WO2020219978A1 - Bcma/cd3 bispecific trivalent t-cell engaging (tce) antibodies and their use to treat hematological malignancies - Google Patents
Bcma/cd3 bispecific trivalent t-cell engaging (tce) antibodies and their use to treat hematological malignancies Download PDFInfo
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- WO2020219978A1 WO2020219978A1 PCT/US2020/029954 US2020029954W WO2020219978A1 WO 2020219978 A1 WO2020219978 A1 WO 2020219978A1 US 2020029954 W US2020029954 W US 2020029954W WO 2020219978 A1 WO2020219978 A1 WO 2020219978A1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/02—Antineoplastic agents specific for leukemia
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/35—Valency
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
Definitions
- the current invention is directed to optimization of bispecific trivalent anti-BCMA/anti-CD3 T cell engaging antibodies (TCE) for the treatment of oncological conditions, hematological malignancies, and autoimmune disorders.
- TCE T cell engaging antibodies
- BCMA B cell maturation antigen
- B cell maturation antigen is a member of the tumor necrosis factor (TNF) receptor superfamily that binds to a proliferation inducing ligand (APRIL) with 100-fold higher affinity than it binds to B cell-activating factor (BAFF) (Day ES, et al.
- TNF tumor necrosis factor
- APRIL proliferation inducing ligand
- BAFF B cell-activating factor
- BCMA BCMA
- BCMA x CD3 T cell bispecific engagers and a BCMA antibody drug conjugate are also currently being investigated in Phase 1 clinical studies in MM (see, e.g., Topp MS, et al., J Clin Oncol.2016;34 (suppl; abstr TPS8067); Girgis S, et al., Blood.2016;128(22):5668; Cohen AD, et al., American Society for Hematology 58th Annual Meeting and Exposition.2016b Dec 3-6; San Diego, CA: Abstract 1148). Although disulfide bonds are important for folding and stability, their optimized formation is difficult to achieve.
- the present invention is directed to an optimized bispecific trivalent anti-BCMA/anti-CD3 T Cell Engaging Antibody (TCE) comprising a heavy chain (anti-BCMA and anti-CD3) A (HC) (SEQ ID NO:1); a heavy chain (anti-BCMA) B (HC') (SEQ ID NO:3); a light chain (anti-CD3) A (LC'') (SEQ ID NO:2); a light chain (anti-BCMA) A (LC''') (SEQ ID NO:4); and, a light chain (anti-BCMA) B (LC'''') (SEQ ID NO:4), wherein the heavy chain (anti-BCMA and anti-CD3) A (HC) (SEQ ID NO:1) comprises at least one intra-chain disulfide bond selected from the group consisting of C22-C96, C143-C199, C252-C320, C368-C424, C485- C545, and C591-C649, and wherein the heavy chain (HC) (
- the present invention is further directed to an optimized bispecific trivalent anti-BCMA/anti- CD3 T Cell Engaging Antibody (TCE) comprising a heavy chain (anti-BCMA and anti-CD3) A (HC) (SEQ ID NO:1); a heavy chain (anti-BCMA) B (HC') (SEQ ID NO:3); a light chain (anti-CD3) A (LC'') (SEQ ID NO:2); a light chain (anti-BCMA) A (LC''') (SEQ ID NO:4); and, a light chain (anti-BCMA) B (LC'''') (SEQ ID NO:4), wherein the heavy chain (anti-BCMA and anti-CD3) A (HC) (SEQ ID NO:1) comprises at least one inter- chain disulfide bond selected from the group consisting of ⁇ C219-C216''' between itself and the light chain (anti-BCMA) A (LC''') (SEQ ID NO:4); ⁇ C444-C232’’ between
- the present invention is also directed to five (5) precursor peptides including signal sequences for production and assembly of the optimized TCE described herein including, (i) for the production of mature heavy chain (anti-BCMA and anti-CD3) A (HC) (SEQ ID NO:1) and assembly of the TCE comprising SEQ ID NO:5, and (ii) for the production of mature heavy chain (anti-BCMA) B (HC') (SEQ ID NO:3) and assembly of the TCE, comprising SEQ ID NO:7, and (iii) for the production of mature light chain (anti- CD3) A (LC'') (SEQ ID NO:2) and assembly of the TCE, comprising SEQ ID NO:6, and (iv) for the production of mature light chain (anti-BCMA) A (LC''') (SEQ ID NO:4) or light chain (anti-BCMA) B (LC''') (SEQ ID NO:4), and assembly of the TCE, comprising SEQ ID NO:8.
- HC mature heavy chain
- the present invention is also directed to whole precursor DNA for optimized CHO cell production, i.e., a nucleic acid sequence for production of mature heavy chain (anti-BCMA and anti-CD3) A (HC) (SEQ ID NO:1) and assembly of the TCE, comprising SEQ ID NO:15; a nucleic acid sequence for production of mature heavy chain (anti-BCMA) B (HC') (SEQ ID NO:3) and assembly of the TCE comprising SEQ ID NO:17; a nucleic acid sequence for production of mature light chain (anti-CD3) A (LC'') (SEQ ID NO:2) and assembly of the TCE, comprising SEQ ID NO:16; and, a nucleic acid sequence for production of mature light chain (anti-BCMA) A (LC''') (SEQ ID NO:4) or mature light chain (anti-BCMA) B (LC'''') (SEQ ID NO:4), and assembly of the TCE, comprising SEQ ID NO:18.
- HC mature heavy chain
- the present invention is further directed to nucleic acid sequence coding region, with signal peptide, translation start and termination codon for production and assembly of the TCE including a nucleic acid sequence for production of mature heavy chain (anti-BCMA and anti-CD3) A (HC) (SEQ ID NO:1) and assembly of the TCE, comprising SEQ ID NO:19; a nucleic acid sequence for production of mature heavy chain (anti-BCMA) B (HC') (SEQ ID NO:3) and assembly of the TCE, comprising SEQ ID NO:21; a nucleic acid sequence for production of mature light chain (anti-CD3) A (LC'') (SEQ ID NO:2) and assembly of the TCE, comprising SEQ ID NO:20; and, a nucleic acid sequence for production of mature light chain (anti-BCMA) A (LC''') (SEQ ID NO:4) or mature light chain (anti-BCMA) B (LC'''') (SEQ ID NO:4), and assembly of the TCE, comprising
- the present invention is also directed to a pharmaceutical composition
- an optimized TCE comprising a heavy chain (anti-BCMA and anti-CD3) A (HC) (SEQ ID NO:1); a heavy chain (anti- BCMA) B (HC') (SEQ ID NO:3); a light chain (anti-CD3) A (LC'') (SEQ ID NO:2); a light chain (anti-BCMA) A (LC''') (SEQ ID NO:4); and, a light chain (anti-BCMA) B (LC'''') (SEQ ID NO:4), wherein the heavy chain (anti-BCMA and anti-CD3) A (HC) (SEQ ID NO:1) comprises at least one inter-chain disulfide bond selected from the group consisting of ⁇ C219-C216''' between itself and the light chain (anti-BCMA) A (LC''') (SEQ ID NO:4); ⁇ C444-C232’’ between itself and the light chain (anti-CD3) A (LC'''
- the present invention is further directed to a method for the treatment of a pathological condition in a patient or mammal selected from the group consisting of a hematological malignancy, a plasma cell disorder, a B-cell malignancy, multiple myeloma (MM), lymphoma, an immune system disorder, an autoimmune disorder, and an inflammation disorder, comprising administering an effective amount of an optimized TCE described herein to a patient or mammal in need thereof.
- a pathological condition in a patient or mammal selected from the group consisting of a hematological malignancy, a plasma cell disorder, a B-cell malignancy, multiple myeloma (MM), lymphoma, an immune system disorder, an autoimmune disorder, and an inflammation disorder
- Figure 1 displays the optimized species' arrangement of five antibody chains as well as the exact linkage of disulfide bridge positions.
- Corresponding SEQ ID Nos are shown in parentheses, e.g., (1) denotes SEQ ID NO:1. All positions are numbered from the N terminus of each chain.
- BCMA B-cell maturation antigen
- BAFF B-cell activating factor (also referred to BLyS)
- APRIL a proliferation- inducing ligand
- BCMA (SEQ ID NO:37), as used herein, refers to human B cell maturation antigen, also known as TR17_HUMAN, TNFRSF17 (UniProt Q02223; Q2TQ40), a member of the tumor necrosis receptor superfamily expressed, for example, in differentiated plasma cells.
- the extracellular domain of BCMA consists according to UniProt of amino acids 1– 54 (or 5-51).
- the terms“anti-BCMA”, “antibody against BCMA” and “anti-BCMA antibody” as used herein refer to an antibody or portion thereof which specifically binds the extracellular domain of BCMA.
- CD3 ⁇ or CD3 refers to human CD3 ⁇ described according to UniProt P07766; A8K997 (CD3 ⁇ _HUMAN) (SEQ ID NO:38).
- the terms“anti- CD3”, “anti-CD3 ⁇ ”, “antibody against CD3”, and “ “antibody against CD3 ⁇ ” as used herein refer to an antibody or portion thereof which specifically binds CD3 ⁇ .
- Position numbering of each antibody chain referred to herein is from the N-terminus of that chain. CDRs and other regions are identified according to Kabat.
- Amino acid sequences of antibody chains referred to herein are deemed to encompass those which effect substantially the same structure and function in substantially the same way.
- nucleic acids specifically referred to herein are deemed to encompass those which encode and substantially encode each corresponding antibody chain referred to herein, including codon-degenerate nucleotide sequences that encode the same polypeptides as the nucleic acids disclosed herein.
- nucleic acids which exhibit at least 94%, 95%, 96%, 97%, 98%, or 99% identity and encode amino acid sequences which effect substantially the same function in substantially the same way as antibody chains otherwise referred to herein are intended to be within the scope of the claims appended hereto.
- nucleic acid sequences that encode the polypeptides (e.g., antibody chains) disclosed herein, wherein the nucleotide sequences are codon-optimized for a particular expression system, e.g., expression in Chinese Hamster Ovary (CHO) cells, insect cells, yeast cells, or bacterial cells (e.g., in a cell-free expression system).
- CC-93269 is a humanized, asymmetric, two-arm, immunoglobulin (Ig)G1-based monoclonal antibody (mAb) that binds bivalently to BCMA and monovalently to the cluster of differentiation 3 epsilon chain (CD3 ⁇ ).
- Ig immunoglobulin
- mAb monoclonal antibody
- TCE structures described herein are highly potent in inducing killing/lysis of all cell types which exhibit the selected target, BCMA. Species described herein bind simultaneously to (a) BCMA- expressing cells, e.g., tumor cells, plasma cells, and B-cells; and, (b) T cells, which results in T cell activation and subsequent target-cell lysis.
- CC-93269 is an immunoglobulin G1-kappa/lambda with domain crossover, anti-BCMA and anti-CD ⁇ humanized monoclonal antibody, bispecific, trivalent trisdisulfide (450- 225’:453-228’:578-348’) dimer. Optimized formation of disulfide bonds, inter alia, critical
- Heavy chain (HC) anti-BCMA VH/anti-CD3e VL crossmab
- C indicates example intra-chain disulfide bridges as shown in FIG.1 and Table II (C22-C96, C143-C199, C252-C320, C368-C424, C485-C545, and C591-C649).
- C indicates example inter-chain disulfide bridges as shown in FIG.1 and Table II (C219-, C444-, C450-, C453-, and C578- (C578 is a disulfide bridge variant)).
- N is glycosylated Asn 521, e.g., Manb1–4GlcNAcb1–4GlcNAcb1-N-Asn 521.
- the glycosylation comprises fucosylated complex bi-antennary CHO glycans.
- the residue is not fucosylated.
- the residue is not glycosylated.
- E146 double underlined
- A458, A459 and G553 are effector attenuating variants.
- W590 (bold and wavy underlined) is a knob-in-hole variant.
- GS linker GGGGSGGGGS (221-230), is in bold.
- Table III Heavy chain (HC’) (anti-BCMA) B chain (SEQ ID NO:3), (Homo sapiens anti-BCMA (1’-446’)):
- C' (bold, underlined) indicates example intra-chain disulfide bridges as shown in FIG.1 and Table III (C22’-C96’, C143’-C199’, C260’-C320’, C366’-C424’).
- C' (bold, italics, underlined) indicates example inter-chain disulfide bridges as shown in FIG.1 and Table III (C225’-, C228’-, C348’- (C348’ is a disulfide bridge variant)).
- E 146' and 212' (double underlined) are charge variants.
- N is glycosylated Asn 296', e.g., Manb1–4GlcNAcb1–4GlcNAcb1-N-Asn 296'.
- the glycosylation comprises fucosylated complex bi-antennary CHO glycans.
- the residue is not fucosylated.
- the residue is not glycosylated.
- a 233', 234'and G 328'(wavy underlined) are effector attenuating variants.
- S 365', A 367' and V 406' are knob-in-hole variants. Table IV.
- Light chain (LC’’) anti-CD3 VH/Kappa crossmab
- C'' indicates example intra-chain disulfide bridges as shown in FIG.1 and Table IV (C22’’-C98’’, C152’’-C212’’).
- C'' indicates an example inter-chain disulfide bridge as shown in FIG.1 and Table IV (C232’’-).
- Table V Light chain (LC’’’) (anti-BCMA) A chain (SEQ ID NO:4), (kappa light chain anti-BCMA (1’’’-216’’’)):
- C''' indicates example intra-chain disulfide bridges as shown in FIG.1 and Table V (C23’’’-C89’’’, C136’’’-C196’’’).
- C''' indicates an example inter-chain disulfide bridge as shown in FIG.1 and Table V (C216’’’-).
- R125''' and K126''' are charge variants. Table VI. Light chain (LC’’’’) (anti-BCMA) B chain (SEQ ID NO:4), (kappa light chain anti-BCMA
- C'''' indicates example intra-chain disulfide bridges as shown in FIG.1 and Table VI (C23’’’’-C89’’’, C136’’’’-C196’’’’’).
- C'''' indicates an example inter-chain disulfide bridge as shown in FIG.1 and Table VI (C216’’’’-C219’).
- R125'''' and K126'''' are charge variants.
- Properly placed disulfide bonds described herein significantly effect physio-chemical and biotherapeutic properties in vivo and provide stability to the optimized species.
- the combination of affinity, avidity and format valence and head-to-tail geometry in the optimization of CC-93269 confers high potency and selective BCMA targeting on the surface of MM cell lines and primary myeloma cells.
- the avidity effect, along with a higher affinity of binding to BCMA (KD » 0.3 nM), enables preferential binding to BCMA expressed on target cells over CD3 ⁇ on T cells.
- the optimized species induces tumor regression in myeloma and promotes myeloma cell death in primary patient bone marrow aspirates.
- Characteristics exhibited by the species described herein include no binding to FcgR and C1q to minimize infusion-related reactions, binding to FcRn is retained for IgG-like PK, no clinically relevant anti-drug antibodies are observed upon administration to a mammal, a clinically acceptable safety profile is observed, as well as dose-dependent efficacy. No binding to cell lines lacking BCMA or CD3 expression is observed. In cell binding assays, CC-93269 bound with approximately 8- to 35-fold more potency to the tumor target BCMA on myeloma cell lines than to CD3 on T cell lines.
- the present invention is directed to an optimized bispecific trivalent anti-BCMA/anti-CD3 T Cell Engaging Antibody (TCE) comprising a heavy chain (anti-BCMA and anti-CD3) A (HC) (SEQ ID NO:1); a heavy chain (anti-BCMA) B (HC') (SEQ ID NO:3); a light chain (anti-CD3) A (LC'') (SEQ ID NO:2); a light chain (anti-BCMA) A (LC''') (SEQ ID NO:4); and, a light chain (anti-BCMA) B (LC'''') (SEQ ID NO:4), wherein the heavy chain (anti-BCMA and anti-CD3) A (HC) (SEQ ID NO:1) comprises at least one, at least two, at least three, at least four, at least five, or all intra-chain disulfide bonds selected from the group consisting of C22-C96, C143-C199, C252-C320, C368-C424, C485-
- the present invention is further directed to an optimized bispecific trivalent anti-BCMA/anti- CD3 T Cell Engaging Antibody (TCE) comprising a heavy chain (anti-BCMA and anti-CD3) A (HC) (SEQ ID NO:1); a heavy chain (anti-BCMA) B (HC') (SEQ ID NO:3); a light chain (anti-CD3) A (LC'') (SEQ ID NO:2); a light chain (anti-BCMA) A (LC''') (SEQ ID NO:4); and, a light chain (anti-BCMA) B (LC'''') (SEQ ID NO:4), wherein the heavy chain (anti-BCMA and anti-CD3) A (HC) (SEQ ID NO:1) comprises at least one, at least two, at least three, at least four, at least five, or all inter-chain disulfide bonds selected from the group consisting of ⁇ C219-C216''' between itself and the light chain (anti-BCMA) A (LC'''''
- CC-93269 nucleic acid and amino acid sequences for CHO cell production for example, e.g., CHO-K1, is described herein.
- the present invention is particularly directed to five (5) precursor peptides including signal sequences for production and assembly of the optimized TCE described herein including, (i) for the production of mature heavy chain (anti-BCMA and anti-CD3) A (HC) (SEQ ID NO:1) and assembly of the TCE comprising SEQ ID NO:5, and (ii) for the production of mature heavy chain (anti-BCMA) B (HC') (SEQ ID NO:3) and assembly of the TCE, comprising SEQ ID NO:7, and (iii) for the production of mature light chain (anti-CD3) A (LC'') (SEQ ID NO:2) and assembly of the TCE, comprising SEQ ID NO:6, and (iv) for the production of mature light chain (anti-BCMA) A (LC''') (SEQ ID NO:4) or light chain (anti-BCMA
- the invention is also particularly directed to whole precursor DNA for optimized CHO cell production, i.e., a nucleic acid sequence for production of mature heavy chain (anti-BCMA and anti-CD3) A (HC) (SEQ ID NO:1) and assembly of the TCE, comprising SEQ ID NO:15; a nucleic acid sequence for production of mature heavy chain (anti-BCMA) B (HC') (SEQ ID NO:3) and assembly of the TCE comprising SEQ ID NO:17; a nucleic acid sequence for production of mature light chain (anti-CD3) A (LC'') (SEQ ID NO:2) and assembly of the TCE, comprising SEQ ID NO:16; and, a nucleic acid sequence for production of mature light chain (anti-BCMA) A (LC''') (SEQ ID NO:4) or mature light chain (anti-BCMA) B (LC'''') (SEQ ID NO:4), and assembly of the TCE, comprising SEQ ID NO:18.
- HC mature heavy chain
- the invention is directed to nucleic acid sequence coding region, with signal peptide, translation start and termination codon for production and assembly of the TCE including a nucleic acid sequence for production of mature heavy chain (anti-BCMA and anti-CD3) A (HC) (SEQ ID NO:1) and assembly of the TCE, comprising SEQ ID NO:19; a nucleic acid sequence for production of mature heavy chain (anti- BCMA) B (HC') (SEQ ID NO:3) and assembly of the TCE, comprising SEQ ID NO:21; a nucleic acid sequence for production of mature light chain (anti-CD3) A (LC'') (SEQ ID NO:2) and assembly of the TCE, comprising SEQ ID NO:20; and, a nucleic acid sequence for production of mature light chain (anti-BCMA) A (LC''') (SEQ ID NO:4) or mature light chain (anti-BCMA) B (LC'''') (SEQ ID NO:4), and assembly of the TCE, comprising SEQ ID NO:
- the present invention is also directed to a pharmaceutical composition
- a pharmaceutical composition comprising an optimized TCE described herein e.g., optimized CC-93269; and, at least one pharmaceutically acceptable excipient.
- CC-93269 is administered, or formulated to be administered, by intravenous (IV) infusion.
- IV intravenous
- a method of treating, controlling or preventing B-cell and/or plasma cell disorders comprising administering an effective amount of an optimized bispecific trivalent anti- BCMA/anti-CD3 T Cell Engaging Antibody (TCE) described herein to a patient in need thereof.
- TCE T Cell Engaging Antibody
- Trivalent antibodies described herein are provided for the treatment and control of hematological malignancies including but not limited to lymphoma and multiple myeloma.
- the present invention is further directed to a method for the treatment of a pathological condition in a patient or mammal selected from the group consisting of a hematological malignancy, a plasma cell disorder, a B-cell malignancy, multiple myeloma (MM), lymphoma, an immune system disorder, an autoimmune disorder, and an inflammation disorder, comprising administering an effective amount of an optimized TCE described herein to a patient or mammal in need thereof.
- a pathological condition in a patient or mammal selected from the group consisting of a hematological malignancy, a plasma cell disorder, a B-cell malignancy, multiple myeloma (MM), lymphoma, an immune system disorder, an autoimmune disorder, and an inflammation disorder
- CC-93269 bispecific trivalent anti-BCMA/anti-CD3 T cell engaging antibody (TCE)
- TCE T cell engaging antibody
- I&I immunological and inflammation
- B cell condition or disorders are selected from the group consisting of multiple myeloma (MM), Waldenstrom's macroglobulinemia (WM), chronic lymphocytic leukemia (CLL), B cell non-Hodgkin's lymphoma, plasmacytoma, Hodgkins' lymphoma, follicular lymphomas, small non-cleaved cell lymphomas, endemic Burkitt's lymphoma, sporadic Burkitt's lymphoma, marginal zone lymphoma, extranodal mucosa-associated lymphoid tissue lymphoma, nodal monocytoid B cell lymphoma, splenic lymphoma, mantle cell lymphoma, large cell lymphoma, diffuse mixed cell lymphoma,
- MM multiple myeloma
- WM Waldenstrom's macroglobulinemia
- CLL chronic lymphocytic leukemia
- the method of the present invention encompasses wherein the B cell condition or disorder is a B cell malignancy.
- the method of the present invention encompasses wherein the B cell condition or disorder is a plasma cell malignancy.
- the current invention is directed to a method for the treatment of a pathological condition in a mammal selected from the group consisting of a hematological malignancy, a plasma cell malignancy, a B-cell malignancy, multiple myeloma (MM), lymphoma, an immune system disorder, an autoimmune disorder, and an inflammation disorder, comprising administering an effective amount of an optimized Bispecific Trivalent anti-BCMA/anti-CD3 T Cell Engaging Antibody (TCE) described herein to a mammal in need thereof.
- TCE Bispecific Trivalent anti-BCMA/anti-CD3 T Cell Engaging Antibody
- the current invention is particularly directed to a method for the treatment of a pathological condition in a human patient selected from the group consisting of a hematological malignancy, a plasma cell malignancy, a B-cell malignancy, multiple myeloma (MM), lymphoma, an immune system disorder, an autoimmune disorder, and an inflammation disorder, comprising administering an effective amount of an optimized Bispecific Trivalent anti-BCMA/anti-CD3 T Cell Engaging Antibody (TCE) described herein to a patient in need thereof.
- TCE Bispecific Trivalent anti-BCMA/anti-CD3 T Cell Engaging Antibody
- the disease to be treated with the optimized CC-93269 is multiple myeloma.
- the multiple myeloma is Stage I, Stage II, or Stage III according to the International Staging System or the Revised International Staging System. In certain embodiments, said multiple myeloma is newly-diagnosed multiple myeloma. In other embodiments, said multiple myeloma is relapsed or refractory multiple myeloma.
- the stages of multiple myeloma are as follows: Stage I: Serum beta-2 microglobulin ⁇ 3.5 mg/L and serum albumin 33.5 g/dL; Stage II: Not stage I or stage III; Stage III: Serum beta-2 microglobulin 3 5.5 mg/L.
- Stage I ISS stage I and standard-risk chromosomal abnormalities by fluorescence in situ hybridization (FISH)(that is, no high-risk) and serum lactate dehydrogenase (LDH) level at or below the upper limit of normal; Stage II: Not R-ISS stage I or III; Stage III: ISS stage III and either high-risk chromosomal abnormalities by FISH (for example, presence of del(17p) and/or translocation t(4;14) and/or translocation t(14;16)) or serum LDH level above the upper limit of normal.
- FISH fluorescence in situ hybridization
- LDH serum lactate dehydrogenase
- Multiple myeloma may also be staged using the Durie-Salmon system. Under this system, multiple myeloma is classified as stage I, II, or III (1, 2, or 3). Each stage is further classified into A or B, depending on whether kidney function has been affected, with the B classification indicating significant kidney damage. Stage I: Patients show no symptoms; however, if the cancer has affected kidney function, the prognosis may be worse regardless of the stage.
- Factors characteristic of stage I include: Number of red blood cells is within or slightly below normal range; normal amount of calcium in the blood; low levels of M protein in the blood or urine; M protein ⁇ 5 g/dL for IgG; ⁇ 3 g/dL for IgA; ⁇ 4 g/24 h for urinary light chain; and/or no bone damage on x-rays or only 1 bone lesion is visible.
- Stage II More cancer cells are present in the body in stage II, and if kidney function is affected, then the prognosis worsens regardless of the stage. Criteria for stage II are defined as those that fit neither stage I nor stage III.
- Stage III Many cancer cells are present in the body at stage III.
- Factors characteristic of this stage include: Anemia, with a hemoglobin ⁇ 8.5 g/dL; hypercalcemia; advanced bone damage (3 or more bone lesions); high levels of M protein in the blood or urine; and/or M protein >7 g/dL for IgG; >5 g/dL for IgA; >12 g/24 h for urinary light chain.
- Optimized CC-93269 described herein is highly potent in inducing killing/lysis of all cell types carrying the selected target, BCMA. Table VII.
- the Heavy Chain human Immunoglobulin (Ig) G1 (SEQ ID NO:1) additionally comprises an N-terminal methionine (M).
- the Heavy Chain human Immunoglobulin (Ig) G1 (SEQ ID NO:3) additionally comprises an N-terminal methionine (M).
- the Light chain’’ Kappa (SEQ ID NO:2) additionally comprises an N- terminal methionine (M).
- the Light chain’’’ Kappa (SEQ ID NO:4) additionally comprises an N- terminal methionine (M).
- the Light Chain’’’’ Kappa (SEQ ID NO:4) additionally comprises an N- terminal methionine (M).
- a prominent embodiment of the optimized assembled entity (CC-93269 (FIG.1)) described herein exhibits no C-terminal Lys (K) residue on SEQ ID NO:1 and/or SEQ ID NO:3 (HC' B).
- a prominent embodiment of CHO-produced optimized assembled entity (CC-93269 (FIG.1)) particularly exhibits 1 G0F (a certain substructure of bi-antennary N-linked oligosaccharides) glycosylation on SEQ ID NO:1 (HC A) Asn 521 and on SEQ ID NO:3 (HC' B) Asn 296'.
- a further prominent embodiment of the optimized assembled entity exhibits 1 G0F glycosylation on SEQ ID NO:1 (HC A) Asn 521 and on SEQ ID NO:3 (HC' B) Asn 296' as well as no C-terminal Lys (K) residue on either of these chains (this structural isoform has a molecular formula of C 8622 H 13310 N 2292 O 2763 S 54 and calculated molecular weight of 195013.1 Da).
- lmmunoglobulin G1 [146-glutamic acid,212-glutamic acid,458-alanine,459-alanine,553- glycine,578-cysteine,590-tryptophan], anti-BCMA (human monoclonal 83A10-42 VH-CH1 fragment) fusion protein with peptide (synthetic GGGGSGGGGS linker) fusion protein with immunoglobulin anti- (human CD3 antigen e-chain) (human-Mus musculus monoclonal CH2527 heavy chain VL(l)-CH1-CH2- CH3), (219®216"')-disulfide with immunoglobulin anti-BCMA (human monoclonal 83A10-42 k-chain [125"'-arginine, 126"'-lysine]) and (444®232'')-disulfide with immunoglobulin anti-(human CD3 antigen e-chain) (human-
- a predominant form of the optimized assembled entity (CC-93269 (FIG.1)) exhibits 1 G0F glycosylation on SEQ ID NO:1 (HC A) Asn 521 and on SEQ ID NO:3 (HC' B) Asn 296' as well as no C- terminal Lys (K) residue on either of these chains (this structural isoform has a molecular formula of C8622H13310N2292O2763S54 and molecular weight of 195013.1 Da).
- Optimized Species with C-terminal lysine (K) (on both HC)
- TCE T Cell engaging antibody
- gamma-kappa heavy chain anti-BCMA and anti-CD3e (VH-CH1-VL-CH1-CH2-CH3) (1-671) [human VH anti-BCMA (Human IGHV3-23D*01 (94.9%) -(IGHD) -IGHJ4*02 (100%)) [8.8.9] (1-116) -Human IGHG1*01, G1m17 (CH1 K120 (213) [K>E26 (146) K>E119 (212)] (117-214), hinge 1-6 (215-220))(117- 220) -10-mer bis(tetraglycyl-seryl) linker (221-230) -humanized VL anti-CD3E (Human (V-LAMBDA (musculus IGLV1*01 (80.4%) -IGLJ1*01 (100%)/Human IGLV7-46*01 (80.9%) -IGLJ3*02 (100%)) [9.3.9] (23
- CC-93269 bound to human BCMA (hu-BCMA) and cynomolgus BCMA (cyno-BCMA) with approximately 10-fold higher affinity than to rodent BCMA.
- CC-93269 bound to human cluster of differentiation 3 epsilon/delta heterodimer (CD3ed) with a KD value of 25 ⁇ 12 nM, whereas bound to cynomolgus CD3ed with KD values of 57 and 59 nM.
- CC-93269 had no detectable complement-dependent cytotoxicity (CDC) on BCMA expressing Pfeiffer cells. CC-93269 did not show any binding affinity to FcgRs expressed on human embryonic kidney (HEK) 293 cells. CC-93269 exhibited BCMA-specific binding on various myeloma cell lines expressing different levels of BCMA (BCMAhigh NCI-H929, BCMAmid L363, BCMAmid JJN-3, BCMAlow RPMI-8226), as well as on stably transfected BCMAhigh HEK293T-huBCMA and
- BCMAhighHEK293T-cynoBCMA Binding half-maximal effective concentration (EC50) values ranged from 3.4 nM to 15.2 nM for the BCMA+ cell lines.
- EC50 half-maximal effective concentration
- the ability of CC-93269 to bind T cells was assessed by flow cytometry.
- the EC50 values for CC- 93269 to CD3 on Jurkat cells ranged between 16.76 nM and 37.42 nM in one study and 121.3 nM in a second study. No specific binding of CC-93269 was detected on primary mouse or rat CD4 and CD8 T cells.
- CC-93269 Upon simultaneous binding of CC-93269 to CD3 on T cells and BCMA on target cells, CC-93269 forces crosslinking of TCR/CD3 leading to major histocompatibility class (MHC)-independent T cell activation and release of cytokines, followed by formation of a cytolytic synapse and secretion of cytolytic enzymes resulting in tumor cell lysis.
- MHC major histocompatibility class
- Co-cultures of T cells derived from healthy human donors with BCMA-expressing MM cell lines in the presence of CC-93269 resulted in MM cell killing.
- CC-93269 did not induce T cell-redirected killing in the absence of BCMA+ target cells, ie, BCMA negative MKN45 gastric cancer and control HEK293T cell lines.
- CC-93269 demonstrated concentration-dependent killing of primary MM plasma cells of bone marrow samples from both newly diagnosed and relapsed MM patients in the presence of autologous T cells as effector cells.
- Two separate methods were employed, either using unmanipulated whole bone marrow or CD138+ partially-depleted bone marrow (manipulated) incubated with CC-93269.
- Multiple myeloma plasma cells were sensitive to CC-93269-dependent T cell-redirected killing and significant lysis was observed in 11 out of 16 patient whole bone marrow samples (unmanipulated) after 48 hours of incubation.
- CD138+ partially-depleted bone marrow aspirates all 13 patient samples responded to CC-93269.
- CC-93269-induced tumor cell killing was accompanied by T cell activation and cytokine release in both cell lines and primary MM patient bone marrow samples. Concentration-dependent upregulation of T cell activation markers (CD69, CD25, and HLA-DR) on both CD4 and CD8 T cells from the patient unmanipulated whole bone marrow was observed after 48 and 96 hours of incubation.
- Cytokines i.e., interferon-gamma [IFN-g]
- cytolytic proteins ie, granzyme B and perforin
- Biochemical and cellular assays were employed to characterize the binding profile of CC-93269.
- the BCMA binder of CC-93269 cross-reacted with the four orthologs of BCMA tested (human, cynomolgus monkey, rat, and mouse).
- CC-93269 binds to human BCMA (hu-BCMA) and cynomolgus BCMA (cyno-BCMA) with approximately 10-fold higher affinity than to rodent BCMA.
- CC-93269 showed no sustained binding to human BAFF receptor (hu-BAFF-R) and human transmembrane activator and calcium modulator and cyclophilin ligand interactor (hu-TACI).
- BCMAhigh NCI-H929, BCMAmid L363, BCMAmid JJN-3, BCMAlow RPMI-8226 as well as stably transfected BCMAhigh HEK293T-huBCMA and BCMAhigh HEK293T-cynoBCMA was evaluated by flow cytometry. Binding EC50 values ranged from 3.4 to 15.2 nM for the BCMA+ cell lines. Concentration- dependent, BCMA-specific binding of CC-93269 was demonstrated in U266 MM cell lines with blocking experiments.
- CC-93269 Increasing concentrations of BCMA/Fc, APRIL, and anti-BCMA murine IgG2a (19F2) proportionately reduced the binding of phycoerythrin (PE)-conjugated BCMA binder (Clone 83A10-42) of CC-93269 on U266 cells.
- PE phycoerythrin
- CC-93269 In addition to BCMA-expressing cell line killing, CC-93269 also demonstrated concentration dependent, autologous T cell-mediated killing of primary MM cells in ex vivo cultures of MM patient bone marrow aspirates. * * * All publications and patents referred to herein are incorporated by reference. Various modifications and variations of the described subject matter will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to these embodiments. Indeed, various modifications for carrying out the invention are obvious to those skilled in the art and are intended to be within the scope of the following claims.
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Abstract
An optimized bispecific trivalent anti-BCMA/anti-CD3 T cell engaging antibody (TCE), related pharmaceutical compositions, and methods of use therefore are disclosed.
Description
BCMA/CD3 BISPECIFIC TRIVALENT T-CELL ENGAGING (TCE) ANTIBODIES AND THEIR USE TO TREAT HEMATOLOGICAL MALIGNANCIES
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] Priority is indicated herein under 35 U.S.C. §119(e) to U.S. Provisional Application No.
62/838,660, filed April 25, 2019, which is hereby incorporated by reference in its entirety.
SEQUENCE LISTING
[0002] This disclosure includes a Sequence Listing submitted in ASCII format via EFS-Web hereby incorporated by reference in its entirety. The ASCII file, created on April 23, 2020, is named 298068- 325_Sequence_Listing.txt and is 59.1 kilobytes in size.
FIELD OF THE INVENTION
[0003] The current invention is directed to optimization of bispecific trivalent anti-BCMA/anti-CD3 T cell engaging antibodies (TCE) for the treatment of oncological conditions, hematological malignancies, and autoimmune disorders.
BACKGROUND OF THE INVENTION
[0004] B cell maturation antigen (BCMA) is primarily expressed on normal plasma cells and on myeloma cells from patients who have multiple myeloma (MM) (Carpenter RO, et al., Clin Cancer Res.2013;
19(8):2048; Frigyesi I, et al., Blood. 2014;123(9):1336; Novak AJ, et al., Blood. 2004;103(2):689;
Seckinger A, et al., Cancer Cell. 2017;31(3):396; Tai YT, et al., Blood. 2014;123(20):3128), and therefore represents an attractive target for immunotherapeutic agents. B cell maturation antigen is a member of the tumor necrosis factor (TNF) receptor superfamily that binds to a proliferation inducing ligand (APRIL) with 100-fold higher affinity than it binds to B cell-activating factor (BAFF) (Day ES, et al.
Biochemistry.2005;44(6):1919). Expression of BCMA is thought to support tumor cell survival and to protect against apoptosis (Bellucci R, et al., Blood. 2005;105(10):3945; Moreaux J, et al., Blood.
2004;103(8):3148; Novak AJ, et al., Blood. 2004;103(2):689).
[0005] Apart from lymphoid tissues and the gastrointestinal (Gl) tract, expression of BCMA in humans appears absent in other non-lymphoid tissues including brain, heart, lung, kidney, liver, and pancreas (Carpenter RO, et al., Clin Cancer Res.2013; 19(8):2048). Evaluation by dual-staining
immunohistochemistry confirms that the BCMA expression in the Gl tract is confined to tissue resident
plasma cells and is not observed on epithelial cells (Carpenter RO, et al., Clin Cancer Res.2013;
19(8):2048). Other studies also reported the presence of BCMA expressing plasma cells in gut- associated lymphoid tissues (GALT) and lamina propria (Barone F, et al., Mucosal Immunol.2009;
2(6):495; Brandtzaeg P., Immunol Invest.2010;39(4-5):303; Spencer J, et al., Mucosal Immunol.
2016;9(5):1113). Plasmablasts and B cells derived from B cells activated in the GALT can circulate via the blood and home back to the gut (Spencer J, et al., Mucosal Immunol.2016;9(5):1113). [0006] At least four (4) BCMA-targeting chimeric antigen receptor (CAR) T cell therapies have shown promising interim results in Phase 1 clinical studies in relapsed and refractory MM (Berdeja JG, et al. , The EORTC-NCI-AACR Molecular Targets and Cancer Therapeutics Symposium.2016 Nov 29-Dec 2; Munich, Germany: Abstract 14LBA.; Ali SA, et al., Blood.2016;128(13):1688; Cohen AD, et al., American Society for Hematology 58th Annual Meeting and Exposition.2016a Dec 3-6; San Diego, CA: Abstract 653; Fan F, et al., 2017 Annual Meeting of American Society of Clinical Oncology; 2017 June 2-6; Chicago IL: J Clin Oncol 35, 2017 (suppl; abstr LBA3001)). At least seven (7) BCMA x CD3 T cell bispecific engagers and a BCMA antibody drug conjugate (ADC) are also currently being investigated in Phase 1 clinical studies in MM (see, e.g., Topp MS, et al., J Clin Oncol.2016;34 (suppl; abstr TPS8067); Girgis S, et al., Blood.2016;128(22):5668; Cohen AD, et al., American Society for Hematology 58th Annual Meeting and Exposition.2016b Dec 3-6; San Diego, CA: Abstract 1148). Although disulfide bonds are important for folding and stability, their optimized formation is difficult to achieve. Properly placed disulfide bonds significantly effect biotherapeutic properties in vivo and provide stability to the entity, decreasing further entropic choices that facilitate folding progression toward the native state by limiting unfolded or improperly folded conformations. [0007] An unmet medical need continues to exist for improved nucleic acid expression systems and optimization of inter- and intra- peptide chain disulfide bonds toward stable and effective tertiary and quaternary assembly of biopharmaceutical therapeutic entities now in development.
SUMMARY OF THE INVENTION
[0008] The present invention is directed to an optimized bispecific trivalent anti-BCMA/anti-CD3 T Cell Engaging Antibody (TCE) comprising a heavy chain (anti-BCMA and anti-CD3) A (HC) (SEQ ID NO:1); a heavy chain (anti-BCMA) B (HC') (SEQ ID NO:3); a light chain (anti-CD3) A (LC'') (SEQ ID NO:2); a light
chain (anti-BCMA) A (LC''') (SEQ ID NO:4); and, a light chain (anti-BCMA) B (LC'''') (SEQ ID NO:4), wherein the heavy chain (anti-BCMA and anti-CD3) A (HC) (SEQ ID NO:1) comprises at least one intra-chain disulfide bond selected from the group consisting of C22-C96, C143-C199, C252-C320, C368-C424, C485- C545, and C591-C649, and wherein the heavy chain (anti-BCMA) B (HC') (SEQ ID NO:3) comprises at least one intra-chain disulfide bond selected from the group consisting of C22’-C96’, C143’-C199’, C260’- C320’, and C366’-C424’, and wherein the light chain (anti-CD3) A (LC'') (SEQ ID NO:2) comprises at least one intra-chain disulfide bond selected from the group consisting of C22’’-C98’’ and C152’’-C212’’, and wherein the light chain (anti-BCMA) A (LC''') (SEQ ID NO:4) comprises at least one intra-chain disulfide bond selected from the group consisting of C23’’’-C89’’’ and C136’’’-C196’’’, and wherein the light chain (anti-BCMA) B (LC'''') (SEQ ID NO:4) comprises at least one intra-chain disulfide bond selected from the group consisting of C23’’’’-C89’’’’ and C136’’’’-C196’’’’. [0009] The present invention is further directed to an optimized bispecific trivalent anti-BCMA/anti- CD3 T Cell Engaging Antibody (TCE) comprising a heavy chain (anti-BCMA and anti-CD3) A (HC) (SEQ ID NO:1); a heavy chain (anti-BCMA) B (HC') (SEQ ID NO:3); a light chain (anti-CD3) A (LC'') (SEQ ID NO:2); a light chain (anti-BCMA) A (LC''') (SEQ ID NO:4); and, a light chain (anti-BCMA) B (LC'''') (SEQ ID NO:4), wherein the heavy chain (anti-BCMA and anti-CD3) A (HC) (SEQ ID NO:1) comprises at least one inter- chain disulfide bond selected from the group consisting of● C219-C216''' between itself and the light chain (anti-BCMA) A (LC''') (SEQ ID NO:4);● C444-C232’’ between itself and the light chain (anti-CD3) A (LC'') (SEQ ID NO:2);● C450-C225’ between itself and the heavy chain (anti-BCMA) B (HC') (SEQ ID NO:3);● C453-C228’ between itself and the heavy chain (anti-BCMA) B (HC') (SEQ ID NO:3); and,● C578- C348’ between itself and the heavy chain (anti-BCMA) B (HC') (SEQ ID NO:3), and wherein the heavy chain (anti-BCMA) B (HC') (SEQ ID NO:3) comprises an inter-chain disulfide bond● C219’-C216’’’’ between itself and the light chain (anti-BCMA) B (LC'''') (SEQ ID NO:4). [0010] The present invention is also directed to five (5) precursor peptides including signal sequences for production and assembly of the optimized TCE described herein including, (i) for the production of mature heavy chain (anti-BCMA and anti-CD3) A (HC) (SEQ ID NO:1) and assembly of the TCE comprising SEQ ID NO:5, and (ii) for the production of mature heavy chain (anti-BCMA) B (HC') (SEQ ID NO:3) and assembly of the TCE, comprising SEQ ID NO:7, and (iii) for the production of mature light chain (anti- CD3) A (LC'') (SEQ ID NO:2) and assembly of the TCE, comprising SEQ ID NO:6, and (iv) for the production
of mature light chain (anti-BCMA) A (LC''') (SEQ ID NO:4) or light chain (anti-BCMA) B (LC'''') (SEQ ID NO:4), and assembly of the TCE, comprising SEQ ID NO:8. [0011] The present invention is also directed to whole precursor DNA for optimized CHO cell production, i.e., a nucleic acid sequence for production of mature heavy chain (anti-BCMA and anti-CD3) A (HC) (SEQ ID NO:1) and assembly of the TCE, comprising SEQ ID NO:15; a nucleic acid sequence for production of mature heavy chain (anti-BCMA) B (HC') (SEQ ID NO:3) and assembly of the TCE comprising SEQ ID NO:17; a nucleic acid sequence for production of mature light chain (anti-CD3) A (LC'') (SEQ ID NO:2) and assembly of the TCE, comprising SEQ ID NO:16; and, a nucleic acid sequence for production of mature light chain (anti-BCMA) A (LC''') (SEQ ID NO:4) or mature light chain (anti-BCMA) B (LC'''') (SEQ ID NO:4), and assembly of the TCE, comprising SEQ ID NO:18. [0012] The present invention is further directed to nucleic acid sequence coding region, with signal peptide, translation start and termination codon for production and assembly of the TCE including a nucleic acid sequence for production of mature heavy chain (anti-BCMA and anti-CD3) A (HC) (SEQ ID NO:1) and assembly of the TCE, comprising SEQ ID NO:19; a nucleic acid sequence for production of mature heavy chain (anti-BCMA) B (HC') (SEQ ID NO:3) and assembly of the TCE, comprising SEQ ID NO:21; a nucleic acid sequence for production of mature light chain (anti-CD3) A (LC'') (SEQ ID NO:2) and assembly of the TCE, comprising SEQ ID NO:20; and, a nucleic acid sequence for production of mature light chain (anti-BCMA) A (LC''') (SEQ ID NO:4) or mature light chain (anti-BCMA) B (LC'''') (SEQ ID NO:4), and assembly of the TCE, comprising SEQ ID NO:22. [0013] The present invention is also directed to a pharmaceutical composition comprising an optimized TCE comprising a heavy chain (anti-BCMA and anti-CD3) A (HC) (SEQ ID NO:1); a heavy chain (anti- BCMA) B (HC') (SEQ ID NO:3); a light chain (anti-CD3) A (LC'') (SEQ ID NO:2); a light chain (anti-BCMA) A (LC''') (SEQ ID NO:4); and, a light chain (anti-BCMA) B (LC'''') (SEQ ID NO:4), wherein the heavy chain (anti-BCMA and anti-CD3) A (HC) (SEQ ID NO:1) comprises at least one inter-chain disulfide bond selected from the group consisting of● C219-C216''' between itself and the light chain (anti-BCMA) A (LC''') (SEQ ID NO:4);● C444-C232’’ between itself and the light chain (anti-CD3) A (LC'') (SEQ ID NO:2);● C450-C225’ between itself and the heavy chain (anti-BCMA) B (HC') (SEQ ID NO:3);● C453-C228’ between itself and the heavy chain (anti-BCMA) B (HC') (SEQ ID NO:3); and,● C578-C348’ between itself and the heavy chain (anti-BCMA) B (HC') (SEQ ID NO:3), and wherein the heavy chain (anti-BCMA) B
(HC') (SEQ ID NO:3) comprises an inter-chain disulfide bond● C219’-C216’’’’ between itself and the light chain (anti-BCMA) B (LC'''') (SEQ ID NO:4); and, at least one pharmaceutically acceptable excipient. [0014] The present invention is further directed to a method for the treatment of a pathological condition in a patient or mammal selected from the group consisting of a hematological malignancy, a plasma cell disorder, a B-cell malignancy, multiple myeloma (MM), lymphoma, an immune system disorder, an autoimmune disorder, and an inflammation disorder, comprising administering an effective amount of an optimized TCE described herein to a patient or mammal in need thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 displays the optimized species' arrangement of five antibody chains as well as the exact linkage of disulfide bridge positions. Corresponding SEQ ID Nos are shown in parentheses, e.g., (1) denotes SEQ ID NO:1. All positions are numbered from the N terminus of each chain.
DETAILED DESCRIPTION OF THE INVENTION
[0016] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. All publications and patents referred to herein are incorporated by reference in their entireties. [0017] BCMA (B-cell maturation antigen) is a well-characterized transmembrane B cell surface receptor which binds ligands BAFF (B-cell activating factor (also referred to BLyS)) and APRIL (a proliferation- inducing ligand) to promote B cell survival. The term BCMA (SEQ ID NO:37), as used herein, refers to human B cell maturation antigen, also known as TR17_HUMAN, TNFRSF17 (UniProt Q02223; Q2TQ40), a member of the tumor necrosis receptor superfamily expressed, for example, in differentiated plasma cells. The extracellular domain of BCMA consists according to UniProt of amino acids 1– 54 (or 5-51). [0018] The terms“anti-BCMA", "antibody against BCMA" and "anti-BCMA antibody” as used herein refer to an antibody or portion thereof which specifically binds the extracellular domain of BCMA.
[0019] “CD3ɛ or CD3” as used herein refers to human CD3ɛ described according to UniProt P07766; A8K997 (CD3ɛ_HUMAN) (SEQ ID NO:38). [0020] The terms“anti- CD3", "anti-CD3ɛ", "antibody against CD3", and " "antibody against CD3ɛ" as used herein refer to an antibody or portion thereof which specifically binds CD3ɛ. [0021] Position numbering of each antibody chain referred to herein is from the N-terminus of that chain. CDRs and other regions are identified according to Kabat. [0022] Amino acid sequences of antibody chains referred to herein are deemed to encompass those which effect substantially the same structure and function in substantially the same way. Accordingly, amino acid sequences which exhibit at least 97%, 98%, or 99% identity and effect substantially the same structure and function in substantially the same way as antibody chains otherwise referred to herein are intended to be within the scope of the claims appended hereto. [0023] Nucleic acids specifically referred to herein are deemed to encompass those which encode and substantially encode each corresponding antibody chain referred to herein, including codon-degenerate nucleotide sequences that encode the same polypeptides as the nucleic acids disclosed herein.
Accordingly, nucleic acids which exhibit at least 94%, 95%, 96%, 97%, 98%, or 99% identity and encode amino acid sequences which effect substantially the same function in substantially the same way as antibody chains otherwise referred to herein are intended to be within the scope of the claims appended hereto. Further provided herein are nucleic acid sequences that encode the polypeptides (e.g., antibody chains) disclosed herein, wherein the nucleotide sequences are codon-optimized for a particular expression system, e.g., expression in Chinese Hamster Ovary (CHO) cells, insect cells, yeast cells, or bacterial cells (e.g., in a cell-free expression system). [0024] Monoclonal antibodies against BCMA, published as WO2017021450 and US 20190352427, is herein incorporated by reference. [0025] CC-93269 is a humanized, asymmetric, two-arm, immunoglobulin (Ig)G1-based monoclonal antibody (mAb) that binds bivalently to BCMA and monovalently to the cluster of differentiation 3 epsilon chain (CD3ɛ). An example Bispecific Trivalent anti-BCMA/anti-CD3 T Cell Engaging Antibody
(TCE), otherwise referred to as BCMA 2+1 T Cell Engager (TCE) CC-93269, which comprises a heavy chain (anti-BCMA and anti-CD3) A chain (HC) (SEQ ID NO:1); a heavy chain (anti-BCMA) B chain (HC') (SEQ ID NO:3); a light chain (anti-CD3) A chain (LC'') (SEQ ID NO:2); a light chain (anti-BCMA) A chain (LC''') (SEQ ID NO:4); and, a light chain (anti-BCMA) B chain (LC'''') (SEQ ID NO:4). Optimization of the bispecific trivalent anti-BCMA/anti-CD3 T cell engaging antibody (TCE), CC-93269, for the treatment of oncological conditions, hematological malignancies, and autoimmune disorders is described herein. See FIG.1. [0026] TCE structures described herein are highly potent in inducing killing/lysis of all cell types which exhibit the selected target, BCMA. Species described herein bind simultaneously to (a) BCMA- expressing cells, e.g., tumor cells, plasma cells, and B-cells; and, (b) T cells, which results in T cell activation and subsequent target-cell lysis. TCE structure disclosed herein (FIG.1) IG class and subclass, IG format [0027] Heavy Chain: human Immunoglobulin (Ig) G1 (SEQ ID NO:1) [0028] Heavy Chain’: human Immunoglobulin (Ig) G1 (SEQ ID NO:3) [0029] Light chain’’: Kappa (SEQ ID NO:2) [0030] Light chain’’’: Kappa (SEQ ID NO:4) [0031] Light Chain’’’’: Kappa (SEQ ID NO:4)
Chain Composition: [0032] Heavy Chain: VH-CH1-linker-VL(lambda)-CH1-hinge-CH2-CH3 (SEQ ID NO:1) [0033] Heavy Chain’: VH-CH1-hinge-CH2-CH3 (SEQ ID NO:3) [0034] LC’’: VH-kappa (SEQ ID NO:2)
[0035] LC’’’: VL(kappa)-Kappa (SEQ ID NO:4) [0036] LC’’’’: VL(kappa)-Kappa (SEQ ID NO:4) [0037] Example optimized CC-93269 is an immunoglobulin G1-kappa/lambda with domain crossover, anti-BCMA and anti-CDɛ humanized monoclonal antibody, bispecific, trivalent trisdisulfide (450- 225’:453-228’:578-348’) dimer. Optimized formation of disulfide bonds, inter alia, critical for folding, stability, properties disclosed herein and efficacy are described as follows. COMPONENTS OF OPTIMIZED CC-93269
Table I. Optimized Disulfide Bridge Locations. Bold indicates inter-chain bonds.
Table II. Heavy chain (HC) (anti-BCMA VH/anti-CD3e VL crossmab) A chain (SEQ ID NO:1), (gamma-kappa heavy chain anti-BCMA and anti-CD3ɛ (VH-CH1-VL-CH1-CH2-CH3) (1-671)):
[0045] C' (bold, underlined) indicates example intra-chain disulfide bridges as shown in FIG.1 and Table III (C22’-C96’, C143’-C199’, C260’-C320’, C366’-C424’). [0046] C' (bold, italics, underlined) indicates example inter-chain disulfide bridges as shown in FIG.1 and Table III (C225’-, C228’-, C348’- (C348’ is a disulfide bridge variant)). [0047] E 146' and 212' (double underlined) are charge variants. [0048] N (underlined) is glycosylated Asn 296', e.g., Manb1–4GlcNAcb1–4GlcNAcb1-N-Asn 296'. In some embodiments, the glycosylation comprises fucosylated complex bi-antennary CHO glycans. In some embodiments the residue is not fucosylated. In some embodiments the residue is not glycosylated. [0049] A 233', 234'and G 328'(wavy underlined) are effector attenuating variants.
[0050] S 365', A 367' and V 406' (bold and wavy underlined) are knob-in-hole variants. Table IV. Light chain (LC’’) (anti-CD3 VH/Kappa crossmab) A chain (SEQ ID NO:2), (VH kappa light chain humanized anti-CD3ɛ (1’’-232’’)):
[0053] C''' (bold, underlined) indicates example intra-chain disulfide bridges as shown in FIG.1 and Table V (C23’’’-C89’’’, C136’’’-C196’’’). [0054] C''' (bold, italics, underlined) indicates an example inter-chain disulfide bridge as shown in FIG.1 and Table V (C216’’’-). [0055] R125''' and K126''' (double underlined) are charge variants.
Table VI. Light chain (LC’’’’) (anti-BCMA) B chain (SEQ ID NO:4), (kappa light chain anti-BCMA
(1’’’’-216’’’’))):
[0060] Optimization of the location of inter- and intra- chain disulfide bonds toward increased stability and efficacy of the CC-93269 entity are particularly disclosed.
[0061] Optimized intra- and inter-chain (●) Disulfide Bridges within CC93269 (see FIG.1):
[0070] Provided herein is optimized CC-93269 (bispecific trivalent anti-BCMA/anti-CD3 T cell engaging antibody (TCE)) and methods of administering this entity for the treatment of hematological malignancies as well as immunological and inflammation (I&I) disorders. Described herein is a method of treating, controlling or preventing these conditions by means of administering an effective amount of the optimized species described herein to a mammal or a human patient in need thereof. Preferred is a method of administering an effective amount of the optimized species to a patient in need thereof. Particularly preferred is a method of administering an effective amount of a bispecific anti-BCMA/anti- CD3 TCE to a patient in need thereof, e.g., optimized CC-93269. [0071] B cell condition or disorders are selected from the group consisting of multiple myeloma (MM), Waldenstrom's macroglobulinemia (WM), chronic lymphocytic leukemia (CLL), B cell non-Hodgkin's lymphoma, plasmacytoma, Hodgkins' lymphoma, follicular lymphomas, small non-cleaved cell lymphomas, endemic Burkitt's lymphoma, sporadic Burkitt's lymphoma, marginal zone lymphoma, extranodal mucosa-associated lymphoid tissue lymphoma, nodal monocytoid B cell lymphoma, splenic lymphoma, mantle cell lymphoma, large cell lymphoma, diffuse mixed cell lymphoma, immunoblastic lymphoma, primary mediastinal B cell lymphoma, pulmonary B cell angiocentric lymphoma, small lymphocytic lymphoma, B cell proliferations of uncertain malignant potential, lymphomatoid granulomatosis, post-transplant lymphoproliferative disorder, an immunoregulatory disorder,
rheumatoid arthritis, myasthenia gravis, idiopathic thrombocytopenia purpura, anti-phospholipid syndrome, Chagas' disease, Grave's disease, Wegener's granulomatosis, poly-arteritis nodosa, Sjogren's syndrome, pemphigus vulgaris, scleroderma, multiple sclerosis, anti-phospholipid syndrome, ANCA associated vasculitis, Goodpasture's disease, Kawasaki disease, autoimmune hemolytic anemia, and rapidly progressive glomerulonephritis, heavy-chain disease, primary or immunocyte-associated amyloidosis, and monoclonal gammopathy. [0072] The method of the present invention encompasses wherein the B cell condition or disorder is a B cell malignancy. [0073] The method of the present invention encompasses wherein the B cell condition or disorder is a plasma cell malignancy. [0074] The current invention is directed to a method for the treatment of a pathological condition in a mammal selected from the group consisting of a hematological malignancy, a plasma cell malignancy, a B-cell malignancy, multiple myeloma (MM), lymphoma, an immune system disorder, an autoimmune disorder, and an inflammation disorder, comprising administering an effective amount of an optimized Bispecific Trivalent anti-BCMA/anti-CD3 T Cell Engaging Antibody (TCE) described herein to a mammal in need thereof. [0075] The current invention is particularly directed to a method for the treatment of a pathological condition in a human patient selected from the group consisting of a hematological malignancy, a plasma cell malignancy, a B-cell malignancy, multiple myeloma (MM), lymphoma, an immune system disorder, an autoimmune disorder, and an inflammation disorder, comprising administering an effective amount of an optimized Bispecific Trivalent anti-BCMA/anti-CD3 T Cell Engaging Antibody (TCE) described herein to a patient in need thereof. [0076] In certain embodiments, the disease to be treated with the optimized CC-93269 is multiple myeloma. In specific embodiments, the multiple myeloma is Stage I, Stage II, or Stage III according to the International Staging System or the Revised International Staging System. In certain embodiments, said multiple myeloma is newly-diagnosed multiple myeloma. In other embodiments, said multiple myeloma is relapsed or refractory multiple myeloma.
[0077] Under the International Staging System (ISS), the stages of multiple myeloma are as follows: Stage I: Serum beta-2 microglobulin < 3.5 mg/L and serum albumin ³3.5 g/dL; Stage II: Not stage I or stage III; Stage III: Serum beta-2 microglobulin ³ 5.5 mg/L. Under the Revised International Staging System (R-ISS), the stages of multiple myeloma are as follows: Stage I: ISS stage I and standard-risk chromosomal abnormalities by fluorescence in situ hybridization (FISH)(that is, no high-risk) and serum lactate dehydrogenase (LDH) level at or below the upper limit of normal; Stage II: Not R-ISS stage I or III; Stage III: ISS stage III and either high-risk chromosomal abnormalities by FISH (for example, presence of del(17p) and/or translocation t(4;14) and/or translocation t(14;16)) or serum LDH level above the upper limit of normal. [0078] Multiple myeloma may also be staged using the Durie-Salmon system. Under this system, multiple myeloma is classified as stage I, II, or III (1, 2, or 3). Each stage is further classified into A or B, depending on whether kidney function has been affected, with the B classification indicating significant kidney damage. Stage I: Patients show no symptoms; however, if the cancer has affected kidney function, the prognosis may be worse regardless of the stage. Factors characteristic of stage I include: Number of red blood cells is within or slightly below normal range; normal amount of calcium in the blood; low levels of M protein in the blood or urine; M protein <5 g/dL for IgG; <3 g/dL for IgA; <4 g/24 h for urinary light chain; and/or no bone damage on x-rays or only 1 bone lesion is visible. Stage II: More cancer cells are present in the body in stage II, and if kidney function is affected, then the prognosis worsens regardless of the stage. Criteria for stage II are defined as those that fit neither stage I nor stage III. Stage III: Many cancer cells are present in the body at stage III. Factors characteristic of this stage include: Anemia, with a hemoglobin <8.5 g/dL; hypercalcemia; advanced bone damage (3 or more bone lesions); high levels of M protein in the blood or urine; and/or M protein >7 g/dL for IgG; >5 g/dL for IgA; >12 g/24 h for urinary light chain. [0079] Optimized CC-93269 described herein is highly potent in inducing killing/lysis of all cell types carrying the selected target, BCMA.
Table VII.
20
22
23
24
IJĵ
26
27
28
29
[0080] In some embodiments, the Heavy Chain: human Immunoglobulin (Ig) G1 (SEQ ID NO:1) additionally comprises an N-terminal methionine (M).
[0081] In some embodiments, the Heavy Chain’: human Immunoglobulin (Ig) G1 (SEQ ID NO:3) additionally comprises an N-terminal methionine (M).
[0082] In some embodiments, the Light chain’’: Kappa (SEQ ID NO:2) additionally comprises an N- terminal methionine (M).
[0083] In some embodiments, the Light chain’’’: Kappa (SEQ ID NO:4) additionally comprises an N- terminal methionine (M).
[0084] In some embodiments, the Light Chain’’’’: Kappa (SEQ ID NO:4) additionally comprises an N- terminal methionine (M).
[0085] A prominent embodiment of the optimized assembled entity (CC-93269 (FIG.1)) described herein exhibits no C-terminal Lys (K) residue on SEQ ID NO:1 and/or SEQ ID NO:3 (HC' B).
[0086] A prominent embodiment of CHO-produced optimized assembled entity (CC-93269 (FIG.1)) particularly exhibits 1 G0F (a certain substructure of bi-antennary N-linked oligosaccharides) glycosylation on SEQ ID NO:1 (HC A) Asn 521 and on SEQ ID NO:3 (HC' B) Asn 296'. A further prominent embodiment of the optimized assembled entity (CC-93269 (FIG.1)) exhibits 1 G0F glycosylation on SEQ ID NO:1 (HC A) Asn 521 and on SEQ ID NO:3 (HC' B) Asn 296' as well as no C-terminal Lys (K) residue on either of these chains (this structural isoform has a molecular formula of C8622H13310N2292O2763S54 and calculated molecular weight of 195013.1 Da).
EXAMPLES
EXAMPLE I Optimized Species Chemical Name and Formula
[0087] lmmunoglobulin G1 [146-glutamic acid,212-glutamic acid,458-alanine,459-alanine,553- glycine,578-cysteine,590-tryptophan], anti-BCMA (human monoclonal 83A10-42 VH-CH1 fragment) fusion protein with peptide (synthetic GGGGSGGGGS linker) fusion protein with immunoglobulin anti- (human CD3 antigen e-chain) (human-Mus musculus monoclonal CH2527 heavy chain VL(l)-CH1-CH2- CH3), (219®216"')-disulfide with immunoglobulin anti-BCMA (human monoclonal 83A10-42 k-chain [125"'-arginine, 126"'-lysine]) and (444®232'')-disulfide with immunoglobulin anti-(human CD3 antigen e-chain) (human-Mus musculus monoclonal CH2527 light chain VH-CL(k)), (450®225'), (453®228'), (578®348')-tris(disulfide) with immunoglobulin G1 [146'-glutamic acid,212'-glutamic acid,233'- alanine,234'-alanine,328'-glycine,348'-cysteine,365'-serine,367'-alanine,406'-valine] anti-BCMA (human monoclonal 83A10-42 g1-chain), (216''''®219')-disulfide with human monoclonal 83A10-42 k-chain [125''''-arginine, 126''''-lysine]. [0088] A predominant form of the optimized assembled entity (CC-93269 (FIG.1)) exhibits 1 G0F glycosylation on SEQ ID NO:1 (HC A) Asn 521 and on SEQ ID NO:3 (HC' B) Asn 296' as well as no C- terminal Lys (K) residue on either of these chains (this structural isoform has a molecular formula of C8622H13310N2292O2763S54 and molecular weight of 195013.1 Da). Optimized Species with C-terminal lysine (K) (on both HC) CC-93269 glycosylated G0F/G0F = C8634 H13334 N2296 O2765 S54 = 195269.5 Da
CC-93269 non-glycosylated = C8522 H13150 N2288 O2687 S54 = 192378.8 Da
Optimized Species without C-terminal lysine (K) (on both HC) CC-93269 glycosylated G0F/G0F = C8622 H13310 N2292 O2763 S54 = 195013.1 Da
CC-93269 non-glycosylated = C8510 H13126 N2284 O2685 S54 = 192122.5 Da
EXAMPLE II Sequence details of the described optimized bispecific trivalent anti-BCMA/anti-CD3 T Cell engaging antibody (TCE) (immunoglobulin G1-kappa/lambda with domain crossover, anti-BCMA and anti-CD3e, humanized monoclonal antibody, bispecific, trivalent dimer (450-225’:453-228’:578- 348’)-trisdisulfide)
HC (SEQ ID NO:1) A Chain:
[0089] gamma-kappa heavy chain anti-BCMA and anti-CD3e (VH-CH1-VL-CH1-CH2-CH3) (1-671) [human VH anti-BCMA (Human IGHV3-23D*01 (94.9%) -(IGHD) -IGHJ4*02 (100%)) [8.8.9] (1-116) -Human IGHG1*01, G1m17 (CH1 K120 (213) [K>E26 (146) K>E119 (212)] (117-214), hinge 1-6 (215-220))(117- 220) -10-mer bis(tetraglycyl-seryl) linker (221-230) -humanized VL anti-CD3E (Human (V-LAMBDA (musculus IGLV1*01 (80.4%) -IGLJ1*01 (100%)/Human IGLV7-46*01 (80.9%) -IGLJ3*02 (100%)) [9.3.9] (231-339))-2-mer biseryl linker -Human IGHG1*01, G1m17,1 (CH1 K120 (438) (342-439), hinge 1-15 (440-454), CH2 [L1.3>A (458), L1.2>A (459), P114>G (553)] (455-564), CH3 D12 (580), L14 (582) [S10>C (578), T22>W (590)] (565-669), CHS (670-671)) (342-671)] HC’ (SEQ ID NO:3) B Chain:
[0090] Human anti-BCMA (1’-446’) [human VH (Human IGHV3-23D*01 (94.9%) -(IGHD) -IGHJ4*02 (100%)) [8.8.9] (1’-116’) -Human IGHG1*01, G1m17,1 (CH1 K120 (213’) [K>E26 (146’), K>E119 (212’)] (117’-214’), hinge 1-15 (215’-229’), CH2 [L1.3>A (233’), L1.2>A (234’), P114>G (328’)] (230’-339’), CH3 D12 (355’), L14 (357’) [Y5>C (348’), T22>S (365’), L24>A (367’), Y86>V (406’)] (340’-444’), CHS (445’- 446’)) (117’-446’)] LC’’ (SEQ ID NO:2) anti-CD3ɛ A Chain:
[0091] VH kappa light chain humanized anti-CD3e (1’’-232’’) [(Human IGHV3-23*03 (86.1%) -(IGHD) - IGHJ4*02 (92.9%))[8.10.16](1’’-125’’) -2-mer Ala-Ser linker (126’’-127’’) -Human IGKC*01 Km3 A45.1 (171’’), V101(209’’) (128’’-232’’)] LC’’’ (SEQ ID NO:4) anti-BCMA A Chain:
[0092] kappa light chain anti-BCMA (1’’’-216’’’) [human V-KAPPA (Human IGKV3-20*01 (92.3%) - IGKJ1*01 (91.7%)) [7.3.10] (1’’’-109’’’) -Human IGKC*01 Km3 A45.1 (155’’’), V101(193’’’) [E12>R(125’’’), Q13>K (126’’’)] (110’’’-216’’’)]
LC’’’’ (SEQ ID NO:4) anti-BCMA B Chain:
[0093] kappa light chain anti-BCMA (1’’’’-216’’’’) [human V-KAPPA (Human IGKV3-20*01 (92.3%) - IGKJ1*01 (91.7%)) [7.3.10] (1’’’’-109’’’’) -Human IGKC*01 Km3 A45.1 (155’’’’), V101(193’’’’)
[E12>R(125’’’’), Q13>K (126’’’’)] (110’’’’-216’’’’)]
EXAMPLE III Activity profile
[0094] A series of in vitro, ex vivo, and in vivo studies have been conducted to investigate the activity profile of CC-93269. Surface plasmon resonance (SPR) assays were used to measure the binding affinity of CC-93269 to BCMA of human, cynomolgus monkey, rat, and mouse origin. The affinity of the BCMA binder of CC-93269 (anti-BCMA IgG and Fab fragment of clone 83A10-42) to human BCMA ranged from equilibrium dissociation constant (KD) values of 0.203 nM to 0.450 nM, with a mean value of 0.431 ± 0.22 nM. CC-93269 bound to human BCMA (hu-BCMA) and cynomolgus BCMA (cyno-BCMA) with approximately 10-fold higher affinity than to rodent BCMA. CC-93269 bound to human cluster of differentiation 3 epsilon/delta heterodimer (CD3ed) with a KD value of 25 ± 12 nM, whereas bound to cynomolgus CD3ed with KD values of 57 and 59 nM.
EXAMPLE IV Binding studies
[0095] In vitro, CC-93269 had no detectable complement-dependent cytotoxicity (CDC) on BCMA expressing Pfeiffer cells. CC-93269 did not show any binding affinity to FcgRs expressed on human embryonic kidney (HEK) 293 cells. CC-93269 exhibited BCMA-specific binding on various myeloma cell lines expressing different levels of BCMA (BCMAhigh NCI-H929, BCMAmid L363, BCMAmid JJN-3, BCMAlow RPMI-8226), as well as on stably transfected BCMAhigh HEK293T-huBCMA and
BCMAhighHEK293T-cynoBCMA. Binding half-maximal effective concentration (EC50) values ranged from 3.4 nM to 15.2 nM for the BCMA+ cell lines. [0096] The ability of CC-93269 to bind T cells was assessed by flow cytometry. The EC50 values for CC- 93269 to CD3 on Jurkat cells ranged between 16.76 nM and 37.42 nM in one study and 121.3 nM in a second study. No specific binding of CC-93269 was detected on primary mouse or rat CD4 and CD8 T cells. Upon simultaneous binding of CC-93269 to CD3 on T cells and BCMA on target cells, CC-93269
forces crosslinking of TCR/CD3 leading to major histocompatibility class (MHC)-independent T cell activation and release of cytokines, followed by formation of a cytolytic synapse and secretion of cytolytic enzymes resulting in tumor cell lysis. Co-cultures of T cells derived from healthy human donors with BCMA-expressing MM cell lines in the presence of CC-93269 resulted in MM cell killing. As expected, CC-93269 did not induce T cell-redirected killing in the absence of BCMA+ target cells, ie, BCMA negative MKN45 gastric cancer and control HEK293T cell lines.
EXAMPLE V Efficacy model
[0097] CC-93269 demonstrated concentration-dependent killing of primary MM plasma cells of bone marrow samples from both newly diagnosed and relapsed MM patients in the presence of autologous T cells as effector cells. Two separate methods were employed, either using unmanipulated whole bone marrow or CD138+ partially-depleted bone marrow (manipulated) incubated with CC-93269. Multiple myeloma plasma cells were sensitive to CC-93269-dependent T cell-redirected killing and significant lysis was observed in 11 out of 16 patient whole bone marrow samples (unmanipulated) after 48 hours of incubation. When tested with manipulated CD138+ partially-depleted bone marrow aspirates, all 13 patient samples responded to CC-93269. Extended incubation of unmanipulated bone marrow samples with CC-93269 demonstrated that there is a tendency for enhanced antibody-dependent tumor cell lysis. An increase in tumor cell lysis was observed in 7 of the 8 MM patient samples tested at 96 hours compared to 48 hours of incubation with CC-93269. [0098] CC-93269-induced tumor cell killing was accompanied by T cell activation and cytokine release in both cell lines and primary MM patient bone marrow samples. Concentration-dependent upregulation of T cell activation markers (CD69, CD25, and HLA-DR) on both CD4 and CD8 T cells from the patient unmanipulated whole bone marrow was observed after 48 and 96 hours of incubation. Cytokines (i.e., interferon-gamma [IFN-g]) and cytolytic proteins (ie, granzyme B and perforin) were released from the T cells in co-culture, which ultimately resulted in lysis of the targeted tumor cell.
EXAMPLE VI Binding studies
[0099] Biochemical and cellular assays were employed to characterize the binding profile of CC-93269. In SPR assays, the BCMA binder of CC-93269 cross-reacted with the four orthologs of BCMA tested
(human, cynomolgus monkey, rat, and mouse). The affinity of the BCMA binder of CC-93269 (anti- BCMA IgG and Fab fragment of clone 83A10-42) to human BCMA ranged from a KD value of 0.203 nM to 0.450 nM, with a mean value of 0.431 ± 0.22 nM (n = 3). CC-93269 binds to human BCMA (hu-BCMA) and cynomolgus BCMA (cyno-BCMA) with approximately 10-fold higher affinity than to rodent BCMA. [0100] CC-93269 showed no sustained binding to human BAFF receptor (hu-BAFF-R) and human transmembrane activator and calcium modulator and cyclophilin ligand interactor (hu-TACI). CC-93269 bound to human CD3ed heterodimer with a KD value of 25 ± 12 nM (n = 3), whereas it bound to cynomolgus CD3ed with KD values of 57 and 59 nM (n = 2).
EXAMPLE VII Binding studies
[0101] Binding of CC-93269 on various myeloma cell lines expressing different levels of BCMA
(BCMAhigh NCI-H929, BCMAmid L363, BCMAmid JJN-3, BCMAlow RPMI-8226) as well as stably transfected BCMAhigh HEK293T-huBCMA and BCMAhigh HEK293T-cynoBCMA was evaluated by flow cytometry. Binding EC50 values ranged from 3.4 to 15.2 nM for the BCMA+ cell lines. Concentration- dependent, BCMA-specific binding of CC-93269 was demonstrated in U266 MM cell lines with blocking experiments. Increasing concentrations of BCMA/Fc, APRIL, and anti-BCMA murine IgG2a (19F2) proportionately reduced the binding of phycoerythrin (PE)-conjugated BCMA binder (Clone 83A10-42) of CC-93269 on U266 cells. Another study using 3 different lots of CC-93269 showed similar binding to NCI- H929 MM cells, with EC50 values ranging between 1.7 nM and 2.8 nM. The ability of CC-93269 to bind mouse and rat T cells was assessed by flow cytometry. No specific binding of CC-93269 was detected on primary mouse or rat CD4 and CD8 T cells up to the highest concentration tested (30 mg/mL [156 nM]).
EXAMPLE VIII Efficacy model
[0102] In addition to BCMA-expressing cell line killing, CC-93269 also demonstrated concentration dependent, autologous T cell-mediated killing of primary MM cells in ex vivo cultures of MM patient bone marrow aspirates. * * *
All publications and patents referred to herein are incorporated by reference. Various modifications and variations of the described subject matter will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to these embodiments. Indeed, various modifications for carrying out the invention are obvious to those skilled in the art and are intended to be within the scope of the following claims.
Claims
WHAT IS CLAIMED IS 1. A bispecific trivalent anti-BCMA/anti-CD3 T Cell Engaging Antibody (TCE) comprising, a heavy chain (anti-BCMA and anti-CD3) A (HC) (SEQ ID NO:1); a heavy chain (anti-BCMA) B (HC') (SEQ ID NO:3); a light chain (anti-CD3) A (LC'') (SEQ ID NO:2); a light chain (anti-BCMA) A (LC''') (SEQ ID NO:4); and, a light chain (anti-BCMA) B (LC'''') (SEQ ID NO:4).
2. A TCE according to claim 1 wherein the heavy chain (anti-BCMA and anti-CD3) A (HC) (SEQ ID NO:1) comprises at least one intra-chain disulfide bond selected from the group consisting of C22-C96, C143- C199, C252-C320, C368-C424, C485-C545, and C591-C649.
3. A TCE according to claim 2 wherein the heavy chain (anti-BCMA) B (HC') (SEQ ID NO:3) comprises at least one intra-chain disulfide bond selected from the group consisting of C22’-C96’, C143’-C199’, C260’- C320’, and C366’-C424’.
4. A TCE according to claim 3 wherein the light chain (anti-CD3) A (LC'') (SEQ ID NO:2) comprises at least one intra-chain disulfide bond selected from the group consisting of C22’’-C98’’ and C152’’-C212’’.
5. A TCE according to claim 4 wherein the light chain (anti-BCMA) A (LC''') (SEQ ID NO:4) comprises at least one intra-chain disulfide bond selected from the group consisting of C23’’’-C89’’’ and C136’’’- C196’’’.
6. A TCE according to claim 5 wherein the light chain (anti-BCMA) B (LC'''') (SEQ ID NO:4) comprises at least one intra-chain disulfide bond selected from the group consisting of C23’’’’-C89’’’’ and C136’’’’- C196’’’’.
7. A TCE according to claim 6 wherein the heavy chain (anti-BCMA and anti-CD3) A (HC) (SEQ ID NO:1) comprises at least one inter-chain disulfide bond selected from the group consisting of● C219-C216''' between itself and the light chain (anti-BCMA) A (LC''') (SEQ ID NO:4);● C444-C232’’ between itself and the light chain (anti-CD3) A (LC'') (SEQ ID NO:2);● C450-C225’ between itself and the heavy chain (anti- BCMA) B (HC') (SEQ ID NO:3);● C453-C228’ between itself and the heavy chain (anti-BCMA) B (HC') (SEQ ID NO:3); and,● C578-C348’ between itself and the heavy chain (anti-BCMA) B (HC') (SEQ ID NO:3).
8. A TCE according to claim 7 wherein the heavy chain (anti-BCMA) B (HC') (SEQ ID NO:3) comprises an inter-chain disulfide bond● C219’-C216’’’’ between itself and the light chain (anti-BCMA) B (LC'''') (SEQ ID NO:4).
9. A TCE according to claim 8 wherein the heavy chain (anti-BCMA and anti-CD3) A (HC) (SEQ ID NO:1) comprises glycosylated N521.
10. A TCE according to claim 9 wherein glycosylated N521 comprises fucosylated complex bi-antennary CHO-type glycans.
11. A TCE according to claim 9 wherein the heavy chain (anti-BCMA) B (HC') (SEQ ID NO:3) comprises glycosylated N296’.
12. A TCE according to claim 10 wherein heavy chain (anti-BCMA) B (HC') (SEQ ID NO:3) N296’ comprises fucosylated complex bi-antennary CHO-type glycans.
13. A precursor peptide including a signal sequence, for the production of mature heavy chain (anti- BCMA and anti-CD3) A (HC) (SEQ ID NO:1) and assembly of the TCE according to claim 1, comprising SEQ ID NO:5.
14. A precursor peptide including a signal sequence, for the production of mature heavy chain (anti- BCMA) B (HC') (SEQ ID NO:3) and assembly of the TCE according to claim 1, comprising SEQ ID NO:7.
15. A precursor peptide including a signal sequence, for the production of mature light chain (anti-CD3) A (LC'') (SEQ ID NO:2) and assembly of the TCE according to claim 1, comprising SEQ ID NO:6.
16. A precursor peptide including a signal sequence, for the production of mature light chain (anti- BCMA) A (LC''') (SEQ ID NO:4) or light chain (anti-BCMA) B (LC'''') (SEQ ID NO:4), and assembly of the TCE according to claim 1, comprising SEQ ID NO:8.
17. A nucleic acid sequence for production of mature heavy chain (anti-BCMA and anti-CD3) A (HC) (SEQ ID NO:1) and assembly of the TCE according to claim 1, comprising SEQ ID NO:15.
18. A nucleic acid sequence for production of mature heavy chain (anti-BCMA) B (HC') (SEQ ID NO:3) and assembly of the TCE according to claim 1, comprising SEQ ID NO:17.
19. A nucleic acid sequence for production of mature light chain (anti-CD3) A (LC'') (SEQ ID NO:2) and assembly of the TCE according to claim 1, comprising SEQ ID NO:16.
20. A nucleic acid sequence for production of mature light chain (anti-BCMA) A (LC''') (SEQ ID NO:4) or mature light chain (anti-BCMA) B (LC'''') (SEQ ID NO:4), and assembly of the TCE according to claim 1, comprising SEQ ID NO:18.
21. A nucleic acid sequence for production of mature heavy chain (anti-BCMA and anti-CD3) A (HC) (SEQ ID NO:1) and assembly of the TCE according to claim 1, comprising SEQ ID NO:19.
22. A nucleic acid sequence for production of mature heavy chain (anti-BCMA) B (HC') (SEQ ID NO:3) and assembly of the TCE according to claim 1, comprising SEQ ID NO:21.
23. A nucleic acid sequence for production of mature light chain (anti-CD3) A (LC'') (SEQ ID NO:2) and assembly of the TCE according to claim 1, comprising SEQ ID NO:20.
24. A nucleic acid sequence for production of mature light chain (anti-BCMA) A (LC''') (SEQ ID NO:4) or mature light chain (anti-BCMA) B (LC'''') (SEQ ID NO:4), and assembly of the TCE according to claim 1, comprising SEQ ID NO:22.
25. A nucleic acid sequence for production of mature heavy chain (anti-BCMA and anti-CD3) A (HC) (SEQ ID NO:1) and assembly of the TCE according to claim 1, comprising SEQ ID NO:27.
26. A nucleic acid sequence for production of mature heavy chain (anti-BCMA) B (HC') (SEQ ID NO:3) and assembly of the TCE according to claim 1, comprising SEQ ID NO:29.
27. A nucleic acid sequence for production of mature light chain (anti-CD3) A (LC'') (SEQ ID NO:2) and assembly of the TCE according to claim 1, comprising SEQ ID NO:28.
28. A nucleic acid sequence for production of mature light chain (anti-BCMA) A (LC''') (SEQ ID NO:4) or mature light chain (anti-BCMA) B (LC'''') (SEQ ID NO:4), and assembly of the TCE according to claim 1, comprising SEQ ID NO:30.
29. A pharmaceutical composition comprising a TCE according to claim 1 and at least one pharmaceutically acceptable excipient.
30. A method for the treatment of a pathological condition in a mammal selected from the group consisting of a hematological malignancy, a plasma cell disorder, a B-cell malignancy, multiple myeloma (MM), lymphoma, an immune system disorder, an autoimmune disorder, and an inflammation disorder, comprising administering an effective amount of a TCE according to claim 1 to a mammal in need thereof.
* * *
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