “simultaneous” or “concurrent delivery.” In some embodiments, the delivery of one treatment is completed before the commencement of the delivery of the other treatment. In some embodiments of any case, the treatments are more effective because they are administered in combination. For example, the second treatment is more effective, for instance, a smaller dose of the second treatment may produce the same effect, or the symptoms are significantly reduced by the second treatment compared to the case where the second treatment is administered without the first treatment, or a similar situation is observed with the first treatment. In some embodiments, delivery is made such that the reduction in symptoms or other parameters related to the disorder is greater than that observed with one treatment delivered in the absence of the other. The effects of the two treatments may be partially additive, fully additive, or more additive. Delivery may be made such that the effect of the delivered first treatment is still detectable when the second treatment is delivered.

[0191] In one embodiment, the present disclosure provides a method for treating a subject by administering a second therapeutic agent in combination with one or more CD19-binding polyspecific binding proteins and / or antibodies disclosed herein.

[0192] Examples of therapeutic agents that may be used as part of combination therapy in cancer treatment include, for example, radiation, mitomycin, tretinoin, ribomustine, gemcitabine, vincristine, etoposide, cladribine, mitobronitol, methotrexate, doxorubicin, carbocon, pentostatin, nitracrine, dinostatin, cetrorelix, letrozole, larcitrexed, daunorubicin, fadrozol, fotemustine, thymalfacin, sobuzoxane, nedaplatin, cytarabine, bicalutamide, vinorelbine, vesnarinone, aminoglutethimide, amsacrine, proglumide, eriptinium acetate, ketanserine, doxifluridine, etretinate, isotretinoin, streptozocin, nimustine, vindesine, flutamide. Examples include drogenil, butosine, carmoflu, razoxane, schizophyllan, carboplatin, mitractol, tegaflu, ifosfamide, prednimustine, picibanil, levamisol, teniposide, improsulfan, enocitabine, lislide, oxymetholone, tamoxifen, progesterone, mepitiostane, epithiostanol, formestan, interferon alpha, interferon-2 alpha, interferon-beta, interferon-gamma, colony-stimulating factor-1, colony-stimulating factor-2, denileukin difuticox, interleukin-2, luteinizing hormone-releasing factor, and variants of the aforementioned drugs that may exhibit different binding to their homologous receptors, as well as increased or decreased serum half-lives.

[0193] An additional class of drugs that may be used as part of combination therapy in the treatment of cancer is immune checkpoint inhibitors. Checkpoint inhibitors may be selected from, for example, PD-1 antagonists, PD-L1 antagonists, CTLA-4 antagonists, adenosine A2A receptor antagonists, B7-H3 antagonists, B7-H4 antagonists, BTLA antagonists, KIR antagonists, LAG3 antagonists, TIM-3 antagonists, VISTA antagonists, or TIGIT antagonists.

[0194] In some embodiments, checkpoint inhibitors are PD-1 or PD-L1 inhibitors. PD-1 is a receptor present on the surface of T cells that acts as a checkpoint in the immune system, inhibiting or otherwise regulating T cell activity at the appropriate time to prevent an excessive immune response. However, cancer cells can exploit this checkpoint by expressing ligands, such as PD-L1, that interact with PD-1 on the surface of T cells to shut down or regulate T cell activity. Exemplary PD-1 / PD-L1-based immune checkpoint inhibitors include antibody-based therapies. Exemplary anti-PD-1 antibodies include, for example, nivolumab (Opdivo®, Bristol-Myers Squibb Co.), pembrolizumab (Keytruda®, Merck Sharp & Dohme Corp.), PDR001 (Novartis Pharmaceuticals), and pizilizumab (CT-11, Cure Tech). Examples of anti-PD-L1 antibodies include, for example, atezolizumab (Tecentriq®, Genentech), duvalumab (AstraZeneca), MEDI4736, avelumab, and BMS 936559 (Bristol Myers Squibb Co.).

[0195] In some embodiments, the methods or compositions described herein are administered in combination with CTLA-4 inhibitors. In the CTLA-4 pathway, the interaction between CTLA-4 on T cells and its ligands (e.g., CD80, also known as B7-1, and CD86) on the surface of antigen-presenting cells (not cancer cells) results in T cell inhibition. Exemplary CTLA-4 antibodies include ipilimumab or tremelimumab.

[0196] In some embodiments, the methods or compositions described herein are administered in combination with (i) a PD-1 or PD-L1 inhibitor, for example, a PD-1 or PD-L1 inhibitor disclosed herein, and (ii) a CTLA-4 inhibitor, for example, a CTLA-4 inhibitor disclosed herein.

[0197] In some embodiments, the methods or compositions described herein are administered in combination with an IDO inhibitor. Exemplary IDO inhibitors include 1-methyl-D-tryptophan (known as indoximod), epacadostat (INCB24360), napoximod (GDC-0919), and BMS-986205.

[0198] Further other drugs that may be used as part of combination therapy in cancer treatment include monoclonal antibody agents that target non-checkpoint targets (e.g., Herceptin) and non-cytotoxic agents (e.g., tyrosine kinase inhibitors).

[0199] Furthermore, other categories of anticancer drugs include, for example, (i) ALK inhibitors, ATR inhibitors, A2A antagonists, base excision repair inhibitors, Bcr-Abl tyrosine kinase inhibitors, Bruton's tyrosine kinase inhibitors, CDC7 inhibitors, CHK1 inhibitors, cyclin-dependent kinase inhibitors, DNA-PK inhibitors, inhibitors of both DNA-PK and mTOR, DNMT1 inhibitors, DNMT1 inhibitors plus 2-chlorodeoxyadenosine, HDAC inhibitors, Hedgehog signaling pathway inhibitors, IDO inhibitors, JAK inhibitors, mTOR inhibitors, ME This includes inhibitors selected from (ii) OX40, CD137, CD40, GITR, CD27, HVEM, TNFRSF25, or ICOS agonists, and (iii) cytokines selected from IL-12, IL-15, GM-CSF, and G-CSF.

[0200] It is understood that antibodies or polyspecific binding proteins disclosed herein, which are designed to activate T lymphocytes, may cause side effects such as neurotoxicity. Therefore, in certain embodiments, a second therapeutic agent that may be used in combination with the antibody or polyspecific binding protein may include an agent that mitigates the side effects of the antibody or polyspecific binding protein, e.g., an agent that reduces neurotoxicity. In some embodiments, the second therapeutic agent inhibits T cell transport, e.g., reduces or inhibits immune cells from crossing the blood-brain barrier. Non-limiting examples of such therapeutic agents include antagonists of adhesion molecules on immune cells (e.g., α4 integrins), such as natalizumab (e.g., antagonist antibodies). In some embodiments, the second therapeutic agent increases the internal translocation of sphingosine-1-phosphate (SIP) receptors (e.g., S1PR1 or S1PR5), e.g., fingolimod or ozanimod. In some embodiments, the second therapeutic agent is a nitric oxide synthase (NOS) inhibitor such as lonopterin, sindunistat, A-84643, ONO-1714, L-NOARG, NCX-456, VAS-2381, GW-273629, NXN-462, CKD-712, KD-7040, or guanidinoethyl disulfide. In some embodiments, the second therapeutic agent is a CSF1 or CSF1R antagonist such as pexidartinib, emuctuzumab, kabilizumab, LY-3022855, JNJ-40346527, or MCS110. Further non-limiting examples of a second type of therapeutic agent include pentosan polysulfate, minocycline, anti-ICAM-1 antibodies, anti-P-selectin antibodies, anti-CD11a antibodies, anti-CD162 antibodies, and anti-IL-6R antibodies (e.g., tocilizumab).

[0201] The present invention also relates to a kit comprising an injection device and instructions for use. The instructions for use may include instructions for subcutaneous administration of a pharmaceutical composition or unit dose to a patient. The instructions for use may specify that the injection device, unit dose, and / or pharmaceutical composition are for the treatment of NHL. The kit may include packaging, which is adapted to hold the injection device and instructions for use. The instructions for use may be attached to the injection device.

[0202] Instructions for use may specify that the administration of the pharmaceutical composition to the patient will treat the patient's NHL.

[0203] The amounts and relative timing of administration of antibodies or polyspecific binding proteins and additional therapeutic agents may be selected to achieve the desired combination therapeutic effect. For example, when combination therapy is administered to a patient requiring such administration, the therapeutic agent, or pharmaceutical composition containing the therapeutic agent, may be administered in any order, such as sequentially, simultaneously, together, or concurrently. Furthermore, for example, antibodies or polyspecific binding proteins may be administered during the time when the additional therapeutic agent(s) exert their prophylactic or therapeutic effect, or vice versa.

[0204] Throughout the description, where a composition is described as having, including, or comprising certain components, or where a process and method is described as having, including, or comprising certain steps, it is intended that, in addition, there exist compositions of the disclosure that are essentially composed of or comprise the components described, and processes and methods of the disclosure that are essentially composed of or comprise the processing steps described.

[0205] Where it is stated in this application that an element or component is included in and / or selected from a list of elements or components described, it should be understood that such element or component may be any of the elements or components described, or that such element or component may be selected from a group consisting of two or more of the elements or components described.

[0206] Furthermore, it should be understood that the elements and / or features of the compositions or methods described herein, whether expressly or implicitly, can be combined in various ways without departing from the spirit and scope of this disclosure. For example, where a particular compound is referred to, that compound can be used in various embodiments of the compositions and / or methods of this disclosure, unless otherwise understood from the context. In other words, while embodiments are described and shown within this application in such a way as to make the application concise and clear, it is intended and understood that embodiments may be combined or separated in various ways without departing from this teaching and disclosure. For example, it will be recognized that all features described and shown herein are applicable to all embodiments of this disclosure described and shown herein.

[0207] The expression "at least one of" should be understood, unless otherwise understood from the context and usage, to include each of the objects described before it and each of the various combinations of two or more of those objects. The expression "and / or" in relation to three or more objects should be understood to have the same meaning, unless otherwise understood from the context.

[0208] The use of the terms "include", "includes", "including", "have", "has", "having", "contain", "contains", or "containing", including their grammatical equivalents, is generally open-ended and non-limiting unless the context specifically dictates otherwise or clearly indicates a different meaning. For example, it should be understood as not excluding additional elements or steps not recited.

[0209] When the term "about" is used before a quantitative value, the present disclosure includes the specific quantitative value itself unless otherwise specifically stated. As used herein, the term "about" refers to a variation of ±10% from the nominal value, unless otherwise indicated or inferred.

[0210] It should be understood that the order of steps or the order for performing certain actions is not critical as long as the present disclosure is practicable. Further, two or more steps or actions may be performed simultaneously.

[0211] All examples or exemplary language herein, such as the use of "such as" or "including", are intended only to better illustrate the present invention and do not limit the scope of the present disclosure unless otherwise claimed. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the present disclosure.

[0212] The above description illustrates multiple aspects and embodiments of the present disclosure. This patent application clearly contemplates all combinations and permutations of aspects and embodiments.

Examples

[0213] The disclosure generally described herein will be more readily understood by reference to the following examples, which are included for the sole purpose of exemplifying certain aspects and embodiments of the disclosure and are not intended to limit the disclosure.

[0214] Example 1: Design and Manufacture of tAB0050 tAB0050 is a recombinant fusion protein composed of a single polypeptide chain containing a humanized anti-serum albumin single domain antibody (sdAb), a humanized anti-CD19 scFv, and a human anti-CD3 epsilon scFv. Using Adimab's yeast-based antibody engineering platform, each binding domain was optimized for affinity, selectivity, cross-species reactivity, thermal stability, and developability. tAB0050 has an apparent molecular size of 65 kDa and was produced from a cloned Chinese hamster ovary (CHO) cell line. The expressed protein was affinity purified by protein A chromatography, followed by additional polishing steps to ensure high purity of the product. Figure 1B illustrates an exemplary design of tAB0050.

[0215] Example 2: Binding Characteristics of tAB0050

[0216] The binding of tAB0050 to human CD19, CD3, and serum albumin was determined by surface plasmon resonance (SPR) (Figure 5A). The equilibrium dissociation constants (KD) of tAB0050 for CD19, CD3, and albumin were 73.8 pM, 7,997 pM, and 596 pM, respectively. Given the binding affinity for albumin and the typical serum concentration of albumin in the range of 500 μM, it is assumed that all of tAB0050 will be bound by albumin in vivo. SPR analysis performed in the presence of albumin showed that the affinity and kinetics of the interaction between tAB0050 and CD3εδ and CD19 decreased by 2-3 fold, which was mainly driven by a decrease in the on-rate (Figure 6).

[0217] To evaluate target binding specificity, tAB0050 was screened for binding to fixed HEK293 cells expressing 6,19 individual full-length human plasma membrane proteins, secreted and cell surface-bound human secretory proteins, and an additional 397 human heterodimers, followed by a series of confirmatory screenings (Retrogenix technology; Report RP1885).

[0218] tAB0050 showed specific interactions with CD19, either alone or in heterodimers with CD81, albumin, and CD3ε. No interactions were observed with other cell surface or secreted human proteins of the test protein, which demonstrates the high specificity of CLN-978 to its primary targets: CD19, CD3ε, and albumin.

[0219] Interspecies cross-reactivity of tAB0050 was evaluated using flow cytometry across multiple donors in human, non-human primate (NHP), and mouse peripheral blood mononuclear cells (PBMCs) by cell surface binding to CD20+ B cells and CD4+ and CD8+ T cells. Cell surface binding of tAB0050 to B and T cells correlated with SPR binding. In human CD20+ B cells, very low EC50 values ​​were calculated, with a narrow range of 0.33–0.42 μg / mL. Binding to human T cells was more moderate. In CD4+ T cells, the EC50 value was 7.93 μg / mL (6.90–8.67 μg / mL). Binding to CD8+ T cells varied, with an EC50 value of 99.5 μg / mL in one donor, compared to 13.55 and 19.52 μg / mL in T cells from two donors. In NHP-derived PBMCs, the EC50 value of tAB0050 binding to B cells was 1.72 μg / mL (1–2.59 μg / mL). Similar EC50 values ​​were observed in CD4+ (28.86 μg / mL) and CD8+ T cells (28.66 μg / mL) in the T cell subpopulation. Minimal binding of tAB0050 was observed in mouse B and T cells at half-effect concentration (EC50) values ​​exceeding 100 μg / mL in the B cell and CD4+ and CD8+ T cell populations (Figure 5B), and saturation was not achieved.

[0220] Overall, tAB0050 showed stronger binding to B cells than to T cells, which was consistent with its higher affinity for CD19 than for CD3. Cross-reactivity with NHP PBMCs was also confirmed; cell binding to CD8+ T cells was similar, but binding to human B cells and CD4+ T cells was approximately 5 times and 3 times lower, respectively.

[0221] The mechanism of action of tAB0050 allows T cells to lyse a wide range of CD19-expressing cells. tAB0050 potently induces redirected lysis of CD19-expressing target cells in vitro and in vivo. It possesses very high affinity binding to CD19 for efficient targeting of malignant B cells expressing very low CD19 levels, and binding to serum albumin for extended serum half-life.

[0222] Example 3: Activity of tAB0050 in co-culture of human T cells and CD19-expressing target cells T cell-dependent cytotoxicity (TDCC), T cell activation, and the release of selected cytokines as pharmacodynamic markers were evaluated. These tests were performed in co-culture experiments with unstimulated human PBMCs as effector cells (E) and human B lymphoma RAMOS cell line as target cells (T), with an E:T ratio of 10:1. RAMOS cells expressed an average of 13,108 CD19 molecules on their cell surface, which is within the range of normal B cells and many primary lymphoma cells. First, the ability of tAB0050 to redirect lysis and activate T cells was evaluated among multiple donors in the absence of albumin. Figure 7A shows the variability of TDCC among six human PBMC donors. The EC50 values ​​for tAB0050 for lysis ranged from 0.26 to 5 pM, but all donors mediated more than approximately 80% lysis at a concentration of 2 nM over 48 hours. The expression of T cell activation markers CD69 and CD25 on CD4+ and CD8+ T cells was evaluated simultaneously. Consistent with TDCC, the EC50 values ​​for T cell activation were found to be below the single-digit pM range (0.2–1 pM and 1.6–4.7 pM for CD69 and CD25, respectively) in the majority of CD8+ and CD4+ T cells (80–100%) expressing CD69 and CD25 at 2 nM tAB0050 (Figure 7B). Of the six donors, only two donors' CD8+ T cells showed less than 50% induction of CD25, and one donor showed CD25 on CD4+ T cells. In summary, these data indicate robust activation of the majority of T cells in co-culture.

[0223] Next, the effect of albumin on the biological activity of tAB0050 was evaluated. Figure 7C shows the maximum half-volume lysis of RAMOS cells at 0.44 pM (+ / -0.7 SEM, n=10) and 2.68 pM tAB0050 (+ / -0.43 SEM: n=10) after 48 hours, in the absence and presence of HSA, respectively. As expected from the observed shift in binding affinity in the presence of HSA, a small shift (<10-fold) in the maximum half-volume concentration of tAB0050 was observed in the presence of HSA, and EC50 remained in the single-order pM range. Complete lysis of RAMOS cells was achieved at tAB0050 concentrations of 55 pM or higher, both in the absence and presence of HSA, suggesting that the presence of HSA is unlikely to affect the antitumor activity of tAB0050 under physiological conditions. Similar results were observed using the Raji cell line (Figure 8A).

[0224] As a pharmacodynamic measure of activated cytotoxic T cells, the release of TNFα and IFNγ (Figure 7D) and IL-6 and IL-1β (Figure 7E) in response to tAB0050 titration in co-cultures of RAMOS human lymphoma cells with human PBMCs in the presence of albumin was examined. Dose-dependent release of TNFα and IFNγ was observed despite higher EC50 values ​​than those observed for TDCC and T cell activation. EC50 values ​​were 23.3 pM (+ / -9.74 SEM, n=2) for TNFα release and 17.6 pM (+ / -2.1 SEM, n=2) for IFNγ. Maximum cytokine release was observed at 333 pM of tAB0050 for both cytokines. In conclusion, tAB0050 is a very potent inducer of redirected lysis / TDCC, T cell activation, and cytokine release by previously unstimulated T cells.

[0225] The effect of TDCC on the endogenous CD19+ B cell population present in the PBMC fraction was also evaluated in this assay. High basal levels of B cell death were observed throughout the assay period. Dose-dependent tAB0050-induced TDCC could still be observed above the high basal levels (Figure 8B). EC50 values ​​were within the same pM range as for lymphoma target cell lines. As seen in lymphoma target cell lines, albumin supplementation reduced B cell TDCC by approximately 25-fold.

[0226] T cell activation was monitored in CD4+ and CD8+ T cell populations using CD69, an early T cell activation marker detectable within hours of TCR ligation, and CD25, a high-affinity subunit of the IL-2 receptor that is induced more slowly after T cell activation than CD69.

[0227] Treatment with tAB0050 resulted in potent T cell activation, as measured by increased cell surface expression of both CD25 and CD69, and EC50 values ​​were in the low pM range for both target cell lines (Figure 8C). In the presence of albumin, both curves shifted to the right, demonstrating a decrease in T cell activation by tAB0050 in the presence of albumin.

[0228] Example 4: In vivo evaluation of tAB0050 activity The in vivo activity of tAB0050-HIS was directly compared with blinatumomab in two different mouse models (Figures 9A and 9B). One was a human CD19-expressing A20 lymphoma model transplanted into the flank of BALB / c mice expressing a human CD3e transgene. The other was a disseminated Raji B. luc luciferase NCG mouse model using transplanted human PBMCs as effector cells. In both models, tAB0050 was superior to blinatumomab. In established treatment trials using A20 flank tumors, a single IV dose of 0.1 mg / kg of tAB0050 completely prevented tumor growth, while an equivalent dose of blinatumomab was ineffective. In the disseminated Raji B. luc model, weekly IV administration of 0.1 mg / kg prevented increased luciferase signaling, but blinatumomab failed to prevent leukemia spread.

[0229] Figure 9E shows the effect on hCD3ε-expressing BALB / c mice inoculated with A20 cells expressing various levels of hCD19. Tumor volume approximately 100 mm². 3 When the mice reached a certain level, they were treated with 0.1 mg / kg of tAB0050 or with a control via IV once weekly. These data demonstrate the efficacy of tAB0050 in a lymphoma model expressing very low levels of CD19.

[0230] Example 5: Efficacy of tAB0050 in in vitro redirection lysis in low CD19-expressing cell lines This example is designed to determine the efficacy of in vitro redirection lysis of the polyspecific binding protein (tAB0050) in low-CD19-expressing cell lines.

[0231] In short, cytotoxic assays were performed using three A20 clones expressing 17,000, 3,700, or 325 copies of human CD19 on the cell surface.

[0232] As shown in FIGS. 3A-3D, tAB0050 targets a range of CD19 expression levels in engineered A20 cell lines, as measured by both the low pM EC50 value of redirected cell lysis and the maximum percentage of lysis. In the co-culture TDCC assay, 100% redirected lysis was observed in all three A20 cell clones in the presence of HSA (FIG. 3A). Consistent with no cross-reactivity of tAB0050 to mouse CD19, no lysis of non-transfected A20 cells was observed. The EC50 values of TDCC were similar in clones overexpressing CD19 by over 3,000 copies (range of 14-27 pM), but the lowest-expressing A20 clone with 325 copies of CD19 required 683 pM for half lysis. However, no differences in maximum solubility were observed between cell lines. All three A20 clones expressing hCD19 activated CD8+ and CD4+ T cells, as measured by the expression of CD25 and CD69 (FIG. 3B), and induced the release of TNFα (FIG. 3C) and IFNγ (FIG. 3D) in a CD19-dependent and dose-dependent manner.

[0233] The data indicate that tAB0050 can be used to treat patient populations with lower levels of CD19 expression and / or patients in whom CD19 expression is downregulated as a resistance mechanism to CD19-targeted therapy. The data also show that potent lysis of target cells was CD19-expression dependent, as the A20 parental cell line lacking CD19 expression was not sensitive to lysis at any of the tAB0050 concentrations tested.

[0234] Example 6: Comparison of tAB0050 and Blinatumomab for Potency of Redirected Lysis In Vitro This example was designed to determine the potency of redirected lysis in vitro of a bispecific binding protein (tAB0050) compared to blinatumomab.

[0235] In short, EMT6 cells were stably transfected to express hCD19 (Figure 2A). The parental, low-CD19 (clone 6), and high-CD19 (clone 5) strains were characterized by flow cytometry. Unstimulated T cells were co-cultured with the indicated EMT6 cell line at a 3:1 E:T ratio for 48 hours, and the cytotoxicity observed in the parental (Figure 2B), low-CD19 (Figure 2C), and high-CD19 (Figure 2D) strains was evaluated by flow cytometry.

[0236] In summary, these data indicate improved efficacy of redirected lysis of tAB0050 in vitro compared to blinatumomab.

[0237] Example 7: Activity of tAB0050 in lymphoma cell lines with low CD19 expression. This example illustrates the activity of tAB0050 in lymphoma cell lines with low CD19 expression.

[0238] To determine the minimum number of CD19 copies on target cells required for redirection lysis by tAB0050, a chemically inducible system was used to adjust the expression level of the CD19 transgene in CHO cells. As a hamster-derived epithelial cell line, CHO cells do not express B cell lineage-specific human CD19 protein. CHO cells transfected with the inducible transgene were incubated with titration of the chemical inducer ABA in the range of 2.4 nM to 10 μM, allowing for CD19 expression levels on CHO cells of 78 to 185,223 copies per cell after 24 hours. At that point, ABA was washed off the medium, and purified CD8+ T cells from four human donors were added with either 225 or 1,770 pM of tAB0050 up to an E:T ratio of 10:1 for 68 hours. For all four T cell donors, signals for TDCC, T cell activation (CD25 expression), proliferation (Ki67), and IFNγ release were observed beyond the background of wild-type CHO cells (Figures 4A–4D, representative donors). The observed cytotoxicity was CD19 density-dependent, ranging from 38% lysis (78 CD19 copies) to 80% lysis (185,000 CD19 copies) at 225 pM tAB0050. At higher tAB0050 concentrations of 1,770 pM, cell lysis was independent of CD19 levels. 1,770 pM tAB0050 mediated nearly maximum lysis of induced CHO cells even at levels of 78 CD19 molecules per cell (Figure 4A). CD25 and Ki67 expression, as well as IFNγ release, increased with increasing levels of CD19 expression in both tAB0050 at 225M and 1,770 pM (Figures 4B-4D). Despite this heightened sensitivity to low levels of CD19, tAB0050 was also highly specific to CD19 expression, and no activity was observed in wild-type control cells lacking CD19 expression.

[0239] A similar assay was performed in this inducible system to compare the minimum CD19 expression levels required for tAB0050 or blinatumomab to induce cytotoxicity and T cell activation, as measured by INFγ production. As shown in Figures 4E-4F, tAB0050 demonstrated potent efficacy at lower CD19 expression levels compared to blinatumomab.

[0240] Example 8: Comparison of antitumor activity of tAB0050 administered IV or SC To minimize systemic toxicity and improve patient convenience, several T-cell engagers have recently been evaluated for SC administration. Since the tAB0050 formulation allows for both IV and SC administration of tAB0050, the antitumor effects of both administration routes were investigated in immunodeficient NCG mice transplanted with Raji B.luc cells and human PBMCs.

[0241] Materials and methods tAB0050 generation

[0242] The individual components of tAB0050 were optimized using Adimab's antibody engineering yeast platform. Full-length tAB0050 was assembled by fusing a humanized antiserum albumin single-domain antibody (sdAb), a humanized anti-CD19 single-chain variable fragment (scFv), and a humanized anti-CD3 epsilon scFv into a single polypeptide chain using a glycine / serine linker. tAB0050-HIS consisted of the same sequence as tAB0050 with a 10× histidine tag at the C-terminus and was produced in HEK-293 cells only for initial characterization testing.

[0243] Biacore evaluation of tAB0050 binding activity

[0244] To determine affinity for CD3εδ dimers and albumin, each protein was immobilized on the CAPture chip sensor surface via biotin capture. Multiple cycles of tAB0050 interaction kinetics were evaluated by injection of appropriate concentrations of a series of test samples. To measure affinity for CD19, human CD19-Fc fusion protein was immobilized on the Series S CM5 sensor chip via surface-immobilized anti-human Fc antibody. Single cycles of protein-sample interaction kinetics were evaluated by injection of appropriate concentrations of a series of test samples. Each protein-sample interaction was analyzed in triplicate, and the Langmuir 1:1 binding model was fitted to the data. To determine the effect of albumin binding on tAB0050 affinity, tAB0050 was initially pre-complexed with HSA or left uncomplexed, then immobilized on the chip, and subjected to recombinant CD3εδ, with binding measured throughout. The global fit algorithm (Biacore Insight Evaluation) was used. D The values ​​of ka and kd were calculated.

[0245] Cell binding of tAB0050

[0246] Cryopreserved PBMCs derived from humans, cynomolgus monkeys, or mouse donors were thawed, and then viability testing dyes, Fc blocks, and staining solutions were added. Briefly, 0.3–1 × 10⁻⁶ 6Individual cells were stained with FVS700 to confirm viability according to the manufacturer's recommendations and blocked with mouse or human Fc blocking antibody. The cells were then incubated with tAB0050 conjugated to APC in the dark at 37°C for 60 minutes and stained with a fluorescently conjugated antibody. Mouse and NHP cells were diluted with Brilliant Stain Buffer and FBS staining buffer (BD Biosciences), while human cells were diluted with Brilliant Stain Buffer and PBS (ThermoFisher). After staining, the cells were fixed with Flourofix buffer (Biolegend) and stored in the dark at 2–8°C until acquisition by flow cytometry. CD4+, CD8+ T cells and CD20+ B cells were identified and gated.

[0247] In vitro T cell-dependent cell-mediated cytotoxicity (TDCC) and T cell activation

[0248] In short, target cells (RAMOS, Raji, and A20-CD19) stained with PBMC (stained with eFluor450) and eFluor670 were subjected to approximately 3 × 10⁻¹⁶ exposures in the presence or absence of 0.15 mg / mL of human serum albumin. 5 Target cell seeding density per cell / well was evaluated using a 10:1 E:T ratio. Eight dilutions of tAB0050 were performed, starting at 2 nM and progressing in 6-fold dilution steps. Cytotoxicity was assessed by flow cytometry using a fixable viability indicator (eFluor780). T cell activation was identified by flow cytometry (CD4, CD8, CD25, and CD69). Cytokine production (IFN-γ and TNFα) was quantified using Luminex Multiplex Analysis as needed. CD19 expression was quantified using Quantibrite beads (BDBiosciences).

[0249] Generation of CHO-CD19-inducible cell lines and in vitro testing

[0250] CHO-K1 cells were transduced with an induction cassette expressing a chemo-inducible proximity (CiP) activated cassette that enables titrable expression of CD19 upon treatment with the chemoinducer ABA, allowing CD19 expression levels ranging from 78 to 185,223 copies per cell in CHO cells after 24, 48, or 96 hours. CD19 expression was quantified by staining with PE anti-CD19 and plotted on curves generated using the Quanti-Brite PE receptor quantification kit.

[0251] In the TDCC study using inducible CHO CD19 cells, CHO-K1 ABA CD19 cells were seeded on xCELLigence RTCA plates and treated with ABA at seven concentration ranges (2.4–10 μM) for 24 hours to induce CD19 expression. Isolated T cells and target cells were evaluated at two fixed tAB0050 concentrations (225 pM and 1,770 pM) and using four individual T cell donors, with an E:T ratio of 10:1. For comparison with blinatumomab, T cells isolated from 10 individual donors were used with an E:T ratio of 10:1, and either 225 pM tAB0050 or blinatumomab was used. CD8+ T cell proliferation was measured by Ki-67 staining and flow cytometry. IFN-γ production by effector cells was measured from the supernatant by ELISA.

[0252] In vivo efficacy trial

[0253] In the A20-CD19 study, hCD3ε-expressing BALB / c mice were inoculated with A20 cells expressing hCD19 produced by stable transfection, and individual clones were selected by limiting dilution. Tumor volume was approximately 100 mm². 3 When the mouse reached a certain stage, it was treated once with tAB0050-HIS or blinatumomab IV. For the Raji B.luc study, immunodeficient NCG mice were given 1 × 10⁶ mice. 5 IV transplantation of 10 Raji B.luc cells was performed the following day (day 1). 2 x 10⁶ cells derived from a normal human donor were transplanted. 7Individual PBMCs were transplanted intraperitoneally (IP). From day 1, mice were treated weekly with tAB0050-HIS or blinatumomab intravenously.

[0254] To compare IV administration and SC administration, mice that received PBMC were divided into seven groups: PBS vehicle control (n=9), or PBMC + tAB0050 administered at doses of 0.3, 3, or 30 μg / kg (n=9), IV, or SC. tAB0050 was administered once a week at a dose of 10 mL / kg adjusted for body weight until the end of the study. On day 14, the number of Raji cells and B cells in peripheral blood was measured by flow cytometry.

[0255] As evident from luciferase fluorescence signals comparable to the background, weekly IV or SC administration of tAB0050 at 3 and 30 mg / kg resulted in a 10 / 10 complete response after 28 days (Figure 9C, Figure 9D). Treatment at 0.3 mg / kg had a minimal effect with SC administration and a moderate effect with IV administration. The percentage of human B cells and Raji B. luc cells in the peripheral blood of mice on day 14 was determined by FACS analysis. Very significant and complete elimination of human B cells and lymphoma cells was observed in peripheral blood samples from mice in all tAB0050-treated groups, regardless of dose level or route of administration (Figure 10A). A dose-dependent trend of decreasing T cell counts in the periphery was also observed on day 14, consistent with the efflux of T cells from peripheral circulation at activation (Figure 10B).

[0256] Following single-dose administration of tAB0050 via either IV or SC, a similar dose-dependent PK / PD study was conducted in the huPBMC Raji B.luc model. Consistent with the multi-dose efficacy study, analysis of Raji cells, normal B cells, and T cells in the bone marrow, measured by FACS on day 8 of the study, showed a statistically significant decrease in Raji B.luc cell counts across all dose levels and administration routes (Figure 10C), and a trend toward a decrease in normal B cell counts in both administration routes. Furthermore, dose-dependent increases in both CD8+ and CD4+ T cell activation, as measured by CD69, were observed in both administration routes.

[0257] Cytokine levels were measured 2, 6, and 24 hours after the initial administration of tAB0050. While it can be difficult to observe a strong human cytokine response in the huPBMC efficacy model, the IV dose group showed a dose-dependent increasing trend in IL-6, IL-8, and GM-CSF at 6 hours post-administration, recovering to baseline at 24 hours, whereas the SC dose group did not show a dose-dependent increase in these cytokines (Figures 11A and 11B). These data suggest that both IV and SC administrations of tAB0050 are highly effective in the Raji lymphoma xenograft model.

[0258] The pharmacokinetics (PK) of tAB0050 were also evaluated. Serum was collected from blood samples at 0.5, 2, 4, 24, 74, and 168 hours after a single dose. tAB0050 concentrations in mouse serum were measured using non-GLP, appropriate methods. After a single dose, tAB0050 concentrations were only detectable above the LLOQ (6.25 ng / mL) at 3 and 30 μg / kg IV and 30 μg / kg SC dose levels. While tAB0050 concentrations at 3 μg / kg IV were below the limit of quantification (BLQ) at 24 hours post-administration, tAB0050 remained detectable up to 72 hours in all mice in the 30 μg / kg dose group (Figure 12A). PK analysis showed that the C0 value of tAB0050 increased almost proportionally with dose increase from 3 to 30 μg / kg IV (Figure 12B). The estimated half-life was 1.85 days after IV administration. Cmax after SC administration was approximately one-tenth lower than Cmax after IV administration (16.7 ng / mL and 126.4 ng / mL, respectively). Tmax with 30 μg / kg SC was approximately 24 hours.

[0259] To evaluate the stability of tAB0050 under relevant physiological conditions, 100 μg / mL of tAB0050 was incubated in human serum at 37°C. Samples were collected at days 0, 7, and 14 and stored at -80°C. At the end of the two-week study, all samples were subjected to the NFAT-RE T cell activation assay in the presence of Raji target cells. EC50 was calculated at each time point.

[0260] As shown in Figure 13, the EC50 values ​​ranged from 0.38 ng / mL on day 0 to 0.52 ng / mL on day 14. Considering that the EC50 was within 2 times, these data suggest that tAB0050 is stable in human serum when incubated at physiological temperature for two weeks.

[0261] Example 9: Comparison of pharmacokinetics, pharmacodynamics, and tolerability of tAB0050 administered intravenously or via seroconcentrate in non-human primates.

[0262] The inventors hypothesized that SC administration could reduce systemic toxicity commonly associated with IV-delivered T-cell engagers by lowering the maximum serum concentration (Cmax), and therefore directly compared tAB0050 at single doses of 0.1 and 1 mg / kg by single intravenous (IV) or subcutaneous (SC) injection in cynomolgus monkeys (n=2). Figure 14A shows the study design. The time points at which PK serum samples were collected from each animal were 0.25, 0.5, 1, 2, 6, 24, 48, 144, 312, 480, and 672 hours relative to the dose. The monkeys were also observed for signs of toxicity for up to 29 days.

[0263] Serum tAB0050 dose levels increased in a dose-dependent manner across both administration routes (Figure 14B). Serum half-lives ranged from 5 to 7 days, suggesting that prolonged half-life supports low-frequency administration. Exposure and half-life were similar across both administration routes. As expected, Cmax values ​​were at least one-fifth lower in the SC route than in the IV route.

[0264] A single dose of tAB0050 reduced circulating B cell counts to background levels throughout the entire 28-day experiment, and B cell counts did not clearly recover (Figure 14C). T cells also transiently disappeared from the periphery two days after administration, but rapidly recovered to pre-administration levels by eight days post-infusion, consistent with observations of other T cell engagers (Figure 14D).

[0265] As expected, administration of tAB0050 induced transient release of various cytokines and chemokines into the peripheral blood (Figure 14E). Following IV infusion of tAB0050, serum levels of TNFα, IL-2, IFNγ, and IL-8 were highest at 2 hours post-infusion and tended to return to baseline by 6 hours, while IL-6 and IL-10 levels peaked at 6 hours. Notably, the release of most cytokines was lower after SC delivery compared to IV infusion, and the kinetics of cytokine release also changed. 24 hours after infusion, all cytokine levels returned to baseline regardless of the delivery route.

[0266] tAB0050 was well tolerated in cynomolgus monkeys at a dose level of 0.1 mg / kg via both the IV and SC pathways, while the 1 mg / kg dose level was tolerated only after SC administration. In contrast, IV administration of 1 mg / kg of tAB0050 resulted in cytokine release symptoms in one of two cynomolgus monkeys, requiring euthanasia on day 1 of the study. The 1 mg / kg dose administered via SC exposed animals to serum concentrations of approximately 1–5 μg / mL (approximately 15–75 nM) of tAB0050 over 300 hours (12.5 days). This was more than 10 times higher than the tAB0050 concentration required for complete target cell lysis in vitro, even in low-CD19-expressing cells.

[0267] The serum concentration-time profiles of individual animals in both dose levels for IV administration are shown in Figures 15A and 15B. The serum concentration-time profiles of individual animals in both dose levels for SC administration are shown in Figures 15C and 15D. In monkeys administered by IV injection, Cmax was observed at 0.25 hours, and t1 / 2 ranged from 43.8 to 113 hours. The mean clearances were 1.18 and 0.697 mL / hour / kg for the 0.1 and 1 mg / kg dose groups, respectively. The mean Vss was 101 and 98.0 mL / kg for the 0.1 and 1 mg / kg dose groups, respectively.

[0268] In monkeys administered via SC injection, Tmax ranged from 24 to 144 hours, and t1 / 2 ranged from 110 to 167 hours. Cmax after SC administration was observed to be four times lower in the 1 mg / kg dose group compared to IV.

[0269] CLN-978(tAB0050) exposure generally increased with dose in female monkeys administered via IV or SC routes. The increase, as indicated by dose-normalized Cmax and AUClast values, was dose-proportional from 0.1 to 1 mg / kg in both IV and SC administrations. The TK parameters determined for this study are summarized in Figures 16A and 16B.

[0270] The parameters evaluated included mortality, cage-side observation, detailed clinical observation, body weight, dietary assessment, clinicopathological parameters (hematology, coagulation, clinical chemistry, and urinalysis), toxicological analysis, cytokine sample analysis, and flow cytometry analysis.

[0271] There were no treatment-related changes in body weight or urinary parameters. One animal treated IV with 1 mg / kg of CLN-978 was euthanized on day 1 due to its mortal condition. All other animals survived until the end of the study. The mortal animal exhibited decreased activity, closed eyelids, a curled posture, moderately delayed response time, hypoglycemia, a slightly elevated heart rate, decreased body temperature, and was cold to the touch. This animal also showed macroscopic findings of gastrointestinal lesions and pulmonary adhesions.

[0272] Clinical observations after a single IV injection included a curled posture, vomiting or vomiting-like substances (approximately 1-2 hours after administration), and decreased appetite at doses of 0.1 mg / kg or higher. Clinical observations after a single SC injection included vomiting or vomiting-like substances (approximately 6-7 hours after administration) at doses of 0.1 mg / kg or higher, and decreased appetite in animals at 1 mg / kg.

[0273] Treatment-related changes were observed in hematology, coagulation, and clinical chemistry. Hematological changes included changes in leukocyte counts, including lymphocytes, monocytes, basophils, large unstained cells, and eosinophils, in all animals. These hematological parameters initially decreased on day 2 in all groups, followed by a general increase at later time points compared to pre-test values. Neutrophil counts fluctuated throughout the study, increasing across all groups on day 29 compared to pre-test values. Coagulation changes were included on day 2, with increased fibrinogen in all groups, further increases on day 8 in the remaining IV-treated animals (1 mg / kg) and SC-treated animals (0.1 mg / kg), and further increases on day 29 in IV-treated animals at 0.1 mg / kg, and in SC-treated and IV-treated animals at 1 mg / kg.

[0274] On day 2, clinical chemistry changes included increased alanine aminotransferase and / or aspartate aminotransferase in all groups compared to baseline values. On day 29, alkaline phosphatase increased in single-dose IV animals at 0.1 mg / kg and in single-dose SC animals at 1 mg / kg. Total bilirubin and urea nitrogen increased in all groups on day 2, and total bilirubin increased in all animals on day 8. Triglycerides increased on day 29 in SC animals at 1 mg / kg CLN-978.

[0275] Flow cytometry data showed B cell depletion in both SC-treated and IV-treated animals. Transient decreases in peripheral T cells and NK cells occurred in both IV-treated and SC-treated animals after administration on day 2, and these generally increased to near or above pre-treatment levels on days 8 and 15. Peripheral lymphocyte counts on day 29 were also decreased compared to pre-treatment levels in IV-treated animals and one animal treated with 1 mg / kg of SC, but to a lesser extent than those observed on day 2.

[0276] Qualitative analysis of cytokine data did not show any changes in IL1 beta after IV or SC administration. In general, with IV administration, the remaining cytokines and chemokines (IL1RA, IL2, IL4, IL5, IL6, IL8, MCP1, MIP1β, TNFα) increased from pre-administration levels 2 and 6 hours after administration and decreased by 24 or 672 hours post-administration. With SC administration, there were no essential changes in IL2, IL4, and IL5, IL6, IL8, MCP1, and MIP1β increased sporadically and transiently, and TNFα decreased over time.

[0277] Overall, single injections of CLN-978 were generally tolerated at 0.1 mg / kg IV or 0.1 and 1 mg / kg SC. With IV administration, one animal died due to substance-related mortality at 1 mg / kg. Generally, treatment-related changes after IV administration included hunched posture, vomiting, decreased appetite, and clinical signs of clinical pathological changes at doses of 0.1 mg / kg and above. Single SC injections were associated with vomiting and decreased appetite at 1 mg / kg, and changes in clinicopathological parameters at doses of 0.1 mg / kg and above.

[0278] Example 11: GLP 4-week toxicity study of CLN-978 by subcutaneous injection in cynomolgus monkeys with a 4-week recovery period.

[0279] The objective of this study was to determine the potential toxicity of CLN-978 when administered weekly by SC injection for 4 weeks in cynomolgus monkeys, and to evaluate the potential reversibility of any findings.

[0280] Cynomolgus monkeys (3-5 males or females per group) received SC injections of CLN-978 at doses of 0, 0.1, 0.3, and 1 mg / kg once weekly (days 1, 8, 15, and 22) for 4 weeks. All primary test animals (3 males or females per group) were observed until day 29 of the study, while a subset of animals in the vehicle control and high-dose groups (2 males or females per group) were maintained for a 4-week recovery period (until day 57 of the study). The study design is shown in Table 7.

[0281] [Table 7]

[0282] The parameters evaluated in this study included mortality, clinical observations (including detailed and post-administration observations), body weight, dietary assessment, respiratory rate, ophthalmology, electrocardiography, neurobehavioral tests, clinicopathological parameters (hematology, coagulation, clinical chemistry, and urinalysis), bioanalysis and toxicological parameters, anti-drug antibody analysis, cytokine analysis, immunophenotyping and flow cytometry, organ weights, and macroscopic and microscopic examinations.

[0283] There were no CLN-978-related changes in body weight, weight gain, ophthalmology, electrocardiogram, respiratory rate, neurobehavioral parameters, macroscopic pathology, cytokines, or urinalysis parameters.

[0284] Based on clinical observations including moderate tremor, moderate dehydration, hunched posture, emaciation, liquid stool, and decreased activity, females that recovered with a single high dose were euthanized on day 31. The clinicopathological findings of this animal were similar to those of animals that survived until the scheduled necropsy. This animal had numerous macroscopic and microscopic abnormalities, and the cause of morbidity was chronic peritonitis, which most likely represents an opportunistic infection secondary to CLN-978-induced immunosuppression. Microscopic findings associated with CLN-978 were identical to those identified at the time of final slaughter.

[0285] Clinical observations associated with CLN-978 included a curled posture and decreased activity in a single male at a dose of 1 mg / kg / dose. No clinical observations were observed during the 4-week recovery period, indicating complete recovery.

[0286] CLN-978-related changes in hematological parameters included minimal to moderate decreases in red blood cell count, hemoglobin, and hematocrit, as well as minimal increases in platelet count at doses of 0.1 mg / kg / day or higher, minimal decreases in reticulocyte count at doses of 0.3 mg / kg / day or higher, and variable changes in white blood cells (neutrophils, monocytes, basophils, large unstained cells, and white blood cell count). Neutrophil counts increased towards the end of the treatment phase (days 23 and 29) at doses of 0.3 mg / kg / day or higher. Monocyte counts decreased significantly at 0.1 and 0.3 mg / kg / day on day 2. Remaining white blood cells decreased minimally to significantly at doses of 0.1 mg / kg / day or higher after the first dose on day 2, followed by an increase on day 8. Lymphocyte counts also decreased after the fourth dose on days 23 and 29.

[0287] In coagulation and clinical chemistry, CLN-978-related changes were consistent with the acute phase response, including a minimal to mild increase in fibrinogen at doses of 0.1 mg / kg / day or higher, a mild prolongation of activated partial thromboplastin time at doses of 0.3 mg / kg / day or higher on day 2 and / or day 23, and a slight decrease in albumin at doses of 0.1 mg / kg / day or higher. Other clinical chemistry changes were generally minimal to mild, including increases (up to moderate) in total bilirubin and urea nitrogen at 0.1 mg / kg / day, increases in creatinine at 0.1 and 1 mg / kg / day, decreases in glucose at 0.1 and 0.3 mg / kg / day, decreases in phosphorus at 0.3 and 1 mg / kg / day, and decreases in cholesterol, sodium, and globulin (up to moderate) at doses of 0.1 mg / kg / day or higher. The decrease in albumin and globulin led to a decrease in total protein and calcium, and varied albumin-to-globulin ratios.

[0288] After a 4-week recovery period, hematological, coagulation, and clinical chemistry changes were observed at 1 mg / kg / day, indicating a lack of recovery. Hematologically, these changes included a mild decrease in red blood cell count, hemoglobin, and hematocrit, a slight increase in neutrophil count, and a slight to moderate decrease in lymphocyte count. In coagulation and clinical chemistry, total protein and calcium decreased, and the albumin-to-globulin ratio varied, resulting from a moderate increase in fibrinogen, a minimal decrease in sodium, and a moderate decrease in albumin and globulin.

[0289] Immunophenotypic analysis revealed that 4 weeks of CLN-978 treatment (0.1, 0.3, and 1 mg / kg / day) resulted in depletion of test substance-related B cells, as well as transient decreases in peripheral T cells and NK cells, at the dose point of 2 days post-administration. The results suggested that the decrease in T cells and NK cells was transient, as these populations recovered to or above pre-administration baseline levels by 8 days (with the exception of a small number of animals [1-2 animals per group] with T cell, T helper, and NK cell populations at 0.1, 0.3, and 1 mg / kg / dose), and decreased moderately again at 23 days after a fourth dose of CLN-978. Peripheral absolute total T cells, T helpers, T cytotoxic cells, and NK cells in all treated animals recovered to their respective pre-administration baseline levels by the post-administration point of 29 days. Absolute B cells remained depleted in animals in all treatment groups in both study cohorts.

[0290] Treatment-related organ weight changes included decreases in spleen weight (absolute value relative to brain weight and body weight) at doses of 0.1 and 1 mg / kg / day in males and ≥ 0.1 mg / kg / day in females, and decreases in thymus weight (absolute value relative to brain weight and body weight) at doses of ≥ 0.1 mg / kg / day in both males and females. These findings were histopathologically correlated with a significant decrease in lymphocyte density in germinal centers within the white pulp and a decrease in lymphocyte density in the thymic cortex. Thymus weight fully recovered in males at 1 mg / kg / day, but not in females until sacrifice during the recovery period, although the decrease in spleen weight persisted in males. Histological changes resulting in the decrease in spleen weight persisted in females during the recovery period at 1 mg / kg / day, although weight increased, but was complicated by coexisting lesions. Thymic changes may be secondary to physiological stress.

[0291] Microscopic findings associated with CLN-978 administration were identified in all evaluated secondary lymphoid tissues, including the spleen, rump, mandibular lymph nodes, mesenteric lymph nodes, and GALT. These findings consisted of marked to severe decreases in lymphocyte density in male and female germinal centers at doses of 0.1 mg / kg / day or higher, and marked to severe decreases in CD20-positive cells (i.e., B cells) in CD20 immunohistochemistry slides in normally B-cell-rich areas of the white pulp, lymph node cortex, GALT, spleen, and thoracic bone marrow (see, e.g., Figures 22A-22C). These findings did not exhibit a clear dose-response and persisted at the same severity throughout the recovery period as the last sacrifice, consistent with no perceptible recovery. These changes are interpreted as pharmacological effects of CLN-978.

[0292] Additional microscopic findings potentially associated with CLN-978 administration included the aforementioned decrease in thymic lymphocyte density and an increase in the bone marrow-to-red blood cell ratio in the sternal bone marrow. These changes may be secondary to the pharmacologically induced immunosuppression response of physiological stress (thymus) and / or the adaptive immune system (bone marrow).

[0293] One male designated for final slaughter at 0.3 mg / kg / day and one female designated for convalescent slaughter at 1 mg / kg / day showed microscopic findings consistent with chronic bacterial peritonitis throughout the abdominal cavity, with the worst inflammation confined to the gastrointestinal tract, which is likely the entry point for the bacteria. These changes are interpreted as opportunistic infections following pharmacological immunosuppression induced by CLN-978.

[0294] In summary, these results indicate that a 4-week administration of 0.3 mg / kg / day of CLN-978 via once-weekly SC injection was tolerable in cynomolgus monkeys. Notable changes included non-harmful CLN-978-related clinicopathological findings, decreased thymic and splenic weight, and microscopic findings of the spleen, rheological, mandibular, and mesenteric lymph nodes, as well as GALT (severe decrease in germinal centers and / or marked to severe decrease in CD20-positive cells). These microscopic changes are interpreted as pharmacological effects of CLN-978. Based on these results, the HNSTD dose was considered to be 0.3 mg / kg / day.

[0295] Example 12: Development of rationale for starting dose and schedule

[0296] Using information from in vitro binding and in vitro cytotoxicity assays, in vivo mouse xenograft models, and cynomolgus monkey PK and toxicity studies, we developed SP models for CLN-978 and used them to predict safe starting doses and effective doses in humans. A total of four SP models were developed within the overall SP modeling approach. In vitro models were used to obtain cytotoxicity data and to predict the cross-linking of CLN-978. on Based on the determination of the threshold and the achievement of 50% cytotoxicity in the assay, a clinical starting dose criterion was established. A mouse SP model developed using mouse xenograft data was used to define the effective dose criterion by comparing the model output with the TGI response. A cynomolgus monkey model was developed to obtain PK test data. Allometric scaling was used to convert the cynomolgus monkey PK parameters to those of humans. A human NHL model was constructed and used to predict the safe starting dose and effective dose of CLN-978 after SC administration. The main output of this model predicted the formation of a tripolecular complex (CD19:CLN-978:CD3, "trimer"). The human model diagram is shown in Figure 17.

[0297] In vitro cytotoxicity assays involved co-culturing PBMCs and Raji cells with different E:T ratios and different concentrations of the drug (CLN-978 or blinatumomab). The binding activity / crosslinking parameter kon2 was determined based on the hypothesis that trimer formation drives cytotoxicity, and that the trimer-versus-cytotoxicity curves for both blinatumomab and CLN-978 should generally overlap. on The binary range was investigated. The relationship between the resulting mean trimer ("TpT") per T cell versus cytotoxicity % over incubation periods across the assay concentration range was also examined. Visual inspection revealed that the trimer-cytotoxicity curves best matched when the association rate constant (k) of both molecules was the same. on 2) was determined to be when it was estimated to be 30 dm2 / nmol / s. This value was applied to subsequent modeling of CLN-978. It was determined that achieving an average TpT ​​resulting in 50% of the cytotoxicity observed in the in vitro assay would be the criterion for the starting dose, and therefore 60 TpT (maximum observed cytotoxicity 48.4%) was initially used to determine the starting dose.

[0298] A pharmacokinetic (PK) study of CLN-978 was conducted in mice, collecting data from both IV and SC administrations. Based on AUC comparisons in non-compartmental analysis, the bioavailability of SC was determined to be 100%, and the bioavailability parameter of the model was fixed to this value. Mouse PKs were used only in the mouse model and were not converted to humans. A mouse TGI study was performed using CLN-978 administered weekly with SC at 0.3 μg / kg, 1 μg / kg, and 10 μg / kg. Using the mouse model, a dose-range TGI study was simulated to predict the corresponding TpT. The 10 μg / kg dose on day 21 resulted in the highest TGI and a clear distinction from other doses, and was therefore selected as the criterion for defining the effective dose in the human model. This model also predicted the highest mean TpT at 10 μg / kg. The resulting mean TpT within the peripheral compartment on day 21 was 472, which was adopted as the criterion for the effective dose.

[0299] A dose-range PK study was conducted in cynomolgus monkeys with four dosing groups (0.1 and 1 mg / kg, both IV and SC, n=2 per dose group). The cynomolgus monkey model was parameterized to include circulating and peripheral T cells and normal B cells, and reported affinity values ​​were used. Since CLN-978 is administered in multiple doses in a clinic setting, B cell depletion was explicitly included in the model to better convert from the single dose administered in this study and to capture differences in TMDD after multiple dosing due to B cell depletion. Central and peripheral volumes were fixed to physiological values. Bioavailability was fixed to 100% based on internal exposure analysis between IV and SC doses. Allometric scaling was used to determine human PK parameters.

[0300] A certain dose range was simulated using a human NHL model. The dosing frequency was quarter-weekly (QW) SC administration. The criteria for the starting dose and effective dose were based on the mean TpT (Time Point Tolerance) of cancer cells in the tumor at day 28, corresponding to the end of the fourth dose. Day 28 was chosen as the end day of the administration cycle because trimer formation approaches a steady state at this time. Based on the results of preclinical modeling, the criteria were set at 60 TpT for the starting dose and 472 TpT for the effective dose. Based on the human model simulation, 240 μg is the recommended starting dose and 1850 μg is the predicted effective dose. Predicted PK values ​​and the actual mean (AUC divided by time) TpT are presented in Figures 18A and 18B, and the exposure results are reported in Table 8.

[0301] [Table 8]

[0302] Sensitivity analysis was also performed to investigate the impact of potential variability in the patient population on the starting dose. T cell count in the tumor, drug distribution within the tumor, and tumor volume are parameters that can vary among NHL patients. For example, a threefold decrease in the half-life of CLN-978 tumor distribution (corresponding to a threefold increase in tumor distribution) might suggest a threefold reduction in the starting dose (from 240 μg to 90 μg). Based on sensitivity analysis and a careful decision to target EC20 (7 mean trimers per T cell in the tumor) instead of EC50, 30 μg of SC QW was defined as the recommended starting dose for the FIH trial.

[0303] Example 14: Clinical trial of tAB0050 in patients with relapsed or refractory (R / R) B-cell non-Hodgkin lymphoma (B-NHL). This example describes the first phase 1, open-label, multicenter, human, dose-escalation, and dose-expansion study of tAB0050 in patients with relapsed or refractory (R / R) B-cell non-Hodgkin lymphoma (B-NHL).

[0304] Adult patients (≥18 years of age) with any of the following CD19+ B-cell histological structures: diffuse large B-cell lymphoma – novel or transformed, high-grade B-cell lymphoma, primary mediastinal large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, or marginal zone lymphoma (nodal, extranodal, or mucosal) who are not available with approved standard therapy. Other key eligibility criteria include two or more lines of prior chemotherapy and an Eastern Cooperative Oncology Group performance status of 0–2. Key exclusion criteria include allogeneic hematopoietic stem cell transplantation or prior treatment with any other trial CD19×CD3 T-cell engager. Previous CAR-T therapy, unconjugated or radioconjugated CD19 mAbs, and CD19 antibody-drug conjugates are permitted.

[0305] This study consists of two parts: 1) a dose escalation phase initiating escalation titration in a single-patient cohort, followed by a conventional 3+3 dose escalation design to identify the recommended Phase 2 dose (RP2D); and 2) a dose expansion phase further characterizing the safety and preliminary efficacy of tAB0050 in disease-specific cohorts at the RP2D. Figure 19 shows the two parts of the study. tAB0050 is administered subcutaneously (SC) once weekly (QW) in 28-day cycles up to a maximum of 24 cycles of treatment for progressive disease, unacceptable toxicity, or other conditions. Based on new data, and with approval from the Institutional Review Board, a lower-frequency dosing schedule may be implemented from cycle 4 onward. Safety, pharmacokinetic (PK), pharmacodynamic, and preliminary efficacy evaluations will guide the selection of doses and schedules for further evaluation. The primary endpoint is safety. The primary secondary endpoints include PK, anti-drug antibodies, overall response rate, duration of response, time to response, time to subsequent anti-lymphoma therapy, progression-free survival, and overall survival.

[0306] Up to approximately 90 patients (30 receiving dose escalation and 60 receiving dose expansion) will be enrolled in about six facilities in the United States, considering both community centers and academic centers.

[0307] The exam is currently open for registration (NCT05879744).

[0308] The following clinical trials will be conducted as described below. [Table 9]

[0309] Evaluation items

[0310] Primary evaluation criteria: 1. Safety and tolerability of tAB0050 Incidence and severity of adverse events (AEs), particularly noteworthy adverse events (AESls), and serious adverse events (SAEs); incidence of dose interruption and administration delays. 2. Specify the dosage regimen for tAB0050. Dose-limiting toxicity (DLTs)

[0311] Secondary evaluation criteria: 1. Preliminary efficacy evaluation of tAB0050 in patients with selected R / R B-NHL histological subtypes. Overall response rate (ORR), complete response (CR), duration of response (DOR) 2. Select the PK parameter for tAB0050. PK parameters: Area under the concentration-time curve, maximum concentration, and half-life. 3. Immunogenicity of tAB0050 and potential effects on drug exposure Incidence of antidrug antibodies against tAB0050

[0312] Eligibility Criteria • Target age for the exam: 18 years and older • Target gender for the test: All Based on gender: No • Acceptance of healthy volunteers: None

[0313] standard

[0314] Selection criteria: · Eastern Cooperative Oncology Group (ECOG) PS≦2 • A diagnostic record of one of the following CD19+ B-cell neoplasms according to the WHO classification (Swerdlow et al., 2016) or WHO classification 2008: a. Diffuse large B-cell lymphoma (DLBCL) - novel or transformed type b. High-grade B-cell lymphoma c. Primary mediastinal large B-cell lymphoma d. Follicular lymphoma (FL) e. Mantle cell lymphoma f. Marginal zone lymphoma (nodal, extranodal, or mucosal-associated) • Relapsed, progressive, and / or refractory disease after at least two lines of treatment. • In the case of the Part B expanded cohort: a. Cohort B1: R / R DLBCL relapsed after at least two prior therapies including CD20 monoclonal antibody and anthracycline. b. Cohort B2: R / R FL (grade 1-3a) that relapsed after at least two prior therapies including CD20 monoclonal antibody and alkylating agent. c. Cohort B3: Other R / R B-NHL (excluding DLBCL and FL). • Measurable disease is present as one or more measurable nodular lesions (long axis > 1.5 cm and short axis > 1.0 cm) or one or more measurable extranodal lesions (long axis > 1.0 cm) on computed tomography (CT) scans or magnetic resonance imaging (MRI), and baseline fluorodeoxyglucose-positron emission tomography (FDG-PET) scans show positive lesion(s) that match the anatomical tumor site defined on CT or MRI. • Test parameters including the following: a. Lymphocyte count < 5 × 10 9 / L b. Platelet count ≧75×10 9 / L c. Absolute neutrophil count ≥ 1.0 × 10 9 If involvement of the bone marrow is confirmed, supportive therapy with growth factors is permitted. d. Hemoglobin ≥ 9 g / dL, with or without blood transfusion. e. Creatinine clearance ≥ 45 mL / min f. Total bilirubin ≤ 1.5 times the upper limit of normal (ULN) (excluding patients with confirmed Gilbert's syndrome) g. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) ≤ 3.0 × ULN (unless due to hepatic involvement in lymphoma)

[0315] Exclusion criteria: • Known involvement of the CNS due to primary CNS lymphoma or lymphoma at the time of screening. • Known past or present malignant tumors other than those diagnosed according to the selection criteria • Known clinically significant heart disease • Serious central nervous system disorders • History of organ allogeneic transplantation • Confirmed history of or ongoing autoimmune disorders or other diseases requiring continued immunosuppression • Active hepatitis C virus (HCV), hepatitis B virus (HBV), or known human immunodeficiency virus (HIV) infection • Live viral vaccines within 28 days of the first dose of tAB0050, during treatment, and until the end of the final dose of tAB0050. • Known active, clinically significant bacterial, viral, fungal, mycobacterial, parasitic, or other infections, including coronavirus infection (COVID-19), at the time of registration or within 7 days of the first dose of tAB0050. • Previous treatment by any of the following: a. Allogeneic HSCT b. Autologous HSCT within 30 days prior to the first administration of tAB0050 Chimeric antigen receptor T-cell therapy (CAR-T) within 30 days prior to the first dose of c.tAB0050 d. Any clinical trial CD19×CD3 T cell engagers (TCEs) Unconjugated CD19 monoclonal antibody four weeks prior to the first dose of e.tAB0050 Radioconjugated or CD19 antibody-drug conjugate 12 weeks prior to the first dose of f.tAB0050 Prior to the first dose of g.tAB0050, for a period of 4 weeks or 5 half-lives, whichever is shorter, a monoclonal antibody, chemotherapy, or other investigational drug used in a clinical trial or standard treatment. h. Radiotherapy (XRT) (excluding local treatment for symptom control), within 4 weeks of the first dose of tAB0050 • Women who are pregnant, breastfeeding, planning to become pregnant, or who may become pregnant. • Male patients who plan to conceive a child or donate semen within 120 days of the last dose of the study drug.

[0316] Clinical safety and efficacy After the first patient treated at a dose level of 30 μg experienced a grade ≥ 2 adverse event, enrollment at that dose level was expanded to three patients, as outlined below.

[0317] Patient 1 was a 25-year-old male with relapsed T-cell-rich B-cell lymphoma. Previous treatments included rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) followed by maintenance rituximab, then rituximab, ifosfamide, carboplatin, and etoposide (R-ICE), and most recently pembrolizumab. The patient received the first dose of CLN-978 (30 μg SC) after premedication with dexamethasone, diphenhydramine, famotidine, and acetaminophen. Approximately 10 hours after administration, the patient developed grade 1 CRS, manifesting as a fever up to 102.9°F (grade 2), accompanied by a dull headache, chills, and severe shivering, for which the patient was treated with acetaminophen and meperidine. The fever resolved within 2.5 hours, and the patient was hospitalized and observed for up to 48 hours post-administration, with no sequelae or recurrence. A fever of up to 100.8°F (grade 1) recurred approximately 50 hours after administration and again improved with acetaminophen. In both cases, tests for infection were negative. Other clinically significant adverse events included transient lymphopenia (most severe at grade 4, 24 hours after administration) and leukopenia (maximum grade 2, C1D5–C1D7), which are consistent with the mechanism of action of CLN-978. The patient also experienced associated grade 1 injection site reactions (C1D13–C2D8), grade 1 intermittent headache (C1D2–continued), and grade 1 fatigue (C1D2–C1D6, C1D9–C1D13). Other TEAEs included grade 1 hyperglycemia (C1D2-C1D3), grade 1 ALT (C1D2-C1D15), grade 1 decreased creatinine (C1D3-C1D4), and grade 1 nausea (C1D10-C1D12), all of which were considered unrelated to the investigational treatment. The patient was readmitted for a second dose of CLN-978 without complications. Treatment was discontinued after several doses of CLN-978.

[0318] Patient 2 was a 75-year-old male with relapsed follicular lymphoma. Previous treatments included bendamustine and rituximab (BR), followed by maintenance rituximab, R-CHOP, and lenalidomide and rituximab. The patient received the first dose of CLN-978 (30 μg SC) after premedication with dexamethasone, diphenhydramine, famotidine, and acetaminophen. Approximately 48 hours after administration, the patient developed grade 1 CRS, manifesting as a fever up to 103.1°F (grade 2), for which acetaminophen was administered. The fever subsided quickly, and tests for infection were negative. Other TEAEs included associated transient lymphopenia (most severe, grade 4, 24 hours after administration) (consistent with the mechanism of action of CLN-978; observed from C1D2), and grade 1 pruritus (C1D22–C2D1), as well as grade 1 intermittent hypomagnesemia (C1D2–C1D8) and grade 1 hypocalcemia at C1D4–C1D8 (both considered unrelated to the study drug). The patient received a total of 24 weekly doses of CLN-978. Imaging studies after 2 and 4 cycles of treatment showed stable disease, and a re-evaluation scan after 6 cycles is planned.

[0319] Patient 3 was a 68-year-old male with relapsed mantle cell lymphoma. Previous treatments included maintenance rituximab and lenalidomide, rituximab, dexamethasone, cytarabine, and cisplatin (R-DHAP) following BR, as well as maintenance rituximab following high-dose chemotherapy and autologous stem cell transplantation. The patient received the first dose of CLN-978 (30 μg SC) after premedication with prednisone, diphenhydramine, famotidine, and acetaminophen. Approximately 26 hours after administration, the patient experienced a fever of 100.4°F (grade 1, which was considered unrelated to the treatment) with chills, for which an additional dose of acetaminophen was administered. The fever subsided quickly, and the injection was tested negative. Transient lymphopenia (consistent with the mechanism of action of CLN-978), most severe at grade 3, began to be observed from C1D2, 24 hours after administration. The remainder of the 48-hour hospital stay was uneventful. At a scheduled outpatient follow-up 96 hours after administration, the patient was asymptomatic but appeared unwell, exhibiting tremors but fully disoriented (immune effector cell encephalopathy [ICE] score 9 / 10), and clinically hypovolemic with a systolic blood pressure in the 90s. The patient was admitted to the hospital for emergency fluid therapy and diagnostic evaluation. Blood pressure normalized rapidly after infusion, and clinical laboratory tests confirmed a diagnosis of acute grade 1 influenza A virus infection (C1D4-C1D6), for which oseltamivir was used. Treatment was initiated and the patient was discharged. The palpable area of ​​mantle cell lymphoma in the right mandible was reported to have recovered from baseline, but there was no clinical or laboratory evidence of tumor lysis syndrome or cytokine release syndrome. Tremor (grade 1, thought to be unrelated to the investigational treatment) was further treated with corticosteroids, improved after 24 hours, and resolved by C1D6. Further treatment was postponed for one week to allow the patient to complete 5 days of oseltamivir and fully recover from influenza. The patient was readmitted to receive a second dose of CLN-978. The patient also experienced associated grade 2 confusion (C2D14-C2D15), but its association with substantial corticosteroid use cannot be ruled out.Other TEAEs included grade 1 GERD (C1D14–present), grade 1 intermittent blurred vision (C1D16–present), grade 2 agitation (C1D22–present), and grade 3 vascular access complications (C2D4; the patient had a history of grade 3 DVT). All of these were considered unrelated to the investigational treatment. Treatment was discontinued after 7 doses of CLN-978 due to grade 2 agitation and grade 2 confusion, but a link to high-dose corticosteroid use cannot be ruled out. Imaging studies obtained after 2 cycles of treatment showed a complete metabolic response.

[0320] Clinical Pharmacokinetics

[0321] Clinical pharmacokinetic data were limited to patients 2 and 3. These data are provided in Figure 20 and show that the preclinical PK model used for B-cell response simulation generally predicts the clinical pharmacokinetic data observed in two patients who received 30 μg of SC on a weekly schedule in the clinical trial.

[0322] Clinical Pharmacodynamics

[0323] Clinical pharmacodynamic data for CLN-978 were generated for each patient. Figures 21A-21F show graphs illustrating B cell depletion, T cell activation, and cytokine analysis for patients 1, 2, and 3.

[0324] Peripheral blood was examined for the presence of B cells using a quantitative flow cytometry assay (TBNK [T cell, B cell, NK cell] panel). In two of the two B-NHL patients (Patient 1 and 3) whose B cells were detectable at baseline, B cell depletion occurred within 96 hours of a 30 μg dose administered subcutaneously once weekly (QW) and remained low but detectable at the remaining time points tested (Figure 21A). Patient 2 had low peripheral blood B cell counts at baseline, which remained low throughout the course of treatment. Interestingly, in Patient 1, the B cell lymphocyte count remained low at the safety follow-up visit on day 149 and showed persistent peripheral B cell depletion 90 days after the last dose of CLN-978 was administered on day 59.

[0325] Peripheral CD3+ cell counts were also assessed using the same TBNK panel. All three patients showed a decrease in CD3+ cell counts within 4 days post-treatment, which was likely caused by the recruitment of peripheral T cells to CD19+ tissue (Figure 21B). Recovery from the initial post-treatment decrease in the CD3+ event was observed, indicating peripheral T cell redistribution.

[0326] Further phenotypic characterization of peripheral T cells showed variability in the overall frequency of CD4+ (Figure 21C) and CD8+ (Figure 21D) T cells across the time points examined, but all three patients returned to baseline frequencies. T cell activation was examined via CD69 and PD-1 expression in both CD4 and CD8 T cells (Figure 21E). In patient 3, the frequency of CD69+ T cells increased before C1D8 administration, but expression returned to baseline levels by C1D15. There was significant inter-patient variability in PD-1 expression on T cells among baseline patients, but no significant intra-patient changes were observed during the course of treatment.

[0327] Changes in serum cytokines were also monitored in patients during the course of treatment. Patient 1 showed transient increases in IL-6, IL-10, IL-1Rα, and MCP-1 levels 6 hours after the first dose of CLN-978 (C1D1 6 hours), which were consistent with the clinical symptoms of Grade 1 CRS (Figure 21F). Cytokine levels returned to baseline at the remaining time points evaluated. Both Patients 2 and 3 showed limited changes in cytokine expression compared to baseline, despite Patient 2 having clinical symptoms of Grade 1 CRS at 48 hours post-administration. Notably, there was no expression of IL-1β, IFNγ, or IL-2 at any of the time points tested (data not shown).

[0328] Taken together, these data demonstrate B cell depletion in two of the two B-NHL patients with detectable B cells at baseline, as well as peripheral T cell recruitment in three patients after treatment with CLN-978.

[0329] The clinical observations of the three patients are further summarized in Figure 23.

[0330] Patient 3 was further analyzed. As shown in Figure 24, a 7.4 × 1.7 cm tumor in the left mandibular ramus and adjacent muscle of Patient 3 was palpable on physical examination and visible in the oral cavity at baseline before treatment. The tumor was no longer recognizable on physical examination 96 hours after the first dose of CLN-978. After seven doses of CLN-978, the mass decreased by 66% (sum of product diameters (SPD)), and the hypermetabolic disease improved (standardized uptake (SUV)), which was consistent with a partial response to treatment.

[0331] Embedding by reference All publications and patents cited throughout this Specified (including all patents, patent applications, scientific publications, manufacturers' specifications, instructions, etc.) are incorporated herein by reference for any purpose, whether above or below. In the event that any material incorporated by reference conflicts with or is inconsistent with this Specified, this Specified shall prevail over all such material.

[0332] Equal parts This disclosure can be embodied in other specific forms without departing from its spirit or essential features. Therefore, the embodiments described above should be considered, in all respects, illustrative rather than limiting, the disclosure described herein. Accordingly, the scope of this disclosure is indicated not by the foregoing description but by the appended claims, and all modifications that fall within the meaning and scope of equivalence of the claims are intended to be encompassed within the claims.

Claims

1. A method for treating a person requiring treatment for relapsed and / or refractory non-Hodgkin lymphoma (NHL), wherein the person: a) A first antigen-binding site comprising a heavy chain variable domain (VH) that binds to human CD19 and includes complementarity-determining regions HCDR1, HCDR2, and HCDR3, and a light chain variable domain (VL) that includes complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of SEQ ID NOs: 4, 5, 7, 8, 9, and 10, and b) The method comprising administering a polyspecific binding protein comprising a second antigen-binding domain comprising a VH that binds to CD3 and includes complementarity-determining regions HCDR1, HCDR2, and HCDR3, and a VL that includes complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences described in SEQ ID NOs. 15, 16, 18, 19, 20, and 21.

2. The method according to claim 1, wherein the individual has relapsed after treatment with CD19-targeted therapy or has shown resistance to the CD19-targeted therapy.

3. The method according to claim 2, wherein the CD19-targeted therapy comprises blinatumomab.

4. The method according to claim 2, wherein the CD19-targeted therapy includes CD19 CAR-T cell therapy.

5. The method according to any one of claims 1 to 4, wherein the VH of the first antigen-binding domain comprises an amino acid sequence identical to that of SEQ ID NO: 1 by at least 85%, at least 90%, at least 95%, at least 99%, or 100%, and the VL of the first antigen-binding domain comprises an amino acid sequence identical to that of SEQ ID NO: 2 by at least 85%, at least 90%, at least 95%, at least 99%, or 100%.

6. The method according to any one of claims 1 to 5, wherein the VH of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 1, and the VL of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO:

2.

7. The method according to any one of claims 1 to 6, wherein the first antigen-binding site includes an amino acid sequence that is at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of SEQ ID NO:

11.

8. The method according to any one of claims 1 to 7, wherein the first antigen-binding site comprises the amino acid sequence of SEQ ID NO:

11.

9. The method according to any one of claims 1 to 8, wherein the VH of the second antigen-binding site comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to that of SEQ ID NO: 12, and the VL of the second antigen-binding site comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to that of SEQ ID NO:

13.

10. The method according to any one of claims 1 to 9, wherein the VH of the second antigen-binding site comprises the amino acid sequence of SEQ ID NO: 12, and the VL of the second antigen-binding site comprises the amino acid sequence of SEQ ID NO:

13.

11. The method according to any one of claims 1 to 10, wherein the second antigen-binding site comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:

22.

12. The method according to any one of claims 1 to 11, wherein the second antigen-binding site includes the amino acid sequence of SEQ ID NO:

22.

13. The method according to any one of claims 1 to 12, wherein the multispecific binding protein further comprises a half-life extension domain.

14. The method according to claim 13, wherein the half-life extension domain includes a third antigen-binding site that binds to human serum albumin.

15. The method according to claim 13 or claim 14, wherein the half-life extension domain is not located between the first antigen-binding site and the second antigen-binding site in the polypeptide chain.

16. The method according to claim 14 or claim 15, wherein the third antigen-binding site is a VH comprising complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 each comprise the amino acid sequences of SEQ ID NOs. 26, 27, and 29.

17. The method according to claim 16, wherein the VH of the third antigen-binding site comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:

24.

18. The method according to claim 16 or claim 17, wherein the VH of the third antigen-binding site comprises the amino acid sequence of SEQ ID NO:

24.

19. The aforementioned polyspecific binding protein is oriented from the N-terminus to the C-terminus. a) A third antigen-binding site comprising an amino acid sequence that binds to human serum albumin and is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 24, b) A first antigen-binding site comprising an amino acid sequence that binds to CD19 and is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 11, and c) The method according to any one of claims 14 to 18, comprising a second antigen-binding site that binds to human CD3 and includes an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:

22.

20. The aforementioned polyspecific binding protein is oriented from the N-terminus to the C-terminus. a) A third antigen-binding site that binds to human serum albumin and contains the amino acid sequence of SEQ ID NO: 24, b) A first antigen-binding site that binds to CD19 and includes the amino acid sequence of SEQ ID NO: 11, and c) The method according to any one of claims 14 to 19, comprising a second antigen-binding site that binds to human CD3 and includes the amino acid sequence of SEQ ID NO:

22.

21. The method according to any one of claims 1 to 20, wherein the polyspecific binding protein comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:

41.

22. The method according to any one of claims 1 to 21, wherein the polyspecific binding protein comprises the amino acid sequence of SEQ ID NO:

41.

23. The method according to any one of claims 1 to 22, wherein the administration results in B cell depletion in the individual within 96 hours of administration of the polyspecific binding protein.

24. The method according to any one of claims 1 to 23, wherein the administration results in persistent B cell depletion that lasts for at least 90 days after the administration of the polyspecific binding protein.

25. The method according to any one of claims 1 to 24, wherein the administration results in persistent B cell depletion that lasts for at least 90 days after the final administration of the polyspecific binding protein.

26. The method according to any one of claims 1 to 25, wherein the individual is administered a dose of at least 20 μg of the polyspecific binding protein.

27. The method according to any one of claims 1 to 25, wherein the individual is administered a dose of at least 30 μg of the polyspecific binding protein.

28. The method according to any one of claims 1 to 25, wherein the individual is administered a dose of at least 40 μg of the polyspecific binding protein.

29. The method according to any one of claims 1 to 25, wherein the individual is administered a dose of at least 50 μg of the polyspecific binding protein.

30. The method according to any one of claims 1 to 25, wherein the individual is administered a dose of at least 60 μg of the polyspecific binding protein.

31. The method according to any one of claims 1 to 30, wherein the multispecific binding protein is administered once a week.

32. The method according to any one of claims 1 to 30, wherein the multispecific binding protein is administered once every two weeks.

33. A method for performing treatment on an individual having a CD19 low-expression cancer that requires treatment, wherein the individual a) A first antigen-binding site comprising a heavy chain variable domain (VH) that binds to human CD19 and includes complementarity-determining regions HCDR1, HCDR2, and HCDR3, and a light chain variable domain (VL) that includes complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of SEQ ID NOs: 4, 5, 7, 8, 9, and 10, and b) The method comprising administering a polyspecific binding protein comprising a second antigen-binding domain comprising a VH that binds to CD3 and includes complementarity-determining regions HCDR1, HCDR2, and HCDR3, and a VL that includes complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences described in SEQ ID NOs. 15, 16, 18, 19, 20, and 21.

34. The method according to claim 33, wherein the CD19 low-expression cancer is classified as low-expression by flow cytometry.

35. The method according to claim 33 or 34, wherein the CD19-low-expressing cancer is classified by having approximately 325 to approximately 17,000 CD19 molecules per cell.

36. The method according to claim 33 or 34, wherein the CD19 low-expression cancer is classified by having fewer than approximately 3,000 CD19 molecules per cell.

37. The method according to any one of claims 33 to 36, wherein the VH of the first antigen-binding domain comprises an amino acid sequence identical to that of SEQ ID NO: 1 by at least 85%, at least 90%, at least 95%, at least 99%, or 100%, and the VL of the first antigen-binding domain comprises an amino acid sequence identical to that of SEQ ID NO: 2 by at least 85%, at least 90%, at least 95%, at least 99%, or 100%.

38. The method according to any one of claims 33 to 37, wherein the VH of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 1, and the VL of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO:

2.

39. The method according to any one of claims 33 to 38, wherein the first antigen-binding site includes an amino acid sequence that is at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of SEQ ID NO:

11.

40. The method according to any one of claims 33 to 39, wherein the first antigen-binding site comprises the amino acid sequence of SEQ ID NO:

11.

41. The method according to any one of claims 33 to 40, wherein the VH of the second antigen-binding site comprises an amino acid sequence identical to that of SEQ ID NO: 12 by at least 85%, at least 90%, at least 95%, at least 99%, or 100%, and the VL of the second antigen-binding site comprises an amino acid sequence identical to that of SEQ ID NO: 13 by at least 85%, at least 90%, at least 95%, at least 99%, or 100%.

42. The method according to any one of claims 33 to 41, wherein the VH of the second antigen-binding site comprises the amino acid sequence of SEQ ID NO: 12, and the VL of the second antigen-binding site comprises the amino acid sequence of SEQ ID NO:

13.

43. The method according to any one of claims 33 to 42, wherein the second antigen-binding site comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:

22.

44. The method according to any one of claims 33 to 43, wherein the second antigen-binding site comprises the amino acid sequence of SEQ ID NO:

22.

45. The method according to any one of claims 33 to 44, wherein the multispecific binding protein further comprises a half-life extension domain.

46. The method according to claim 45, wherein the half-life extension domain includes a third antigen-binding site that binds to human serum albumin.

47. The method according to claims 33 to 46, wherein the half-life extension domain is not located between the first antigen-binding site and the second antigen-binding site in the polypeptide chain.

48. The method according to claim 46 or claim 47, wherein the third antigen-binding site is a VH comprising complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 each comprise the amino acid sequences of SEQ ID NOs. 26, 27, and 29.

49. The method according to claim 48, wherein the VH of the third antigen-binding site comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:

24.

50. The method according to claim 48 or claim 49, wherein the VH of the third antigen-binding site comprises the amino acid sequence of SEQ ID NO:

24.

51. The aforementioned polyspecific binding protein is oriented from the N-terminus to the C-terminus. a) A third antigen-binding site comprising an amino acid sequence that binds to human serum albumin and is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 24, b) A first antigen-binding site comprising an amino acid sequence that binds to CD19 and is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 11, and c) The method according to any one of claims 48 to 50, comprising a second antigen-binding site that binds to human CD3 and includes an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:

22.

52. The aforementioned polyspecific binding protein is oriented from the N-terminus to the C-terminus. a) A third antigen-binding site that binds to human serum albumin and contains the amino acid sequence of SEQ ID NO: 24, b) A first antigen-binding site that binds to CD19 and includes the amino acid sequence of SEQ ID NO: 11, and c) The method according to any one of claims 48 to 51, comprising a second antigen-binding site that binds to human CD3 and includes the amino acid sequence of SEQ ID NO:

22.

53. The method according to any one of claims 33 to 52, wherein the polyspecific binding protein comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:

41.

54. The method according to any one of claims 33 to 53, wherein the polyspecific binding protein comprises the amino acid sequence of SEQ ID NO:

41.

55. The method according to any one of claims 33 to 54, wherein the administration results in B cell depletion in the individual within 96 hours of administration of the polyspecific binding protein.

56. The method according to any one of claims 33 to 55, wherein the administration results in persistent B cell depletion that lasts for at least 90 days after the administration of the polyspecific binding protein.

57. The method according to any one of claims 33 to 56, wherein the administration results in persistent B cell depletion that lasts for at least 90 days after the final administration of the polyspecific binding protein.

58. The method according to any one of claims 33 to 57, wherein the individual is administered a dose of at least about 30 μg of the polyspecific binding protein.

59. The method according to any one of claims 33 to 57, wherein the individual is administered a dose of at least about 40 μg of the polyspecific binding protein.

60. The method according to any one of claims 33 to 57, wherein the individual is administered a dose of at least about 50 μg of the polyspecific binding protein.

61. The method according to any one of claims 33 to 57, wherein the individual is administered a dose of at least about 60 μg of the polyspecific binding protein.

62. The method according to any one of claims 33 to 61, wherein the multispecific binding protein is administered once a week.

63. The method according to any one of claims 33 to 61, wherein the multispecific binding protein is administered once every two weeks.

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