WO2014151438A1 - Anticorps anti-cd37 multispécifiques, compositions et méthodes s'y rapportant - Google Patents

Anticorps anti-cd37 multispécifiques, compositions et méthodes s'y rapportant Download PDF

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WO2014151438A1
WO2014151438A1 PCT/US2014/025729 US2014025729W WO2014151438A1 WO 2014151438 A1 WO2014151438 A1 WO 2014151438A1 US 2014025729 W US2014025729 W US 2014025729W WO 2014151438 A1 WO2014151438 A1 WO 2014151438A1
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seq
antibody
amino acid
acid sequence
muitispecific
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Philip Tan
John Blankenship
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Emergent Product Development Seattle, Llc
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    • 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
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • 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
    • 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
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation
    • 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
    • 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
    • C07K2317/524—CH2 domain
    • 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
    • C07K2317/64—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
    • C07K2317/71—Decreased effector function due to an Fc-modification

Definitions

  • the present invention relates to anti-CD37 multispecific antibodies that specifically target B ceils expressing CD37 and are useful for the treatment of B-cei! malignancies and disorders.
  • the anti-CD37 multispecific antibodies bind both CD37- expressing cells and the T ceil receptor complex on T cells to induce target-dependent T cell cytotoxicity, T cell activation and proliferation.
  • B lymphocytes produce antibodies that bind to, and in some cases mediate destruction of, a foreign substance or pathogen.
  • the human immune system and specifically the B lymphocytes of the human immune system go awry and disease results.
  • B cell malignancies include Chronic Lymphocytic Leukemia (CLL) and Non-Hodgkin's lymphoma (NHL).
  • CLL Chronic Lymphocytic Leukemia
  • NHL Non-Hodgkin's lymphoma
  • Rituximab marketed by Biogen targets the B-celi antigen CD20 and is approved as first-line therapy in CLL and NHL (as well as in the immune disorders rheumatoid arthritis, Wegener's granulomatosis and microscopic polyangiitis).
  • rituximab in combination with CHOP (cyclophosphamide plus doxorubicin plus vincristine plus prednisone) is the standard therapy for patients newly diagnosed with diffuse large B- celi lymphoma, one of the most common forms of NHL, Even so, around one-third of patients will develop relapsed or refractory disease, which has a poor prognosis.
  • CHOP cyclophosphamide plus doxorubicin plus vincristine plus prednisone
  • CLL is a heterogeneous disease, primarily afflicting the elderly. For many years treatment for this disease has focused on palliative chemotherapy based approaches as monotherapy or in combination.
  • Non-randomized and randomized trials have shown that
  • chemoimmunotherapy combining rituximab with fludarabine and cyclophosphamide offers a survival advantage. See, for instance, Haliek et a/., 2008, Blood. 1 1 1 :5446-56; Hailek et a/., 2010, Lancet. 376:1 164-74; Keating et al., 2005, J. Clin. Oncol. 23:4079-88; Robak et a!., 2010, J. Clin, Oncol. 28:1756-65; Tarn et al., 2008, Blood. 1 12:975-80; and Wierda et al., 2005, J. Clin. Oncol. 23:4070-8. Although the therapy is often successful, around half of patients are either unresponsive or experience early relapse as is common with fludarabine- based chemotherapy. Moreover, many elderly patients are not candidates for
  • CD37 is one such potential alternative target for antibody directed therapy.
  • CD37 is a member of the tetraspanin superfamily of molecules which as a class of proteins are generally implicated in diverse processes, including cellular activation and proliferation, cell motility, and cell-ceil adhesion.
  • CD37 is a heavily glycosylated ceil surface protein expressed constitutiveiy at high levels on mature human B ceils and transformed mature human B-cell leukemia and lymphoma ceils.
  • CD37 is not expressed on pro-B cells or terminally differentiated plasma cells.
  • CD37 is either absent or expressed weakly on normal T ceils, monocytes, and neutrophils, and is absent from natural killer (NK) cells, platelets, and erythrocytes.
  • NK natural killer
  • CD37 is considered to be a lineage-specific marker of mature human B cells restricted to the surface of B lymphocytes and therefore represents a unique therapeutic target. Because normal mature B-celis also express CD37, normal B-ceils are depleted by an anti-CD37 antibody (Press et a/., 1989, J. Clin. OncoL 7(3):1027-1038). After anti-CD37 treatment is completed, however, normal B-cei!s can be regenerated from CD37- negative B-celi precursors; therefore, patients treated with anti-CD37 therapy do not experience significant immunosuppression. [007] Until recently, only minimal effort has been directed toward CD37 immune therapy.
  • MB-1 a murine igG1 monoclonal antibody labeled with 131 i and tested in clinical trials for therapy of NHL. See Press et a!., J, Clin. Oncol., 7(3): 1027-1038 (1989); Bernstein et al., Cancer Res. (SuppL), 50: 1017-1021 (1990); Press et al., Front. Radiat. Ther. Oncol., 24: 204-213 (1990); Press et al., Adv. Exp. Med. Biol., 303: 91 -98 (1991 ) and Brown et al., Nucl. Med. Biol., 24: 657-663 (1997).
  • MB-1 lacked Fc effector functions such as antibody-dependent cellular cytotoxicity (ADCC), and it did not inhibit tumor growth in an in vivo xenograft model unless labeled with an isotope (Buchsbaum et al., Cancer Res., 52(83): 6476-6481 (1992).
  • ADCC antibody-dependent cellular cytotoxicity
  • 131 1- MB-1 was seen in lymphoma patients who had lower tumor burdens ( ⁇ 1 kg) and therapy of these patients resulted in complete tumor remissions lasting from 4 to 1 1 months (Press et al., 1989 and Bernstein et al. 1990).
  • TRU-016 is a CD37-specific antibody-like therapeutic protein comprising, from amino to carboxyl terminus, a binding domain derived from G28-1 (i.e., scFv), an
  • SMI P-016 has been shown to induce apoptosis of CLL cells in vitro in a tyrosine phosphoryiation-dependent manner that suggests an alternative signaling mechanism of action compared to rituximab.
  • a recent publication demonstrated that CD37 has both !T!M and ITAM-like signaling activity, and ligation of this antigen by SMI P-016 prompts recruitment of the phosphatase SHP1 , inhibition of the PI3-kinase pathway, and up- regulation of BIM, which is responsible for apoptosis mediated by this agent.
  • the unique mechanism of killing through CD37 distinct from CD20 selective binding of TRU-016 to mature B-cells and promising in vivo activity, the fully humanized TRU-016 was moved into the clinic for testing.
  • TRU-016 is the furthest along in the clinic of anti-CD37 therapeutics currently in development, in one trial, fifty-seven patients were treated in the dose-escalation phase and 26 in the expansion phase. A maximum tolerated dose (MTD) was not identified. Pharmacokinetics of TRU-016 was dose-proportionai with a median terminal haif-!ife of 8 days. Clinical activity was observed with partial responses in untreated and relapsed patients including individuals with del(17p13.1 ). Specifically, lymphocyte reduction 50% was observed in 55% (46/83) of all patients treated and 19 (23%) attained a response by NCI-98 criteria. All responses were partial responses and occurred more commonly in patients with symptomatic untreated CLL (8/7) or 1 -2 prior therapies (12/29) compared to those with 3 or more therapies (1/47). TRU-018 demonstrated a favorable safety profile.
  • chimeric and humanized anti-CD37 antibodies derived from murine antibody G28-1 have been developed with engineered CH2 domains for improved binding to human Fey receptors.
  • One such chimeric antibody, mAb 37.1 has been reported to show high intrinsic proapoptotic activity on malignant B cells accompanied by homotypic aggregation, if has also been reported to exhibit Ab-mediated high Ab-dependent cell- mediated cytotoxicity (ADCC) on lymphoma and primary CLL ceils. It has been reported that mAb 37.1 strongly depleted normal B cells as well as spiked B-!ymphoma cells in blood samples from healthy donors as well as malignant B ceils in blood from CLL patients.
  • ADCC Ab-mediated high Ab-dependent cell- mediated cytotoxicity
  • a single dose of mAb CD37.1 administered to human CD37-transgenic mice resulted in a reversible, dose-dependent reduction of peripheral B cells.
  • administration of mAb 37.1 strongly suppressed tumor growth. See, for instance, Heider ei a/., 201 1 , Blood. 1 18(15):4159-69.
  • ISV1GN529 Another anti-CD37 antibody-like polypeptide in development is ISV1GN529, an antibody-drug conjugate targeting hCD37 that consists of the CD37-targeting K7153A antibody linked to the maytansinoid DM1 via the thioether S CC linker.
  • I GN529 has been reported to exhibit anti-ieukemic effects in a murine model of aggressive B-cell malignancy. Based on data from an engraftment model, it is believed that IMGN529 is capable of eliminating widespread and highly proliferative mouse leukemia by a mechanism that is both CD37 antigen and conjugate dependent. See, for instance,
  • TRU-016 and other antibodies and antibody-like polypeptides currently in the clinic are monospecific therapies that depend, at least partially, on ADCC activity directed through Natural Killer (NK) cells.
  • NK Natural Killer
  • the molecules of the current invention have a different mechanism of action from the anti-CD37 molecules currently in the clinic.
  • the multispecific anti-CD37 molecules disclosed herein contain an anti-CD37 domain and an anti-CD3 domain that acts to redirect cytotoxic T cells to CD37 expressing B cells.
  • the invention includes a mu!tispecific anti-CD37 antibody comprising a CD37 binding domain and a CDS domain, wherein said muitispecific antibody is capable of redirecting T cell cytotoxicity to CD37 expressing cells.
  • the invention includes a muitispecific or bispecific antibody with a CD37 binding domain that binds CD37 on a B ceil with specificity and a CD3 binding domain that concurrently binds CD3 on a T cell with specificity, in this embodiment, the proximity of the bound CD37 and bound CDS is such that the T ceil is capable of killing the B cell.
  • the muitispecific anti-CD37 antibody is capable of T ceil activation and T cell proliferation.
  • the muitispecific antibody is produced using recombinant genetic techniques, in one embodiment, the muitispecific antibody of the invention is not synthesized using chemical techniques to cross-link binding domains.
  • the antibodies, compositions and methods of the invention do not include a muitispecific antibody created by chemically cross!inking binding domains with maleimide and SH groups following treatment with pheny!enedimaieimide.
  • the muitispecific anti-CD37 antibody comprising a CDS binding domain and a CD37 binding domain is a bispecific antibody and is sufficient to activate T cells.
  • the antibody it is not necessary that the antibody contain a third binding domain that specifically targets an accessory molecule other than CD3 and CD37 (for instance, CD8, CD4, CDS, CD2 and / or T1 1 ) for T cell activation, in some embodiments, a muitispecific antibody does not contain a third binding domain that specificaily targets an accessory molecule.
  • the muitispecific antibody does not comprise a binding domain that binds CDS, CD4, CDS, CD2 and / or T1 1 with specificity.
  • the muitispecific antibody comprising a CD37 binding domain and a CD3 binding domain and capable of redirecting T cell cytotoxicity can be in a variety of formats.
  • the muitispecific antibody comprises, from amino to carboxyl terminus, a CD3 binding domain (e.g., scFv), a linker domain and a CD37 binding domain (e.g., scFv).
  • the muitispecific antibody comprises, from amino to carboxyl terminus, a CD37 binding domain (e.g., a scFv), a linker domain and a CDS binding domain (e.g., scFv).
  • the muitispecific antibody comprises, from amino to carboxyl terminus, a CD3 binding domain (e.g., scFv), an N-terminus linker, an
  • the muitispecific antibody of the invention comprises, from amino to carboxyi terminus, a CD37 binding domain (e.g., scFv), an N-terminus linker, an immunoglobulin constant region, a C-terminus linker and a CD37 binding domain (e.g., scFv).
  • the C-terminus linker may comprise an immunoglobulin hinge region or domain.
  • the N-terminus linker may comprise a linker derived from the stalk region of a type ⁇ c-iectin or an immunoglobulin hinge core sequence.
  • the constant region is modified so that the antibody exhibits little to no effector function.
  • the mu!tispecific antibody exhibits increased half-life and / or reduced cytokine release as compared to a multispecific antibody in the scFv - linker - scFv format (e.g., bispecific single chain antibody format).
  • a multispecific antibody is a dimer composed of two single chain polypeptides, each polypeptide comprising, from amino to carboxyi terminus, a CD3 binding domain (e.g., scFv), an N-terminus linker, an immunoglobulin constant region, a C- terminus linker and a CD37 binding domain (e.g., scFv).
  • a CD3 binding domain e.g., scFv
  • an N-terminus linker e.g., an immunoglobulin constant region
  • C- terminus linker e.g., scFv
  • a multispecific antibody is a dimer composed of two single chain polypeptides, each polypeptide comprising, from amino to carboxyi terminus, a CD37 binding domain (e.g., scFv), an N- terminus linker, an immunoglobulin constant region, a C-terminus linker and a CD3 binding domain (e.g., scFv).
  • the C-terminus linker may comprise an
  • the N-terminus linker may comprise a linker derived from the stalk region of a type I I c-iectin or an immunoglobulin hinge core sequence.
  • the constant region is modified so that the antibody exhibits little to no effector function.
  • the multispecific antibody exhibits increased half-life and / or reduced cytokine release as compared to a multispecific antibody in the scFv - linker - scFv format (e.g., bispecific single chain antibody format).
  • the invention includes an anti-CD37 antibody with an amino acid of at least about 90% identity, at least about 95% identity or at least about 99% identity to an amino acid sequence of SEQ I D NO: 46, SEQ ID NO:48, SEQ I D NO:50, SEQ I D NO:52, SEQ I D NO:54; SEQ I D NO:58; SEQ ID NO:58; SEQ I D NO:60; or SEQ ID NO:63.
  • the invention includes multispecific anti-CD37 antibodies comprising a single chain polypeptide in the format anti-CD37 scFv - linker - anti ⁇ CD3 scFv (or alternatively, anti-CD3 scFv - linker - anti-CD37 scFv).
  • the multispecific antibody of the invention may comprise multiple formats so long as the molecule is capable of binding CD3 with specificity and CD37 with specificity and is able to activate T ceils in close proximity to CD37 expressing B ceils.
  • the CD37 binding domain comprises a variable heavy chain and a variable light chain derived from an antibody that binds CD37 with specificity.
  • variable heavy chain and variable light chain may be derived from an anti-CD37 antibody selected from the group consisting of G28-1 , B371 , BL14, N N46, IP024, HH1 , WR17, HD28, BM4, F93G8, RFB-7, Y29/55, MB-1 , M-B371 , IPO-24, S-B3 and K7153A.
  • the CD37 binding domain may comprise a humanized version of a known murine or other animal anti-CD37 antibody (e.g., G28-1 ).
  • the CD37 binding domain contains an amino acid sequence comprising SEQ ID NO: 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 30, 31 , 32, 33, 24 and 35.
  • variable heavy chain of the CD37 binding domain comprises CDR1 , CD2 and CDS.
  • variable heavy chain CDR1 , CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO: 8, SEQ ID NO: 1 1 and SEQ ID NO:14.
  • the variable heavy chain CDR1 , CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO: 9 or 10, SEQ ID NO: 12 or 13, and SEQ ID NO: 15, 18 and 17.
  • the CD37 binding domain variable heavy chain region contains CDR1 , CDR2 and CDR3 comprising SEQ ID NOs: 9, 12 and 15.
  • the heavy chain contains CDRs comprising SEQ ID NOs: 9, 13, and 15; SEQ ID NOs: 9, 12, and 18; SEQ ID NOs: 9, 12, and 17; SEQ ID NOs: 9, 13, and 16; SEQ ID NOs: 9, 13 and 17; SEQ ID NOs: 10, 12, and 15; SEQ ID NOs: 10, 12 and 16; SEQ ID NOs: 10, 12 and 17; SEQ ID NOs: 10, 13, and 15; SEQ ID NOs: 10, 13 and 16 or SEQ ID NOs: 10, 13 and 17. in yet another
  • the heavy chain contains CDRs comprising SEQ ID NOs: 30, 31 and 32.
  • variable light chain of the CD37 binding domain comprises CDR1 , CDR2 and CDR3.
  • the variable light chain CDR1 , CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NOs: 18, 22 and 24.
  • the variable light chain CDR1 , CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NOs: 19, 20 or 21 ; SEQ ID NO: 23 and SEQ ID NO: 25.
  • the variable light chain CDR1 , CDR2 and CDR3 comprises SEQ ID NOs: 19, 23 and 25; SEQ ID NOs: 20, 23 and 25; or SEQ ID NOs: 21 , 23 and 25.
  • the variable light chain CDR1 , CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NOs: 33, 34 and 35.
  • CD37binding domain comprises a variable heavy chain CDR1 , CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 8, 1 1 and 14 and a variable light chain CDR1 , CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 18, 22 and 24, in another embodiment, the CD37 binding domain comprises a variable heavy chain CDR1 , CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NQs: 9 or 10, SEQ ID NOs: 12 or 13; and SEQ ID NOs: 15, 16 or 17, and a variable light chain CDR1 , CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 19, 20, or 21 , SEQ ID NO:23 and SEQ ID NO: 25.
  • the CD37 binding domain comprises a variable heavy CDR1 , CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NQs: 30, 31 and 32 and a variable light CDR1 , CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 33, 34 and 35.
  • the CD37 binding domain contains a variable heavy domain comprising at least about 90% identity or at least about 95% identity to an amino acid sequence of SEQ ID NO: 5, 27, 38 or 39. In another embodiment, the CD37 binding domain contains a variable heavy domain comprising an amino acid sequence of SEQ ID NO: 5, 27, 38 or 39.
  • the CD37 binding domain contains a variable light chain comprising at least about 90% identity or at least about 95% identity to an amino acid sequence of SEQ ID NO: 7, 29 or 43. in another embodiment, the CD37 binding domain contains a variable light domain comprising an amino acid sequence of SEQ ID NO: 7, 29 or 43.
  • the CD37 binding domain contains a variable heavy domain comprising at least about 90% identity or at least about 95% identity to an amino acid sequence of SEQ ID NO: 5 and a variable light chain comprising at least about 90% identity or at least about 95% identity to SEQ ID NO: 7.
  • the invention includes a CD37 binding domain containing a variable heavy domain comprising SEQ ID NO: 5 and a variable light domain comprising SEQ ID NO: 7.
  • the CD37 binding domain contains a variable heavy domain comprising at least about 90% identify or at least about 95% identity to an amino acid sequence of SEQ ID NO: 27 and a variable light chain comprising at least about 90% identity or at least about 95% identity to SEQ ID NO: 29.
  • the invention includes a CD37 binding domain containing a variable heavy domain comprising SEQ ID NO: 27 and a variable light domain comprising SEQ ID NO: 29.
  • the CD37 binding domain contains a variable heavy domain comprising at least about 90% identity or at least about 95% identity to an amino acid sequence of SEQ ID NO: 38 or 39 and a variable light chain comprising at least about 90% identity or at least about 95% identity to SEQ ID NO: 43.
  • the invention includes a CD37 binding domain containing a variable heavy domain comprising SEQ ID NO: 38 or 39 and a variable light domain comprising SEQ ID NO: 43.
  • the invention includes an antibody with a CD37 binding domain comprising an amino acid with at least about 90% or about 95% identity to SEQ ID NO: 3 that is capable of binding CD37 with specificity, in one embodiment of the invention, the CD37 binding domain comprises SEQ ID NO: 3.
  • the multispecific anti-CD37 antibodies of the invention include molecules with a CD3 binding domain derived from X35-3, VIT3, BMA030 (BW264/56), BMA031 , G19-4, 145-2C1 1 , OKT3, BC3, CLB-T3/3, CRIS7, YTH12.5, F1 1 1 -409, CLB-T3A2, WT31 , WT32, SPv-T3b, 1 1 D8, XIII-141 , XIII-46, ⁇ -87, 12F6, T3/RW2-8C8, T3/RW2-4B6, OKT3D, M- T301 , SMC2, RIV9, I2C and F101.01 . in one embodiment of the invention, the CD3 binding domain is derived from CRIS-7, in another embodiment, the CD3 binding domain is not derived from OKT3.
  • the multispecific anti-CD37 antibody of the invention includes an antibody with a CD3 binding domain comprising an amino acid sequence of SEQ ID NO:90, SEQ ID NO: 91 , SEQ ID NO: 92, SEQ ID NO:93, SEQ ID NO:94 or SEQ ID NO:95.
  • the CD3 binding domain comprises an amino acid sequence with at least about 90% identity or at least about 95% identity to an amino acid sequence of SEQ ID NO:88 or SEQ ID NO:89.
  • the CD3 binding domain comprises HCDR1 , HCDR2, HCDR3, LCDR1 , LCDR2 and LCDR3.
  • the invention includes a CD3 binding domain comprising an HCDR1 of SEQ ID NO:90, an HCDR2 of SEQ ID NO:91 and an HCDR3 of SEQ ID NO:92, in this embodiment, the LCDR1 may comprise an amino acid of SEQ ID NO:93, the LCDR2 may comprise an amino acid of SEQ ID NO:94 and the LCDR3 may comprise an amino acid of SEQ ID NO:95.
  • the CDS binding domain comprises HCDR1 , HCDR2, HCDR3, LCDR1 , LCDR2 and LCDR3.
  • the invention includes a CDS binding domain comprising an HCDR1 of SEQ ID NO:105, an HCDR2 of SEQ ID NO:106 and an HCDR3 of SEQ ID NO:107.
  • the LCDR1 may comprise an amino acid of SEQ ID NO: 108
  • the LCDR2 may comprise an amino acid of SEQ ID NO: 109
  • the LCDR3 may comprise an amino acid of SEQ ID NO:1 10.
  • the CD3 binding domain comprises HCDR1 , HCDR2, HCDR3, LCDR1 , LCDR2 and LCDR3.
  • the invention includes a CD3 binding domain comprising an HCDR1 of SEQ ID NO:1 1 1 , an HCDR2 of SEQ ID NO:1 12 and an HCDR3 of SEQ ID NO:1 13.
  • the LCDR1 may comprise an amino add of SEQ ID NO: 1 14
  • the LCDR2 may comprise an amino acid of SEQ ID NO:1 15
  • the LCDR3 may comprise an amino acid of SEQ ID NO:1 18.
  • the CD37 binding domain and CDS binding domain of the mu!tispecific antibody each comprise a variable heavy chain and a variable light chain separated by a linker.
  • the linker comprises an amino acid sequence of about 3 to 35 amino acids.
  • the linker comprises an amino acid sequence of about 5 to 35 amino acids.
  • the invention includes a linker separating the variable domains of about 10 to 25 amino acids, about 10 to 35 amino acids, about 12 to 25 amino acids, about 12 to about 35 amino acids, about 15 to 25 amino acids, about 15 to 35 amino acids, or about 15 to 20 amino acids in length.
  • the present disclosure provides a composition comprising any of the muitispecific anti-CD37 antibodies as set forth herein and a pharmaceutically acceptable carrier, diluent, or excipient.
  • the present disclosure provides a method for inducing redirected T cell cytotoxicity (RTCC) against a cell expressing CD3 such as B-cells.
  • a method for inducing RTCC against the cell expressing CD37 includes contacting the CD37-expressing ceil with a muitispecific anti-CD37 antibody of the invention.
  • the present disclosure provides a method for treating a disorder in a subject, wherein the disorder is characterized by overexpression of CD37, an elevated number of B-cells or malignant B-ceils.
  • the method includes administering to the subject a therapeutically effective amount of a muitispecific anti-CD37 antibody of the invention to a patient.
  • the antibody comprises dimerized polypeptide chains and the dimeric molecule induces redirected T cell cytotoxicity (RTCC) in the subject.
  • the anti-CD37 antibody may be homodimeric or heterodimeric and may comprise, from amino to carboxyl terminus, a first binding domain, an N-terminus linker, a modified immunoglobulin constant region, a C- terminus linker and a second binding domain, such that one of the first and second binding domains is a CDS binding domain and one of the first and second binding domains is a CD37 binding domain.
  • Figure 1A is an annotated nucleic acid sequence encoding muitispecific antibody CAS105 (SEQ ID NO:45).
  • Figure 1 B is an annotated amino acid sequence of muitispecific antibody CAS105 (SEQ ID NO:46).
  • Figure 2 shows specific binding of the anti-CD37 domain of bispecific molecules on Ramos cells [CD37(+) / CD3(-)] ( Figure 2A) and C4 ⁇ 2 cells [CD37( ⁇ ) / CD3(-)] ( Figure 2B) while binding properties of the anti-CD3 domain was explored on Jurkat ceils [CD37(-) / CD3(+)] ( Figure 2C).
  • Figure 3 shows specific redirected T-ceil cytotoxicity observed with various concentrations of bi-specific molecules on Ramos (CD37-positive) ceils ( Figure 3A) and C4-2 (CD37-negative) ceils ( Figure 3B).
  • Target ceils were loaded with 51 Cr and incubated with bispecific molecules and human T cells at an effector to target ratio of 5:1.
  • Figure 4 shows that anti-CD37 x anti-CD3 bispecific molecules induced division of both CD4+ ( Figure 4A) and CD8+ T ( Figure 4B) cells in the presence of CD37-positive target cells.
  • CFSE-labeied T cells were incubated for 4 days with various concentrations of bispecific molecules and Ramos target ceils.
  • Plots show the number of T cells in a fixed volume from each sample that divided at least once as determined by dilution of intracellular CFSE.
  • Figure 5 shows that anti-CD37 x anti-CD3 bispecific molecules induced
  • the invention provides muitispecific anti-CD37 antibodies and compositions that specifically bind CD37 (e.g., CD37+ B ceils).
  • Administration of a therapeutically effective amount of a CD37-binding antibody of the invention to a patient in need thereof is useful for treatment of B-cel! malignancies and disorders, including, for instance, leukemias and autoimmune disease, in one embodiment, the recombinant antibodies of the invention simultaneously bind a B-ce!! expressing CD37 and a T ceil, thereby "cross-linking" the B-ceil and the T ceil.
  • RTCC potent target-dependent redirected T ceil cytotoxicity
  • any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
  • “about” means ⁇ 20% of the indicated range, value, or structure, unless otherwise indicated.
  • the terms “a” and “an” as used herein refer to “one or more” of the enumerated components unless otherwise indicated.
  • the use of the alternative should be understood to mean either one, both, or any combination thereof of the alternatives.
  • polypeptides comprising the various combinations of the components (e.g., domains or regions) and substituents described herein, are disclosed by the present application to the same extent as if each polypeptide was set forth individually. Thus, selection of particular components of individual polypeptides is within the scope of the present disclosure.
  • multispecific anti-CD37 antibody refers to a recombinant antibody or polypeptide that binds to both human CD37 with specificity and human TCR complex (i.e., CD3) with specificity.
  • multispecific anti-CD37 antibody includes antibody derivatives. The term also includes molecules comprising functional antibody fragments or derivatives of fragments which retain binding specificity. For instance, the invention includes fusion proteins and other polypeptides that contain variable heavy and / or light chain domains. The antibodies of the invention are all recombinant, non-naturaliy occurring molecules.
  • the multispecific anti-CD37 antibodies of the invention can be tested for binding to both a T cell and a CD37+ cell using assays and methods disclosed herein.
  • the invention includes a multispecific anti-CD37 antibody comprising dimerized single chain polypeptides, each single chain polypeptide comprising, from amino to carboxyl terminus, a CD37 binding domain, an N-terminus linker, an immunoglobulin constant region, a C-terminus linker and a CD3 binding domain.
  • the invention includes a multispecific anti-CD37 antibody comprising dimerized single chain polypeptides, each single chain polypeptide comprising, from amino to carboxyl terminus, a CD3 binding domain (or other binding domain that binds a T ceil antigen with specificity), an N-terminus linker, an immunoglobulin constant region, a C-terminus linker and a CD-37 binding domain.
  • the N-terminus linker may comprise or may consist essentially of an
  • the muitispecific anti-CD37 antibody comprises a single chain polypeptide comprising, from amino to carboxyl terminus, a CD37 binding domain, an N-terminus linker, an immunoglobulin constant region, a C-terminus linker and a CD3 binding domain.
  • the invention includes a muitispecific anti-CD37 antibody comprising, from amino to carboxyl terminus, a CD3 binding domain, an N-terminus linker, an immunoglobulin constant region, a C-terminus linker and a CD-3 binding domain.
  • the invention includes a muitispecific anti-CD37 antibody comprising a binding domain linked via a linker domain to a second binding domain (e.g., a scFv linked via a linker to another scFv).
  • a muitispecific anti-CD37 antibody comprising a CD37 binding domain (in Vi-i-linker-VL or V L -linker-Vn orientation) linked via a peptide linker domain to a CD3 binding domain (in V H -linker-V L or V L -linker-V H orientation).
  • a bispecific antibody in the scFv-linker-scFv format may comprise variable heavy and variable light domains derived from any anti-CD37 antibody and antibody to a T cell antigen (such as CD3) including, but not limited to, the variable domains disclosed herein.
  • the muitispecific anti-CD37 antibodies are scFv dimers or diabodies rather than whole antibodies.
  • Diabodies and scFv can be constructed without an Fc region, using only variable domains.
  • Diabodies are bivalent, bispecific antibodies in which V H and V L domains are expressed on a single polypeptide chain, but using a peptide linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Hoiliger, P., et al. (1993) Proc. Natl. Acad. ScL USA 90:6444-6448; Poijak, R.
  • a muitispecific antibody is a disulfide - stabilized diabody.
  • a muitispecific antibody may comprise two distinct polypeptides that are coexpressed to generate a covalently linked heterodimeric complex with one binding site for each of 2 specificities.
  • each Fv is formed by the association of a V L partner on one chain with a V H partner on the second chain in a V LA - V H B (first chain) and V L. B-V H A (second chain) configuration.
  • the antibody is stabilized by either of two alternative carboxy terminal heterodimerization domains: a pairing of VEPKSC on one chain and FNRGEC on the other or a pairing of oppositely charged, coiied-coil domains. See, for instance, Moore et a/., 201 1 , Blood. 1 17:4542-4551.
  • the multispecific anti-CD37 antibody may comprise a first chain with a CD3 binding domain V H linked to a CD37 binding domain V L and the second chain comprises a CD3binding domain V L linked to a CD37 binding domain V H, and the two chains are linked via a disulfide bond at the c-termini.
  • a disu!fide-stabilized diabody may be designed using variable heavy and light chains derived from known anti-CD37 and anti-CD3 antibodies including, for instance, the variable heavy and light chains disclosed herein.
  • the multispecific anti-CD37 antibody is a dual variable domain binding proteins capable of binding CD37 and TCR complex with specificity
  • the recombinant antibody comprises a polypeptide chain, wherein said polypeptide chain comprises VD1 -(X1 )n-VD2-C— (X2)n, wherein VD1 is a first variable domain, VD2 is a second variable domain, C is a constant domain, X1 is a linker (e.g., a polypeptide linker of about 10 to 20 amino acids in length), X2 represents an Fc region and n is 0 or 1 . See, for instance, US 8,258,268.
  • a multispecific anti-CD37 antibody comprises one, two, three or more polypeptide chains.
  • the invention includes a multispecific anti-CD37 antibody with a first chain comprising VHI-V L 2, a second chain comprising CH2-CH3-Vu-V H 2 and a third chain comprising CH2-CH3.
  • the Vi-ii and V L i may correspond to anti-CD37 variable domains
  • V ⁇ and V L2 may correspond to anti-CD3 (or other T cell antigen) variable domains
  • the V H i and V L may correspond to anti-CD3 (or other T cell antigen) variable domains
  • the V H 2 and VL . 2 may correspond to anti-CD37 variable domains.
  • binding domain refers to the domain, region, portion, or site of a protein, polypeptide, oligopeptide, or peptide that possesses the ability to specifically recognize and bind to a target molecule, such as an antigen, ligand, receptor, substrate, or inhibitor (e.g., CD3, CD37).
  • exemplary binding domains include single-chain antibody variable regions (e.g., domain antibodies, sFv, scFv, scFab), receptor ectodomains. and ligands (e.g., cytokines, chemokines).
  • the binding domain comprises or consists of an antigen binding site (e.g., comprising a variable heavy chain sequence and variable light chain sequence or three light chain complementary determining regions (CDRs) and three heavy chain CDRs from an antibody placed into alternative framework regions (FRs) (e.g., human FRs optionally comprising one or more amino acid substitutions).
  • an antigen binding site e.g., comprising a variable heavy chain sequence and variable light chain sequence or three light chain complementary determining regions (CDRs) and three heavy chain CDRs from an antibody placed into alternative framework regions (FRs) (e.g., human FRs optionally comprising one or more amino acid substitutions).
  • FRs alternative framework regions
  • a muitispecific antibody comprises a "first binding domain” and a "second binding domain.”
  • the "first binding domain” is a CD37-binding domain, in certain embodiments comprising both the first and second binding domains, the second binding domain is a T ceil binding domain such as a scFv derived from a mouse monoclonal antibody (e.g., CRiS-7) that binds to a T cell surface antigen (e.g., CD3).
  • the first binding domain binds to a T cell surface antigen and the second binding domain binds to CD37.
  • a "binding domain” comprises a scFv.
  • a "scFv” refers to a variable heavy domain linked via a peptide linker to a variable light domain.
  • a scFv can be in the V H -peptide linker- V L or V L -peptide linker-V H orientation.
  • a binding domain "specifically binds" a target if it binds the target with an affinity or K a ⁇ i.e., an equilibrium association constant of a particular binding interaction with units of 1/M) equal to or greater than 10 5 '1 , while not significantly binding other components present in a test sample. Binding domains can be classified as “high affinity” binding domains and “low affinity” binding domains. "High affinity” binding domains refer to those binding domains with a K a of at least 10 7 M " ⁇ at least 10 8 M “1 , at least 10 9 M '1 , at least 10 10 M “1 , at least 10 11 M “1 , at least 10 12 M “1 , or at least 10 13 M “1 .
  • “Low affinity” binding domains refer to those binding domains with a K a of up to 10' M “1 , up to 10 6 M “1 , up to 10 b M “1 .
  • affinity can be defined as an equilibrium dissociation constant (K d ) of a particular binding interaction with units of M ⁇ e.g., 10 "5 M to 10 ⁇ 13 M).
  • K d equilibrium dissociation constant
  • Affinities of binding domain polypeptides and single chain polypeptides according to the present disclosure can be readily determined using conventional techniques (see, e.g., Scatchard et al. (1949) Ann. N.Y. Acad. Sci. 51 :660; and U.S. Patent Nos. 5,283,173, 5,468,614, or the equivalent).
  • CD3 is known in the art as a multi-protein complex of six chains ⁇ see, e.g., Abbas and Lichtman, 2003; Janeway et ai., p. 172 and 178, 1999), which are subunits of the T cell receptor complex.
  • the CD3 subunits of the T ceil receptor complex are a GD3y chain, a CD35 chain, two CD3e chains, and a homodimer of CD3 chains.
  • the CD3y, CD35, and CD3e chains are highly related ceil surface proteins of the immunoglobulin superfamily containing a single immunoglobulin domain.
  • the transmembrane regions of the CD3y, CD35, and CD3e chains are negatively charged, which is a characteristic that allows these chains to associate with the positively charged T ceil receptor chains.
  • the intracellular tails of the CD3y, CD35, and CD3e chains each contain a single conserved motif known as an immunoreceptor tyrosine-based activation motif or ITAM, whereas each ⁇ 3 ⁇ chain has three. It is believed the ITAMs are important for the signaling capacity of a TCR complex.
  • CD3 as used in the present disclosure can be from various animal species, including human, monkey, mouse, rat, or other mammals.
  • a "conservative substitution” is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties.
  • exemplary conservative substitutions are weli-known in the art (see, e.g., WO 97/09433, page 10, published March 13, 1997; Lehninger, Biochemistry, Second Edition; Worth Publishers, inc. NY:NY (1975), pp.71-77; Lewin, Genes IV, Oxford University Press, NY and Ceil Press, Cambridge, MA (1990), p. 8).
  • a conservative substitution includes a leucine to serine substitution.
  • derivative refers to a modification of one or more amino acid residues of a peptide by chemical or biological means, either with or without an enzyme, e.g., by glycosylation, alkylation, acyiation, ester formation, or amide formation.
  • a multispecific antibody “derived from” an antibody refers to the origin of the multispecific antibody.
  • a multispecific antibody may comprise an amino acid sequence which is derived from a particular sequence (sometimes referred to as the "starting" or “parent” or “parental” or “reference” sequence) has an amino acid sequence that is essentially identical to the starting sequence or a portion thereof, wherein the portion consists of at least 10-20 amino acids, at least 20-30 amino acids, or at least 30-50 amino acids, or at least 50-150 amino acids, or which is otherwise identifiable to one of ordinary skill in the art as having its origin in the starting sequence, in one embodiment, "derived from” means that the CDRs are identical to or highly similar to that of the reference antibody.
  • a multispecific antibody may be derived from a reference antibody if it contains differences in CDRs as compared to the reference antibody that do not adversely affect binding specificity.
  • CDRs may be modified to improve binding as compared to the reference antibody
  • a multispecific antibody is derived from a reference antibody if it comprises variable heavy and / or variable light chain with at least about 90% identity or at least about 95% identity as compared to that of the reference antibody. "Derived from” can also signify that a variable heavy and / or light chain from a reference antibody has been humanized or otherwise improved as compared to the reference antibody (for instance, to improve stability or manufacturabi!ity of the multispecific antibody).
  • Polypeptides derived from another polypeptide can have one or more mutations relative to the starting polypeptide, e.g., one or more amino acid residues which have been substituted with another amino acid residue or which has one or more amino acid residue insertions or deletions.
  • the polypeptide can comprise an amino acid sequence which is not naturally occurring. Such variations necessarily have less than 100% sequence identity or similarity with the starting polypeptide. In one embodiment, the variant will have an amino acid sequence from about 60% to less than 100% amino acid sequence identity or similarity with the amino acid sequence of the starting polypeptide.
  • the variant will have an amino acid sequence from about 75% to less than 100%, from about 80% to less than 100%, from about 85% to less than 100%, from about 90% to less than 100%, from about 95% to less than 100% amino acid sequence identity or similarity with the amino acid sequence of the starting polypeptide.
  • a position of an amino acid residue in a variable region of an immunoglobulin molecule is numbered according to the Kabat numbering convention (Kabat, Sequences of Proteins of immunological interest, 5 ih ed. Bethesda, MD: Public Health Service, National Institutes of Health (1991 )), and a position of an amino acid residue in a constant region of an immunoglobulin molecule is numbered according to EU nomenclature (Ward et aL, 1995 Therap. Immunol. 2:77-94).
  • the term "dimer” refers to a biological entity that consists of two subunits associated with each other via one or more forms of intramolecular forces, including cova!ent bonds (e.g., disulfide bonds) and other interactions (e.g., electrostatic interactions, salt bridges, hydrogen bonding, and hydrophobic interactions), and is stable under appropriate conditions (e.g., under physiological conditions, in an aqueous solution suitable for expressing, purifying, and/or storing recombinant proteins, or under conditions for non- denaturing and/or non-reducing electrophoresis).
  • cova!ent bonds e.g., disulfide bonds
  • other interactions e.g., electrostatic interactions, salt bridges, hydrogen bonding, and hydrophobic interactions
  • heterodimer or “heterodimeric protein,” as used herein, refers to a dimer formed from two different polypeptides.
  • a heterodimer does not include an antibody formed from four polypeptides (i.e., two light chains and two heavy chains).
  • a “homodimer” or “homodimeric protein,” as used herein, refers to a dimer formed from two identical polypeptides.
  • a "hinge region” or a “hinge” refers to a polypeptide derived from (a) an interdomain region of a transmembrane protein (e.g., a type I transmembrane protein).
  • a hinge region can be derived from an interdomain region of an immunoglobulin superfamiiy member; suitable hinge regions within this particular class include immunoglobulin hinge regions (made up of. for example, upper and/or core region(s)) or functional variants thereof, including wild-type and altered immunoglobulin hinges.
  • a wild-type immunoglobulin hinge region refers to a naturally occurring upper and middle hinge amino acid sequences interposed between and connecting the CH1 and CH2 domains (for IgG, IgA, and IgD) or interposed between and connecting the CH1 and CHS domains (for igE and IgM) found in the heavy chain of an antibody.
  • a wild type immunoglobulin hinge region sequence is human, and can comprise a human IgG hinge region,
  • an "altered wild-type immunoglobulin hinge region” or “altered immunoglobulin hinge region” refers to (a) a wild type immunoglobulin hinge region with up to 30% amino acid changes ⁇ e.g., up to 25%, 20%, 15%, 10%, or 5% amino acid substitutions or deletions), or (b) a portion of a wild type immunoglobulin hinge region that has a length of about 5 amino acids (e.g., about 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids) up to about 120 amino acids (for instance, having a length of about 10 to about 40 amino acids or about 15 to about 30 amino acids or about 15 to about 20 amino acids or about 20 to about 25 amino acids), has up to about 30% amino acid changes ⁇ e.g., up to about 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1 % amino acid substitutions or deletions or a combination thereof), and has an IgG core hinge region as disclosed in
  • the term "humanized” refers to a process of making an antibody derived from a non-human species (e.g., mouse or rat) less immunogenic to humans, while still retaining antigen-binding properties of the original antibody, using genetic engineering techniques.
  • the binding domain(s) of an antibody or immunoglobulin binding proteins and polypeptides e.g., light and heavy chain variable regions, Fab, scFv
  • Non-human binding domains can be humanized using techniques known as CDR grafting (Jones ef a/., Nature 321 :522 (1986)) and variants thereof, including
  • an "immunoglobulin dimerization domain” or “immunoglobulin heterodimerization domain”, as used herein, refers to an immunoglobulin domain of a polypeptide chain that preferentially interacts or associates with a different immunoglobulin domain of a second polypeptide chain, wherein the interaction of the different immunoglobulin heterodimerization domains substantially contributes to or efficiently promotes heterodimerization of the first and second polypeptide chains (i.e., the formation of a dimer between two different polypeptide chains, which is also referred to as a "heterodimer").
  • immunoglobulin heterodimerization domains "substantia!ly contributes to or efficiently promotes" the heterodimerization of first and second polypeptide chains if there is a statistically significant reduction in the dimerization between the first and second polypeptide chains in the absence of the immunoglobulin heterodimerization domain of the first polypeptide chain and/or the immunoglobulin heterodimerization domain of the second polypeptide chain.
  • the first and second polypeptide chains when the first and second polypeptide chains are co-expressed, at least 60%, at least about 60% to about 70%, at least about 70% to about 80%, at least 80% to about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the first and second polypeptide chains form heterodimers with each other.
  • immunoglobulin heterodimerization domains include an immunoglobulin CH1 domain, an immunoglobulin CL domain (e.g., CK or CA isotypes), or derivatives thereof, including wild type immunoglobulin CH 1 and CL domains and altered (or mutated) immunoglobulin CH1 and CL domains, as provided therein.
  • immunoglobulin CH1 domain an immunoglobulin CH1 domain
  • immunoglobulin CL domain e.g., CK or CA isotypes
  • derivatives thereof including wild type immunoglobulin CH 1 and CL domains and altered (or mutated) immunoglobulin CH1 and CL domains, as provided therein.
  • an "immunoglobulin constant region” or “constant region” is a term defined herein to refer to a peptide or polypeptide sequence that corresponds to or is derived from part or all of one or more constant region domains.
  • the immunoglobulin constant region corresponds to or is derived from part or ail of one or more constant region domains, but not ail constant region domains of a source antibody, in certain embodiments, the constant region comprises IgG CH2 and CH3 domains, e.g., igG1 CH2 and CH3 domains.
  • the constant region does not comprise a CH1 domain.
  • the constant region domains making up the constant region are human.
  • the constant region domains lack or have minimal effector functions of antibody- dependent cell-mediated cytotoxicity (ADCC) and complement activation and complement- dependent cytotoxicity (CDC), while retaining the ability to bind some F c receptors (such as F c Rn, the neonatal Fc receptor) and retaining a relatively long half-life in vivo.
  • ADCC antibody- dependent cell-mediated cytotoxicity
  • CDC complement activation and complement- dependent cytotoxicity
  • F c receptors such as F c Rn, the neonatal Fc receptor
  • a fusion protein of this disclosure includes constant domains that retain such effector function of one or both of ADCC and CDC.
  • a binding domain of this disclosure is fused to a human igG1 constant region, wherein the igG1 constant region has one or more of the following amino acids mutated: leucine at position 234 (L234), leucine at position 235 (L235), glycine at position 237 (G237), glutamate at position 318 (E318), lysine at position 320 (K320), lysine at position 322 (K322), or any combination thereof (numbering according to EU). For example, any one or more of these amino acids can be changed to alanine.
  • an lgG1 Fc domain has each of L234, L235, G237, E318, K320, and K322 (according to EU numbering) mutated to an alanine ⁇ i.e., L234A, L235A, G237A, E318A, K320A, and K322A, respectively), and optionally an N297A mutation as well ⁇ i.e., essentially eliminating giycosyiation of the CH2 domain).
  • Fc region or “Fc domain” refers to a polypeptide sequence corresponding to or derived from the portion of a source antibody that is responsible for binding to antibody receptors on cells and the C1 q component of complement.
  • Fc stands for "fragment crystalline," the fragment of an antibody that will readily form a protein crystal. Distinct protein fragments, which were originally described by proteolytic digestion, can define the overall general structure of an immunoglobulin protein. As originally defined in the literature, the Fc fragment consists of the disu!fide-Iinked heavy chain hinge regions, CH2, and CH3 domains.
  • the "stalk region" of a type II C-iectin refers to the portion of the extracellular domain of the type II C-lectin that is located between the C-type lectin-!ike domain (CTLD; e.g., similar to CTLD of natural killer ceil receptors) and the transmembrane domain.
  • C-type lectin-!ike domain C-type lectin-!ike domain
  • the extracellular domain corresponds to amino acid residues 34- 179
  • the CTLD corresponds to amino acid residues 81-176.
  • the stalk region of the human CD94 molecule includes amino acid residues 34-60, which is found between the membrane and the CTLD (see Boyington et ai, Immunity 10:75, 1999; for descriptions of other stalk regions, see also Beavii ef a/., Proc. Natl. Acad. Sci. USA 89:753, 1992; and Figdor ef ai., Nature Rev. Immunol. 2:77, 2002).
  • These type II C-lectins can also have from six to 10 junction amino acids between the stalk region and the transmembrane region or the CTLD.
  • the 233 amino acid human NKG2A protein GenBank Accession No.
  • P26715.1 PRI June 15, 2010
  • the CTLD is comprised of amino acids 1 19-231 , and the stalk region comprises amino acids 99-1 16, which is flanked by junctions of five and two amino acids.
  • Other type II C- lectins, as well as their extracellular ligand-bind domains, interdomain or stalk regions, and CTLDs are known in the art (see, e.g., GenBank Accession Nos. NP 001993.2; AAH07037.1 , PR! July 15, 2006; NP_001773.1 , PRI June 20, 1010; AAL65234.1 , PR!
  • a mu!tispecific antibody comprises a linker derived from the stalk region of a type ⁇ c-lectin.
  • a multispecific antibody in the format comprising dimerized single chain polypeptides, each polypeptide comprising from amino to carboxyl terminus, a first binding domain, an N-terminus linker, a constant region, a C- terminus linker and a second binding domain may contain a linker derived from the stalk region of a type II c-!ectin as either the N-terminus linker or C-terminus linker.
  • interdomain region of a transmembrane protein refers to a portion of the extracellular domain of the transmembrane protein that is located between two adjacent domains.
  • interdomain regions include regions linking adjacent ig domains of immunoglobulin superfami!y members (e.g., an immunoglobulin hinge region from IgG, IgA, IgD, or IgE; the region linking the IgV and igC2 domains of CD2; or the region linking the IgV and igC domains of CD80 or CD88).
  • an interdomain region is the region linking the non-lg and lgC2 domain of CD22, a type I sialic acid-binding Ig-like lectin.
  • a linker such as an N-terminus or C-terminus linker in the format of a dimerized molecule comprising two polypeptide chains, each polypeptide comprising, from amino to carboxyl terminus, a first binding domain, an N-terminus linker, a constant region, a C-terminus linker and a second binding domain
  • a transmembrane protein interdomain region e.g., an immunoglobulin hinge region.
  • a polypeptide region "derived from” a stalk region of a type II C-lectin, or “derived from” a transmembrane protein interdomain region refers to an about five to about 150 amino acid sequence, an about 5 to about 100 amino acid sequence, an about 5 to about 50 amino acid sequence, an about 5 to about 40 amino acid sequence, an about 5 to about 30 amino acid sequence, an about 5 to about 25 amino acid sequence, an about 5 to about 20 amino acid sequence, an about 10 to about 25 amino acid sequence, an about 10 to about 20 amino acid sequence, about 8 to about 20 amino acid sequence, about 9 to about 20 amino acid sequence, about 10 to about 20 amino acid sequence, about 1 1 to about 20 amino acid sequence, about 12 to about 20 amino acid sequence, about 13 to about 20 amino acid sequence, about 14 to about 20 amino acid sequence, about 15 to about 20 amino acid sequence, or an about 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid sequence, wherein all or at
  • N-terminus linker refers, in one embodiment, to a linker in an antibody format comprising dimerized single chain polypeptides, each single chain comprising, from amino to carboxyi terminus, a first binding domain, an N-terminus linker, a constant region, a C-terminus linker and a second binding domain, in this format, the N-terminus may comprise, for instance, a linker derived from a transmembrane protein interdomain region (e.g., an immunoglobulin hinge region).
  • an N-terminus linker may comprise an immunoglobulin hinge or a portion of an immunoglobulin hinge.
  • an N-terminus linker may also be used in
  • a "C-terminus linker” or a “carboxyi terminus linker” refers, in one embodiment, to a linker in an antibody format comprising dimerized single chain polypeptides, each single chain comprising, from amino to carboxyi terminus, a first binding domain, an N-terminus linker, a constant region, a C-terminus linker and a second binding domain.
  • the C-terminus may comprise, for instance, a linker derived from a stalk region of a type ⁇ c- lectin.
  • an N-terminus linker may comprise an immunoglobulin hinge or a portion of an immunoglobulin hinge.
  • a C- terminus linker may also be used in recombinant antibody formats in addition to the muitispecific heterodimer and homodimer antibody formats as described herein.
  • junction amino acids refers to one or more (e.g., about 2-10) amino acid residues between two adjacent regions or domains of a polypeptide, such as between a hinge and an adjacent immunoglobulin constant region or between a hinge and an adjacent binding domain or between a peptide linker that links two immunoglobulin variable domains and an adjacent immunoglobulin variable domain.
  • Junction amino acids can result from the construct design of a polypeptide (e.g., amino acid residues resulting from the use of a restriction enzyme site during the construction of a nucleic acid molecule encoding a polypeptide).
  • a linker between CH3 and CH1 or CL refers to one or more (e.g., about 2-12, about 2-10, about 4-10, about 5-10, about 6-10, about 7-10, about 8- 10, about 9-10, about 8-12, about 9-12, or about 10-12) amino acid residues between the C- terminus of a CH3 domain (e.g., a wild type CH3 or a mutated CHS) and the N-terminus of a CH1 domain or CL domain (e.g., Ck).
  • a CH3 domain e.g., a wild type CH3 or a mutated CHS
  • the term "patient in need” refers to a patient at risk of, or suffering from, a disease, disorder or condition that is amenable to treatment or amelioration with a mu!tispecific anfi-CD37 antibody.
  • peptide linker may refer to an amino acid sequence that connects a heavy chain variable region to a light chain variable region and provides a spacer function compatible with interaction of the two sub-binding domains so that the resulting polypeptide retains a specific binding affinity to the same target molecule as an antibody that comprises the same light and heavy chain variable regions.
  • a linker is comprised of three to about 35 amino acids, for instance, about 15 to about 25 amino acids.
  • the CD37 binding domain and CD3 binding domain each comprise a variable heavy chain and a variable light chain separated by a linker (in either V H ⁇ peptide !inker-V L or V L -peptide iinker-V H orientation).
  • the linker comprises an amino acid sequence of about 3 to 35 amino acids.
  • the linker comprises an amino acid sequence of about 5 to 35 amino acids.
  • the invention includes a linker separating the variable domains of about 10 to 25 amino acids, about 10 to 35 amino acids, about 12 to 25 amino acids, about 12 to about 35 amino acids, about 15 to 25 amino acids, about 15 to 35 amino acids, or about 15 to 20 amino acids in length.
  • the term "pharmaceutically acceptable” refers to molecular entities and compositions that do not produce allergic or other serious adverse reactions when administered using routes well known in the art. Molecular entities and compositions approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans are considered to be “pharmaceutically acceptable.”
  • promoter refers to a region of DNA involved in binding RNA polymerase to initiate transcription.
  • nucleic acid As used herein, the terms “nucleic acid,” “nucleic acid molecule,” or
  • polynucleotide refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the terms encompass nucleic acids containing analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon
  • nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.
  • nucleic acid As used herein, the terms “nucleic acid,” “nucleic acid molecule,” or “polynucleotide” are intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof.
  • DNA molecules e.g., cDNA or genomic DNA
  • RNA molecules e.g., mRNA
  • analogs of the DNA or RNA generated using nucleotide analogs e.g., mRNA
  • expression refers to the biosynthesis of a product encoded by a nucleic acid.
  • expression involves transcription of the nucleic acid segment into mRNA and the translation of mRNA into one or more polypeptides.
  • expression unit and "expression cassette” are used interchangeably herein and denote a nucleic acid segment encoding a polypeptide of interest and capable of providing expression of the nucleic acid segment in a host cell.
  • An expression unit typically comprises a transcription promoter, an open reading frame encoding the polypeptide of interest, and a transcription terminator, all in operable configuration.
  • an expression unit can further include other nucleic acid segments such as, e.g., an enhancer or a polyadenyiation signal.
  • expression vector refers to a nucleic acid molecule, linear or circular, comprising one or more expression units.
  • an expression vector can also include additional nucleic acid segments such as, for example, one or more origins of replication or one or more selectable markers.
  • Expression vectors are generally derived from plasmid or viral DNA, or can contain elements of both.
  • sequence identity refers to a relationship between two or more polynucleotide sequences or between two or more polypeptide sequences. When a position in one sequence is occupied by the same nucleic acid base or amino acid residue in the corresponding position of the comparator sequence, the sequences are said to be “identical” at that position. The percentage “sequence identity” is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of "identical” positions.
  • the number of "identical” positions is then divided by the total number of positions in the comparison window and multiplied by 100 to yield the percentage of "sequence identity.” Percentage of "sequence identity” is determined by comparing two optimally aligned sequences over a comparison window.
  • the comparison window for nucleic acid sequences can be, for instance, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 or more nucleic acids in length.
  • the comparison window for polypeptide sequences can be, for instance, at least 20, 30, 40, 50, 80, 70, 80, 90, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300 or more amino acids in length.
  • the portion of a polynucleotide or polypeptide sequence in the comparison window can comprise additions or deletions termed gaps while the reference sequence is kept constant.
  • An optimal alignment is that alignment which, even with gaps, produces the greatest possible number of "identical" positions between the reference and comparator sequences.
  • Sequence identify between two sequences can be determined using the version of the program "BLAST 2 Sequences" which was available from the National Center for Biotechnology information as of September 1 , 2004, which program incorporates the programs BLASTN (for nucleotide sequence comparison) and BLASTP (for polypeptide sequence comparison), which programs are based on the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90(12):5873-5877, 1993).
  • BLASTN for nucleotide sequence comparison
  • BLASTP for polypeptide sequence comparison
  • nucleotide or amino acid sequences are considered to have "substantially similar sequence identity” or “substantial sequence identity” if the two sequences have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity relative to each other.
  • polypeptide or “polypeptide chain” is a single, linear and contiguous arrangement of covalently linked amino acids. Polypeptides can have or form one or more intrachain disulfide bonds. With regard to polypeptides as described herein, reference to amino acid residues corresponding to those specified by SEQ ID NO includes post-translational modifications of such residues.
  • a "protein” is a macromoiecule comprising one or more polypeptide chains.
  • a protein can also comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents can be added to a protein by the ceil in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.
  • ammo-terminal and “carboxyi-termina! are used herein to denote positions within mu!tispecific antibodies and polypeptides. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a certain sequence positioned carboxy!-terminai to a reference sequence within a polypeptide is located proximal to the carboxyi-terminus of the reference sequence, but is not necessarily at the carboxyi-terminus of the complete polypeptide.
  • T cell receptor is a molecule found on the surface of T cells that, along with CD3, is generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules. It consists of a disulfide-linked heterodimer of the highly variable a and ⁇ chains in most T cells. In other T cells, an alternative receptor made up of variable ⁇ and ⁇ chains is expressed. Each chain of the TCR is a member of the immunoglobulin superfami!y and possesses one N-termina! immunoglobulin variable domain, one
  • TCR as used in the present disclosure can be from various animal species, including human, mouse, rat, or other mammals.
  • TCR complex refers to a complex formed by the association of CD3 chains with other TCR chains.
  • a TCR complex can be composed of a CD3y chain, a CD36 chain, two CD3e chains, a homodimer of CD3( chains, a TCRa chain, and a TCR chain.
  • a TCR complex can be composed of a CD3y chain, a CD35 chain, two CD3E chains, a homodimer of CD3( chains, a TCRy chain, and a TCR5 chain.
  • a component of a TCR complex refers to a TCR chain (i.e., TCRa, TCR , TCRy or TCR5), a CDS chain (i.e., CD3 , CD35, CD3e or CD3 ), or a complex formed by two or more TCR chains or CD3 chains (e.g., a complex of TCRa and TCRP, a complex of TCRy and TCR5, a complex of CD3e and CD36, a complex of CD3Y and CD3e, or a sub-TCR complex of TCRa, TCRP, CD3y, CD35, and two CD3e chains).
  • Antibody-dependeni cell-mediated cytotoxicity and "ADCC,” as used herein, refer to a cell-mediated process in which nonspecific cytotoxic cells that express FcyRs (e.g., monocytic cells such as Natural Killer (NK) cells and macrophages) recognize bound antibody (or other protein capable of binding FcyRs) on a target cell and subsequently cause lysis of the target ceil.
  • FcyRs e.g., monocytic cells such as Natural Killer (NK) cells and macrophages
  • NK Natural Killer
  • any effector cell with an activating FcyR can be triggered to mediate ADCC.
  • the primary cells for mediating ADCC are N K cells, which express only FcyRIIL whereas monocytes, depending on their state of activation, localization, or differentiation, can express FcyRI, FcyRII, and FcyRIIL
  • N K cells which express only FcyRIIL
  • monocytes depending on their state of activation, localization, or differentiation, can express FcyRI, FcyRII, and FcyRIIL
  • the term "having ADCC activity,” as used herein in reference to a polypeptide or protein, means that the polypeptide or protein (for example, one comprising an
  • immunoglobulin hinge region and an immunoglobulin constant region having CH2 and CH3 domains is capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC) through binding of a cytolytic Fc receptor (e.g., FcyRIII) on a cytolytic immune effector cell expressing the Fc receptor (e.g., an NK cell).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • Complement-dependent cytotoxicity and “CDC,” as used herein, refer to a process in which components in normal serum (“complement”), together with an antibody or other C1 q-complement-binding protein bound to a target antigen, exhibit lysis of a target cell expressing the target antigen.
  • Complement consists of a group of serum proteins that act in concert and in an orderly sequence to exert their effect.
  • classical complement pathway and “classical complement system,” as used herein, are synonymous and refer to a particular pathway for the activation of complement.
  • the classical pathway requires antigen-antibody complexes for initiation and involves the activation, in an orderly fashion, of nine major protein components designated C1 through C9.
  • the product is an enzyme that catalyzes the subsequent step. This cascade provides amplification and activation of large amounts of complement by a relatively small initial signal.
  • the term "having CDC activity,” as used herein in reference to a polypeptide or protein, means that the polypeptide or protein (for example, one comprising an
  • immunoglobulin hinge region and an immunoglobulin constant region having CH2 and CH3 domains is capable of mediating complement- dependent cytotoxicity (CDC) through binding of Cl q complement protein and activation of the classical complement system.
  • CDC complement- dependent cytotoxicity
  • "Redirected T-ceii cytotoxicity" and "RTCC,” as used herein, refer to a T-ce!l- mediated process in which a T-ceii is recruited to a target vii using a multi-specific protein that is capable of specifically binding both the T-ce!i and the target cell, and whereby a target-directed T-celi cytotoxic response is elicited against the target cell.
  • RTCC is applicable to any T-cells, including, but not limited to CD8+ or cytotoxic T-celis (CTL), CD4+ or helper T-celis, regulatory T-celis (Treg), and natural killer T-celis (NKT).
  • CTL cytotoxic T-celis
  • Treg regulatory T-celis
  • NKT natural killer T-celis
  • treatment refers to either a therapeutic treatment or prophy!actic/preventative treatment.
  • a treatment is therapeutic if at least one symptom of disease in an individual receiving treatment improves or a treatment can delay worsening of a progressive disease in an individual, or prevent onset of additional associated diseases.
  • a therapeutically effective amount (or dose) or “effective amount (or dose)” of a specific binding molecule or compound refers to that amount of the compound sufficient to result in amelioration of one or more symptoms of the disease being treated in a statistically significant manner.
  • a therapeutically effective dose refers to that ingredient alone.
  • a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered serially or simultaneously (in the same formulation or concurrently in separate formulations).
  • the term “transformation,” “transfection,” and “transduction” refer to the transfer of nucleic acid (i.e., a nucleotide polymer) into a cell.
  • the term “genetic transformation” refers to the transfer and incorporation of DNA, especially recombinant DNA, into a cell.
  • the transferred nucleic acid can be introduced into a ceil via an expression vector.
  • variants refers to a nucleic acid or polypeptide differing from a reference nucleic acid or polypeptide, but retaining essential properties thereof. Generally, variants are overall closely similar, and, in many regions, identical to the reference nucleic acid or polypeptide. For instance, a variant may exhibit at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity compared to the active portion or full length reference nucleic acid or polypeptide.
  • variable region also referred to as “light chain variable domain” or “V L “ or “VL”
  • heavy chain variable region also referred to as “heavy chain variable domain” or “V H “ or “VH”
  • CDRs complementarity determining regions
  • FRs frame regions
  • CL refers to an "immunoglobulin light chain constant region” or a "light chain constant region,” i.e., a constant region from an antibody light chain.
  • CH refers to an antibody light chain constant region
  • immunoglobulin heavy chain constant region or a “heavy chain constant region,” which is further divisible, depending on the antibody isotype into CH 1 , CH2, and CHS (IgA, igD, igG), or CH1 , CH2, CH3, and CH4 domains (IgE, IgM).
  • a "Fab” fragment antigen binding is the part of an antibody that binds to antigens and includes the variable region and CH 1 domain of the heavy chain linked to the light chain via an inter-chain disulfide bond.
  • the present disclosure provides multivalent anti-CD37 antibodies comprising binding domains, in particular, at least one binding domain that specifically binds CD37 and at least one binding domain that specifically binds a target on a T ceil.
  • the multivalent anti- CD37 antibodies comprising binding domains of this disclosure can further comprise immunoglobulin constant regions, linker peptides, hinge regions, immunoglobulin
  • dimerization/heterodimerization domains dimerization/heterodimerization domains, junctional amino acids, tags, ete.
  • a multispecific antibody of the invention may take a variety of different formats so long as the antibody comprises a CD37 binding domain and a CD3 binding domain, and the scaffold of the antibody allows a bound B-cell to be in close proximity to a bound T ceil.
  • a multispecific anti-CD37 antibody includes dimerized single chain polypeptides, each polypeptide comprising, from amino to carboxyi terminus, a binding domain, an N-terminus linker (e.g., immunoglobulin hinge domain), a constant region, a C- terminus linker and another binding domain, in one embodiment, the N-terminus linker is an immunoglobulin hinge region.
  • single chain polypeptides as disclosed in this paragraph, are capable of homodimerization, typically through disulfide bonding, via the immunoglobulin constant region and/or hinge region (e.g., via an immunoglobulin constant region comprising IgG CH2 and CHS domains and an IgG hinge region).
  • two identical CD37-binding polypeptides are capable of homodimerization, typically through disulfide bonding, via the immunoglobulin constant region and/or hinge region (e.g., via an immunoglobulin constant region comprising IgG CH2 and CHS domains and an IgG hinge
  • the CD37 binding domain may comprise a scFv in the Vn-V L or V L -V H orientation.
  • the CD3 binding domain may comprise a scFv in the V H -V L or V L -V H orientation, in one embodiment, the CD3 binding domain scFv comprises an anti-CDS scFv disclosed herein or known in the art. In another embodiment, the CD37 binding domain scFv comprises an anti-CD37 scFv disclosed herein or known in the art.
  • the CDS binding scFv and the CD37 binding scFv may comprise variable heavy and variable light chain polypeptides as disclosed herein or known in the art.
  • the CD3 binding scFv and the CD37 binding scFv comprises CDRs as disclosed herein or that are known in the art.
  • the antibody may comprise an amino acid sequence with at least about 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity to an amino acid sequence of SEQ ID NO: 46, 48, 50, 52, 54, 56, 58, 60, or 63.
  • the multispecific antibody of the invention comprises an amino acid sequence of SEQ ID NO: 46, 48, 50, 52, 54, 56, 58, 60, or 63.
  • an antibody in the multispecific homodimer format, multispecific heterodimer format (described in more detail below) or a multispecific antibody comprising a constant region modified to exhibit little to no effector function exhibits an increased half-life as compared to a multispecific anti-CD37 antibody with no constant region or a multispecific anti-CD37 antibody in the scFv - linker ⁇ ⁇ scFv format (e.g., bispecific single chain antibody format; described in detail below).
  • heterodimer antibody or other multispecific antibody format comprising a modified constant region exhibits a half-life that is about 1 fold, 2 fold, 3 fold, 4 fold, or 5 fold or more greater than that of a multispecific anti-CD37 antibody in the scFv - linker - scFv format (e.g., bispecific single chain format).
  • the multispecific anti- CD37 antibody is not in the bispecific single chain format and comprises a molecule with a half-life at least about 1 fold, 2 fold, 3 fold, 4 fold or 5 fold greater than a multispecific anti- CD37 antibody in the bispecific single chain format.
  • the multispecific anti-CD37 antibody in the multispecific homodimer antibody or heterodimer antibody format is capable of activating T cells at an activation level comparable to that of a multispecific anti-CD37 antibody in the bispecific single chain format (described in detail below) but has the added benefit of causing a lower level of cytokine release in a patient when administered as compared to an antibody in the bispecific single chain format.
  • the invention includes, for instance a multispecific anti-CD37 antibody in the multispecific homodimer or heterodimer formats that is capable of activating T ceils and when administered to a patient in need, results in a release of cytokines that is about 1 fold, 2 fold, 3 fold, 4 fold or 5 fold or less than that which typically results with the administration of an anti-CD37 antibody in the bispecific single chain format.
  • Multispecific antibodies of the invention like an antibody in the multispecific homodimer antibody format that comprise a constant region or Fc region are preferably modified to knock-out or make null effector function so as to reduce the risk of eliciting a cytokine storm when administered to a patient. Modifications that can be made to the constant region or Fc region to abate effector function are discussed elsewhere in the specification.
  • an anti-CD37 multispecific antibody composed of dimerized single chain polypeptide chains comprises different single chain polypeptides (i.e., is a heterodimer).
  • each polypeptide chain comprising a binding domain, N-terminus linker, constant region, C-terminus linker and another binding domain further includes a beterodimerization domain
  • the second polypeptide chain for heterodimerization includes additional binding domains.
  • the heterodimeric anti-CD37 recombinant antibody may contain two, three or four binding different binding domains.
  • the invention includes a multispecific anti-CD37 antibody comprising a binding domain linked via a linker domain to a second binding domain (e.g., a scFv linked via a linker to another scFv).
  • a multispecific anti-CD37 antibody comprising a CD37 binding domain (in V H -linker-V L or V L -linker-V H orientation) linked via a peptide linker domain to a CDS binding domain (in V H -linker-V L or V L -iinker-V H orientation).
  • a bispecific antibody in the scFv-!inker-scFv format may comprise variable heavy and variable light domains derived from any anti-CD37 antibody and antibody to a T cell antigen (such as CDS) including, but not limited to, the variable domains disclosed herein.
  • the linker is ((Gly 4 )Ser) 3 .
  • the linker may also comprise about 8-12 amino acids.
  • An antibody of the invention in this format (“bispecific single chain antibody”), does not comprise an Fc region and as a result, has no Fc-related effector function.
  • a multispecific antibody is a disulfide - stabilized diabody (referred to herein as “disulfide-stabilized diabody antibody”).
  • a multispecific antibody may comprise two distinct polypeptides that are
  • each Fv is formed by the association of a V L partner on one chain with a ⁇ ⁇ partner on the second chain in a V LA -V H B (first chain) and LB- HA (second chain) configuration.
  • the diabody is stabilized by either of two alternative carboxyl terminal heterodimerization domains: a pairing of VEPKSC on one chain and FNRGEC on the other or a pairing of oppositely charged, coiled-coil domains. See, for instance, Moore et a/., 201 1 , Blood.
  • the muitispecific anti-CD37 antibody may comprise a first chain with a CDS binding domain V H linked to a CD37 binding domain V L and the second chain comprises a CD3 binding domain V L linked to a CD37 binding domain V H, and the two chains are linked via a disulfide bond at the c-termini.
  • a disuifide-stabilized diabody may be designed using variable heavy and light chains derived from known anti-CD37 and anti-CD3 antibodies including, for instance, the variable heavy and light chains disclosed herein.
  • the muitispecific anti-CD37 antibody is a dual variable domain binding protein capable of binding CD37 and TCR complex with specificity.
  • the recombinant antibody comprises a polypeptide chain, wherein said polypeptide chain comprises VD1 -(X1 )n-VD2-C-(X2)n, wherein VD1 is a first variable domain, VD2 is a second variable domain, C is a constant domain, X1 is a linker (e.g., a polypeptide linker of about 10 to 20 amino acids in length), X2 represents an Fc region and n is 0 or 1.
  • VD1 may be a variable anti ⁇ CD37 domain and VD2 may be a variable anti-CD3 domain.
  • VD1 may be a variable anti-CD3 domain and VD2 may be a variable anti-CD37 domain.
  • This format of antibody is referred to herein as "dual variable domain antibody" format.
  • a recombinant muitispecific antibody of the present disclosure specifically binds CD37.
  • the CD37-binding domain is capable of competing for binding to CD37 with an antibody having V H and V L regions having amino acid sequences as shown in SEQ ID NO:5 or 27 and SEQ ID NO:7 or 29, respectively (e.g., variable regions derived from murine monoclonal antibody G28-1], or with a single-chain Fv (scFv) having an amino acid sequence as shown in SEQ ID NO:3.
  • a binding domain is a single-chain Fv fragment (scFv) that comprises V H and V L regions specific for a target of interest, in certain embodiments, the V H and V L regions are human. In another embodiment, the variable regions are humanized, for instance, by modifying framework regions to more closely resemble human germline sequences.
  • scFv single-chain Fv fragment
  • a CD37-binding domain comprises or is a scFv that is at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100% identity to an amino acid sequence of a scFv of SEQ ID NO: 3.
  • the CD37 binding domain comprises a variable heavy chain and a variable light chain derived from an antibody that binds CD37 with specificity.
  • variable heavy chain and variable light chain may be derived from an anti-CD37 antibody selected from the group consisting of G28-1 , MB371 , BL14, NMN46, IP024, HH 1 , WR17, HD28, BM 4, F93G6, RFB-7, Y29/55, MB-1 , M-B371 , I PO-24, S-B3 and K7153A.
  • an anti-CD37 antibody selected from the group consisting of G28-1 , MB371 , BL14, NMN46, IP024, HH 1 , WR17, HD28, BM 4, F93G6, RFB-7, Y29/55, MB-1 , M-B371 , I PO-24, S-B3 and K7153A.
  • a multispecific antibody with a CD37 binding domain binds the same or overlapping epitope as an anti-CD37 antibody selected from the group consisting of G28-1 , MB371 , BL14, NMN46, I P024, HH 1 , WR17, HD28, B114, F93G6, RFB-7, Y29/55, MB-1 , M-B371 , ! PO-24, S-B3 and K7153A.
  • a multispecific antibody with a CD37 binding domain competes for binding with an anti-CD37 antibody selected from the group consisting of G28-1 , MB371 , BL14, NMN46, I P024, HH 1 , WR17, HD28, BI 14, F93G6, RFB-7, Y29/55, MB-1 , M-B371 , I PO-24, S-B3 and K7153A.
  • the CD37 binding domain may comprise a humanized version of a known murine or other animal anti-CD37 antibody (e.g., G28-1 ).
  • the CD37-binding domain comprises (i) an
  • Suitable CD37-binding domains include those having CDR sequences derived from mAb G28-1 .
  • the CD37 binding domain contains an amino acid sequence comprising SEQ I D NO: 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 30, 31 , 32, 33, 24 or 35.
  • variable heavy chain of the CD37 binding domain comprises CDR1 , CD2 and CD3.
  • variable heavy chain CDR1 , CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO: 8, SEQ I D NO: 1 1 and SEQ ID NO: 14.
  • the variable heavy chain CDR1 , CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO: 9 or 10, SEQ I D NO: 12 or 13, and SEQ I D NO: 15, 16 and 17.
  • the CD37 binding domain variable heavy chain region contains CDR1 , CDR2 and CDRS comprising SEQ I D NOs: 9, 12 and 15.
  • the heavy chain contains CDRs comprising SEQ ID NOs: 9, 13, and 15; SEQ ID NOs: 9, 12, and 16; SEQ I D NOs: 9, 12, and 17; SEQ I D NOs: 9, 13, and 16; SEQ I D NOs: 9, 13 and 17; SEQ I D NOs: 10, 12, and 15; SEQ I D NOs: 10, 12 and 16; SEQ I D NOs: 10, 12 and 17; SEQ I D NOs: 10, 13, and 15; SEQ ID NOs: 10, 13 and 16 or SEQ I D NOs: 10, 13 and 17. in yet another
  • the heavy chain contains CDRs comprising SEQ I D NOs: 30, 31 and 32.
  • the variable light chain of the CD37 binding domain comprises CDR1 , CDR2 and CDR3.
  • the variable light chain CDR1 , CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NQs: 18, 22 and 24.
  • the variabie light chain CDR1 , CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NQs: 19, 20 or 21 ; SEQ ID NO: 23 and SEQ ID NO: 25.
  • variable light chain CDR1 , CDR2 and CDR3 comprises SEQ ID NOs: 19, 23 and 25; SEQ ID NOs: 20, 23 and 25; or SEQ ID NOs: 21 , 23 and 25.
  • variable light chain CDR1 , CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NOs: 33, 34 and 35.
  • a CD37 binding domain comprises a variabie heavy chain CDR1 , CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 8, 1 1 and 14 and a variable light chain CDR1 , CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NQs: 18, 22 and 24.
  • the CD37 binding domain comprises a variable heavy chain CDR1 , CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 9 or 10, SEQ ID NOs: 12 or 13; and SEQ ID NOs: 15, 16 or 17, and a variabie light chain CDR1 , CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 19, 20, or 21 , SEQ ID NO:23 and SEQ ID NO: 25.
  • the CD37 binding domain comprises a variable heavy CDR1 , CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 30, 31 and 32 and a variable light CDR1 , CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 33, 34 and 35.
  • the CD37 binding domain contains a variabie heavy domain comprising at least about 90% identity or at least about 95% identity to an amino acid sequence of SEQ ID NO: 5, 27, 38 or 39. In another embodiment, the CD37 binding domain contains a variable heavy domain comprising an amino acid sequence of SEQ ID NO: 5, 27, 38 or 39.
  • the CD37 binding domain contains a variabie light chain comprising at least about 90% identity or at least about 95% identity to an amino acid sequence of SEQ ID NO: 7, 29 or 43. in another embodiment, the CD37 binding domain contains a variabie light domain comprising an amino acid sequence of SEQ ID NO: 7, 29 or 43.
  • the CD37 binding domain contains a variable heavy domain comprising at least about 90% identity or at least about 95% identity to an amino acid sequence of SEQ ID NO: 5 and a variabie light chain comprising at least about 90% identity or at least about 95% identity to SEQ ID NO: 7.
  • the invention includes a CD37 binding domain containing a variable heavy domain comprising SEQ ID NO: 5 and a variable light domain comprising SEQ ID NO: 7.
  • the CD37 binding domain contains a variable heavy domain comprising at least about 90% identity or at least about 95% identity to an amino acid sequence of SEQ ID NO: 27 and a variable light chain comprising at least about 90% identity or at least about 95% identity to SEQ ID NO: 29.
  • the invention includes a CD37 binding domain containing a variable heavy domain comprising SEQ ID NO: 27 and a variable light domain comprising SEQ ID NO: 29.
  • the CD37 binding domain contains a variable heavy domain comprising at least about 90% identity or at least about 95% identity to an amino acid sequence of SEQ ID NO: 38 or 39 and a variable light chain comprising at least about 90% identity or at least about 95% identity to SEQ ID NO: 43.
  • the invention includes a CD37 binding domain containing a variable heavy domain comprising SEQ ID NO: 38 or 39 and a variable light domain comprising SEQ ID NO: 43.
  • the invention includes a CD37 binding domain comprising an amino acid with at least about 90% or about 95% identity to SEQ ID NO: 3 that is capable of binding CD37 with specificity, in one embodiment of the invention, the CD37 binding domain comprises SEQ ID NO: 3.
  • a binding domain may comprise noncontiguous amino acids based on the format of the multispecific antibody.
  • the invention includes antibodies comprising a variable heavy domain on a different polypeptide than the corresponding variable light domain.
  • the binding domain is composed of two or more polypeptide chains that are joined to form antibody-like binding domains.
  • the invention includes antibodies comprising a CD37 binding domain and a CD3 binding domain wherein the CD37 binding domain comprises two polypeptide chains and the CD3 binding domain comprises two polypeptide chains - for instance, in a V L(CD3 7 BJR , D ; NG) ⁇ H( CD3 inding) (first chain) and VL(CD3 inding) -VH(CD37 inding) (second chain configuration.
  • each CDR comprises no more than one, two, or three substitutions, insertions or deletions, as compared to that from a monoclonal antibody or fragment or derivative thereof that specifically binds to a target of interest (e.g., CD37 or CD3).
  • a target of interest e.g., CD37 or CD3
  • the multispecific antibodies of the invention comprise a TCR binding domain for recruitment of T ceils to target cells expressing CD37.
  • the recombinant antibodies of the invention comprise a binding domain that specifically binds a TCR complex or a component thereof (e.g., TCRa, TCRp, CD3Y, CD35, and CDSE) and another binding domain that specifically binds to CD37.
  • the TCR binding domain is a CD3 binding domain
  • the TCR binding domain is a CD3 ⁇ binding domain.
  • the VH and VL regions of the TCR binding domain or CD3 binding domain are derived from a reference anti-CD3 antibody selected from the group consisting of X35-3, ViT3, BMA030 (BW264/56), CLB-T3/3, CRIS7,
  • CD3- specific antibodies are well known in the art and, inter alia, described in Tunnacliffe (1989), int. Immunol. 1 , 548-550.
  • said VH and VL regions of said CD3 specific domain are derived from OKT-3 or TR-66.
  • the TCR binding domain or CD3 binding domain is derived from a CD3 specific antibody other than OKT3 (i.e., the binding domain is not derived from OKT3).
  • V H and V L regions are or are derived from an antibody/antibody derivative specifically directed against CDS described by
  • V H and V L regions are derived from antibodies/antibody derivatives and the like which are capable of specifically recognizing human CDS epsilon in the context of other TCR subunits, e.g., in mouse T cells transgenic for human CDS epsilon.
  • the CD3 binding domain is a CDS binding (e.g., CD3 ⁇ binding domain) as disclosed in US 201 1 /0262439, US 2006/0193852, US 2012/0034228, US 2010/0150918, and US 2009/0022738, each of which is herein incorporated by reference in its entirety.
  • the invention includes CDS binding domains that are optimized for cross-reactivity in other species such as non-human primates.
  • Reference anti-CD3 antibodies from which the binding domain of this disclosure can be derived include CRlS-7 monoclonal antibody (Reinherz, E. L. et ai. (eds.), Leukocyte typing I I., Springer Verlag, New York, (1986); VL and VH amino acid sequences respectively shown in SEQ I D NO:97
  • An exemplary anti-TCR antibody is the BMA031 monoclonal antibody (Borst et al. (1990) Human immunology 29:175-188).
  • a second binding domain (or first binding domain if CD37 binding domain is second binding domain) specifically binds CD3e, and the second binding domain competes for binding to CD3E with the CRIS-7 or HuM291 monoclonal antibody.
  • a multispecific antibody of the invention comprising a CD3 binding domain binds to the same epitope or an overlapping epitope as X35-3, VIT3, BMA030 (BW264/56), BMA031 , G19-4, 145-2C1 1 , OKT3, BC3, CLB-T3/3, CRIS7, YTH12.5, F1 1 1-409, CLB-T3.4.2, WT31 , WT32, SPv-T3b, 1 1 D8, XIII-141 , XIII-46, XIII-87, 12F6, T3/RW2-8C8, T3/RW2-4B6, OKT3D, M-T301 , S C2, RIV9 or F101.01 . in another embodiment, a multispecific antibody of the invention comprising a CD3 binding domain competes with an antibody selected from the list consisting of X35-3, VIT3, B A030
  • the CD3-binding domain comprises an immunoglobulin heavy chain variable region (V H ) and an immunoglobulin light chain variable region (V L ) derived from the CRIS-7 or Hu 291 monoclonal antibody ⁇ e.g., the V H and V L of the second binding domain can be humanized variable regions comprising, respectively, the heavy chain CDRs and the light chain CDRs of the monoclonal antibody).
  • the V H and V L regions derived from CRIS-7 can be selected from a V L region comprising an amino acid sequence that is at least 95% identical or 100% to the amino acid sequence set forth in SEQ ID NO:67, 81 , 83, 85, 87 or 97.
  • the V H and V L regions derived from CRIS-7 can be selected from a V H region comprising an amino acid sequence that is at least 95% identical or 100% to the amino acid sequence set forth in SEQ ID NO: 65, 69, 71 , 73, 75, 77, 79, or 96.
  • amino acids of a CD3 binding domain and adjacent amino acid sequence are mutated to modify the isoelectric point of the molecule. This may be done, for instance, to reduce clipping of the molecule during expression and to increase half-life.
  • the invention envisions multispecific antibodies with modifications of the binding domain and/or surrounding sequences to reduce clipping during expression and / or increase half-life of the molecule. See, for instance, commonly owned US provisional patent applications
  • a V H and/or V L region contains about one or more (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, about one or more (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, about one or more (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid
  • the insertion(s), deletion(s) or substitution(s) can be anywhere in the V H and/or V L region, including at the amino- or carboxyl-terminus or both ends of this region, provided that each CDR comprises zero changes or at most one, two, or three changes and provided a binding domain containing the modified V L and/or Vn region can stili specifically bind its target with an affinity similar or greater to the wild type binding domain.
  • the binding domain is a single-chain Fv (scFv) comprising immunoglobulin V H and V L regions joined by a peptide linker.
  • scFv single-chain Fv
  • a widely used peptide linker is a 15mer consisting of three repeats of a Gly-Gly-Gly-Gly-Ser amino acid sequence ((Giy 4 Ser) 3 ).
  • Other linkers have been used, and phage display technology, as well as selective infective phage technology, has been used to diversify and select appropriate linker sequences (Tang ei ai, J. Biol. Chem.
  • a binding domain comprises humanized immunoglobulin V H and/or V L regions. Techniques for humanizing immunoglobulin V H and V L regions are known in the art and are discussed, for example, in United States Patent Application Publication No. 2006/0153837 which is incorporated by reference in its entirety.
  • Humanization is expected to result in an antibody that is less immunogenic, with complete retention of the antigen-binding properties of the original molecule. In order to retain ail of the antigen-binding properties of the original antibody, the structure of its antigen binding site should be reproduced in the "humanized” version.
  • humanization by CDR grafting involves recombining only the CDRs of a non-human antibody onto a human variable region framework and a human constant region. Theoretically, this should substantially reduce or eliminate immunogenicity (except if allotypic or idiotypic differences exist). However, it has been reported that some framework residues of the original antibody also may need to be preserved (Reichmann et a!., Nature, 332:323 (1988); Queen et ai, Proc. Natl. Acad. Sci. USA, 86:10,029 (1989)).
  • framework residues that need to be preserved are amenable to identification through computer modeling.
  • critical framework residues can potentially be identified by comparing known antigen-binding site structures (Padian, Molec. Immunol., 31 (3):169-217 (1994), incorporated herein by reference).
  • the residues that potentially affect antigen binding fall into several groups.
  • the first group comprises residues that are contiguous with the antigen site surface, which could therefore make direct contact with antigens. These residues include the amino-termina! residues and those adjacent to the CDRs.
  • the second group includes residues that could alter the structure or relative alignment of the CDRs, either by contacting the CDRs or another peptide chain in the antibody.
  • the third group comprises amino acids with buried side chains that could influence the structural integrity of the variable domains.
  • the residues in these groups are usually found in the same positions (Padian, 1994, supra) although their positions as identified may differ depending on the numbering system (see Kabat ef a/., "Sequences of proteins of immunological interest, 5th ed., Pub. No. 91-3242, U.S. Dept. Health & Human Services, NIH, Bethesda, Md., 1991 ).
  • the multispecific antibodies of the invention are not traditional monoclonal antibodies, the multispecific antibodies of the invention contain variable regions and can be humanized according to methods known in the art for humanizing monoclonal antibodies. In one embodiment, the invention comprises humanized multispecific antibodies.
  • a recombinant antibody comprises single chain
  • polypeptides each polypeptide containing an immunoglobulin hinge region (e.g., a multispecific homodimer antibody or a multispecific heterodimer antibody format).
  • a hinge is a wild-type human immunoglobulin hinge region.
  • one or more amino acid residues can be added at the amino- or carboxyl- terminus of a wild type immunoglobulin hinge region as part of a fusion protein construct design.
  • additional junction amino acid residues at the hinge amino-terminus can be "RT,” “R8S,” “TG,” or “T,” or at the hinge carboxyi-terminus can be "SG”, or a hinge deletion can be combined with an addition, such as ⁇ with "SG” added at the carboxyi- terminus.
  • an N-ferminus linker or amino-terminus linker comprises or consists essentially of an immunoglobulin hinge region (see, for instance, description of multispecific homodimer antibody and multispecific heterodimer antibody), in certain other embodiments, a C-terminus linker or carboxyl terminus linker comprises or consists essentially of an immunoglobulin hinge region.
  • a hinge is an altered immunoglobulin hinge in which one or more cysteine residues in a wild type immunoglobulin hinge region is substituted with one or more other amino acid residues (e.g., serine or alanine).
  • Exemplary altered immunoglobulin hinges include an immunoglobulin human lgG1 hinge region having one, two or three cysteine residues found in a wild type human igG1 hinge substituted by one, two or three different amino acid residues (e.g., serine or alanine). For instance, in one embodiment, the first cysteine residue of the hinge is substituted with another amino acid (e.g., serine).
  • An altered immunoglobulin hinge can additionally have a proline substituted with another amino acid (e.g., serine or alanine).
  • another amino acid e.g., serine or alanine
  • the above-described altered human lgG1 hinge can additionally have a proline located carboxyl-terminai to the three cysteines of wild type human lgG1 hinge region substituted by another amino acid residue (e.g., serine, alanine), in one embodiment, the prolines of the core hinge region are not substituted.
  • a hinge polypeptide comprises or is a sequence that is at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a wild type immunoglobulin hinge region, such as a wild type human igG1 hinge, a wild type human lgG2 hinge, or a wild type human lgG4 hinge.
  • a hinge present in a muitispecific anti-CD37 antibody can be a hinge that is not based on or derived from an immunoglobulin hinge (i.e., not a wild-type immunoglobulin hinge or an altered immunoglobulin hinge).
  • Examples for such hinges include peptides of about five to about 150 amino acids derived from an interdomain region of a transmembrane protein.
  • the invention includes linkers derived from, comprising or consisting essentially of an interdomain region of a transmembrane protein, for instance, an N-terminus linker or a C-terminus linker of a muitispecific homodimer antibody or a muitispecific heterodimer antibody.
  • the invention includes linkers derived from stalk region of a type ⁇ C-Iectin, for instance, peptides of about eight to 25 amino acids and peptides of about seven to 18 amino acids.
  • a muitispecific antibody of the invention for instance, an antibody in the muitispecific homodimer antibody format or muitispecific heterodimer antibody format may contain an N- terminus linker comprising or consisting essentially of an amino acid sequence derived from a stalk region of a type II C-lectin and / or a C-terminus linker comprising or consisting essentially of an amino acid sequence derived from a stalk region of a type II C-iectin.
  • an interdomain region or stalk region have seven to 18 amino acids and can form an a-heiicai coiled coil structure.
  • interdomain or stalk region hinges contain 0, 1 , 2. 3, or 4 cysteines.
  • Exemplary interdomain or stalk region hinges are peptide fragments of the interdomain or stalk regions, such as ten to 150 amino acid fragments from the stalk regions of CD69, CD72, CD94, NKG2A and NKG2D.
  • hinge sequences have about 5 to 150 amino acids, 5 to 10 amino acids, 10 to 20 amino acids, 20 to 30 amino acids, 30 to 40 amino acids, 40 to 50 amino acids, 50 to 60 amino acids, 5 to 60 amino acids, 5 to 40 amino acids, 8 to 20 amino acids, or 10 to 15 amino acids.
  • the hinge can be primarily flexible, but can also provide more rigid characteristics or can contain primarily cs-heiicai structure with minimal ⁇ -sheet structure.
  • the lengths or the sequences of the hinges can affect the binding affinities of the binding domains to which the hinges are directly or indirectly (via another region or domain, such as an heterodimerization domain) connected as well as one or more activities of the Fc region portions to which the hinges are directly or indirectly connected.
  • hinge sequences are stable in plasma and serum and are resistant to proteolytic cleavage.
  • the first lysine in the igG1 upper hinge region can be mutated to minimize proteolytic cleavage, for instance, the lysine can be substituted with methionine, threonine, alanine or glycine, or is deleted.
  • the CD37-binding antibody comprises a first polypeptide that is capable of forming a heterodimer with a second polypeptide chain and comprises a hinge region (a) immediately amino-termina! to an immunoglobulin constant region (e.g., amino-terminal to a CH2 domain wherein the immunoglobulin constant region includes CH2 and CHS domains, or amino-terminal to a CH3 domain wherein the immunoglobulin constant region (e.g., amino-terminal to a CH2 domain wherein the immunoglobulin constant region includes CH2 and CHS domains, or amino-terminal to a CH3 domain wherein the
  • immunoglobulin sub-regions includes CH3 and CH4 domains), (b) interposed between and connecting a binding domain (e.g., scFv) and a immunoglobulin heterodimerization domain, (c) interposed between and connecting a immunoglobulin heterodimerization domain and an immunoglobulin constant region (e.g., wherein the immunoglobulin constant region includes CH2 and CH3 domains or CH3 and CH4 domains), (d) interposed between and connecting an immunoglobulin constant region and a binding domain, (e) at the amino-terminus of a polypeptide chain, or (f) at the carboxy!-terminus of a polypeptide chain, in one embodiment of the invention, a polypeptide chain comprising a hinge region as described herein will be capable of associating with a different polypeptide chain to form a heterodimeric protein provided herein, and the heterodimer formed will contain a binding domain that retains its target specificity or its specific target binding affinity.
  • a hinge present in a polypeptide that forms a heterodimer with another polypeptide chain can be an immunoglobulin hinge, such as a wild-type immunoglobulin hinge region or an altered immunoglobulin hinge region thereof.
  • a hinge of one polypeptide chain of a heterodimeric protein is identical to a corresponding hinge of the other polypeptide chain of the heterodimer. in certain other embodiments, a hinge of one chain is different from that of the other chain (in their length or sequence).
  • a heterodimeric antibody has a CD3- or TCR-binding domain in one chain and a CD37-binding domain in another chain. Having two different hinges in the two chains may allow the heterodimer to bind to the CD37 first, and then to a CD3 or other TCR component second. Thus, the heterodimer may recruit CD3 + T ceils to CD37-expressing B-ceils, which in turn may damage or destroy the B-cells.
  • Exemplary linker for instance, N-terminus linker and C-terminus linker
  • hinge regions suitable for use in accordance with the present invention are shown in the Tables 1 and 2, Additional exemplary linker and hinge regions are set forth in SEQ ID NOs: 241-244, 601 , 78, 763-791 , 228, 379-434, 618-749 of WO201 1/090762 (said sequences incorporated by reference herein).
  • a mu!tispecific ani ibody of the invention can comprise
  • immunoglobulin dimerization domain or “immunoglobulin heterodimenzation domain,”
  • the interactions between immunoglobulin heterodimenzation domains “substantially contributes to or efficiently promotes” the heterodimenzation of first and second polypeptide chains if there is a statistically significant reduction in the
  • the first and second polypeptide chains when the first and second polypeptide chains are co-expressed, at least 80%, at least about 60% to about 70%, at least about 70% to about 80%, at least 80% to about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the first and second polypeptide chains form heterodimers with each other.
  • Representative immunoglobulin heterodimenzation domains include an immunoglobulin CH 1 domain, an immunoglobulin CL1 domain (e.g., C or CA isotypes), or derivatives thereof, including wild-type
  • Dimerization/heterodimerization domains can be used where it is desired to form heterodimers from two non-identical polypeptide chains, where one or both polypeptide chains comprises a binding domain, in certain embodiments, one polypeptide chain member of certain heterodimers described herein does not contain a binding domain.
  • a muitispecific heterodimer antibody of the present disclosure comprises an immunoglobulin heterodimerization domain in each polypeptide chain.
  • immunoglobulin heterodimerization domains in the polypeptide chains of a heterodimer are different from each other and thus can be differentially modified to facilitate heterodimerization of both chains and to minimize homodimerization of either chain.
  • heterodimerization domains provided herein allow for efficient heterodimerization between different polypeptides and facilitate purification of the resulting muitispecific heterodimer antibody.
  • immunoglobulin heterodimerization domains useful for promoting heterodimerization of two different single chain polypeptides include immunoglobulin CH 1 and CL domains, for instance, human CH 1 and CL domains.
  • an immunoglobulin heterodimerization domain is a wild-type CH 1 domain, such as a wild type igG1 , lgG2, lgG3, lgG4, igA1 , igA2, igD, IgE, or IgM CH 1 domain.
  • an immunoglobulin heterodimerization domain is a wild-type human lgG1 , lgG2, igG3, igG4, lgA1 , lgA2, IgD, IgE, or IgM CH 1 domain as set forth in SEQ I D NOS: 1 14, 186-192 and 194, respectively, of PCT Publication No. WO201 1 /090762 (said sequences incorporated by reference herein), in certain embodiments, an immunoglobulin heterodimerization domain is a wild-type human igG1 CH 1 domain as set forth in SEQ ID NO: 1 14 of WO201 1 /090762 (said sequence incorporated by reference herein).
  • an immunoglobulin heterodimerization domain is an altered immunoglobulin CH 1 domain, such as an altered igG1 , lgG2, lgG3, igG4, IgAI , lgA2 IgD, IgE, or IgM CH 1 domain.
  • an immunoglobulin CH 1 domain such as an altered igG1 , lgG2, lgG3, igG4, IgAI , lgA2 IgD, IgE, or IgM CH 1 domain.
  • heterodimerization domain is an altered human lgG1 , igG2, igG3, lgG4, lgA1 , lgA2, IgD, IgE, or IgM CH 1 domain.
  • a cysteine residue of a wild-type CH 1 domain e.g., a human CH 1 ) involved in forming a disulfide bond with a wild type
  • immunoglobulin CL domain e.g., a human CL
  • immunoglobulin CH 1 domain is deleted or substituted in the altered immunoglobulin CH 1 domain such that a disulfide bond is not formed between the altered CH 1 domain and the wild-type CL domain.
  • an immunoglobulin heterodimerization domain is a wild- type CL domain, such as a wild type CK domain or a wild type CA domain.
  • an immunoglobulin heterodimerization domain is a wild type human CK or human CA domain as set forth in SEQ I D NOS: 1 12 and 1 13, respectively, of WO201 1 /090762 (said sequences incorporated by reference herein), in further
  • an immunoglobulin heterodimerization domain is an altered immunoglobulin CL domain, such as an altered CK or CA domain, for instance, an altered human CK or human CA domain.
  • a cysteine residue of a wild-type CL domain (e.g., a human CL) involved in forming a disulfide bond with a wild type immunoglobulin CH 1 domain (e.g., a human CH 1 ) is deleted or substituted in the altered immunoglobulin CL domain,
  • Such altered CL domains can further comprise an amino acid deletion at their amino-termini.
  • An exemplary CK domain is set forth in SEQ I D NO:141 of VVO201 1/090762 (said sequence incorporated by reference herein ), in which the first arginine and the last cysteine of the wild type human Ck domain are both deleted.
  • only the last cysteine of the wild type human Ck domain is deleted in the altered Ck domain because the first arginine deleted from the wild type human Ck domain can be provided by a linker that has an arginine at its carboxyi-terminus and links the amino-terminus of the altered Ck domain with another domain (e.g., an immunoglobulin sug-region, such as a sub-region comprising immunoglobulin CH2 and CH3 domains).
  • a linker that has an arginine at its carboxyi-terminus and links the amino-terminus of the altered Ck domain with another domain (e.g., an immunoglobulin sug-region, such as a sub-region comprising immunoglobulin CH2 and CH3 domains).
  • An exemplary CA domain is set forth in SEQ I D NQ:140 of WQ201 1 /090762 (said sequence incorporated by reference herein), in which the first arginine of a wild type human CA domain is deleted and the cysteine involved in forming a disulfide bond with a cysteine in a CH 1 domain is substituted by a serine.
  • an immunoglobulin heterodimerization domain is an altered CK domain that contains one or more amino acid substitutions, as compared to a wild type CK domain, at positions that may be involved in forming the interchain-hydrogen bond network at a CK-CK interface.
  • an immunoglobulin heterodimerization domain is an altered human CK domain having one or more amino acids at positions N29, N30, Q52, V55, T56, S68 or T70 that are substituted with a different amino acid.
  • an immunoglobulin heterodimerization domain is an altered human CK domain having one, two, three or four amino acid
  • amino acid used as a substitute at the above-noted positions can be an alanine, or an amino acid residue with a bulk side chain moiety such as arginine, tryptophan, tyrosine, giutamate, glutamine, or lysine.
  • Additional amino acid residues that can be used to substitute amino acid residues of the wild type human Ck sequence at the above noted positions include aspartate, methionine, serine and phenylalanine.
  • Exemplary altered human CK domains are set forth in SEQ ID NOS: 142-178 of WO201 1 /090762 (said sequences incorporated by reference herein).
  • Altered human CK domains are those that facilitate heterodimerization with a CH 1 domain, but minimize homodimerization with another CK domain.
  • Representative altered human CK domains are set forth in SEQ ID NOS:160 (N29W V55A T70A), 161 (N29Y V55A T70A), 202 (T70E N29A N30A V55A), 167 (N30R V55A T70A), 168 (N30K V55A T70A), 170 (N30E V55A T70A), 172 (V55R N29A N3QA), 175 (N29W N30Y V55A T70E), 176 (N29Y N30Y V55A T70E), 177 (N30E V55A T7QE), 178 (N30Y V55A T70E), 838 (N30D V55A T7QE), 839 (N30M V55A T70E), 840 (N30S V55A T70E), and 841 (IM30F
  • both the immunoglobulin heterodirnerization domains i.e., immunoglobulin CH 1 and CL domains
  • the immunoglobulin heterodirnerization domains of a mu!tispecific heterodimer antibody have mutations so that the resulting immunoglobulin heterodirnerization domains form salt bridges (i.e., ionic interactions) between the amino acid residues at the mutated sites.
  • the immunoglobulin heterodirnerization domains of a multispecific heterodimer antibody can be a mutated CH 1 domain in combination with a mutated Ck domain.
  • valine at position 68 (V68) of the wild type human CH 1 domain is substituted by an amino acid residue having a negative charge (e.g., aspartate or glutamate), whereas leucine at position 29 (L29) of a mutated human Ck domain in which the first arginine and the last cysteine have been deleted is substituted by an amino acid residue having a positive charge (e.g., lysine, arginine or histidine).
  • a negative charge e.g., aspartate or glutamate
  • leucine at position 29 (L29) of a mutated human Ck domain in which the first arginine and the last cysteine have been deleted is substituted by an amino acid residue having a positive charge (e.g., lysine, arginine or histidine).
  • V68 of the wild type CH 1 can be substituted by an amino acid residue having a positive charge
  • L29 of a mutated human Ck domain in which the first arginine and the last cysteine have been deleted can be substituted by an amino acid residue having a negative charge
  • Exemplary mutated CH 1 sequences in which V68 is substituted by an amino acid with either a negative or positive charge are set forth in SEQ ID NQS:844 and 845 of WO201 1 /090762 (said sequences incorporated by reference herein).
  • Exemplary mutated Ck sequences in which L29 is substituted by an amino acid with either a negative or positive charge are set forth in SEQ ID NOS:842 and 843 of WO201 1/090762 (said sequences incorporated by reference herein).
  • Positions other than V68 of human CH I domain and L29 of human Ck domain can be substituted with amino acids having opposite charges to produce ionic interactions between the amino acids in addition or alternative to the mutations in V68 of CH 1 domain and L29 of Ck domain.
  • Such positions can be identified by any suitable method, including random mutagenesis, analysis of the crystal structure of the CH 1-Ck pair to identify amino acid residues at the CH1-Ck interface, and further identifying suitable positions among the amino acid residues at the CHI -Ck interface using a set of criteria (e.g., propensity to engage in ionic interactions, proximity to a potential partner residue, etc.).
  • muitispecific heterodimer antibodies of the present disclosure contain only one pair of immunoglobulin heterodimerization domains.
  • a first chain of a muitispecific heterodimer antibody can comprise a CH1 domain as an immunoglobulin heterodimerization domain, while a second chain can comprise a CL domain (e.g., a CK or CA) as an immunoglobulin heterodimerization domain.
  • a first chain can comprise a CL domain (e.g., a CK or CA) as an immunoglobulin
  • heterodimerization domain while a second chain can comprise a CH1 domain as an immunoglobulin heterodimerization domain.
  • the immunoglobulin heterodimerization domains of the first and second chains are capable of associating to form a heterodimeric protein of this disclosure.
  • muitispecific heterodimer antibodies of the present disclosure can have two pairs of immunoglobulin heterodimerization domains.
  • a first chain of a heterodimer can comprise two CH1 domains, while a second chain can have two CL domains that associate with the two CH1 domains in the first chain.
  • a first chain can comprise two CL domains, while a second chain can have two CH1 domains that associate with the two CL domains in the first chain
  • a first polypeptide chain comprises a CH1 domain and a CL domain
  • a second polypeptide chain comprises a CL domain and a CH1 domain that associate with the CH1 domain and the CL domain, respectively, of the first polypeptide chain.
  • the immunoglobulin heterodimerization domain of each chain can be located amino-terminal to the immunoglobulin constant region of that chain.
  • the immunoglobulin heterodimerization domain in each chain can be located carboxyl-terminal to the
  • both immunoglobulin heterodimerization domains in each chain can be located amino-terminal to the immunoglobulin constant region of that chain.
  • both immunoglobulin heterodimerization domains in each chain can be located carboxyl-terminal to the immunoglobulin constant region of that chain.
  • one immunoglobulin heterodimerization domain in each chain can be located amino-terminal to the immunoglobulin constant region of that chain, while the other immunoglobulin heterodimerization domain of each chain can be located carboxyl-terminal to the
  • the immunoglobulin constant region is interposed between the two immunoglobulin
  • a mu!tispecific anti ⁇ CD37 antibody of the present disclosure comprises an immunoglobulin constant region (also referred to as an constant region or Fc region) in each polypeptide chain.
  • an immunoglobulin constant region slows clearance of the antibodies (for instance, the bispecific homodimer antibodies and multispecific heterodimer antibodies of the invention) from circulation after administration to a subject.
  • Effector functions e.g., ADCC, ADCP, CDC, complement fixation, and binding to Fc receptors
  • an immunoglobulin constant region of one or both of the polypeptide chains of a multispecific antibody of the present disclosure will be capable of mediating one or more of these effector functions.
  • one or more of these effector functions are reduced or absent in an immunoglobulin constant region of one or both of the polypeptide chains of an antibody of the of the present disclosure (e.g., multispecific homodimer antibody or multispecific heterodimer antibody), as compared to a corresponding wild-type immunoglobulin constant region.
  • the invention specifically includes a multispecific anti-CD37 antibody to elicit RTCC, wherein the antibody contains no immunoglobulin constant region or contains an immunoglobulin constant region with reduced or no effector function relative to a corresponding wild-type immunoglobulin constant region.
  • a constant region exhibiting a reduction or lack of effector function may reduce the likelihood that the multispecific antibody will cause a cytokine storm when administered to a patient.
  • an immunoglobulin constant region optionally present in multispecific anti-CD37 antibodies may comprise or be derived from part or all of: a CH2 domain, a CH3 domain, a CH4 domain, or any combination thereof.
  • an immunoglobulin constant region may comprise a CH2 domain, a CH3 domain, both CH2 and CH3 domains, both CH3 and CH4 domains, two CH3 domains, a CH4 domain, two CH4 domains, and a CH2 domain and part of a CH3 domain.
  • the immunoglobulin constant region does not contain a CH1 domain.
  • the multispecific homodimer antibody of the invention does not contain a CH1 domain.
  • a CH2 domain can be a wild type immunoglobulin CH2 domain or an altered immunoglobulin CH2 domain thereof from certain immunoglobulin classes or subclasses (e.g., igG1 , lgG2, lgG3, lgG4, lgA1 , igA2, or IgD) and from various species (including human, mouse, rat and other mammals).
  • immunoglobulin classes or subclasses e.g., igG1 , lgG2, lgG3, lgG4, lgA1 , igA2, or IgD
  • a CH2 domain is a wild type human immunoglobulin CH2 domain, such as wild type CH2 domains of human lgG1 , igG2, igG3, igG4, lgA1 , lgA2, or IgD, as set forth in SEQ ID NOS:1 15, 199-201 and 195-197, respectively, of PCT Publication VVO201 1/090762 (said sequences incorporated by reference herein), in certain
  • the CH2 domain is a wild type human lgG1 CH2 domain as set forth in SEQ ID NG:1 15 of WO201 1/090782 (said sequence incorporated by reference herein).
  • a CH2 domain is an altered immunoglobulin CH2 region (e.g., an altered human lgG1 CH2 domain) that comprises an amino acid substitution at the asparagine of position 297 (e.g., asparagine to alanine).
  • an amino acid substitution reduces or eliminates giycosyiation at this site and abrogates efficient Fc binding to FcyR and C1 q.
  • the sequence of an altered human lgG1 CH2 domain with an Asn to Ala substitution at position 297 is set forth in SEQ ID NG:324 of WO201 1/090762 said
  • a CH2 domain is an altered immunoglobulin CH2 region (e.g., an altered human lgG1 CH2 domain) that comprises at least one substitution or deletion at positions 234 to 238.
  • an immunoglobulin CH2 region can comprise a substitution at position 234, 235, 236, 237 or 238, positions 234 and 235, positions 234 and 236, positions 234 and 237, positions 234 and 238, positions 234-236, positions 234,
  • an altered CH2 region can comprise one or more (e.g., two, three, four or five) amino acid deletions at positions 234-238, for instance, at one of position
  • the amino acid residues at one or more of positions 234- 238 has been replaced with one or more alanine residues.
  • only one of the amino acid residues at positions 234-238 have been deleted while one or more of the remaining amino acids at positions 234-238 can be substituted with another amino acid (e.g., alanine or serine).
  • a CH2 domain is an altered immunoglobulin CH2 region (e.g., an altered human lgG1 CH2 domain) that comprises one or more amino acid substitutions at positions 253, 310, 318, 320, 322, and 331 .
  • an altered immunoglobulin CH2 region e.g., an altered human lgG1 CH2 domain
  • immunoglobulin CH2 region can comprise a substitution at position 253, 310, 318, 320, 322, or 331 , positions 318 and 320, positions 318 and 322, positions 318, 320 and 322, or any other combination of two, three, four, five or six amino acids at positions 253, 310, 318, 320, 322, and 331 .
  • the above-noted mutation(s) decrease or eliminate the complement- dependent cytotoxicity (CDC) of a polypeptide heterodimer that comprises the altered CH2 domain.
  • an altered CH2 region in addition to the amino acid substitution at position 297, can further comprise one or more (e.g., two, three, four, or five) additional substitutions at positions 234-238.
  • an immunoglobulin CH2 region can comprise a substitution at positions 234 and 297, positions 234, 235, and 297, positions 234, 236 and 297, positions 234-236 and 297, positions 234, 235, 237 and 297, positions 234, 236, 238 and 297, positions 234, 235, 237, 238 and 297, positions 236-238 and 297, or any combination of two, three, four, or five amino acids at positions 234-238 in addition to position 297.
  • an altered CH2 region can comprise one or more (e.g., two, three, four or five) amino acid deletions at positions 234-238, such as at position 236 or position 237.
  • the additional mutation(s) decreases or eliminates the antibody-dependent cell-mediated cytotoxicity (ADCC) activity or Fc receptor-binding capability of a polypeptide heterodimer that comprises the altered CH2 domain.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the amino acid residues at one or more of positions 234-238 have been replaced with one or more alanine residues, in further embodiments, only one of the amino acid residues at positions 234-238 has been deleted while one or more of the remaining amino acids at positions 234-238 can be substituted with another amino acid (e.g., alanine or serine).
  • a mutated CH2 region in addition to one or more (e.g., 2, 3, 4, or 5) amino acid substitutions at positions 234-238, a mutated CH2 region (e.g., an altered human !gG1 CH2 domain) in a fusion protein of the present disclosure can contain one or more (e.g., 2, 3, 4, 5, or 6) additional amino acid substitutions (e.g., substituted with alanine) at one or more positions involved in complement fixation (e.g., at positions I253, H310, E318, K320, K322, or P331 ).
  • additional amino acid substitutions e.g., substituted with alanine
  • mutated immunoglobulin CH2 regions include human !gG1 , igG2, igG4 and mouse lgG2a CH2 regions with alanine substitutions at positions 234, 235, 237 (if present), 318, 320 and 322.
  • An exemplary mutated Immunoglobulin CH2 region is mouse IGHG2c CH2 region with alanine substitutions at L234, L235, G237, E318, K320, and K322.
  • an altered CH2 region in addition to the amino acid substitution at position 297 and the additional deietion(s) or substitution(s) at positions 234-238, an altered CH2 region (e.g., an altered human lgG1 CH2 domain) can further comprise one or more (e.g., two, three, four, five, or six) additional substitutions at positions 253, 310, 318, 320, 322, and 331.
  • an immunoglobulin CH2 region can comprise a (1 ) substitution at position 297, (2) one or more substitutions or deletions or a combination thereof at positions 234-238, and one or more (e.g., 2, 3, 4, 5, or 6) amino acid substitutions at positions I253, H310, E318, K320, K322, and P331 , such as one, two, three substitutions at positions E318, K320 and K322.
  • the amino acids at the above-noted positions can be substituted by alanine or serine.
  • an immunoglobulin CH2 region polypeptide comprises: (i) an amino acid substitution at the asparagines of position 297 and one amino acid
  • substitution at position 234, 235, 236 or 237 (ii) an amino acid substitution at the asparagine of position 297 and amino acid substitutions at two of positions 234-237; (ill) an amino acid substitution at the asparagine of position 297 and amino acid substitutions at three of positions 234-237; (iv) an amino acid substitution at the asparagine of position 297, amino acid substitutions at positions 234, 235 and 237, and an amino acid deletion at position 238; (v) amino acid substitutions at three of positions 234-237 and amino acid substitutions at positions 318, 320 and 322; or (vi) amino acid substitutions at three of positions 234-237, an amino acid deletion at position 236, and amino acid substitutions at positions 318, 320 and
  • Exemplary altered immunoglobulin CH2 regions with amino acid substitutions at the asparagine of position 297 include: human igG1 CH2 region with alanine substitutions at L234, L235, G237 and N297 and a deletion at G236 (SEQ ID NO:325 of WO201 1/090762, said sequence incorporated by reference herein), human lgG2 CH2 region with alanine substitutions at V234, G236, and N297 (SEQ ID NO:326 of VVQ201 1/090762, said sequence incorporated by reference herein), human lgG4 CH2 region with alanine substitutions at F234, L235, G237 and N297 and a deletion of G236 (SEQ ID NO:322 of WO201 1/090762, said sequence incorporated by reference herein), human lgG4 CH2 region with alanine substitutions at F234 and N297 (SEQ ID NQ:343 of WO201 1/
  • an altered CH2 region in addition to the amino acid substitutions described above, can contain one or more additional amino acid substitutions at one or more positions other than the above- noted positions.
  • Such amino acid substitutions can be conservative or non-conservative amino acid substitutions.
  • P233 can be changed to E233 in an altered igG2 CH2 region (see, e.g., SEQ ID NO:326 of WO201 1/090762, said sequence incorporated by reference herein).
  • the altered CH2 region can contain one or more amino acid insertions, deletions, or both. The insertion(s).
  • deietion(s) or substitution(s) can be anywhere in an immunoglobulin CH2 region, such as at the N- or C-terminus of a wild type immunoglobulin CH2 region resulting from linking the CH2 region with another region (e.g., a binding domain or an immunoglobulin heterodimerization domain) via a hinge.
  • another region e.g., a binding domain or an immunoglobulin heterodimerization domain
  • an altered CH2 region in a polypeptide of the present disclosure comprises or is a sequence that is at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to a wild type immunoglobulin CH2 region, such as the CH2 region of wild type human lgG1 , lgG2, or lgG4, or mouse lgG2a (e.g., IGHG2c).
  • a wild type immunoglobulin CH2 region such as the CH2 region of wild type human lgG1 , lgG2, or lgG4, or mouse lgG2a (e.g., IGHG2c).
  • An altered immunoglobulin CH2 region can be derived from a CH2 region of various immunoglobulin isotypes, such as lgG1 , lgG2, lgG3, igG4, igA igA2, and IgD, from various species (including human, mouse, rat, and other mammals).
  • various immunoglobulin isotypes such as lgG1 , lgG2, lgG3, igG4, igA igA2, and IgD, from various species (including human, mouse, rat, and other mammals).
  • an altered immunoglobulin CH2 region can be derived from a CH2 region of human lgG1 , lgG2 or lgG4, or mouse lgG2a (e.g., IGHG2c), whose sequences are set forth in SEQ ID NOS:1 15, 199, 201 , and 320 of VVQ201 1/090762 (said sequences incorporated by reference herein).
  • an altered CH2 domain is a human IgG1 CH2 domain with alanine substitutions at positions 235, 318, 320, and 322 (i.e., a human lgG1 CH2 domain with L235A, E318A, K320A and K322A substitutions) (SEQ ID NO:595 of WQ201 1/090762, said sequence incorporated by reference herein), and optionally an N297 mutation (e.g., to alanine).
  • an altered CH2 domain is a human lgG1 CH2 domain with alanine substitutions at positions 234, 235, 237, 318, 320 and 322 (i.e., a human lgG1 CH2 domain with L234A, L235A, G237A, E318A, K320A and K322A substitutions) (SEQ ID NO:596 of VVO201 1/090762, said sequence incorporated by reference herein), and optionally an N297 mutation (e.g., to alanine).
  • an altered CH2 domain is an altered human lgG1 CH2 domain with mutations known in the art that enhance immunological activities such as ADCC, ADCP, CDC, complement fixation, Fc receptor binding, or any combination thereof,
  • the CH3 domain can be a wild type immunoglobulin CH3 domain or an altered immunoglobulin CH3 domain thereof from certain immunoglobulin classes or subclasses (e.g., lgG1 , igG2, igG3, lgG4, lgA1 , lgA2, IgD, IgE, IgM) of various species (including human, mouse, rat, and other mammals).
  • immunoglobulin classes or subclasses e.g., lgG1 , igG2, igG3, lgG4, lgA1 , lgA2, IgD, IgE, IgM
  • a CH3 domain is a wild type human immunoglobulin CH3 domain, such as wild type CH3 domains of human igG1 , lgG2, igG3, lgG4, lgA1 , lgA2, IgD, IgE, or IgM as set forth in SEQ I D NOS: 1 16, 208-210, 204-207, and 212, respectively of WO201 1 /090762 (said sequences incorporated by reference herein).
  • the CH3 domain is a wild type human lgG1 CH3 domain as set forth in SEQ ID NO: 1 16 of WO201 1 /090762 (said sequence incorporated by reference herein).
  • a CH3 domain is an altered human immunoglobulin CHS domain, such as an altered CH3 domain based on or derived from a wild-type CH3 domain of human lgG1 , igG2, igG3, igG4, lgA1 , lgA2, IgD, IgE, or IgM antibodies.
  • an altered CH3 domain can be a human igG1 CH3 domain with one or two mutations at positions H433 and N434 (positions are numbered according to EU numbering).
  • an altered CH3 domain can be a human igG1 CH3 domain but with one or two amino acid substitutions at position F405 or Y407.
  • the amino acids at such positions are involved in interacting with another CH3 domain, in certain embodiments, an altered CH3 domain can be an altered human lgG1 CH3 domain with its last lysine deleted.
  • the sequence of this altered CH3 domain is set forth in SEQ I D NO:761 of WO201 1/090762 (said sequence incorporated by reference herein).
  • multispecific heterodimer antibodies may comprise a CHS pair that comprises so called "knobs-into-holes" mutations (see, Marvin and Zhu, Acta Pharmacoiogica Sinica 26:649-58, 2005; Ridgway et al., Protein Engineering 9:617-21 , 1966). More specifically, mutations can be introduced into each of the two CH3 domains of each polypeptide chain so that the steric complementarity required for CH3/CH3 association obligates these two CH3 domains to pair with each other.
  • a CH3 domain in one single chain polypeptide of a polypeptide heterodimer can contain a T366W mutation (a "knob” mutation, which substitutes a small amino acid with a larger one), and a CHS domain in the other single chain polypeptide of the polypeptide heterodimer can contain a Y407A mutation (a "hole” mutation, which substitutes a large amino acid with a smaller one).
  • knobs-into-holes mutations include (1 ) a T366Y mutation in one CHS domain and a Y407T in the other CH3 domain, and (2) a T366W mutation in one CH3 domain and T368S, L388A and Y407V mutations in the other CH3 domain.
  • the antibody in an antibody of the invention in a format comprising a constant region, may optionally comprise a CH4 domain.
  • a CH4 domain can be a wild type immunoglobulin CH4 domain or an altered immunoglobulin CH4 domain thereof from IgE or IgM moiecuies.
  • the CH4 domain is a wild type human
  • a CH4 domain is an altered human immunoglobulin CH4 domain, such as an altered CH4 domain based on or derived from a CH4 domain of human IgE or IgM molecules, which have mutations that increase or decrease an immunological activity known to be associated with an IgE or IgM Fc region.
  • a mu!tispecific anti-CD37 antibody of the present disclosure comprises a combination of CH2, CH3 or CH4 domains (i.e., more than one constant region domain selected from CH2, CHS and CH4).
  • CH2, CH3 or CH4 domains i.e., more than one constant region domain selected from CH2, CHS and CH4.
  • immunoglobulin constant region can comprise CH2 and CH3 domains or CH3 and CH4 domains. In certain other embodiments, the immunoglobulin constant region can comprise two CH3 domains and no CH2 or CH4 domains (i.e., only two or more CH3).
  • the multiple constant region domains that form an immunoglobulin constant region can be based on or derived from the same immunoglobulin molecule, or the same class or subclass
  • the immunoglobulin constant region is an igG CH2CH3 (e.g., lgG1 CH2CH3, igG2 CH2CH3, and lgG4 CH2CH3) and can be a human (e.g., human lgG1 , lgG2, and igG4) CH2CH3.
  • igG CH2CH3 e.g., lgG1 CH2CH3, igG2 CH2CH3, and lgG4 CH2CH3
  • human e.g., human lgG1 , lgG2, and igG4 CH2CH3.
  • the immunoglobulin constant region comprises (1 ) wild type human lgG1 CH2 and CH3 domains, (2) human lgG1 CH2 with N297A substitution (i.e., CH2(N297A)) and wild type human !gG1 CHS, or (3) human !gG1 CH2(N297A) and an altered human lgG1 CH3 with the last lysine deleted.
  • the multiple constant region domains can be based on or derived from different immunoglobulin molecules, or different classes or subclasses immunoglobulin molecules.
  • an immunoglobulin constant region comprises both human IgM CH3 domain and human lgG1 CHS domain.
  • the multiple constant region domains that form an immunoglobulin constant region can be directly linked together or can be linked to each other via one or more (e.g., about 2-10) amino acids.
  • Exemplary immunoglobulin constant regions are set forth In SEQ ID NOS:305-309, 321 , 323, 341 , 342, and 762 of WO201 1/090782 (said sequences incorporated by reference herein).
  • the immunoglobulin constant regions of both chains of a muitispecific antibody are identical to each other.
  • the immunoglobulin constant region of one polypeptide chain of a muitispecific antibody is different from the immunoglobulin constant region of the other polypeptide chain (e.g., an antibody in the muitispecific heterodimer antibody format).
  • one immunoglobulin constant region of a heterodimeric antibody can contain a CHS domain with a "knob" mutation, whereas the other immunoglobulin constant region can contain a CHS domain with a "hole” mutation.
  • the invention also includes nucleic acids (e.g., DNA or RNA) encoding a muitispecific antibody as described herein, or one or more polypeptide chains of a muitispecific antibody described herein.
  • Nucleic acids of the invention include nucleic acids having a region that is substantially identical to a polynucleotide as listed in Table 3, infra.
  • a nucleic acid in accordance with the present invention has at least 80%, typically at least about 90%, and more typically at least about 95% or at least about 98% identity to a polypeptide-encoding polynucleotide as listed in Table 3.
  • Nucleic acids of the invention also include complementary nucleic acids.
  • sequences will be fully complementary (no mismatches) when aligned, in other instances, there can be up to about a 20% mismatch in the sequences.
  • the nucleic acid sequences provided herein can be exploited using codon optimization, degenerate sequence, silent mutations, and other DNA techniques to optimize expression in a particular host, and the present invention encompasses such sequence modifications.
  • Multispecific homodimer EVQLVQSGAEVKKPGESLKISCKGSGYSFTGYNMNWVR antibody CAS1 1 anti-CD37 QMPGKGLEWMGNIDPYYGGTTYNRKFKGQVT!SADKSiS domain derived from G28-1 ; TAYLQWSSLKASDTAMYYCARSVGPFDSWGQGTLVTVS anti-CD3 domain derived from SGGGGSGGGGSGGGGSGGGGSGGGGSEiVLTQSPATL CRIS-7) SLSPGERATLSCRASENVYSYLAWYQQKPGQAPRLLIYF
  • NA encoding anti-CD3 caggtccagctggtggagtctgggggcggagtggtgcagcctgggcggtcact variable heavy chain (derived gaggctgtcctgcaaggcttctggctacacctttactagatctacgatgcactggg from CRIS-7) ta
  • NA encoding anti-CD3 gcacaagacatccagatgacccagtciccaagcagcctgictgcaagcgtggg variable light chain (derived ggacagggtcaccatgacctgcagtgccagctcaagtgtaagttacatgaactg from CRIS-7) gtaccagcagaagccg
  • NA encoding anti-CD3 caggtccagctggtgcagtctggggcigaagtgaagaagcctggggcctcagt variable heavy domain gaaggtgtcctgcaaggcttctggctacacctttactagatctacgatgcactggg (derived from CRIS-7) taaaacaggcccctggacagggtctggaatggatggattggatacattaatcctagca gtgcttatactaattacaatcagaaattcaaggacaaggccacattgactgcag acaaatcctccagiacagcctacaigcaactgagiagcctgaggtctgaggac accgcagictattactgigcacggccccaagtccactatgaitacaacgggittcc ttactggggccaag
  • Antl-CD3 variable heavy qvqlvqsgaevkkpgasvkvsckasgytftrstmhwvkqapgqglewigyin domain (derived from CRIS-7) pssaytnynqkfkdkatltadkssstaymqlsslrsedtavyycarpqvhydyn gfpywgqgtlvtvss
  • NA encoding anti-CD3 caggtccagctggtgcagtctgggggcggagtggtgcagcctgggcggtcact variable heavy domain gaggctgtcctgcaaggcttctggciacacctttactagatctacgatgcactggg (derived from CR!S-7) taaggcaggcccctggaaagggtctggaatggatggattggatacattaatcctagca gtgcitatactaattacaatcagaaattcaaggacaaggccacattgactgcag acaaatccaagaacacagcctacatggagctgagtagcctgaggtctgaggaggtctgagga caccgcagtctattactgtgcacggccccaagtccactatgattacaacgggtttactggggcca
  • Antl-CD3 variable heavy- qvq!vqsgggvvqpgrs!risckasgytftrstmhwvrqapgkglewlgylnpss domain (derived from CRIS-7) aytnynqkfkdkatltadkskntaymelssirsedtavyycarpqvhydyngfp ywgqgtlvtvss
  • NA encoding anti-CD3 caggtccagctggtgcagtctgggggcggagtggtgcagcctgggcggtcact variable heavy domain gaggctgtcctgcaaggcttctggctacacctttactagatctacgatgcactggg (derived from CRIS-7) taaggcaggcccctggaaagggtctggaatggatggattggatacattaatcctagca gtgcttatactaattacaatcagaaattcaaggacaggitcacaaicagcgcag acaaatccaagagcacagccttcctgcagatggacagcctgaggccctgagg acaccggcgtctatttctgtgcacggccccaagtccactatgattacaacgggtttactggggccaa
  • Antl-CD3 variable heavy qvq!vqsgggvvqpgrslrlsckasgytftrstmhwvrqapgkgie igyinpss domain (derived from CRIS-7) ayinynqkfkdrftisadkskstaflqmdsirpedtgvyfcarpqvhydyngfpy wgqgtpvtvss CRIS-7) Gly Arg Ser Leu Arg Leu Ser Cys Lys Ala Ser Giy Tyr Thr
  • Antl-CD3 scFv (derived from QVQLVESGGGWQPGRSLRLSCKASGYTFTRSTMHWV
  • Polynucleotide molecules comprising a desired polynucleotide sequence are propagated by placing the molecule in a vector.
  • Viral and non-viral vectors are used, including plasmids.
  • the choice of p!asmid will depend on the type of cell in which propagation is desired and the purpose of propagation. Certain vectors are useful for amplifying and making large amounts of the desired DNA sequence.
  • Other vectors are suitable for expression in cells in culture.
  • Still other vectors are suitable for transfer and expression in cells in a whole animal or person. The choice of appropriate vector is well within the skill of the art. Many such vectors are available commercially.
  • the partial or full- length polynucleotide is inserted into a vector typically by means of DNA ligase attachment to a cleaved restriction enzyme site in the vector.
  • the desired nucleotide sequence can be inserted by homologous recombination in vivo. Typically this is accomplished by attaching regions of homology to the vector on the flanks of the desired nucleotide sequence. Regions of homology are added by ligation of oligonucleotides, or by polymerase chain reaction using primers comprising both the region of homology and a portion of the desired nucleotide sequence, for example.
  • an expression cassette or system may be employed.
  • a nucleic acid molecule encoding the polypeptide operabiy linked to regulatory sequences that control transcriptional expression in an expression vector, is introduced into a host cell.
  • expression vectors can include translational regulatory sequences and a marker gene which is suitable for selection of ceils that carry the expression vector.
  • the gene product encoded by a polynucleotide of the invention is expressed in any convenient expression system, including, for example, bacterial, yeast, insect, amphibian and mammalian systems.
  • the polypeptide- encoding polynucleotide is linked to a regulatory sequence as appropriate to obtain the desired expression properties.
  • a regulatory sequence can include promoters, enhancers, terminators, operators, repressors, and inducers.
  • the promoters can be regulated (e.g., the promoter from the steroid inducible pIND vector (Invitrogen)) or constitutive (e.g., promoters from CMV, SV40, Elongation Factor, or LTR sequences). These are linked to the desired nucleotide sequence using the techniques described above for linkage to vectors. Any techniques known in the art can be used.
  • the expression vector will generally provide a transcriptional and translational initiation region, which can be inducible or constitutive, where the coding region is operabiy linked under the transcriptional control of the transcriptional initiation region, and a transcriptional and translational termination region.
  • An expression cassette (“expression unit”) can be introduced into a variety of vectors, e.g., p!asmid, BAC, YAC, bacteriophage such as lambda, P1 , M13, etc., plant or animal viral vectors (e.g., retrovirai-based vectors, adenovirus vectors), and the like, where the vectors are normally characterized by the ability to provide selection of cells comprising the expression vectors.
  • the vectors can provide for extrachromosomal maintenance, particularly as piasmids or viruses, or for integration into the host chromosome.
  • an origin sequence is provided for the replication of the p!asmid, which can be low- or high copy-number.
  • markers are available for selection, particularly those which protect against toxins, more particularly against antibiotics.
  • the particular marker that is chosen is selected in accordance with the nature of the host, where in some cases, complementation can be employed with auxotrophic hosts.
  • Introduction of the DNA construct can use any convenient method, including, e.g., conjugation, bacterial transformation, calcium-precipitated DNA,
  • the recombinant mu!tispecific antibodies of the invention can be produced in genetically engineered host ceils according to conventional techniques.
  • Suitable host cells are those ceil types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal ceils, and cultured higher eukaryotic cells (including cultured ceils of multicellular organisms), particularly cultured mammalian ceils. Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are disclosed by Sambrook and Russell,
  • an expression vector will generally include a nucleic acid segment encoding the CD37-binding polypeptide, operably linked to a promoter.
  • a muitispecific heterodirrser antibody comprising different first and second polypeptide chains
  • the first and second polypeptide chains can be co- expressed from separate vectors in the host cell for expression of the entire heterodimeric protein.
  • the first and second polypeptide chains can be co-expressed from separate expression units in the same vector in the host cell for expression of the entire muitispecific heterodimer antibody.
  • the expression vector(s) are transferred to a host cell by conventional techniques, and the transfected cells are then cultured by conventional techniques to produce the encoded polypeptide(s) to produce the corresponding binding domains and/or other domains (e.g., hinge domain, constant region) of the antibodies of the invention.
  • the transfected cells are then cultured by conventional techniques to produce the encoded polypeptide(s) to produce the corresponding binding domains and/or other domains (e.g., hinge domain, constant region) of the antibodies of the invention.
  • a secretory signal sequence (also known as a leader sequence) is provided in the expression vector.
  • the secretory signal sequence can be that of the native form of the recombinant protein, or can be derived from another secreted protein or synthesized cfe novo.
  • the secretory signal sequence is operably linked to the poiypeptide-encoding DNA sequence, i.e., the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host ceil.
  • Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the polypeptide of interest, although certain signal sequences can be positioned elsewhere in the DNA sequence of interest (see, e.g., Welch et al., U.S. Patent No. 5,037,743; Holland et al., U.S. Patent No. 5,143,830).
  • a secretory signal sequence for use in accordance with the present invention has the amino acid sequence
  • MEAPAQLLFLLLLWLPDTTG amino acids 1 -20 of SEQ ID NO:1 and coded for in, e.g., nucleotides 1-60 of SEQ ID NO:45).
  • Cultured mammalian cells are suitable hosts for production of recombinant antibodies and antibody domains for use within the present invention.
  • Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et a/., Cell 14:725, 1978; Corsaro and Pearson, Somatic Celt Genetics 7:603, 1981 : Graham and Van der Eb, Virology 52:456, 1973), electroporation (Neumann et a!., EMBO J.
  • suitable mammalian host cells include African green monkey kidney ceils (Vero; ATCC CRL 1587), human embryonic kidney cells (293-HEK; ATCC CRL 1573), baby hamster kidney cells (BHK-21 , BH -S70; ATCC CRL 8544, ATCC CRL 10314), canine kidney ceils ( DCK; ATCC CCL 34), Chinese hamster ovary cells (CHO-K1 ; ATCC CCL61 ; CHO DG44; CHO DXB1 1 (Hycione, Logan, UT); see a/so, e.g., Chasin et a!., Sorn.
  • African green monkey kidney ceils Vero; ATCC CRL 1587
  • human embryonic kidney cells (293-HEK; ATCC CRL 1573
  • baby hamster kidney cells BHK-21 , BH -S70; ATCC CRL 8544, ATCC CRL 10314
  • canine kidney ceils DCK; ATCC CCL 34
  • Ceil. Molec. Genet. 12:555, 1986) rat pituitary ceils (GH1 ; ATCC CCL82), HeLa S3 cells (ATCC CCL2.2), rat hepatoma ceils (H-4-II-E; ATCC CRL 1548) SV40-transformed monkey kidney cells (COS-1 ; ATCC CRL 1650) and murine embryonic cells (NIH-3T3; ATCC CRL 1658).
  • Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Manassas, Virginia. Strong transcription promoters can be used, such as promoters from SV-40 or cytomegalovirus. See, e.g., U.S. Patent No. 4,956,288.
  • Other suitable promoters include those from metaliothionein genes (U.S. Patents Nos. 4,579,821 and 4,601 ,978) and the adenovirus major late promoter.
  • Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such ceils are commonl referred to as “transfectants.” Ceils that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as “stable transfectants.”
  • Exemplary selectable markers include a gene encoding resistance to the antibiotic neomycin, which allows selection to be carried out in the presence of a neomycin-type drug, such as G-418 or the like; the gpt gene for xanthine-guanine phosphoribosyl transferase, which permits host cell growth in the presence of mycopheno!ic acid/xanthine; and markers that provide resistance to zeocin, bleomycin, blastocidin, and hygromycin (see, e.g., Gatignoi et a!., Mo!.
  • Amplification is carried out by cuituring transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for ceils that produce high levels of the products of the introduced genes.
  • An exemplary amp!ifiab!e selectable marker is dihydrofolate reductase, which confers resistance to methotrexate.
  • Other drug resistance genes e.g., hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
  • drug resistance genes e.g., hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
  • Insect ceils can be infected with recombinant baculovirus, commonly derived from Autographa ca!ifornica nuclear poiyhedrosis virus (AcNPV). See King and Possee, The Baculovirus Expression System: A Laboratory Guide (Chapman & Hall, London); O'Reilly et ai., Baculovirus Expression Vectors: A Laboratory Manual (Oxford University Press., New York 1994); and Baculovirus Expression Protocols. Methods in Molecular Biology
  • Recombinant baculovirus can also be produced through the use of a transposon-based system described by Luckow et al. ⁇ J. Virol. 67:4566-4579, 1993).
  • This system which utilizes transfer vectors, is commercially available in kit form (BAC-TO-BAC kit; Life Technologies, Gaithersburg, D).
  • the transfer vector e.g., PFASTBAC1 ; Life Technologies
  • transfer vectors can include an in-frame fusion with DNA encoding a polypeptide extension or affinity tag as disclosed above.
  • a transfer vector containing a protein-encoding DNA sequence is transformed into E. coil host cells, and the ceils are screened for bacmids which contain an interrupted lacZ gene indicative of recombinant baculovirus.
  • the bacmid DNA containing the recombinant baculovirus genome is isolated, using common techniques, and used to transfect Spodoptera frugiperda ceils, such as Sf9 cells. Recombinant virus that expresses the protein or interest is subsequently produced. Recombinant viral stocks are made by methods commonly used in the art.
  • the recombinant virus is used to infect host cells, typically a ceil line derived from the fall armyworm, Spodoptera frugiperda (e.g., Sf9 or Sf21 cells) or Trichoplusia ni (e.g., HIGH FIVE cells; invitrogen, Carlsbad, CA), See generally Giick and Pasternak, Molecular Biotechnology, Principles & Applications of Recombinant DNA (ASM Press, Washington, D.C., 1994). See also U.S. Patent No. 5,300,435. Serum-free media are used to grow and maintain the ceils. Suitable media formulations are known in the art and can be obtained from commercial suppliers.
  • the cells are grown up from an inoculation density of approximately 2-5 x 10 5 ceils to a density of 1 -2 x 10 s cells, at which time a recombinant viral stock is added at a multiplicity of infection (MOI) of 0.1 to 10, more typically near 3.
  • MOI multiplicity of infection
  • Fungal cells including yeast ceils, can also be used within the present invention.
  • Yeast species of in this regard include, e.g., Saccharomyces cerevisiae, Pichia pastoris, and Pichia meihano!ica.
  • Methods for transforming S. cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Patent No. 4,599,31 1 ; Kawasaki et ai., U.S. Patent No. 4,931 ,373; Brake, U.S. Patent No. 4,870,008; Welch et ai, U.S. Patent No.
  • Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine).
  • An exemplary vector system for use in Saccharomyces cerevisiae is the POT1 vector system disclosed by Kawasaki et ai. (U.S. Patent No. 4,931 ,373), which allows transformed cells to be selected by growth in glucose-containing media.
  • Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Patent No.
  • Pichia methanolica, Pichia guiiierrnondii, and Candida maltose are known in the art. See, e.g., Gleeson et ai., J. Gen. Microbiol. 132:3459-3465, 1986; Gregg, U.S. Patent No. 4,882,279; and Raymond et ai, Yeast 14:1 1-23, 1998.
  • Aspergillus cells can be utilized according to the methods of McKnight et ai., U.S. Patent No. 4,935,349.
  • the protein can be retained in the cytoplasm, typically as insoluble granules, or can be directed to the periplasmic space by a bacterial secretion sequence, in the former case, the cells are lysed, and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea.
  • the denatured protein can then be refolded and dimerized by diluting the denaturant, such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution, in the alternative, the protein can be recovered from the cytoplasm in soluble form and isolated without the use of denaturants.
  • the protein is recovered from the cell as an aqueous extract in, for example, phosphate buffered saline.
  • the extract is applied directly to a chromatographic medium, such as an immobilized antibody or heparin-Sepharose column.
  • Secreted proteins can be recovered from the periplasmic space in a soluble and functional form by disrupting the ceils (by, for example, sonication or osmotic shock) to release the contents of the periplasmic space and recovering the protein, thereby obviating the need for denaturation and refolding.
  • Antibodies, including single-chain antibodies can be produced in bacterial host cells according to known methods.
  • Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host ceils.
  • suitable media including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. Media can also contain such components as growth factors or serum, as required.
  • the growth medium will generally select for cells containing the exogenously added DNA by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker carried on the expression vector or co-transfected into the host cell.
  • Mu!tispecific antibodies can be purified by conventional protein purification methods, typically by a combination of chromatographic techniques. See generally Affinity Chromatography: Principles & Methods (Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988); Scopes, Protein Purification: Principles and Practice (Springer- Verlag, New York 1994). Proteins comprising an immunoglobulin Fc region can be purified by affinity chromatography on immobilized protein A or protein G. Additional purification steps, such as gel filtration, can be used to obtain the desired level of purity or to provide for desalting, buffer exchange, and the like.
  • the present invention provides a method for treating a disorder characterized by overexpression of CD37, too many B-celis and / or the presence of malignant B-ceils.
  • such methods include administering to a patient or a subject in need of such treatment a therapeutically effective amount of a multispecific anti-CD37 antibody as described herein.
  • the multispecific anti-CD37 antibody of the invention comprises a second binding domain that specifically binds a T cell (e.g., to a TCR complex or component thereof, such as GD3e), and the multispecific antibody is capable of inducing redirected T ceil cytotoxicity (RTCC) against CD37-expressing B-celis in the subject.
  • T cell e.g., to a TCR complex or component thereof, such as GD3e
  • the disorder is a B-celi malignancy.
  • a B-ceil malignancy or disorder is one associated with (e.g., causing or resulting from) aberrant B-cell activity.
  • the B-celi malignancy is a B-celi cancer that includes B-ceil lymphomas, such as various forms of Hodgkin's disease, non- Hodgkins lymphoma (NHL) or central nervous system lymphomas, small lymphocytic lymphoma, ieukemias such as prolymphocyte leukemia, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (A L), chronic lymphocytic leukemia (CLL), hairy cell leukemia and chronic myobiastic leukemia and myelomas (such as multiple myeloma).
  • B-ceil lymphomas such as various forms of Hodgkin's disease, non- Hodgkins lymphoma (NHL) or central nervous system lymphomas, small lymphoc
  • B-celi cancers include small lymphocytic lymphoma, B-ceil prolymphocyte leukemia, iymphoplasmacytic lymphoma (including Waldenstrom's macrogiobulinemia), marginal zone lymphomas (including splenic marginal zone lymphoma and nodal marginal zone B-cell lymphoma), plasma cell myeloma/plasmacytoma, solitary plasmacytoma of bone, extraosseous plasmacytoma, nodal marginal zone lymphoma, extra-nodal marginal zone B-ceil lymphoma of mucosa-associated (MALT) lymphoid tissue), follicular lymphoma, mantle ceil lymphoma (MCL), diffuse large B-cell lymphoma, transforming large B-celi lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, Burkitt's !ymphoma
  • the invention includes methods for treating a patient with a relapsed or refractory B-ceil malignancy comprising administering a multispecific anti-CD37 antibody (e.g., a multispecific homodimer antibody, multispecific heterodimer antibody, bispecific single chain antibody, disu!fide-siabi!ized diabody antibody and dual variable domain antibody formats) to a patient in need thereof.
  • a multispecific anti-CD37 antibody e.g., a multispecific homodimer antibody, multispecific heterodimer antibody, bispecific single chain antibody, disu!fide-siabi!ized diabody antibody and dual variable domain antibody formats
  • the invention includes methods of treating a patient with a relapsed or refractory B-ceil malignancy comprising administering a composition comprising a multispecific anti-CD37 antibody to a patient in need thereof.
  • methods of the invention include treating a patient with relapsed or refractory CLL.
  • the methods aiso include treating a patient with relapsed or refractory NHL,
  • a patient with a relapsed or refractory B-celi malignancy is refractory to fludarabine treatment
  • a patient with a relapsed or refractory B- celi malignancy is non-responsive to rituximab treatment.
  • the invention includes patients with a relapsed or refractory B-cell malignancy with one or more genetic markers indicative of a poor prognosis such a TP53 mutation or 17p deletion.
  • the B-ceil malignancy or condition is a disorder characterized by autoantibody production (e.g., autoimmune diseases).
  • autoimmune diseases e.g., autoimmune diseases
  • the B-ceil malignancy or condition is an autoimmune disease such as arthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis,
  • a multispecific anti-CD37 antibody is delivered in a manner consistent with conventional methodologies associated with management of the disease or disorder for which treatment is sought, in accordance with the disclosure herein, an effective amount of a multispecific anti-CD37 antibody (or a pharmaceutical composition comprising a multispecific anti-CD37 antibody) is administered to a subject in need of such treatment for a time and under conditions sufficient to prevent or treat the disease or disorder.
  • Subjects for administration of a multispecific anti-CD37 antibody as described herein include patients at high risk for developing a particular disorder characterized by CD37 overexpression, elevated levels of B-cells and/or malignant B-ce!ls as well as patients presenting with an existing such disorder.
  • the subject has been diagnosed as having the disorder for which treatment is sought. Further, subjects can be monitored during the course of treatment for any change in the disorder (e.g., for an increase or decrease in clinical symptoms of the disorder).
  • compositions or medicaments are administered to a patient susceptible to, or otherwise at risk of, a particular disorder in an amount sufficient to eliminate or reduce the risk or delay the onset of the disorder (including, in the instance of B-ceil malignancies, disease relapse), in therapeutic applications, compositions or medicaments are administered to a patient suspected of, or already suffering from such a disorder in an amount sufficient to cure, or at least partially arrest, the symptoms of the disorder and its complications.
  • An amount adequate to accomplish this is referred to as a therapeutically effective dose or amount.
  • agents are usually administered in several dosages until a sufficient response (e.g., reduction of malignant B-celis, reduction in tumor volume or number) has been achieved. Typically, the response is monitored and repeated dosages are given if the desired response starts to fade.
  • accepted screening methods can be employed to determine risk factors associated with specific disorders or to determine the status of an existing disorder identified in a subject. Such methods can include, for example, determining whether an individual has relatives who have been diagnosed with a particular disorder. Screening methods can also include, for example, conventional work-ups to determine familial status for a particular disorder known to have a heritable component. For example, various cancers are also known to have certain inheritable components.
  • Inheritable components of cancers include, for example, mutations in multiple genes that are transforming (e.g., Ras, Raf, EGFR, cMet, and others), the presence or absence of certain HLA and killer inhibitory receptor (KIR) molecules, or mechanisms by which cancer cells are able to modulate immune suppression of cells like NK ceils and T cells, either directly or indirectly (see, e.g., Ljunggren and Malmberg, Nature Rev. Immunol. 7:329-339, 2007; Boyton and Altmann, C!in. Exp. Immunol.
  • transforming e.g., Ras, Raf, EGFR, cMet, and others
  • KIR HLA and killer inhibitory receptor
  • patients likely to relapse or develop a particularly aggressive form of a B-ceii disease are identified, for instance, by the presence of a TP53 mutation or 17p3 deletion.
  • nucleotide probes can be routinely employed to identify individuals carrying genetic markers associated with a particular disorder of interest.
  • immunological methods are known in the art that are useful to identify markers for specific disorder. For example, various ELISA immunoassay methods are available and well-known in the art that employ monoclonal antibody probes to detect antigens associated with specific tumors. Screening can be implemented as indicated by known patient symptomology, age factors, related risk factors, etc. These methods allow the clinician to routinely select patients in need of the methods described herein for treatment, in accordance with these methods, targeting pathological, B-celis can be implemented as an independent treatment program or as a follow-up, adjunct, or coordinate treatment regimen to other treatments.
  • a multispecific anti-CD37 antibody (e.g., a multispecific homodimer antibody, multispecific heterodimer antibody, bispecific single chain antibody, disulfide-stabilized diabody antibody and dual variable domain antibody formats) is formulated as a pharmaceutical composition.
  • a pharmaceutical composition comprising a multispecific anti-CD37 antibody can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the therapeutic molecule is combined in a mixture with a pharmaceutically acceptable carrier.
  • a composition is said to be a
  • compositions can further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent protein loss on vial surfaces, etc.
  • a pharmaceutical composition comprising a multispecific anti-CD37 antibody is administered to a subject in a therapeutically effective amount.
  • a multispecific anti-CD37 antibody can be administered to subjects by a variety of administration modes, including, for example, by intramuscular, subcutaneous, intravenous, antra-atrial, intra-articular, parenteral, intranasal, intrapulmonary, transdermal, intrapleural, intrathecal, and oral routes of administration.
  • an antagonist can be administered to a subject in a single bolus delivery, via continuous delivery (e.g., continuous transdermal delivery) over an extended time period, or in a repeated administration protocol (e.g., on an hourly, daily, or weekly basis).
  • a therapeutically effective amount is determined in part by the format of the recombinant, multispecific antibody (e.g., a multispecific homodimer antibody, multispecific heterodimer antibody, bispecific single chain antibody, disulfide-stabilized diabody antibody and dual variable domain antibody).
  • an antibody format that lacks a constant region e.g., a bispecific single chain antibody
  • a "therapeutically effective amount" of a composition is that amount that produces a statistically significant effect in amelioration of one or more symptoms of the disorder, such as a statisticaily significant reduction in disease progression or a statistically significant improvement in organ function.
  • the exact dose will be determined by the clinician according to accepted standards, taking into account the nature and severity of the condition to be treated, patient traits, etc. Determination of dose is within the level of ordinary skill in the art.
  • Effective dosages of the compositions of the present invention vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, whether treatment is prophylactic or therapeutic, as well as the specific activity of the composition itself and its ability to elicit the desired response in the individual.
  • the patient is a human, but in some diseases, the patient can be a nonhuman mammal.
  • dosage regimens are adjusted to provide an optimum therapeutic response, i.e.., to optimize safety and efficacy.
  • a therapeutically effective amount is also one in which any undesired collateral effects are outweighed by the beneficial effects of administering a multispecific anti-CD37 antibody as described herein.
  • a dosage typically ranges from about 0.1 g to 100 mg/kg or 1 pg/kg to about 50 mg/kg, and more usually 10 pg to 5 mg/kg of the subject's body weight.
  • an effective amount of the agent is between about 1 pg/kg and about 20 mg/kg, between about 10 pg/kg and about 10 mg/kg, or between about 0.1 mg/kg and about 5 mg/kg.
  • Dosages within this range can be achieved by single or multiple administrations, including, e.g., multiple administrations per day or daily, weekly, bi-weekly, or monthly administrations.
  • a regimen consists of an initial administration followed by multiple, subsequent administrations at weekly or bi-weekly intervals.
  • Another regimen consists of an initial administration followed by multiple, subsequent administrations at monthly or bi-monthly intervals.
  • administrations can be on an irregular basis as indicated by monitoring clinical symptoms of the disorder.
  • Dosage of the pharmaceutical composition can be varied by the attending clinician to maintain a desired concentration at a target site.
  • local concentration of the agent in the bloodstream at the target tissue can be between about 1-50 nanomoies of the composition per liter, sometimes between about 1 .0 nanomoie per liter and 10, 15, or 25 nanomoies per liter depending on the subject's status and projected measured response.
  • Higher or lower concentrations can be selected based on the mode of delivery, e.g., trans-epidermal delivery versus delivery to a mucosal surface.
  • Dosage should also be adjusted based on the release rate of the administered formulation, e.g., nasal spray versus powder, sustained release oral or injected particles, transdermal formulations, etc.
  • the release rate of the administered formulation e.g., nasal spray versus powder, sustained release oral or injected particles, transdermal formulations, etc.
  • slow-release particles with a release rate of 5 nanomolar would be administered at about twice the dosage of particles with a release rate of 10 nanomolar.
  • compositions as described herein can also be used in the context of combination therapy.
  • combination therapy is used herein to denote that a subject is administered at least one therapeutically effective dose of a muitispecific anti- CD37 antibody and another therapeutic agent.
  • a muitispecific anti ⁇ CD37 antibody of the present invention e.g., a muitispecific hornodimer antibody, muitispecific heterodimer antibody, bispecific single chain antibody, disulfide-stabiiized diabody antibody and dual variable domain antibody formats
  • a muitispecific anti-CD37 antibody as described herein can work in synergy with conventional types of chemotherapy or radiation.
  • a muitispecific anti- CD37 antibody of the invention can further reduce tumor burden and allow more efficient killing by a chemotherapeutic.
  • a composition comprising a muitispecific anti- CD37 antibody (e.g., a muitispecific hornodimer antibody, muitispecific heterodimer antibody, bispecific single chain antibody, disulfide-stabiiized diabody antibody and dual variable domain antibody formats) is administered to a patient in combination with a purine analog (e.g., fiudarabine), an anti-CD20 antibody (e.g., rituximab), a PI3K inhibitor or a BTK inhibitor (e.g., ibrutinib) for treatment of a B-ceil malignancy.
  • a purine analog e.g., fiudarabine
  • an anti-CD20 antibody e.g., rituximab
  • a PI3K inhibitor or a BTK inhibitor e.g., ibrutinib
  • compositions of the present invention can also be used in combination with immunomodulatory compounds including various cytokines and co-stimulatory/inhibitory molecules. These can include, but are not limited to, the use of cytokines that stimulate anticancer immune responses (e.g., IL-2, IL-12, or IL-21 ).
  • cytokines that stimulate anticancer immune responses e.g., IL-2, IL-12, or IL-21
  • muitispecific anti-CD37 antibodies can be combined with reagents that co-stimulate various cell surface molecules found on immune-based effector cells, such as the activation of CD137 (see Wilcox ef a/., J. Clin, invest. 109:651 -9, 2002) or inhibition of CTLA4 (see Chambers et a!., Ann. Rev.
  • muitispecific anti-CD37 antibodies of the invention could be used with reagents that induce tumor cell apoptosis by interacting with TNF superfamiiy receptors (e.g., TRAIL-reiated receptors, DR4, DR5, Fas, or CD37).
  • TNF superfamiiy receptors e.g., TRAIL-reiated receptors, DR4, DR5, Fas, or CD37.
  • Such reagents include iigands of TNF superfamiiy receptors, including ligand-lg fusions, and antibodies specific for TNF superfamiiy receptors [e.g., TRAIL ligand, TRAIL ligand-lg fusions, anti-TRAIL antibodies, and the like).
  • Non-measurable disease means the disease comprises of lesions ⁇ 20mm with conventional techniques or ⁇ 10mm with spiral CT scan, and truly non-measurable lesions (too small to accurately measure).
  • Non-measureable disease includes pleural effusions, ascites, and disease documented by indirect evidence.
  • the criteria for objective status are required for protocols to assess solid tumor response. Representative criteria include the following: (1 ) Complete Response (CR), defined as complete disappearance of all measurable disease; no new lesions; no disease related symptoms; no evidence of non-measurable disease; (2) Partial Response (PR) defined as 30% decrease in the sum of the longest diameter of target lesions (3) Progressive Disease (PD), defined as 20% increase in the sum of the longest diameter of target lesions or appearance of any new lesion; (4) Stable or No Response, defined as not qualifying for CR, PR, or Progressive Disease, (See Therasse et a!., supra,)
  • Additional endpoints that are accepted within the oncology art include overall survival (OS), disease-free survival (DFS), objective response rate (ORR), time to OS
  • OS overall survival
  • DFS disease-free survival
  • ORR objective response rate
  • TTP progression
  • PFS progression-free survival
  • compositions comprising a multispecific anti-CD37 antibody of the invention can be supplied as a kit comprising a container and / or written information on indications and usage (e.g., label).
  • a pharmaceutical composition can be provided, for example, in the form of an injectable solution for single or multiple doses, or as a sterile powder that will be reconstituted before injection, in one embodiment, a kit can include instructions and materials for administering a multi-specific anti-CD37 antibody by infusion. Such a kit can further comprise written information on indications and usage of the pharmaceutical composition.
  • EXAMPLE 1 Construction of anti-CD37 x anti-CD3 multispecific homodirner antibody molecules
  • SEQ ID NO:1 As a starting sequence (from amino to carboxyl terminus, a humanized scFv derived from anti-CD37 antibody G28-1 , a modified immunoglobulin igG1 hinge and wild-type lgG1 CH2 and CH3 regions). Wild-type IgGi hinge contains three cysteine residues.
  • the modified !gG1 hinge of SEQ ID NQ:1 (with cysteine to serine mutations at first two cysteines) was changed to a modified lgG1 hinge with a cysteine to serine mutation at the first cysteine residue only (i.e., SCC).
  • the wild-type lgG1 Fc sequence of SEQ ID NO: 1 was modified to either igG1 null2 Fc (SEQ ID NO: 102) or lgG4 N297A ADCC- Fc (SEQ ID IMG: 104) and a C-terminus linker ("H75" derived from NKG2A; SEQ ID NG:132) was added at the C-terminus of each Fc.
  • These two starting sequences are named anti-CD37lgG1 nuil2 H75 and anti-CD37igG4 N297A ADCC- H75, respectively.
  • These two molecules also contained the signal peptide with Hindill restriction site at the N-terminus and EcoRI restriction site at the C-terminus of the H75 linker.
  • a total of 6 fragments were generated: 3 for anti-CD37 lgG1 nuN2 fragments with 3 different linkers and another 3 for anti-CD37 lgG4 N297A ADCC- fragments containing the 3 different linkers.
  • These 6 PGR generated fragments were then digested with Hindi II and EcoRI restriction enzymes and ligated into the PD28 vector along with a humanized anti-CD3 scFv (SEQ ID NO:89) that had been previously digested with EcoR! and Notl in the 3 way ligation reactions.
  • SEQ ID NO:89 humanized anti-CD3 scFv
  • EXAMPLE 2 Binding of multispecific anti-CD37 antibodies to CD37(+ . CD37 . CD3(+V and/or CD3(-) cell lines
  • binding studies could be performed on human cancer cell lines expressing CD37 (such as RajL Ramos, Daudi, DoHH2, SU-DHL-6, Rec-1 and Mino), expressing CDS (such as Jurkat and TALL-1 Q4), or negative for CD37 or CDS expression and binding determined by standard FACS-based staining procedures, A typical experiment blocks 200,000-500,000 cells per well in a 96-wel! plate with human serum or unlabeled human IgG prior to labeling cells with various concentrations of binding molecule on ice.
  • CD37 such as RajL Ramos, Daudi, DoHH2, SU-DHL-6, Rec-1 and Mino
  • CDS such as Jurkat and TALL-1 Q4
  • a typical experiment blocks 200,000-500,000 cells per well in a 96-wel! plate with human serum or unlabeled human IgG prior to labeling cells with various concentrations of binding molecule on ice.
  • Bound molecule is detected with a labeled (e.g., fluorescent), biotinyiated, or unlabeled secondary antibody directed against either the Fc region or one idiotype of the binding molecule. If the secondary antibody is not labeled, a labeled (e.g., fluorescent) streptavidin molecule or tertiary antibody is employed. Cells may also be labeled with a viability dye to exclude unhealthy ceils from the analysis. Signal from bound molecules is detected on a flow cytometer. Signal above the background derived from secondary/tertiary molecules alone indicates binding of molecules to the cell surface. Specificity of binding is demonstrated through the use of negative control binding molecules, cell lines negative for CD37 or CDS expression, or by preventing specific binding with an excess of a competitive binder targeting CD37 or CDS.
  • a labeled e.g., fluorescent
  • Ramos cells a CD37(+) / CD3( ⁇ ) American Burkitt's lymphoma human cell line
  • Jurkat ceils a CD37(-) / CD3(+) acute T-ce!l leukemia human cell line
  • Both Ramos and Jurkat ceils were from the ATCC (Manassas, VA).
  • C4-2 cells a sub-line of the LNCaP human prostate cancer ceil line (negative for both CD37 and CD3 expression) was included as a negative binding control to confirm specificity of binding interactions.
  • C4-2 cells were from The University of Texas MD Anderson Cancer Center (Houston, TX). Binding was assessed by standard flow cytometry staining procedures.
  • RTCC T-cell cytotoxicity
  • PBMC Peripheral blood mononuclear cells
  • T ceils were used without stimulation and added at a 5:1 ratio (T cells : target ceils).
  • CD37-expressing Ramos and CD37-negative C4-2 target ceils were loaded in culture medium with sodium chromate in saline at 0.05 mCi/million ceils. Ceils were washed and plated at 10,000 51 Cr-loaded target cells per well into 96-weli U-bottom untreated polystyrene assay plates together with T ceils and molecules in a final volume of 200 ⁇ RPMI 1640 growth medium supplemented with 10% heat-inactivated fetal bovine serum, 20 mM HEPES, 1X non-essential amino acids, 1 mM sodium pyruvate, and 55 ⁇ 2- mercaptoethano!. Cell cultures were incubated in a C0 2 incubator at 37°C for approximately 20 hours.
  • Ail anti-CD37 x anti-CD3 bispecific molecules tested were capable of specifically redirecting T-ceil cytotoxicity (Figure 3). Results are representative of two independent experiments. The molecules mediated CD37-positive target ceil lysis above background in the presence of T ceils and induced only very low cytotoxicity at high concentrations of molecule on CD37 ⁇ negative target cells.
  • EXAMPLE 4 Target-dependent T cell activation and proliferation induced agasnst CD37( ⁇ ) cell lines directed by muitispecific anti-CD37 antibodies
  • anti-CD37 x anti-CD3 bispecific molecules were assessed in four to five day assays.
  • PBMC Peripheral blood mononuclear cells
  • T cells were isolated from human blood obtained from healthy donors using standard density gradient centrifugation. T cells were purified from isolated PBMC with the Pan T-cei! Isolation Kit II (Miltenyi Biotec). T cells were labeled using the CELLTRACETM CFSE Cell Proliferation Kit (Molecular Probes, Eugene, OR), an amine-reactive intracellular labeling kit used to assess proliferation by loss of fluorescence in cells that have undergone division. CFSE-labeled T cells were added to the assays without prior stimulation.
  • Target cells included both CD37-expressing Ramos cells and CD37-negative C4- 2B human prostate cancer cells (The University of Texas MD Anderson Cancer Center). Target ceils were rendered non-pro!iferative by x-ray irradiation.
  • CFSE-labeled T cells were plated in tissue culture treated U-bottom 96-weil plates at 100,000 cells/well with 30,000 target tumor cells/well, to achieve an effector to target cell ratio of roughly 3:1.
  • test molecules were added to the ceil mixtures in a total volume of 200 ⁇ /well in RPMI 1640 growth media supplemented with 10% human serum, 1 X sodium pyruvate, 1 X non-essential amino acids, 1X L-glutamine, and 20 mM HEPES. Assay plates were incubated in humidified C0 2 incubators at 37°C. [0261] After four or five days, cells were labeled with antibodies for analysis by flow cytometry. Ceils were labeled and washed in their original plates to minimize ceil loss, and ail labeling was performed in DPBS with 0.2% bovine serum albumin and 2 m EDTA. First, cells were blocked with 100 ug/ml human IgG on ice for 20 min. and washed once.
  • Ail anti-CD37 x anti-CD3 bsspecific molecules tested were capable of supporting both CD4+ and CD8+ T-ceil proliferation in the presence of CD37-positive target ceils (Figure 4).
  • a subset of anti-CD37 x anti-CD3 bispecific molecules tested with T cells alone and with both CD37-positive (Ramos) and CD37 ⁇ negative (C4-2B) target cells demonstrated that non-specific T-ceil proliferation in the absence of target cells or on CD37-negative target cells was negligible (Figure 5).
  • the cell division data provided in Figures 4 and 5 was measured independently from CD25 expression. Up-reguiation of cell surface CD25 is associated with T-cell activation and was observed on divided T cells (data not shown).
  • EXAMPLE 5 Inhibition of tumor growth in vivo using muitispecific anti-CD37 antibodies
  • the molecules are evaluated in one or more mouse models of tumor growth.
  • Prophy!actic treatment, or prevention of tumor engraftment of subcutaneous tumors Cultured, CD37-expressing tumor cell lines (such as Raji, Ramos, Daudi, DoHH2, SU-DHL-6, Rec-1 and Mino) are mixed with human lymphocytes (either PBMC or purified T cells) and injected subcutaneously into immunodeficient mice (such as SCiD, NOD/SCI D, etc.).
  • a muitispecific anti-CD37 antibody is injected intravenously (i.v.) or intraperitonealiy (i.p.) on the day of tumor implantation and on several subsequent days. Dose-dependent inhibition of tumor outgrowth, as assessed by tumor volume, indicates that the respective molecule has efficacy against CD37-expressing tumors in vivo.
  • CD37-expressing tumor ceil lines are injected subcutaneously into immunodeficient mice.
  • human lymphocytes PBMC or purified T cells
  • a multispecific anti-CD37 antibody is injected, by the same or separate routes of administration (i.v. or i.p.).
  • the test molecule is also administered on several subsequent days.
  • CD37-expressing tumor cell lines are injected subcutaneousiy into immunodeficient mice. Tumor growth is monitored, and the study is initiated when tumors show signs of established growth (typically a volume of -200 mm 3 ), On the day of study initiation, human lymphocytes (PB C or purified T cells) and a multispecific anti-CD37 antibody is injected, by the same or separate routes of administration (i.v. or i.p.). The test molecule is also dosed on several subsequent days, and additional administration of human lymphocytes may be provided. Dose-dependent inhibition of tumor growth, as assessed by tumor volume, indicates that the respective molecule has efficacy against CD37-expressing tumors in vivo.
  • Prophy!actic treatment, or prevention of tumor engraftment of disseminated tumors Cultured, CD37-expressing tumor cell lines (such as Raji, Ramos, Daudi, DoHH2, SU-DHL-6, Rec-1 and Mino) are injected i.v. into immunodeficient mice (such as SCID, NOD/SCI D, etc.). On the same day, human lymphocytes (PBMC or purified T cells) and a multispecific anti-CD37antibody is injected, by the same or separate routes of administration (i.v. or i.p.). The test molecule is also administered on several subsequent days. Dose- dependent inhibition of tumor growth, as assessed by serum biomarkers, radiography, fluorescent imaging, weight loss, and other proxy measurements of tumor volume, indicates that the respective molecule has efficacy against CD37-expressing tumors in vivo.
  • CD37-expressing tumor cell lines such as Raji, Ramos, Daudi, DoHH2, SU-DHL-6, Rec-1 and Mino
  • CD37-expressing tumor ceil lines such as Raji, Ramos, Daudi, DoHH2, SU-DHL-6, Rec-1 and Mino
  • immunodeficient mice such as SCID, NOD/SCI D, etc.
  • the study is initiated when tumor burden is established, but before animals begin to reach tumor-associated endpoints. Tumor burden is monitored by serum biomarkers, radiography, fluorescent imaging, weight loss, and other proxy measurements.
  • human lymphocytes PBMC or purified T ceils
  • a multispecific anti-CD37 antibody is injected, by the same or separate routes of administration (i.v.
  • test molecule is also dosed on several subsequent days, and additional administration of human lymphocytes may be provided.
  • Dose-dependent inhibition of tumor growth as assessed by serum biomarkers, radiography, fluorescent imaging, weight loss, and other proxy measurements of tumor volume, indicates that the respective molecule has efficacy against CD37-expressing tumors in vivo,
  • EXAMPLE 8 Minimal cytokine release following redirected T cell cytotoxicity with in vitro
  • PBMC peripheral blood mononuclear cells
  • T ceils are purified from isolated PBMC using the Pan T ceil Isolation Kit ii from Mi!tenyi Biotec (Bergisch Gladbach, Germany) and following the manufacturer's protocol. Whole PBMC or purified T ceils are plated in U-bottom 96-we!l plates at 200,000 to 400,000 cells/well with 60,000 to 120,000 CD37-expressing tumor cell lines (such as Raji, Ramos, Daudi, DoHH2, SU-DHL-6, Rec-1 and Mino), to achieve approximate T cell to tumor ratios of 3:1.
  • tumor cell lines such as Raji, Ramos, Daudi, DoHH2, SU-DHL-6, Rec-1 and Mino
  • Cells are mixed in in a total volume of 200 ⁇ /well in standard growth media supplemented with 10% human or bovine serum, sodium pyruvate, non-essential amino acids, L-giutamine, and HEPES. Assay plates are incubated in humidified C0 2 incubators at 37°C, for periods of time ranging from 4 to 48 hours. Supernatants are collected and frozen or tested immediately using a multiplex assay system (Milliplex cytokine kits from Miilipore, USA) to measure cytokines predicted to be released by activated T ceils and myeloid cells.
  • a multiplex assay system Milliplex cytokine kits from Miilipore, USA
  • the cytokines in the analysis include but are not limited to: IL-2, IL-4, IL-5, IL-6, !L-8, !L-10, IL-12, IL-17, TNFa and IFNy. Twenty-five ⁇ volumes from each well are tested in duplicate samples and run following the manufacturer's instructions. The molecule is evaluated for the levels and types of cytokines released compared to control bispecific molecules evaluated with target cells and T ceils or PBMC and control, untreated T cells or PBMC alone.
  • cytokine levels may be low compared to other control multispecific moiecuies in other formats (such as bispecific single chain antibody) that induce similar levels of T cell activation. Presence of normal B cells in cultures initiated with whole PBMC leads to some level of T cell activation in the presence of target, and therefore cytokine secretion.
  • EXAMPLE 7 Tolerable, dose-dependent cytokine release following dosing of Non-Human Primates (NHP) with high doses of muitispecific anti-CD37 antibody
  • the ability of the muitispecific anti-CD37 antibody to trigger RTCC function by macaque T ceils is determined on a mixture of whole NHP PBMC in the presence or absence of human CD37 + targets ceils, as described above.
  • NHP T cells do not need to be purified from whole PBMC because the anti-CD37 binding domain does not cross-react with macaque CD37.
  • Bispecific antibodies and other muitispecific antibodies are capable of specifically redirecting T cell cytotoxicity by the tested species if they mediate CD37-positive target cell lysis above background in the presence of T cells but not on CD37-negative target cells or in the absence of T cells.
  • a comparison of the concentration of a particular muitispecific anti-CD37 antibody required to elicit half-maximum lysis by human and NHP T cells allows calculation of a multiplier to correct for functional equivalence between NHP and human RTCC function for the tested molecule.
  • the molecules are tested in a mixture of PBMC in the presence or absence of human CD37 + targets cells.
  • Supernatants are collected at time points ranging from 4 to 48 hrs., and frozen or tested immediately using a multiplex assay system (e.g., Miliiplex cytokine kits from Millipore, USA) to measure cytokines predicted to be released by activated T ceils and myeloid ceils.
  • the cytokine release assays are performed as described above. The levels of cytokines induced by the bispecific molecule are low compared with those induced by a positive control.
  • a range of doses is selected based on results from the in vitro studies.
  • the maximum dose is selected based on the expected human therapeutic dose, the multiplier of functional equivalence between human and NHP, and a minimum 10-fold excess safety margin.
  • the selected doses of a muitispecific anti-CD37 antibody are injected intravenously into groups of NHP.
  • a vehicle control group is included to control for changes induced by formulation and animal handling. Animals are monitored for at least 2 hours following dosing for signs of cytokine release. Blood samples are drawn before dosing and periodically following dosing to obtain serum for assessment of circulating cytokines.
  • cytokines in serum are determined using a multiplex assay system (e.g., Milliplex cytokine kits from Millipore, USA) as described above.
  • the levels of cytokines induced by the multispecific antibody are drug-dependent, dose- dependent, and tolerable.

Abstract

Cette invention concerne des anticorps multispécifiques recombinés qui se lient spécifiquement à CD3 et à CD37. Dans un mode de réalisation, les anticorps multispécifiques induisent une cytotoxicité, une activation et une prolifération des lymphocytes T dépendantes de la cible. L'invention concerne également des méthodes de traitement d'un patient atteint d'une tumeur maligne ou d'un trouble des lymphocytes B, qui consiste à administrer au patient des compositions comprenant des anticorps qui se lient spécifiquement à CD3 et CD37.
PCT/US2014/025729 2013-03-15 2014-03-13 Anticorps anti-cd37 multispécifiques, compositions et méthodes s'y rapportant WO2014151438A1 (fr)

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US11352426B2 (en) 2015-09-21 2022-06-07 Aptevo Research And Development Llc CD3 binding polypeptides
WO2023219120A1 (fr) * 2022-05-12 2023-11-16 アステラス製薬株式会社 Anticorps bispécifique anti-ｃｄ３７ et anti-ｃｄ３

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Publication number Priority date Publication date Assignee Title
US11352426B2 (en) 2015-09-21 2022-06-07 Aptevo Research And Development Llc CD3 binding polypeptides
WO2021006199A1 (fr) 2019-07-05 2021-01-14 小野薬品工業株式会社 Traitement du cancer hématologique avec une protéine à double spécificité pd-1/cd3
WO2023219120A1 (fr) * 2022-05-12 2023-11-16 アステラス製薬株式会社 Anticorps bispécifique anti-ｃｄ３７ et anti-ｃｄ３

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