WO2020237173A1 - Anti-ror1/anti-cd3 bispecific binding molecules - Google Patents

Anti-ror1/anti-cd3 bispecific binding molecules Download PDF

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WO2020237173A1
WO2020237173A1 PCT/US2020/034281 US2020034281W WO2020237173A1 WO 2020237173 A1 WO2020237173 A1 WO 2020237173A1 US 2020034281 W US2020034281 W US 2020034281W WO 2020237173 A1 WO2020237173 A1 WO 2020237173A1
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seq
amino acid
nos
antigen
binding domain
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French (fr)
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Jeffry D. Watkins
Katti JESSEN
Mira KO
Brian Lannutti
Thanh-Trang VO
J. Monty WATKINS
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Velosbio Inc
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Velosbio Inc
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Priority to BR112021023026A priority Critical patent/BR112021023026A2/pt
Priority to KR1020217041982A priority patent/KR20220012313A/ko
Priority to JP2021569109A priority patent/JP7611171B2/ja
Priority to CA3139111A priority patent/CA3139111A1/en
Priority to CN202080038180.6A priority patent/CN113874399B/zh
Priority to MX2021014193A priority patent/MX2021014193A/es
Priority to US17/613,251 priority patent/US20220227866A1/en
Priority to AU2020277489A priority patent/AU2020277489A1/en
Priority to EP20732061.5A priority patent/EP3972999A1/en
Publication of WO2020237173A1 publication Critical patent/WO2020237173A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], 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 [IG], 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • C07K2317/35—Valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/526—CH3 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/624—Disulfide-stabilized antibody (dsFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
    • 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
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • 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
    • C07K2317/77—Internalization into the cell
    • 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

  • 024651_WO004_SL.txt is 258,844 bytes in size.
  • Receptor tyrosine kinase-like orphan receptor 1 is a cell surface protein that mediates signals from its ligand, the secreted glycoprotein Wnt5a. Consistent with its role in influencing the fate of stem cells during embryogenesis, ROR1 expression is observed on invasive malignancies that revert to an embryonic transcriptional program, but is not observed on normal adult tissues, offering a favorable selectivity profile as a therapeutic target.
  • ROR1 is commonly expressed on the malignant cells of patients with acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphoblastic leukemia (CLL), mantle cell lymphoma (MCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), diffuse large B- cell lymphoma (DLBCL), and Richter's transformation or Richter's syndrome (RS).
  • ALL acute lymphoblastic leukemia
  • AML acute myeloid leukemia
  • CLL chronic lymphoblastic leukemia
  • MCL mantle cell lymphoma
  • FL follicular lymphoma
  • MZL marginal zone lymphoma
  • DLBCL diffuse large B- cell lymphoma
  • RS Richter's transformation or Richter's syndrome
  • CD3 Cluster of differentiation 3
  • TCR T cell receptor complex
  • Functional CD3 is formed from the dimeric association of two of four distinct chains— epsilon, gamma, delta, and zeta— into one of the following three pairs of dimers: gamma/epsilon, delta/epsilon and zeta/zeta.
  • Antibodies against CD3 have been shown to cluster CD3 on T cells, causing T cell activation. Accordingly, anti-CD3 antibodies have been proposed for therapeutic purposes involving the activation of T cells.
  • bispecific antibodies that are capable of binding CD3 and a target antigen have been proposed for therapeutic uses involving targeting T cell immune responses to tissues and cells expressing the target antigen.
  • bispecific binding molecules Numerous formats and compositions have been described for bispecific binding molecules. Multiple variables impact the in vivo potency of these molecules, including PK, targeted antigen epitopes, the relative affinities of the antigen-binding components, and the valency and spatial configuration of the paratopes.
  • PK in vivo potency of these molecules
  • targeted antigen epitopes the relative affinities of the antigen-binding components
  • valency and spatial configuration of the paratopes Currently, there is no reliable method for designing a priori a single molecule that optimizes all these parameters for a bi specific binding molecule, such as an antibody that specifically binds to both ROR1 and CD3. Bispecific constructs must be designed, made, and tested to systematically evaluate the impact of many of these parameters and to select optimal bispecific binding molecules for therapeutic uses such as cancer treatment.
  • the present invention is directed to novel bispecific binding molecules targeting ROR1 and CD3, as well as pharmaceutical compositions comprising one or more of these antibodies, and use of the antibodies and pharmaceutical compositions for treating cancer. Compared to currently available cancer treatments, including antibody treatments, it is contemplated that the bispecific binding molecules of the invention may provide a superior clinical response.
  • the present disclosure provides a bispecific binding molecule comprising a first antigen-binding domain that specifically binds to an extracellular domain of human ROR1 and a second antigen-binding domain that specifically binds to an extracellular domain of human CD3.
  • the first antigen-binding domain competes for binding to human ROR1 with, or binds to the same epitope of human ROR1 as, an antibody comprising a heavy chain amino acid sequence set forth in SEQ ID NO: 82 and a light chain amino acid sequence set forth in SEQ ID NO: 83.
  • the first antigen-binding domain comprises:
  • H-CDR1-3 and L-CDR1-3 comprising the amino acid sequences of SEQ ID NOs: 97, 61, 62, 63, 64, and 65, respectively,
  • H-CDR1-3 and L-CDR1-3 comprising the amino acid sequences of SEQ ID NOs: 60, 61, 62, 63, 64, and 65, respectively;
  • VH heavy chain variable domain
  • VL light chain variable domain
  • VH comprising the amino acid sequence of SEQ ID NO: 72 and a VL comprising the amino acid sequence of SEQ ID NO: 73;
  • an HC comprising the amino acid sequence of SEQ ID NO: 82 and an LC comprising the amino acid sequence of SEQ ID NO: 83;
  • an HC comprising the amino acid sequence of SEQ ID NO: 86 and an LC comprising the amino acid sequence of SEQ ID NO: 83.
  • the second antigen-binding domain comprises:
  • H-CDR1-3 and L-CDR1-3 comprising the amino acid sequences of SEQ ID NOs: 54, 55, 56, 57, 58, and 59, respectively;
  • H-CDR1-3 and L-CDR1-3 comprising the amino acid sequences of SEQ ID NOs: 47, 48, 49, 50, 51, and 52, respectively;
  • H-GDR1 -3 and L-CDR1-3 comprising the amino acid sequences of SEQ ID NOs: 47, 53, 49, 50, 51, and 52, respectively;
  • VH heavy chain variable domain
  • VL light chain variable domain
  • VH comprising an amino acid sequence at least 90% identical to SEQ ID NO: 66 and a VL comprising an amino acid sequence at least 90% identical to SEQ ID NO: 67;
  • VH comprising an amino acid sequence at least 90% identical to SEQ ID NO: 68 and a VL comprising an amino acid sequence at least 90% identical to SEQ ID NO: 69;
  • VH comprising the amino acid sequence of SEQ ID NO: 70 and a VL comprising the amino acid sequence of SEQ ID NO: 71;
  • VH comprising the amino acid sequence of SEQ ID NO: 66 and a VL, comprising the amino acid sequence of SEQ ID NO: 67;
  • VH comprising the amino acid sequence of SEQ ID NO: 68 and a VL comprising the amino acid sequence of SEQ ID NO: 69;
  • VH comprising the amino acid sequence of SEQ ID NO: 80 and a VL, comprising the amino acid sequence of SEQ ID NO: 81;
  • VH comprising the amino acid sequence of SEQ ID NO: 76 and a VL comprising the amino acid sequence of SEQ) ID NO: 77;
  • VH comprising the amino acid sequence of SEQ ID NO: 78 and a VL comprising the amino acid sequence of SEQ ID NO: 79.
  • the present disclosure provides a bispecific binding molecule comprising:
  • the present disclosure provides a bispecific binding molecule comprising:
  • VH heavy chain variable domain
  • VL light chain variable domain
  • a first antigen-binding domain that comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 74 and 73, respectively, and a second antigen-binding domain that comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 66 and 67, respectively;
  • a first antigen-binding domain that comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 72 and 73, respectively, and a second antigen-binding domain that comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 66 and 67, respectively.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • a first antigen-binding domain that comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 72 and 73, respectively, and a second antigen-binding domain that comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 76 and 77, respectively.
  • a first antigen-binding domain that comprises an HC comprising the amino acid sequence of SEQ ID NO: 86, an HC comprising the amino acid sequence of SEQ ID NO: 106 or 109, and an LC comprising the amino acid sequence of SEQ ID NO: 83, and a second antigen-binding domain that comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 70 and 71, respectively:
  • a first antigen-binding domain that comprises an HC comprising the amino acid sequence of SEQ ID NO: 86, an HC comprising the amino acid sequence of SEQ ID NO: 106 or 109, and an LC comprising the amino acid sequence of SEQ ID NO: 83, and a second antigen-binding domain that comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 68 and 69, respectively;
  • a first antigen-binding domain that comprises an HC comprising the amino acid sequence of SEQ ID NO: 86, an HC comprising the amino acid sequence of SEQ ID NO: 106 or 109, and an LC comprising the amino acid sequence of SEQ ID NO: 83, and a second antigen-binding domain that comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 76 and 77, respectively.
  • a bispecific binding molecule described herein may have any of the following valencies for the first and second antigen-binding domains:
  • the first antigen-binding domain has a valency of 2 and the second antigen-binding domain has a valency of 2;
  • a bispecific binding molecule described herein may comprise a human IgG1 constant region; said constant region may contain the amino acid substitutions L234A, L235A, and/or G237A, wherein the residues are numbered according to the EU system.
  • the second antigen-binding domain of a bispecific binding molecule described herein is an scFv.
  • a heavy or light chain amino acid sequence of the second antigen-binding domain is fused to a heavy or light chain amino acid sequence of the first antigen-binding domain via a peptide linker.
  • the peptide linker has the amino acid sequence of GGGGSGGGGS (SEQ ID NO: 93).
  • the heavy or light chain amino acid sequence of the second antigen-binding domain may be fused to, e.g. :
  • a bispecific binding molecule described herein has at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) of the following properties:
  • a) has a KD for immobilized ROR1 of 0.5 nM or less as determined by ELISA;
  • c) demonstrates reduced internalization in ROR1 -transfected MEC cells and/or Jurkat cells as compared to an antibody comprising the heavy and light chain amino acid sequences of SEQ ID NOs: 82 and 83, respectively;
  • d) induces LDH release in PBMC-exposed ROR1 -transfected MEC cells at 1 mg/mL or less; e) induces LDH release in PBMC-exposed JeKo-1 cells at 1 mg/mL or less;
  • the present disclosure also provides an immunoconjugate comprising a bispecific binding molecule described herein conjugated to a cytotoxic agent.
  • the present disclosure also provides a pharmaceutical composition comprising a bispecific binding molecule described herein and a pharmaceutically acceptable excipient.
  • FIG. 2 is a pair of graphs illustrating the binding to human ROR1 of a parental Ab1 Fab (WT) and three heavy chain variants (Ab2 (T32A), Ab3 (T32E), and W110Y), as determined by ELISA (Panel A) and by flow cytometry analysis of binding to live ROR1 -transfected MEC cells (Panel B).
  • WT parental Ab1 Fab
  • T32A three heavy chain variants
  • T32E Ab3
  • W110Y W110Y
  • FIG. 9 is a pair of graphs showing cell surface levels of bispecific binding molecules bound to ROR1 (“Surface biAb”), cell surface levels of free ROR1 not bound by bispecific binding molecules (“Unoccupied epitope”), and total cell surface ROR1 (“Total ROR1”), for ROR1 -transfected MEC cells incubated with construct 19 (Panel A) and construct 20 (Panel B).
  • Surface biAb cell surface levels of bispecific binding molecules bound to ROR1
  • Unoccupied epitope cell surface levels of free ROR1 not bound by bispecific binding molecules
  • Total ROR1 total cell surface ROR1
  • FIG. 13 is a graph quantitating levels of bispecific constructs 1-3, 7-9, and 13-15 on the surface of Jurkat cells over time following binding to CD3 in the absence of ROR1.
  • FIG. 26 is a set of graphs quantitating release of IL-4 from JeKo-1 cells (Panel A), ROR1 -transfected MEC cells (Panel B), and from mock-transfected, ROR1-MEC cells (Panel C) following activation of T cells with designated target cells and bispecific constructs 7, 9, 18, and 20 (Panel A) or 9, 18, and 20 (Panels B and C). Mock-transfected, ROR1-MEC cells (Panel C) were treated with a single concentration (1 mg/mL) of the bispecific constructs.“Control 1” is an unrelated ROR1 ⁇ CD3 bispecific construct.
  • the first antigen-binding domain competes for binding to human ROR1 with, and/or binds to the same epitope of human RORI as, an anti-ROR1 antibody described in PCX Patent Application PCT/US2013/32572. In some embodiments, the first antigen-binding domain competes for binding to human RORI with, and/or binds to the same epitope of human ROR1 as, an antibody comprising a heavy chain amino acid sequence set forth in SEQ ID NO: 82 and a light chain amino acid sequence set forth in SEQ ID NO: 83 (“Ab1”).
  • VH comprising the amino acid sequence of SEQ ID NO: 75 and a VL comprising the amino acid sequence of SEQ ID NO: 73;
  • an HC comprising the amino acid sequence of SEQ ID NO: 88, an HC comprising the amino acid sequence of SEQ ID NO: 109, and an LC comprising the amino acid sequence of SEQ ID NO: 83;
  • the second antigen-binding domain comprises:
  • the second antigen-binding domain comprises the six CDRs and/or the heavy and light chain variable domains of an anti-CD 3 antibody as described in U.S. Patent Publication 2018/011201 1.
  • first antigen-binding domain that comprises H-CDR1-3 and L-CDR1-3 comprising the amino acid sequences of SEQ ID NOs: 98, 61, 62, 63, 102, and 65, respectively
  • second antigen- binding domain that comprises H-CDR1-3 and L-CDR1-3 comprising the amino acid sequences of SEQ ID NOs: 47, 48, 49, 50, 51, and 52, respectively;
  • first antigen-binding domain that comprises a VH and a VL at least 90% (e.g., at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences of SEQ ID NOs: 72 and 103, respectively
  • second antigen-binding domain that comprises a VH and a VL at least 90% (e.g., at least 92%, at least 95*%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences of SEQ ID NOs: 68 and 69, respectively;
  • first antigen-binding domain that comprises a VH and a VL at least 90% (e.g., at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences of SEQ ID NOs: 74 and 100, respectively
  • second antigen-binding domain that comprises a VH and a VL at. least. 90% (e.g., at least 92%, at. least. 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences of SEQ ID NOs: 68 and 69, respectively;
  • first antigen-binding domain that comprises a VH and a VL at least 90% (e.g., at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences of SEQ ID NOs: 75 and 100, respectively
  • second antigen-binding domain that comprises a VH and a VL at least 90% (e.g., at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences of SEQ ID NOs: 70 and 71 , respectively;
  • first antigen-binding domain that comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 74 and 73, respectively
  • second antigen-binding domain that comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 70 and 71 or 80 and 81, respectively;
  • first antigen-binding domain that comprises an HC and an LC comprising the amino acid sequences of SEQ ID NOs: 82 and 104, respectively, and a second antigen-binding domain that comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 66 and 67 or 76 and 77, respectively;
  • first antigen-binding domain that comprises an HC and an LC comprising the amino acid sequences of SEQ ID NOs: 7 and 101, respectively, and a second antigen-binding domain that comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 70 and 71 or 80 and 81, respectively;
  • first antigen-binding domain that comprises an HC and an LC comprising the amino acid sequences of SEQ ID NOs: 7 and 101, respectively, and a second antigen-binding domain that comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 66 and 67 or 76 and 77, respectively;
  • first antigen-binding domain that comprises an HC and an LC comprising the amino acid sequences of SEQ ID NOs: 7 and 101, respectively
  • second antigen-binding domain that comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 68 and 69 or 78 and 79, respectively
  • a first antigen-binding domain that comprises an HC comprising the amino acid sequence of SEQ ID NO: 87, an HC comprising the amino acid sequence of SEQ ID NO: 109, and an LC comprising the amino acid sequence of SEQ ID NO: 83, and a second antigen-binding domain that comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 68 and 69 or 78 and 79, respectively;
  • a first antigen-binding domain that comprises an HC comprising the amino acid sequence of SEQ ID NO: 88, an HC comprising the amino acid sequence of SEQ) ID NO: 109, and an LC comprising the amino acid sequence of SEQ ID NO: 101, and a second antigen-binding domain that comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 70 and 71 or 80 and 81, respectively;
  • a nucleic acid molecule of the present disclosure comprises:
  • bispecific binding molecules of the present disclosure are used to treat solid tumors.
  • the cancer to be treated may be selected from, e.g., lymphoma, small lymphocytic lymphoma, marginal zone lymphoma, marginal cell B-cell lymphoma, Burkitf s lymphoma, mantle cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, a non-Hodgkin lymphoma that has undergone Richter's transformation, T cell non-Hodgkin lymphoma, lymphoplasmacytoid lymphoma, Waldenstrom macroglobulinemia, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, T cell leukemia, sarcoma, osteosarcoma, Ewing sarcoma, renal cell carcinoma, hepatocellular carcinoma, colon cancer, colorectal cancer, breast cancer, epithelial squamous cell cancer, glioblastom
  • Three anti-CD3 sequences were used as scFv constructs in anti-ROR1/anti-CD3 bispecific binding molecules.
  • Two of the sequences are different humanized versions of OKT3 (Ab8 and Ab9) while the third sequence is a third party humanized version of a different anti- CD3 antibody, SP34 (AblO).
  • OKT3 and SP34 bind distinct epitopes, and anti- CD3 antibodies have been classified based on distinct characteristics of their epitopes (Tunnacliffe et al., International Immunology 1:546-550 (1989)).
  • SP34 is classified as Group I while OKT3 is classified as Group II.
  • the SP34-based sequence, but not the OKT3-based sequences, are expected to be cross-reactive with cynomolgus CD3.
  • the MSD ® V-PLEX Cytokine Panel 1 Human Kit was used to concurrently measure IFN-g, IL-2, IL-4, IL-6, IL-10 and TNF-a.
  • the assay was conducted per manufacturer's instructions. Briefly, the V-plex Cytokine Panel 1 plate was washed 3 times with 150 mL/wel olf Wash Buffer (PBS with 0.05% Tween-20). Next, 50 mL of diluted samples and calibrators were added per well. The plate was sealed with an adhesive plate seal and incubated on a shaker overnight at 4°C, 500 rpm.

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PCT/US2020/034281 2019-05-23 2020-05-22 Anti-ror1/anti-cd3 bispecific binding molecules Ceased WO2020237173A1 (en)

Priority Applications (9)

Application Number Priority Date Filing Date Title
BR112021023026A BR112021023026A2 (pt) 2019-05-23 2020-05-22 Moléculas de ligação biespecíficas anti-ror1/anti-cd3
KR1020217041982A KR20220012313A (ko) 2019-05-23 2020-05-22 항-ror1/항-cd3 이중특이적 결합 분자
JP2021569109A JP7611171B2 (ja) 2019-05-23 2020-05-22 抗ｒｏｒ１／抗ｃｄ３二重特異性結合分子
CA3139111A CA3139111A1 (en) 2019-05-23 2020-05-22 Anti-ror1/anti-cd3 bispecific binding molecules
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