NZ762170B2 - Novel anti-cd3epsilon antibodies - Google Patents

Novel anti-cd3epsilon antibodies Download PDF

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Publication number
NZ762170B2
NZ762170B2 NZ762170A NZ76217018A NZ762170B2 NZ 762170 B2 NZ762170 B2 NZ 762170B2 NZ 762170 A NZ762170 A NZ 762170A NZ 76217018 A NZ76217018 A NZ 76217018A NZ 762170 B2 NZ762170 B2 NZ 762170B2
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New Zealand
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seq
antibody
antigen
binding
cd3epsilon
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NZ762170A
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NZ762170A (en
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Jing Li
Qin Mei
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WuXi Biologics Ireland Limited
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Priority claimed from PCT/CN2018/106618 external-priority patent/WO2019057099A1/en
Publication of NZ762170A publication Critical patent/NZ762170A/en
Publication of NZ762170B2 publication Critical patent/NZ762170B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/7051—T-cell receptor (TcR)-CD3 complex
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors

Abstract

Provided are isolated monoclonal anti-CD3epsilon antibodies or antigen-binding fragments thereof comprising heavy and light chain CDR sequences selected from the group consisting of SEQ ID NOs 1-6, 7-12, 13-18, 19-24, 25-30, 31-36, 27-42, and 43-48. The antibodies may also be humanised or chimeric. Further provided are isolated polynucleotides encoding the same, pharmaceutical compositions comprising the same, and the use thereof.

Description

NOVEL ANTI-CD3EPSILON ANTIBODIES FIELD OF THE INVENTION The present disclosure generally relates to novel anti—human CD3 epsilon antibodies.
BACKGROUND The CD3 (cluster of differentiation 3) T-cell co-receptor is a n complex and is composed of four distinct chains, a CD3gamma chain, a CD3 delta chain, and two CD3epsilon chains. These chains associate with a molecule known as the T-cell or (TCR) and the zeta-chain to generate activation signal in T lymphocytes. The TCR, zeta-chain, and CD3 molecules together comprise the TCR complex, in which TCR as a subunit recognizes and binds to antigen, and CD3 as a subunit ers and conveys the n—stimulation to signaling pathway, and ultimately regulates T-cell activity. The CD3 protein is virtually present in all T cells.
The CD3 together with TCR forms a CD3-TCR complex, which plays pivotal role in ting T cell vast functions in both innate and adoptive immune response, as well as cellular and humoral immune ons. These include eliminating pathogenic organisms and controlling tumor growth by broad range of cytotoxic effects.
Mouse monoclonal antibodies speci?c for human CD3, such as OKT3 (Kung et al. (1979) Science 206: 347-9), were the ?rst generation CD3 antibodies for treatment. Although OKT3 has strong immunosuppressive potency, its clinical use was hampered by serious side effects linked to its immunogenic and nic potentials (Chatenoud (2003) Nature Reviews 3:123-132). OKT3 induced an anti-globulin response, promoting its own rapid clearance and neutralization (Chatenoud et al. (1982) Eur. J. Immunol. 0-8). In addition, OKT3 induced T-cell proliferation and cytokine production in vitro, and led to a large scale release of cytokine in vivo (Hirsch et al. (1989) J. Immunol 142: 737-43, 1989). The cytokine release (also ed to as "cytokine storm") in turn led to a "?u—like" syndrome, terized by fever, , headaches, nausea, vomiting, diarrhea, respiratory distress, septic meningitis and hypotension (Chatenoud, 2003). Such serious side effects limited the more widespread use of OKT3 in transplantation as well as the extension of its use to other al fields such as autoimmunity (Id).
There is a signif1cant need for novel anti-CD3 antibodies.
BRIEF SUMMARY OF THE INVENTION Throughout the present disclosure, the articles CL 77 (L a an," and "the" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.
By way of example, "an antibody" means one antibody or more than one antibody.
The present disclosure provides novel monoclonal anti-CD3epsilon dies, amino acid and nucleotide sequences thereof, and uses thereof.
In one aspect, the present disclosure provides isolated dies or antigen-binding fragments thereof, comprising 1, 2, or 3 heavy chain CDR sequences ed from the group consisting of: SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, and 47, and/or 1, 2, or 3 kappa light chain CDR sequences selected from the group consisting of: SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,44, 46 and 48.
In certain ments, the antibodies or antigen-binding fragments thereof comprise l, 2, or 3 heavy chain CDR sequences having at least 80% (e.g. at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to the sequence of SEQIDNOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, or 47. In certain embodiments, the antibodies or antigen—binding fragments thereof comprise l, 2, or 3 light chain CDR sequences having at least 80% (e. g. at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to the sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 or 48.
In certain embodiments, the antibodies or n-binding fragments thereof comprise a heavy chain variable region selected from the group ting of: a) a heavy chain variable region sing 1, 2, or 3 CDR sequences selected from SEQ ID NO: 1, SEQ ID NO: 3, and SEQ IDNO: 5; b) a heavy chain le region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 7, SEQ ID NO: 9, and SEQ IDNO: 11; c) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:13, SEQ ID N015, and SEQ ID NO: 17; d) a heavy chain variable region sing 1, 2, or 3 CDR sequences selected from SEQ ID NO: 19, SEQ ID NO: 21, and SEQ ID NO: 23; e) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 25, SEQ ID NO: 27, and SEQ lD NO: 29; f) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 31, SEQ ID NO: 33, and SEQ ID NO: 35; g) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 37, SEQ ID NO: 39, and SEQ ID NO: 41; and h) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 43, SEQ ID NO: 45, and SEQ ID NO: 47.
In certain embodiments, the dies or antigen-binding fragments thereof comprise a kappa light chain variable region ed from the group consisting of: a) a kappa light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6; b) a kappa light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 8, SEQ ID NO: 10, and SEQ ID NO: 12; c) a kappa light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 14, SEQ ID NO: 16 and/or SEQ ID NO: 18; d) a kappa light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 20, SEQ ID NO: 22, and SEQ ID NO: 24; e) a kappa light chain variable region comprising 1, 2, or 3 CDR ces selected from SEQ ID NO: 26, SEQ ID NO: 28, and SEQ ID NO: 30; f) a kappa light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 32, SEQ ID NO: 34, and SEQ IDNO: 36; g) a kappa light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 38, SEQ ID NO: 40, and SEQ ID NO: 42; and h) a kappa light chain le region sing 1, 2, or 3 CDR ces selected from SEQ ID NO: 44, SEQ ID NO: 46, and SEQ ID NO: 48.
In certain ments, the antibodies or antigen-binding fragments thereof comprise a heavy chain CDR3 sequence selected from the group consisting of SEQ ID NOs: , 11, 17, 23, 29, 35, 41, and 47.
In certain embodiments, the antibodies or antigen-binding fragments thereof comprise: a) a heavy chain CDRl sequence selected from SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO: 25, SEQ ID NO: 31, SEQ ID NO: 37, and SEQ ID NO: 43; b) a heavy chain CDR2 sequence selected from SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 27, SEQ ID NO: 33, SEQ ID NO: 39, and SEQ ID NO: 45; and c) a heavy chain CDR3 sequence selected from SEQ ID NO: 5, SEQ ID NO: 11, SEQ ID NO: 17, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 35, SEQ ID NO: 41, and SEQ ID NO: 47.
In certain embodiments, the antibodies or n-binding fragments thereof comprise: a) a light chain CDR1 sequence selected from SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 20, SEQ ID NO: 26, SEQ ID NO: 32, SEQ ID NO: 38, and SEQ ID NO: 44, b) a light chain CDR2 sequence selected from SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 16, SEQ ID NO: 22, SEQ ID NO: 28, SEQ ID NO: 34, SEQ ID NO: 40, and SEQ ID NO: 46; and c) a light chain CDR3 sequence selected from SEQ ID NO: 6, SEQ ID NO: 12, SEQ ID NO: 18, SEQ ID NO: 24, SEQ ID NO: 30, SEQ ID NO: 36, SEQ ID NO: 42, and SEQ ID NO: 48.
In certain embodiments, the antibodies or antigen-binding fragments f comprises: a) a heavy chain CDR1 sequence selected from SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO: 25, SEQ ID NO: 31, SEQ ID NO: 37, and SEQ ID NO: 43, b) a heavy chain CDR2 sequence selected from SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 27, SEQ ID NO: 33, SEQ ID NO: 39, and SEQ ID NO: 45, c) a heavy chain CDR3 sequence selected from SEQ ID NO: 5, SEQ ID NO: 11, SEQ ID NO: 17, SEQ ID NO: 23, SEQ ID NO: 29, SEQ IDNO: 35, SEQ ID NO: 41, and SEQ ID NO: 47, d) a light chain CDR1 sequence selected from SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 20, SEQ ID NO: 26, SEQ ID NO: 32, SEQ ID NO: 38, and SEQ ID NO: 44, e) a light chain CDR2 ce selected from SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 16, SEQ ID NO: 22, SEQ ID NO: 28, SEQ ID NO: 34, SEQ ID NO: 40, and SEQ ID NO: 46; and f) a light chain CDR3 sequence selected from SEQ ID NO: 6, SEQ ID NO: 12, SEQ ID NO: 18, SEQ ID NO: 24, SEQ ID NO: 30, SEQ ID NO: 36, SEQ ID NO: 42, and SEQ ID NO: 48.
In certain embodiments, the antibodies or antigen-binding fragments thereof comprises: a) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5; and a kappa light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6; b) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 11; and a kappa light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 8, SEQ ID NO: 10, and SEQ ID NO: 12; c) a heavy chain le region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 13, SEQ ID NO: 15, and SEQ ID NO: 17, and a kappa light chain variable region comprising 1, 2, or 3 CDR ces selected from SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO:18; d) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 19, SEQ ID NO: 21, and SEQ ID NO: 23, and a kappa light chain variable region comprising 1, 2, or 3 CDR sequences ed from SEQ ID NO: 20, SEQ ID NO: 22, and SEQ ID NO: 24, e) a heavy chain le region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 25, SEQ ID NO: 27, and SEQ ID NO: 29, and a kappa light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 26, SEQ ID NO: 28, and SEQ ID NO: 30; f) a heavy chain variable region sing 1, 2, or 3 CDR sequences selected from SEQ ID NO: 31, SEQ ID NO: 33, and SEQ ID NO: 35, and a kappa light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 32, SEQ ID NO: 34, and SEQ ID NO: 36; g) a heavy chain variable region comprising 1, 2, or 3 CDR sequences ed from SEQ ID NO: 37, SEQ ID NO: 39, and SEQ ID NO: 41; and a kappa light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 38, SEQ ID NO: 40, and SEQ ID NO: 42; or h) a heavy chain variable region comprising 1, 2, or 3 CDR ces selected from SEQ ID NO: 43, SEQ ID NO: 45, and SEQ ID NO: 47; and a kappa light chain variable region comprising 1, 2, or 3 CDR sequences ed from SEQ ID NO: 44, SEQ ID NO: 46, and SEQ ID NO: 48.
In certain embodiments, the antibodies or antigen-binding fragments thereof further comprises 1, 2, 3 or 4 heavy chain framework region (FR) sequences selected from the group consisting of: SEQ ID NO: 57, 59, 61, 63, 73, 75, 77 and 79, and/or 1, 2, 3, or 4 light chain framework region (FR) sequences selected from SEQ ID NO: 58, 60, 62, 64, 74, 76, 78 and 80.
In certain embodiments, the antibodies or antigen-binding fragments f further comprises heavy chain FR1 sequence selected from SEQ ID NO: 57 and 73; heavy chain FR2 sequence selected from SEQ ID NO: 59 and 75; heavy chain FR3 sequence selected from SEQ ID NO: 61 and 77; and heavy chain FR4 sequence selected from SEQ ID NO: 63 and 79.
In certain ments, the dies or antigen-binding nts thereof further comprises light chain FRl sequence selected from SEQ ID NO: 58 and 74; light chain FR2 sequence selected from SEQ ID NO: 60 and 76; light chain FR3 sequence selected from SEQ ID NO: 62 and 78; and light chain FR4 sequence selected from SEQ ID NO: 64 and 80.
In certain ments, the antibodies or antigen-binding fragments thereof comprises a heavy chain le region selected from the group consisting of: SEQ ID NO: 81, SEQ 1]) NO: 85, SEQ ID NO: 89, SEQ ID NO: 93, SEQ ID NO: 97, SEQ ID NO: 101, SEQ ID NO: 105, SEQ ID NO: 109, SEQ ID NO: 113, SEQ ID NO: 117 and a homologous sequence f having at least 80% (e.g. at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity.
In certain embodiments, the dies or antigen-binding fragments thereof comprises a light chain variable region selected from the group consisting of: SEQ ID NO: 83, SEQ ID NO: 87, SEQ ID NO: 91, SEQ 1]) NO: 95, SEQ 1]) NO: 99, SEQ ID NO: 103, SEQ ID NO: 107, SEQ ID NO: 111, SEQ IDNO: 115, SEQ ID NO: 119 and a homologous sequence thereof having at least 80% (eg. at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity.
In some embodiments, the antibodies or antigen-binding fragments thereof ses all or a portion of the heavy chain variable region sequence selected from the group consisting of: SEQ ID NO: 81, 85, 89, 93, 97, 101, 105, 109, 113, and 117; and/or, all or a portion ofthe light chain variable region sequence selected from the group consisting of: SEQ ID NO: 83, 87, 91, 95, 99, 103, 107, 111, 115, and 119. In one embodiment, the antibodies or antigen- binding fragments thereof is a single domain antibody which consists of all or a portion of the heavy chain variable region selected from the group ting of: SEQ ID NO: 81, 85, 89, 93, 97, 101, 105, 109, 113, and 117.
In certain embodiments, the antibodies or antigen—binding fragments thereof comprises: a) a heavy chain variable region comprising SEQ ID NO: 81 and a kappa light chain variable region comprising SEQ ID NO: 83; b) a heavy chain variable region comprising SEQ ID NO: 85 and a kappa light chain variable region comprising SEQ ID NO: 87; c) a heavy chain variable region comprising SEQ ID NO: 89 and a kappa light chain variable region comprising SEQ ID NO: 91; d) a heavy chain le region comprising SEQ ID NO: 93 and a kappa light chain variable region comprising SEQ ID NO: 95; e) a heavy chain variable region comprising SEQ ID NO: 97 and a kappa light chain variable region comprising SEQ ID NO: 99; f) a heavy chain le region comprising SEQ ID NO: 101 and a kappa light chain variable region comprising SEQ ID NO: 103; g) a heavy chain variable region comprising SEQ ID NO: 105 and a kappa light chain variable region comprising SEQ ID NO: 107; h) a heavy chain variable region comprising SEQ ID NO: 109 and a kappa light chain variable region comprising SEQ ID NO: 111; i) a heavy chain variable region comprising SEQ ID NO: 113 and a kappa light chain variable region comprising SEQ ID NO: 115; or j) a heavy chain variable region comprising SEQ ID NO: 117 and a kappa light chain le region comprising SEQ ID NO: 119.
In certain embodiments, the antibody or antigen-binding fragment thereof comprises one or more amino acid residue substitutions yet retains c binding affinity to CD3 n.
In certain embodiments, the substitution is in one or more CDR sequences, or in one or more FR sequences, or in one or both variable region sequences, or in Fc region. In some embodiments, at least one (or all) of the substitution(s) in the CDR sequences, FR sequences, variable region ces or Fc region comprises a conservative substitution.
In certain embodiments, the antibody or antigen-binding fragment thereof comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue substitutions in one or more CDR sequences selected from SEQ ID NO: 1-48. In certain embodiments, the antibody or antigen- binding fragment thereof comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue substitutions in one or more FR sequences selected from SEQ ID NO: 57—80. In certain embodiments, the antibody or n-binding fragment thereof comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitutions in total in CDR sequences and/or FR sequences of a heavy chain variable region sequences selected from SEQ ID NO: 81, 85, 89, 93, 97, 101, 105, 109, 113, and 117. In certain embodiments, the antibody or antigen-binding fragment thereof comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue substitutions in all the FRs of a light chain variable region sequences selected from SEQ ID NO: 83, 87, 91, 95, 99, 103, 107, 111, 115, and 119.
In certain ments, the substitution confers one or more desirable ties selected from: a) improving binding af?nity to CD3epsilon, b) introducing or ng a glycosylation site, c) introducing a free cysteine residue, d) enhancing or reducing ADCC or CDC, e) increasing serum half—life, and f) increasing FcRn binding.
In certain embodiments, the antibody or antigen-binding fragment thereof further comprises an immunoglobulin nt region, In n embodiments, the antibody or antigen-binding nt thereof comprises a constant region of IgG. In certain embodiment, the antibody or antigen-binding fragment thereof comprises a constant region of human IgG1.
] In certain embodiments, the antibodies or antigen-binding fragments thereof is a murine antibody or a humanized dy.
In certain embodiments, the antibodies or n-binding fragments thereof are a camelized single domain antibody, a diabody, a scFv, an scFv dimer, a Bst, a dst, a (dst)2, a dst-dst', an Fv fragment, a Fab, a Fab', a F(ab')2, a bispeci?c dy, a ds diabody, a nanobody, a domain antibody, or a bivalent domain antibody.
In certain embodiments, the antibodies or antigen-binding fragments thereof is iflc. In certain embodiments, the antibody or an antigen-binding nt f has a first antigenic specificity for CD3epsilon, and a second antigenic speci?city. In certain embodiments, the second antigenicity is for a second antigen different from CD3epsilon, wherein presence of the second antigen in proximity to a CD3epsilon—expressing T cells is desirable for the second antigen to be recognized by immune system. In certain embodiments, the ?rst antigenic speci?city is for CD3epsilon, and the second antigenic speci?city is for a tumor ated antigen.
In n ments, the antibodies or antigen-binding fragments thereof is linked to one or more conjugates. In certain embodiments, the conjugate can be a chemotherapeutic agent, a toxin, a radioactive isotope, a nide, a luminescent label, a ?uorescent label, or an enzyme-sub strate label.
In certain embodiments, the dy or an antigen-binding fragment thereof is capable of speci?cally binding to CD3epsilon. In certain embodiments, the CD3epsilon are derived from mouse, rat, monkey or human. In certain embodiments, the CD3epsilon is a recombinant CD3epsilon or a CD3 epsilon expressed on a cell surface.
In certain embodiments, the antibodies or antigen-binding fragments thereof is e of speci?cally binding to human CD3 epsilon expressed on a cell surface at a KD value of no more than 5x10'9M, no more than 4x10'9M, no more than 3x10'9M, no more than 2x10' 9M, no more than 10'9M, no more than 5x10'10M, no more than 4x10'10M, no more than 3x10' 10M, no more than 0M, no more than 10'10M, no more than 5x10'11 M, no more than 4x10' 11 M, no more than 3x10'11 M, no more than 2x10'11 M, or no more than 10'11 M as measured by ?ow cytometry assay. In certain embodiments, the antibodies or antigen-binding fragments thereof is capable of speci?cally binding to human CD3epsilon sed on surface of cells at an ECso of no more than 0.50 nM, or no more than 1.10 nM by ?ow cytometry assay.
In certain embodiments, the antibodies or antigen-binding fragments thereof is e of speci?cally binding to inant Cynomolgus monkey CD3 epsilon with an EC50 of no more than 0.001 nM, no more than 0.005 nM, no more than 0.01 nM, no more than 0.02 nM, no more than 0.03 nM, no more than 0.04 nM, or no more than 0.05 nM as measured by ELISA.
In certain embodiments, the antibodies or n-binding fragments thereof is capable of speci?cally binding to recombinant human CD3epsilon at an ECso of no more than 0.01 nM, no more than 0.02 nM, no more than 0.03 nM, no more than 0.04 nM, no more than 0.05 nM, no more than 0.06 nM, no more than 0.07 nM or no more than 0.08 nM as measured by ELISA.
In certain embodiments, the antibodies or antigen-binding fragments thereof is e of speci?cally binding to human CD3epsilon expressed on a CD3-expressing cell surface at an ECso of no more than 0.5 nM, no more than 0.6 nM, no more than 0.7 nM, no more than 0.8 nM, no more than 0.9 nM, no more than 1 nM, no more than 2 nM no more than 3 nM no more than 4 nM no more than 5 nM no more than 6 nM, no more than 7 nM, , , , no more than 8 nM, no more than 9 nM or no more than 10 nM as measured by ?ow cytometry assay.
] In certain embodiments, the antibodies or antigen-binding fragments thereof is a humanized dy, which is capable of speci?cally g to human CD3 epsilon expressed on a pressing cell e at an ECso of no more than 0.50 nM, or no more than 110 nM as measured by ?ow cytometry assay.
In one aspect, the present disclosure provides an antibody or an antigen-binding fragment f, which competes for the same epitope with the antibody or antigen-binding fragment thereof provided herein.
In one aspect, the present disclosure further provides a pharmaceutical composition comprising the antibody or antigen-binding nt thereof provided herein and a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical ition further comprises a second agent which is capable of enhancing a therapeutic effect of the antibody or antigen-binding fragment thereof and/or is capable of reducing a side effect of the antibody or n-binding fragment thereof, In one aspect, the present disclosure further provides an isolated polynucleotide encoding the antibody or an antigen-binding fragment thereof ed herein. In certain embodiments, the isolated polynucleotide comprises a nucleotide sequence selecting from a group consisting of SEQ ID NO: 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110,112,114,116,118 and 120.
In one aspect, the present disclosure further provides a vector comprising said isolated polynucleotide.
In one aspect, the present sure further provides a host cell comprising said vector.
In one aspect, the present disclosure further provides a method of expressing the antibody or antigen-binding fragment thereof provided , comprising culturing said host cell under the condition at which said polynucleotide is expressed.
In one aspect, the present disclosure further provides a method of treating a disease or condition in a t that would bene?t from tion of CD3epsilon activity, comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof provided herein or the pharmaceutical composition provided herein. In certain embodiments, said subject is human. In certain embodiments, said e or condition is a CD3 related disease or condition. In certain embodiments, said e or condition is cancer, autoimmune disease, in?ammatory disease, or infectious disease.
] In one aspect, the present disclosure further provides a method of activating CD3epsilon-expressing T cells in Vivo or in vitro, sing contacting the CD3epsilon - expressing T cells with the antibody or antigen—binding fragment thereof ed herein.
In one aspect, the present disclosure further provides a method of modulating CD3 activity in a CD3epsilon-expressing cell, comprising exposing the CD3 epsilon-expressing cell to the antibody or antigen-binding fragment thereof provided herein.
In one aspect, the present disclosure further provides a method of promoting in vivo or in vitro processing of a second antigen by CD3epsilon-expressing T cells, comprising ting the CD3epsilon-expressing T cells with a bispeciflc antibody or n-binding fragment thereof ed herein, wherein the bispeciflc antibody or antigen-binding nt is capable of specifically binding to both the CD3epsilon-expressing T cells and a second antigen y bringing both in close ity.
In one aspect, the present sure r provides a method of ing presence or amount of CD3 epsilon in a sample, comprising contacting the sample with the antibody or antigen-binding fragment thereof provided herein, and determining the presence or the amount of CD3epsilon in the sample.
] In one aspect, the present disclosure further provides a method of diagnosing a CD3 related disease or condition in a subject, comprising: a) obtaining a sample from the subject; b) contacting the sample with the antibodies or antigen-binding fragments thereofprovided herein; c) determining presence or amount ofCD3 epsilon in the sample; and d) correlating the presence or the amount of CD3epsilon to a e or condition in the subject.
In one aspect, the present disclosure further provides use of the antibody or antigen— g fragment thereof provided herein in the manufacture of a medicament for treating a CD3 related disease or condition in a subject.
In one aspect, the present sure further provides use of the antibody or antigen- binding fragment thereof provided herein in the manufacture of a diagnostic reagent for diagnosing a CD3 related disease or condition.
In one aspect, the present disclosure r provides a kit comprising the antibody or antigen—binding nt thereof provided herein, useful in detecting CD3epsilon. In certain embodiments, the kit comprises antibodies or antigen—binding fragment thereof useful in detecting recombinant CD3epsilon, CD3epsiIon expressed on cell surface, or CD3epsiIon— expresing cells. [00051Al In one aspect, the t disclosure further provides an antibody or antigen— binding fragment thereof as described herein for use in the treatment of a CD3 related e or condition.
BRIEF PTION OF FIGURES Figure 1 shows binding of the monoclonal dies, WBP331172.166.48—ngGlK, WBP331172.3O6.4 - ngGlK, and WBP331172.166.48, to recombinant Cynomolgus Monkey CD3epsilon protein as measured by ELISA assay.
Figure 2 shows binding of the onal antibodies, WBP331172.166.48—ulg llK, WBP331172.3O6.4 - ngGlK, WBP331172.166.48, and WBP331172.306.4, to human CD4 T cells as measured by flow cytometry assay.
Figure 3 shows binding affinity ofeight mouse antibodies (W331 1—2.166.48, W331 1—2.3064, W3311-2.383.47, W3311-2.400.5, W3311-2.482.5, W3311-2.488.33, W3311-2.615.8, and W3311- 2844.8) to human CD3 expressing cells ('Jurkat cells) as measured by flow cytometry assay.
Figure 4A shows binding affinity of humanized antibody, WBP331172.166.48—zl—ngG1K to human CD3 expressing cells (Jurkat cells) as measured by flowcytometry assay.
Figure 4B shows the result of binding affinity of humanized antibody, WBP331 172.306.4— zl -u1gG1 K to human CD3 expressing cells (Jurkat cells) as measured by flow cytometry assay.
Figure 4C shows the result of binding af?nity of the positive control, OKT3 to human CD3 expressing cells (Jurkat cells) as measured by flow cytometry assay.
Figure 5 shows cell alization rate ofeight mouse dies (W331 1-2.16648, W3311- 2.3064, W3311-2.383.47, W3311-2.400.5, W3311-2.482.5, W3311-2.488.33, 2.615.8, and W331 48) to human CD3 sing cells (Jurkat cells) as measured by ?ow cytometry assay .
] Figure 6 shows the result of human T cell activation by eight mouse antibodies (W331 l- 2.166.48, W3311-2.306.4, W3311-2.383.47, W3311-2.400.5, W3311-2.482.5, W3311-2.488.33, W331 1-2.6158, and W3311-2.844.8) as measured by intracellular cytokine TNFalpha and IFNgamma staining.
Figure 7 shows the result of human T cell activation by two humanized antibodies (WBP33ll_2.l66.48—zl-ngGlK and l_2.306.4-zl-ngGlK) as measured by intracellular cytokine TNFalpha and IFNgamma staining.
Figure 8 shows the result of epitope g of seven mouse antibodies (W33ll- 2.166.48, W331 l-2.306.4, W331 l-2.400.5, W331 l-2.482.5, W331 1-2.48833, W3311-2.615.8, and W331 l-2.844.8) against the clone WBP33 l l_2.3 83.47.
DETAILED PTION OF THE INVENTION The following description of the disclosure is merely intended to illustrate various embodiments of the disclosure. As such, the speci?c ations discussed are not to be construed as limitations on the scope of the disclosure. It will be apparent to one skilled in the art that various equivalents, changes, and modi?cations may be made without departing from the scope of the disclosure, and it is understood that such equivalent embodiments are to be ed herein. All references cited herein, including publications, s and patent applications are incorporated herein by reference in their entirety.
Definitions The term "antibody" as used herein includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, nt antibody, monovalent antibody, peci?c antibody, or bispeci?c antibody that binds to a speci?c antigen. A native intact antibody comprises two heavy (H) chains and two light (L) chains. Mammalian heavy chains are ?ed as alpha, delta, epsilon, gamma, and mu, each heavy chain consists of a le region (VH) and a ?rst, second, and third constant region (CH1, CH2, CH3, respectively); ian light chains are classi?ed as 9» or K, while each light chain consists of a variable region (VL for 9» light chain or VK for K light chain, respectively) and a constant region(CL for 9» light chain or CK for K light chain, respectively). The dy has a "Y" shape, with the stem of the Y consisting of the second and third constant regions of two heavy chains bound together via disul?de bonding. Each arm ofthe Y includes the variable region and ?rst constant region of a single heavy chain bound to the variable and constant s of a single light chain.
The variable regions of the light and heavy chains are responsible for antigen g. The variable regions in both chains generally contain three highly variable loops called the complementarity determining regions (CDRs) (light chain CDRs including LCDRl, LCDRZ, and LCDR3, heavy chain CDRs including HCDRl, HCDRZ, HCDR3). CDR boundaries for the antibodies and antigen-binding fragments disclosed herein may be de?ned or identi?ed by the conventions of Kabat, lMGT, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, A. M., J. Mol. Biol, 273(4), 927 (1997); Chothia, C. et al, J Mol Biol. Dec 5;186(3):651-63 (1985); Chothia, C. and Lesk, A.M., J.Mol.Biol., 196,901 (1987); Chothia, C. et al., Nature.
Dec 2l-28;342(6252):877-83 (1989) ; Kabat EA. et a]., National Institutes of Health, Bethesda, Md. (1991)). The three CDRs are interposed between ?anking stretches known as framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops. The constant s of the heavy and light chains are not ed in n-binding, but exhibit various effector functions. Antibodies are ed to s based on the amino acid sequence of the constant region of their heavy chain. The ?ve major classes or isotypes of dies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of alpha, delta, epsilon, gamma, and mu heavy chains, respectively. Several of the major antibody classes are divided into subclasses such as IgGl (gammal heavy chain), IgG2 (gamma2 heavy chain), IgG3 (gamma3 heavy chain), IgG4 (gamma4 heavy chain), IgAl (alphal heavy chain), or IgA2 (alpha2 heavy chain).
] The term "bivalent" as used herein refers to an antibody or an antigen-binding fragment having two antigen-binding sites; the term "monovalent" refers to an antibody or an antigen-binding fragment having only one single antigen-binding site; and the term valent" refers to an antibody or an n-binding fragment having multiple antigen- binding sites. In some embodiments, the dy or antigen-binding fragment thereof is bivalent.
] As used herein, a "bispeci?c" antibody refers to an arti?cial antibody which has fragments derived from two different monoclonal antibodies and is capable of binding to two different epitopes. The two epitopes may present on the same antigen, or they may present on two different antigens.
The term "antigen-binding fragment" as used herein refers to an antibody fragment formed from a portion of an antibody comprising 1, 2 or 3 CDRs, or any other antibody fragment that binds to an antigen but does not comprise an intact native antibody structure.
Examples of antigen-binding fragment include, without limitation, a diabody, a Fab, a Fab', a F(ab')2, an Fv fragment, a disul?de stabilized Fv nt (dst), a (dst)2, a bispecific dst (dst—dst'), a disul?de stabilized y (ds diabody), a single-chain antibody molecule (scFv), an scFv dimer (bivalent diabody), a bispecific antibody, a multispecific antibody, a camelized single domain antibody, a nanobody, a domain antibody, and a bivalent domain antibody. An antigen-binding fragment is capable of binding to the same antigen to which the parent antibody binds.
"Fab" with regard to an antibody refers to that portion of the antibody consisting of a single light chain (both variable and constant regions) bound to the variable region and first constant region of a single heavy chain by a disul?de bond.
"Fab'" refers to a Fab fragment that includes a portion of the hinge .
"F(ab')2"refers to a dimer of Fab’. "Fv" with regard to an antibody refers to the st fragment of the dy to bear the complete antigen—binding site. An Fv fragment consists of the variable region of a single light chain bound to the variable region of a single heavy chain.
A "dst" refers to a disul?de-stabilized Fv fragment that the linkage n the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond. In some embodiments, a "(dst)2" or "(dst-dst')" comprises three e chains: two VH moieties linked by a peptide linker (e.g., a long ?exible linker) and bound to two VL moieties, respectively, via disul?de bridges. In some embodiments, dst-dst' is bispecific in which each disulfide paired heavy and light chain has a ent antigen speci?city, "Single-chain Fv antibody" or "scFv" refers to an engineered antibody ting of a light chain variable region and a heavy chain variable region ted to one r directly or via a e linker sequence (Huston JS et al. Proc Natl Acad Sci USA, 85:5879(l988)).
"Fc" with regard to an antibody refers to that portion of the antibody ting of the second and third constant regions of a first heavy chain bound to the second and third constant regions of a second heavy chain via disulf1de bonding. The Fc portion of the antibody is sible for various effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), and complement dependent cytotoxicity (CDC), but does not function in antigen binding.
"Single-chain Fv-Fc antibody" or "scFv-Fc" refers to an engineered antibody consisting of a scFv connected to the Fc region of an antibody.
"Camelized single domain antibody," "heavy chain antibody," or "HCAb" refers to an antibody that contains two VH domains and no light chains (Riechmann L. and Muyldermans S., J Immunol Methods. Dec lO;231(l-2):25-38 (1999); Muyldermans S., J Biotechnol.
Jun,74(4):277—302 , WO94/04678, WO94/2559l, U.S. Patent No. 6,005,079). Heavy chain antibodies were originally derived from Camelidae (camels, dromedaries, and llamas).
Although devoid of light chains, camelized antibodies have an authentic antigen-binding repertoire (Hamers-Casterman C. et al., Nature. Jun 3;363(6428):446-8 (1993); Nguyen VK. el al. "Heavy-chain antibodies in Camelidae, a case of evolutionary innovation," Immunogenetics. Apr;54(1):39-47 (2002), Nguyen VK. er alImmunology. May,109(1):93- 101 ). The variable domain of a heavy chain antibody (VHH domain) represents the smallest known antigen-binding unit ted by adaptive immune responses Nolte F. el 61]., FASEB J. Nov;2l(13):3490-8. Epub 2007 Jun 15 (2007)) A "nanobody" refers to an antibody fragment that consists of a VHH domain from a heavy chain antibody and two constant domains, CH2 and CH3.
"Diabodies" or "dAbs" include small antibody fragments with two n-binding sites, wherein the fragments se a VH domain ted to a VL domain in the same polypeptide chain (VH—VL or VL—VH) (see, e.g., Holliger P. et al, Proc Natl Acad Sci U S A.
Jul 15;90(14):6444-8 (1993); EP404097, WO93/11161). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of r chain, thereby creating two antigen-binding sites. The antigen—binding sites may target the same or different ns (or epitopes). In certain embodiments, a "bispecific ds diabody" is a diabody target two different antigens (or epitopes).In certain embodiments, an "scFv dimer" is a bivalent diabody or bivalent ScFv (Bst) comprising VH-VL (linked by a peptide linker) dimerized with another VH-VL moiety such that VH's of one moiety coordinate with the VL's of the other moiety and form two g sites which can target the same antigens (or eptipoes) or different antigens (or es). In other embodiments, an "scFv dimer" is a ific diabody comprising VH1-VL2 (linked by a peptide linker) associated with VLi-VH2 (also linked by a peptide linker) such that VH1 and VLi coordinate and VH2 and VL2 coordinate and each coordinated pair has a different antigen speci?city.
A "domain antibody" refers to an dy fragment containing only the variable region of a heavy chain or the variable region of a light chain. In certain instances, two or more VH domains are covalently joined with a peptide linker to create a bivalent or multivalent domain antibody. The two VH domains of a bivalent domain dy may target the same or different antigens.
The term "chimeric" as used herein, means an antibody or antigen-binding fragment, having a portion of heavy and/or light chain derived from one species, and the rest of the heavy and/or light chain d from a different species. In an illustrative example, a chimeric dy may comprise a constant region d from human and a variable region from a non-human animal, such as from mouse. In some embodiments, the non-human animal is a mammal, for example, a mouse, a rat, a rabbit, a goat, a sheep, a guinea pig, or a hamster.
] The term "humanized" as used herein means that the antibody or antigen-binding fragment comprises CDRs derived from non-human animals, FR regions derived from human, and when applicable, the constant regions derived from human.
"CD3" as used herein, refers to the r of Differentiation 3 n derived from any vertebrate source, including mammals such as es (e.g. humans, monkeys) and s (e.g., mice and rats). In mammals, the CD3 molecule is a multi-protein complex of six chains, including: a CD3gamma chain, a CD3delta chain, two CD3epsilon chains, and a homodimer of CD3zeta chains, wherein the CD3zeta chain is the intracellular tail of CD3 molecule, and the CD3gamma, ta and CD3epsilon chains all contain extracellular domain (ECD) expressed on surface of T cells. Exemplary sequence of human CD3 includes human CD3 epsilon protein (NCBI Ref Seq No. NP_OOO724), human CD3 delta protein (NCBI Ref Seq No. NP_OOO723), and human CD3gamma protein (NCBI Ref Seq No. NP_000064).
Exemplary sequence of non-human CD3 includes Macaca fascicularis (monkey) CD3epsilon protein (NCBI Ref Seq No. NP_001270544), Macaca fascicularis (monkey) CD 3delta protein (NCBI Ref Seq No. 274617), Macaca fascicularis (monkey) CD3gamma protein (NCBI Ref Seq No. NP_001270839); mouse CD3epsilon protein (NCBI Ref Seq No.
NP_031674), mouse CD3delta protein (NCBI Ref Seq No. NP_038515), mouse CD3gamma protein (NCBI Ref Seq No. 00); Rattus norvegicus (Rat) CD3epsilon protein (NCBI Ref Seq No. NP_001101610), Rattus norvegicus (Rat) CD3delta protein (NCBI Ref Seq No.
RP_037301), Rattus norvegicus (Rat) ma protein (NCBI Ref Seq No. 1071114). In certain embodiments, CD3 used herein can also be recombinant CD3, for example, including recombinant CD3epsilon protein, recombinant CD3 delta protein, and recombinant CD3gamma protein, which may optionally be sed as a recombinant CD3 complex. The recombinant CD3 complex may be expressed on a cell surface, or alternatively may be expressed as a soluble form which is not associated on a cell surface.
The term "CD3epsilon" as used herein is intended to encompass any form of CD3epsilon, for example, 1) native essed CD3epsilon molecule, "full-length" CD3epsilon chain or naturally occurring variants of CD3 epsilon, including, for example, splice variants or allelic variants; 2) any form of CD3 epsilon that results from processing in the cell; or 3) full length, a fragment (e.g., a truncated form, an ellular/transmembrane domain) or a modi?ed form (e. g. a mutated form, a glycosylated/PEGylated, a His- tag/immuno?uorescence fused form) of CD3 epsilon t generated through recombinant method.
The term "anti-CD3epsilon antibody" refers to an antibody that is e of speci?c binding CD3epsilon (e. g. human or monkey CD3epsilon).
The term "speci?c binding" or "speci?cally binds" as used herein refers to a non- random binding reaction between two molecules, such as for e between an antibody and an antigen. In certain embodiments, the antibodies or antigen-binding fragments ed herein specifically bind to human and/or CD3epsilon with a binding affinity (KB) of 310'6 M (e.g., SleO'7 M, SleO'7 M, 310-7 M, SleO'S M, SleO'S M, :10—8 M, :5x10-9 M, :4xio-9M, S3x10‘9M,£2x10'9 M, or 310'9 M). KD used herein refers to the ratio of the dissociation rate to the association rate (koff/kon), which may be determined by using any conventional method known in the art, including but are not limited to surface plasmon resonance , microscale therrnophoresis method, HPLC-MS method and ?ow cytometry (such as FACS) method. In certain embodiments, the K13 value can be appropriately determined by using ?ow cytometry.
The ability to "block binding" or te for the same epitope" as used herein refers to the ability of an antibody or antigen—binding fragment to inhibit the g ction between two les (e.g. human CD3epsilon and an anti-CD3epsilon antibody) to any detectable degree. In certain embodiments, an antibody or antigen-binding fragment that blocks binding between two molecules inhibits the binding interaction between the two molecules by at least 85%, or at least 90%. In certain embodiments, this inhibition may be greater than 85%, or greater than 90%.
The term pe" as used herein refers to the specific group of atoms or amino acids on an n to which an antibody binds. Two antibodies may bind the same or a closely related epitope within an antigen if they exhibit itive binding for the antigen. For example, if an antibody or antigen-binding nt blocks binding of a reference antibody to the antigen (e. g., recombinant monkey CD3 epsilon or CD3 epsilon expressed on surface of cells in the present disclosure) by at least 85%, or at least 90%, then the antibody or antigen- binding fragment may be considered to bind the same/closely related epitope as the reference antibody.
Those skilled in the art will recognize that it is possible to determine, t undue experimentation, if a human monoclonal antibody binds to the same epitope as the antibody of present disclosure (erg, mouse monoclonal antibodies WBP3311_2.166.48, WBP33ll_2.306.4, WBP33ll_2.383.47, WBP33ll_2.400.5, WBP33ll_2.482.5, WBP331_2.488.33, WBP3311_2.615.8, WBP3311_2.844.8, and humanized antibodies WBP331 l_2.166.48-zl and WBP3311_2.306.4-zl) by aining whether the former prevents the latter from binding to a CD3epsilon n polypeptide. If the test antibody competes with the antibody of present disclosure, as shown by a decrease in binding by the antibody of present sure to the CD3 epsilon n ptide, then the two antibodies bind to the same, or a closely related, e. Or if the binding of a test antibody to the CD3epsilon antigen polypeptide was inhibited by the dy of present disclosure, then the two antibodies bind to the same, or a closely related, epitope.
The various symbols used in the antibody names as provided herein are of different representation: "mIgG2" refers to an antibody with mouse constant region of IgG2 isotype; "ngGl" refers an antibody with human constant region of IgG1 isotype; "K" or "L" refers to an antibody using the kappa or lambda light chain.
A "conservative substitution" with reference to amino acid sequence refers to replacing an amino acid residue with a different amino acid residue having a side chain with similar physiochemical properties. For example, conservative substitutions can be made among amino acid es with hydrophobic side chains (e. g. Met, Ala, Val, Leu, and He), among es with neutral hydrophilic side chains (e.g. Cys, Ser, Thr, Asn and Gln), among residues with acidic side chains (e. g. Asp, Glu), among amino acids with basic side chains (e. g.
His, Lys, and Arg), or among residues with aromatic side chains (e. g. Trp, Tyr, and Phe). As known in the art, vative tution usually does not cause significant change in the protein conformational structure, and ore could retain the biological activity of a protein.
The term "homologue" and "homologous" as used herein are interchangeable and refer to nucleic acid sequences (or its complementary strand) or amino acid sequences that have sequence identity of at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) to another sequences when optimally aligned.
"Percent (%) sequence identity" with respect to amino acid sequence (or nucleic acid sequence) is de?ned as the percentage of amino acid (or c acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues in a reference sequence, after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum number of cal amino acids (or c acids). Conservative substitution of the amino acid residues may or may not be considered as identical residues. Alignment for purposes of determining percent amino acid (or nucleic acid) sequence ty can be achieved, for example, using publicly available tools such as BLASTN, BLASTp (available on the website ofUS. National Center for Biotechnology Information (NCBI), see also, ul S.F. et al, J.
Mol. Biol, 215:403—410 (1990), Stephen F. et al, Nucleic Acids Res, 25:3389—3402 (1997)), lW2 (available on the website of European ormatics Institute, see also, Higgins D.G. et al, Methods in Enzymology, 266:383-402 (1996), Larkin MA. et al, Bioinformatics (Oxford, England), 23(21): 2947-8 (2007)), and ALIGN or Megalign (DNASTAR) software.
Those skilled in the art may use the default parameters ed by the tool, or may customize the parameters as appropriate for the alignment, such as for example, by ing a suitable algorithm. tor functions" as used herein refer to biological activities attributable to the binding of Fc region of an antibody to its effectors such as Cl complex and Fc receptor.
Exemplary effector functions include: complement dependent cytotoxicity (CDC) induced by interaction of antibodies and Clq on the C1 complex; antibody—dependent cell-mediated cytotoxicity (ADCC) induced by binding of Fc region of an antibody to PC receptor on an effector cell; and phagocytosis.
"Treating" or "treatment" of a condition as used herein includes preventing or alleviating a condition, g the onset or rate of development of a condition, reducing the risk of developing a condition, preventing or ng the development of symptoms associated with a ion, reducing or ending symptoms associated with a condition, generating a complete or partial regression of a condition, curing a condition, or some combination thereof.
An "isolated" substance has been altered by the hand of man from the natural state.
If an "isolated" composition or nce occurs in nature, it has been d or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living animal is not "isolated," but the same polynucleotide or polypeptide is "isolated" if it has been iently separated from the coexisting materials of its natural state so as to exist in a substantially pure state. An "isolated c acid sequence" refers to the sequence of an isolated c acid molecule. In certain embodiments, an "isolated antibody or antigen-binding fragment thereof’ refers to the antibody or antigen-binding fragments having a purity of at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% as determined by electrophoretic methods (such as SDS—PAGE, isoelectric focusing, capillary electrophoresis), or chromatographic methods (such as ion exchange chromatography or reverse phase HPLC).
] The term "vector" as used herein refers to a e into which a polynucleotide encoding a n may be operably inserted so as to bring about the expression of that protein.
A vector may be used to transform, transduce, or transfect a host cell so as to bring about expression of the genetic element it carries within the host cell. es of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial arti?cial chromosome (BAC), or Pl—derived arti?cial chromosome (PAC), iophages such as lambda phage or M13 phage, and animal viruses. Categories of animal viruses used as vectors include retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus), us, baculovirus, papillomavirus, and papovavirus (e.g., SV40). A vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer ces, selectable elements, and reporter genes. In addition, the vector may contain an origin of replication. A vector may also include materials to aid in its entry into the cell, including but not limited to a viral particle, a me, or a n coating. A vector can be an expression vector or a cloning vector. The t disclosure provides vectors (e.g., expression vectors) ning the nucleic acid sequence provided herein encoding the antibody or antigen-binding fragment thereof, at least one promoter (e.g., SV40, CMV, EF-l 0t) operably linked to the nucleic acid sequence, and at least one selection marker. Examples of vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno- associated virus, herpesvirus (e.g., herpes simplex virus), us, baculovirus, papillomavirus, virus (e.g., SV40), lambda phage, and M13 phage, plasmid pcDNA3.3, pMDl8-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, PsgSL, pBABE, pWPXL, pBI, plSTV-L, , pTD, pRSlO, pLexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNAl.l/amp, pcDNA3.1, pRc/RSV, PCR 2.1, pEF-l, pFB, pSGS, pXTl, pCDEF3, pSVSPORT, pEF-Bos etc.
The phrase "host cell" as used herein refers to a cell into which an ous polynucleotide and/or a vector has been introduced.
A "CD3 related e or condition" as used herein refers to any disease or condition caused by, exacerbated by, or otherwise linked to increased or decreased expression or activities of CD3. In some embodiments, the CD3 related condition is immune-related disorder, such as, for example, cancer, autoimmune disease, in?ammatory disease or infectious disease.
"Cancer" as used herein refers to any l condition characterized by malignant cell growth or neoplasm, abnormal proliferation, in?ltration or metastasis, and includes both solid tumors and non—solid cancers (hematologic malignancies) such as leukemia. As used herein "solid tumor" refers to a solid mass of neoplastic and/or malignant cells.
The term "pharmaceutically acceptable" indicates that the designated carrier, vehicle, diluent, excipient(s), and/or salt is generally chemically and/or ally compatible with the other ingredients sing the formulation, and physiologically compatible with the recipient thereof.
Anti-CD3epsilon antibody The present sure provides anti-CD3epsilon antibodies and antigen-binding fragments thereof comprising one or more (e. g. 1, 2, 3, 4, 5, or 6) CDR sequences of an anti— CD3epsilon antibody WBP33 l l_2. 166.48, WBP33 l l_2.306.4, WBP331 l_2.383.47, WBP331 l_2.400.5, WBP3311_2.482.5, _2.488.33, WBP331 l_2.615.8, or WBP3311_2.844.8. Throughout the present disclosure, the term "WBP33 l l" with respect to the antibody names is used interchangeably with "W33 1 1". For e, antibody WBP33 l l_2.166.48 is also referred to as W33ll_2.l66.48 and such names refer to the same antibody.
"WBP3311_2.166.48" as used herein refers to a mouse monoclonal antibody having a heavy chain variable region of SEQ ID NO: 81, and a kappa light chain variable region of SEQ ID NO: 83.
"WBP33 l l_2.306.4" as used herein refers to a mouse monoclonal antibody having a heavy chain variable region of SEQ ID NO: 85, and a kappa light chain variable region of SEQ ID NO: 87.
"WBP3311_2.383.47" as used herein refers to a mouse monoclonal antibody having a heavy chain variable region of SEQ ID NO: 89, and a kappa light chain variable region of SEQ ID NO: 91.
] "WBP3311_2.400.5" as used herein refers to a mouse onal antibody having a heavy chain variable region of SEQ ID NO: 93, and a kappa light chain variable region of SEQ ID NO: 95.
"WBP3311_2.482.5" as used herein refers to a mouse onal antibody having a heavy chain variable region of SEQ ID NO: 97, and a kappa light chain variable region of SEQ ID NO: 99.
"WBP331_2.488.33" as used herein refers to a mouse monoclonal dy having a heavy chain variable region of SEQ ID NO: 101, and a kappa light chain variable region of SEQ ID NO: 103.
"WBP3311_2.615.8" as used herein refers to a mouse monoclonal antibody having a heavy chain variable region of SEQ ID NO: 105, and a kappa light chain variable region of SEQ ID NO: 107.
"WBP3311_2.844.8" as used herein refers to a mouse monoclonal antibody having a heavy chain variable region of SEQ ID NO: 109, and a kappa light chain variable region of SEQ ID NO: 111.
Table 1 shows the CDR sequences of these 8 anti-CD3epsilon antibodies. The heavy chain and light chain variable region sequences are also provided below.
Table 1. _-———o;SEQIDNO:1 SEQ ID \'0: 3 SEQ ID\ WBP3311_2.166.48 GYSFTTYYIH NIKYSE DSVSIYYFDY SEQ ID NO: 2 SEQ ID \'0: 4 SEQ ID \0: 6 WBP3311_2.166.48 VK §EiQSLLNSRTRKN WASTRKS TQSFILRT SEQ ID NO: 7 SEQ ID \‘0; 9 SEQ ID \0; 11 WBP3311_23064 GFAFTDYYIH ESEECENWTKYN DGYSLYYFDY SEQ ID NO. 8 SEQ ID \‘0; 10 SEQ ID \0; 12 WBP3311_23064 giiQSLLNSRTRKN WASTRQS TQSHTLRT SEQ ID NO: 13 SEQ ID \'O: 15 SEQ ID \0: 17 1_2.383.47 GFTFTNYYIH EPIEIBERGNTKYNE DGYSLYYFDY SEQ ID NO: 14 SEQ ID \'0: 16 SEQ ID \0: l8 WBP3311_2.383.47 VK KSSQSLLNSRTRKN WASIRVS TQSHTLRT SEQ ID NO: 19 SEQ ID \0: 21 SEQ ID \0: 23 DNTKYNE WBP3311_2.400.5 GYSFTNYYLH FFDF SEQ ID NO: 20 SEQ ID \IO: 22 SEQ ID \'0: 24 1_2400.5 §ESAQSLVNNVNNRTRKN WASTRES AQSFILRT SEQ ID NO: 25 SEQ ID \0: 27 SEQ ID \'0: 29 WIFPGSDNIKYNE WBP3311_2.482.5 GYTFTTYYIH DSVSRYYFDY SEQ ID NO: 26 SEQ ID \IO: 28 SEQ ID \'0: 30 WBP3311_2.482.5 §ESAQSLWDRTRKN WASTRES AQSFILRT - SEQ ID NO: 31 SEQ ID \‘0; 33 SEQ ID \‘0; 35 WBP331 2 488 33 GFSFTNYYIH EEEGTVNTKYNE DSVGIYYFDF SEQ ID NO: 32 SEQ ID \IO: 34 SEQ ID \'0: 36 _2.488.33 I;ESAQSLLNNRTRKN WASTRES TQSFILRT SEQ ID NO: 37 SEQ ID \:0 39 SEQ ID \0: 41 DNIKYNE WBP3311_2.615.8 GYSFTDFYTH DSVSVYYFDY SEQ ID NO: 38 SEQ ID \:0 40 SEQ ID \0: 42 VK KSSQSLLNIRTRKNY WBP3311_2.615.8 WASTRDS TQSFILRT SEQ ID I\O: 43 SEQ ID \0: 45 SEQ ID \0: 47 VH WISPGNVNTKYN WBP3311 2844.8 GFAFTDYYIH DGYSLYYFDY — ENFKG - SEQ ID I\O 44 SEQ ID\0; 46 SEQIDo 48 WBP3311_2.844.8 giiQSLLNSRTRKN WASTRES TQSHTLRT Heavy or kappa light chain variable region sequences of WBP3311_2.166.48, WBP3311_2.306.4, WBP3311_2.383.47, WBP3311_2.400.5, WBP3311_2.482.5, WBP331_2.488.33, WBP3311_2.615.8, and WBP3311_2.844.8, and humanized WBP33 1 1_2. 166.48 and 1_2.306.4 antibodies are provided below.
WBP3311_2.166.48—VH Amino acid sequence (SEQ ID NO: 81): QVQLQQSGPELVKPGASVKIACKASGYSFTTYYIHWVKQRPGQGLEWIGWIFPGND NIKYSEKFKGKATLTADTSSSTAYMQLSSLTSEDSAVYFCAIDSVSIYYFDYWGQGTT LTVSS Nucleic acid sequence (SEQ ID NO: 82): CAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAACCTGGGGCTTCAGTG AAGATTGCCTGCAAGGCTTCTGGCTACAGCTTCACAACCTACTATATACACTGGG TGAAGCAGAGGCCTGGACAGGGACTTGAGTGGATTGGATGGATTTTTCCTGGAA ATGATAATATTAAGTACAGTGAGAAGTTCAAGGGCAAGGCCACACTGACGGCAG ACACTTCCTCCAGTACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTC TGCTGTCTATTTCTGTGCTATAGACTCCGTTAGTATCTACTACTTTGACTATTGGG GCCAAGGCACCACTCTCACAGTCTCCTCA WBP3311_2.166.48—VK Amino acid sequence (SEQ ID NO: 83): DIVMSQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIY? ASTRKSGVPDRFTGSGSGTDFTLTINSVQAEDLAVYYCTQSFILRTFGGGTKLEIK Nucleic acid sequence (SEQ ID NO: 84): GACATTGTGATGTCACAGTCTCCATCCTCCCTGGCTGTGTCAGCAGGAGAGAAGG TCACTATGAGCTGCAAATCCAGTCAGAGTCTGCTCAACAGTAGAACCCGAAAGA ACTACTTGGCTTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTA CTGGGCATCCACTAGGAAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCT GGGACAGATTTCACTCTCACCATCAACAGTGTGCAGGCTGAAGACCTGGCAGTTT ATTACTGCACGCAATCTTTTATTCTTCGGACGTTCGGTGGAGGCACCAAGCTGGA AATCAAA WBP3311_2.306.4-VH Amino acid sequence (SEQ ID NO: 85): SGPELVKPGASVRISCKASGFAFTDYYHIWVKQRPGQGLEWIGWISPGNVN TKYNENFKGRATLTADLSSSTAYMQLSSLTSEDSAVYFCARDGYSLYYFDYWGQGT TLTVSS Nucleic acid ce (SEQ ID NO: 86): CAGGTCCAGCTGCAGCAGTCTGGACCTGAATTGGTGAAGCCTGGGGCTTCCGTGA GGATATCCTGCAAGGCTTCTGGCTTCGCCTTCACAGACTACTATATACACTGGGT GAAGCAGAGGCCTGGACAGGGTCTTGAGTGGATTGGATGGATTTCTCCTGGAAA TGTTAATACTAAATACAATGAAAACTTCAAGGGCAGGGCCACACTGACTGCAGA CCTATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACCTCTGAGGACTCT GCGGTCTATTTCTGTGCAAGAGATGGATATTCCCTGTATTACTTTGACTACTGGG GCCAAGGCACCACTCTCACAGTCTCCTCA WBP3311_2.306.4-VK Amino acid sequence (SEQ ID NO: 87): DIVMSQSPSSLTVSAGEKVTMSCKSSS RTRKNYLAWYQQKPGQSPKLLIYW_A STRS 2SGVPDRFTGSGSGTAFTLTISGVQAEDLAVYFCTS QSHTLRTFGGGTKLElK Nucleic acid sequence (SEQ ID NO: 88): GACATTGTGATGTCACAGTCTCCATCCTCCCTGACTGTGTCAGCAGGAGAGAAGG TCACTATGAGCTGCAAATCCAGTCAGAGTCTGCTCAACAGTAGAACCCGAAAGA ACTACTTGGCTTGGTACCAGCAGAAGCCAGGGCAGTCTCCTAAACTACTAATCTA ATCCACTAGGCAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCT GGGACAGCTTTCACTCTCACCATCAGCGGTGTGCAGGCTGAAGACCTGGCAGTTT ATTTCTGCACGCAATCTCATACTCTTCGGACGTTCGGTGGAGGCACCAAGCTGGA AATCAAA 1_2.383.47—VH Amino acid sequence (SEQ ID NO: 89): QVQLQQSGPELVKPGASVRISCKTSGFTFTNYYIHWVIQRPGQGLEWIGWISPENGNT KYNENFS QDKATLTADISSSTAYMHLSSLTSEDSAVYFCARDGYSLYYFDYWGQGTT LTVSS Nucleic acid sequence (SEQ ID NO: 90): CAGGTCCAGCTGCAGCAGTCTGGACCTGAATTGGTGAAGCCTGGGGCTTCAGTG AGGATATCCTGCAAGACTTCTGGCTTCACCTTCACAAACTACTATATACACTGGG TGATACAGAGGCCTGGACAGGGACTTGAGTGGATTGGTTGGATTTCTCCTGAAAA TGGTAATACTAAATACAATGAAAACTTCCAGGACAAGGCCACACTGACTGCAGA CATATCGTCCAGCACAGCCTACATGCACCTCAGCAGCCTGACCTCTGAGGACTCT GCGGTCTATTTCTGTGCAAGAGATGGGTATTCCCTTTACTACTTTGACTACTGGGG CCAAGGCACCACTCTCACAGTCTCCTCA WBP3311_2.383.47—VK Amino acid sequence (SEQ ID NO: 91): SPSSLTVSAGEKVTMSCKSSS 2SLLNSRTRKNYLAWYQQKPGQSPKLLIYXLA SIRVSGVPDRFTGSGSGTTFTLTISGVQAEDLAVYYCTSQSHTLRTFGGGTKLEIK c acid sequence (SEQ ID NO: 92): GACATTGTGATGTCACAGTCTCCATCCTCCCTGACTGTGTCAGCAGGAGAGAAGG TCACTATGAGCTGCAAATCCAGTCAGAGTCTGCTCAACAGTAGAACCCGAAAGA ACTACTTGGCTTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAGCTACTGATCTA CTGGGCATCCATTAGGGTATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCT GGGACAACTTTCACTCTCACCATCAGCGGTGTGCAGGCTGAAGACCTGGCAGTTT ATTATTGCACGCAATCTCATACTCTTCGGACGTTCGGTGGAGGCACCAAGCTGGA AATCAAA [0001 19] WBP331 1_2.400.5-VH Amino acid sequence (SEQ ID NO: 93): QVQLQQSGPELVNPGASVKISCKASGYSFTNYYLHWVKQRPGQGLEWIGWIFPESD NTKYNEKLKGKATLTADTSSDTAYMHLSSLTFEDSAVYFCARDSVGNYFFDFWGQG TTLTVSS Nucleic acid sequence (SEQ ID NO: 94): CAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAATCCTGGGGCTTCAGTGA AGATATCCTGCAAGGCTTCTGGCTACAGTTTCACAAACTACTATTTACACTGGGT GAAACAGAGGCCTGGACAGGGACTTGAGTGGATTGGATGGATTTTTCCTGAAAG TGATAATACCAAGTACAATGAGAAATTGAAGGGCAAGGCCACACTGACGGCAGA CTCCGATACAGCCTACATGCACCTCAGCAGCCTGACATTTGAGGACTCT GCAGTCTATTTCTGTGCAAGAGACTCCGTTGGAAACTACTTCTTTGACTTCTGGG GCCAAGGCACCACTCTCACAGTCTCCTCA 0] WBP3311_2.400.5-VK Amino acid sequence (SEQ ID NO: 95): DIVMSQSPSSLAVSAGEKVTMRCKSSQSLVNNRTRKNYLAWYQQKPGQPPKLLIYE ASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCAQSFILRTFGGGTKLElK Nucleic acid sequence (SEQ ID NO: 96): GTGATGTCACAGTCTCCATCCTCCCTGGCTGTGTCAGCGGGAGAGAAGG TCACTATGAGGTGCAAATCCAGTCAGAGTCTGGTCAACAATAGAACCCGAAAGA ACTACTTGGCATGGTACCAGCAGAAACCAGGGCAGCCTCCTAAACTATTGATCTA CTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCT GGGACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTT ATTACTGCGCGCAATCTTTTATTCTTCGGACGTTCGGTGGAGGCACCAAACTGGA AATCAAA 1_2.482.5-VH Amino acid sequence (SEQ ID NO: 97): QVQLQQSGPELVKPGSSVKISCKPSGYTFTTYYlHWVKQRPGQGLEWIGWIFPGSDNI KYNENFKDKATLTADTSSSTAYMQLSSLTSEDSAVYFCARDSVSRYYFDYWGQGTI LTVSS Nucleic acid sequence (SEQ ID NO: 98): CAGGTTCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAACCTGGGTCTTCAGTGA AGATATCCTGCAAACCTTCTGGCTACACCTTCACAACTTACTATATACATTGGGT GAAGCAGAGGCCTGGACAGGGACTTGAGTGGATTGGATGGATTTTTCCTGGAAG TGATAATATTAAATACAATGAGAATTTCAAGGACAAGGCCACACTGACGGCAGA CACATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAAGACTCT GCAGTCTATTTCTGTGCAAGAGACTCCGTCAGTAGGTACTACTTTGACTACTGGG GCCAAGGCACCATTCTCACAGTTTCTTCA WBP3311_2.482.5-VK Amino acid sequence (SEQ ID NO: 99): SPSSLAVSAGEKVTMSCKSSS 2SLVNDRTRKNYLAWYQQKPGLSPKLLIYE GVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCAS QSFILRTFGGGTKLEIK Nucleic acid sequence (SEQ ID NO: 100): GACATTGTGATGTCACAGTCTCCATCCTCCCTGGCTGTGTCAGCAGGAGAGAAGG TCACTATGAGCTGCAAATCCAGTCAGAGTCTGGTCAATGATAGAACCCGAAAAA ACTACTTGGCTTGGTACCAGCAGAAACCAGGGCTGTCTCCTAAACTGCTGATCTA CTGGGCTTCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCT GGGACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCTGTTT ATTACTGCGCGCAATCTTTTATTCTTCGGACGTTCGGTGGAGGCACCAAGCTGGA AATCAAA WBP331_2.488.33-VH Amino acid sequence (SEQ ID NO: 101): QVQLQQSGPELVKPGTSVKISCKASGFSFTNYYIHWVKQRPGQGPEWIGWIFPGTVN TKYNEKFKGKATLTADTSSNTAFMQLSSLTSADSAVYFCARDSVGI Y Y FDFWGLGTT LTVSS c acid sequence (SEQ ID NO: 102): CAGGTCCAGCTGCAACAGTCTGGACCTGAACTGGTGAAACCTGGGACTTCAGTG AAGATATCCTGCAAGGCTTCTGGCTTCAGCTTCACAAACTACTATATACACTGGG TGAAGCAGAGGCCTGGACAGGGACCTGAGTGGATTGGATGGATTTTTCCTGGAA CTGTTAATACTAAGTACAATGAGAAGTTCAAGGGTAAGGCCACACTGACGGCAG ACACATCCTCCAATACAGCCTTCATGCAGCTCAGCAGCCTGACTTCTGCGGACTC TGCAGTCTATTTCTGTGCAAGAGACTCCGTTGGTATCTACTACTTTGACTTCTGGG GCCTAGGCACCACTCTCACAGTCTCCTCA WBP331_2.488.33-VK Amino acid sequence (SEQ ID NO: 103): DIVMSQSPSSLAVSAGEKVTVSCKSSS 2SLLNNRTRKNYLAWYQQKPGQSPKLLIY? ASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCTEQSFILRTFGGGTKLEIK Nucleic acid sequence (SEQ ID NO: 104): GACATTGTGATGTCACAGTCTCCATCCTCCCTGGCTGTGTCAGCAGGAGAGAAGG TCACTGTGAGTTGCAAATCCAGTCAGAGTCTGCTCAACAATAGAACCCGAAAAA ACTACTTGGCTTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTACTAATCTA CTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCT GATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTT ATTACTGCACGCAATCTTTTATTCTTCGGACGTTCGGTGGAGGCACCAAGCTGGA GATCAAA WBP3311_2.615.8-VH Amino acid sequence (SEQ ID NO: 105): QVQLQQSGPELVKPGTSMKISCKASGYSFTDFYTHWVRQRPGQGLEWIGWIFPGSDN IKYNEKFKGKATLTADTSSSTAYMQLSSLTSEDSAVYFCARDSVSVYYFDYWGQGT TLTVSS Nucleic acid sequence (SEQ ID NO: 106): CAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTAAAACCTGGGACTTCAATG AAAATATCCTGCAAGGCTTCTGGCTACAGTTTCACAGACTTCTATACACACTGGG TGAGGCAGAGGCCTGGACAGGGACTTGAGTGGATTGGATGGATTTTTCCTGGAA GTGATAATATTAAATACAATGAGAAGTTCAAGGGCAAGGCCACACTGACGGCAG ACACATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTC TGCAGTCTATTTCTGTGCAAGAGACTCCGTTAGTGTCTACTACTTTGACTATTGGG GCCAAGGCACCACTCTCACAGTCTCCTCA WBP3311_2.615.8-VK Amino acid sequence (SEQ ID NO: 107): DIVMSQSPSSLAVTAGEKVTMSCKSSQSLLNIRTRKNYLAWYQQKPGQSPKLLIYW_A STRDSGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCTQSFILRTFGGGTKLEIK Nucleic acid sequence (SEQ ID NO: 108): GACATCGTGATGTCACAGTCTCCATCCTCCCTGGCTGTGACAGCAGGAGAGAAG GTCACTATGAGCTGCAAATCCAGTCAGAGTCTGCTCAACATTAGAACCCGAAAG AACTACTTGGCTTGGTACCAACAGAAACCAGGGCAGTCTCCTAAACTGCTGATCT ACTGGGCATCCACTAGGGACTCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATC TGGGACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTT TATTACTGCACGCAATCTTTTATTCTTCGGACGTTCGGTGGAGGCACCAAGCTGG AAATCAAA WBP3311_2.844.8-VH Amino acid sequence (SEQ ID NO: 109): QVQLQQSGPELVKPGASVRISCKASGFAFTDYYIHWVKQRPGQGLEWIGWISPGNVN TKYNENFKGRATLTADLSSSTAYMQLSSLTSEDSAVYFCARDGYSLYYFDYWGQGT TLTVSS Nucleic acid sequence (SEQ ID NO: 110): CAGGTCCAGCTGCAGCAGTCTGGACCTGAATTGGTGAAGCCTGGGGCTTCCGTGA GGATATCCTGCAAGGCTTCTGGCTTCGCCTTCACAGACTACTATATACACTGGGT GAAGCAGAGGCCTGGACAGGGTCTTGAGTGGATTGGATGGATTTCTCCTGGAAA TGTTAATACTAAATACAATGAAAACTTCAAGGGCAGGGCCACACTGACTGCAGA CCTATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACCTCTGAGGACTCT TATTTCTGTGCAAGAGATGGATATTCCCTGTATTACTTTGACTACTGGG GCCAAGGCACCACTCTCACAGTCTCCTCA WBP3311_2.844.8-VK Amino acid sequence (SEQ ID NO: 111): DIVMSQSPSSLTVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYW_A STRESGVPDRFTGSGSGTAFTLTISGVQAEDLAVYFCTQSHTLRTFGGGTKLEK Nucleic acid sequence (SEQ ID NO: 112): GACATTGTGATGTCACAGTCTCCATCCTCCCTGACTGTGTCAGCAGGAGAGAAGG TCACTATGAGCTGCAAATCCAGTCAGAGTCTGCTCAACAGTAGAACCCGAAAGA ACTACTTGGCTTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAGCTACTAATCTA CTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCT GGGACAGCTTTCACTCTCACCATCAGCGGTGTGCAGGCTGAAGACCTGGCAGTTT ATTTCTGCACGCAATCTCATACTCTTCGGACGTTCGGTGGAGGCACCAAGCTGGA AATCAAA CDRs are known to be responsible for antigen binding, however, it has been found that not all of the 6 CDRs are indispensable or unchangeable. In other words, it is possible to replace or change or modify one or more CDRs in anti-CD3 epsilon dy WBP3311_2.166.48, WBP3311_2.306.4, WBP3311_2.383,47, WBP3311_2.400.5, WBP3311_2.482.5, WBP331_2.488.33, 1_2.615.8, or WBP3311_2.844.8, yet substantially retain the speci?c g af?nity to CD3epsilon.
In certain embodiments, the anti-CD3 epsilon antibodies and the antigen-binding fragments provided herein comprise a heavy chain CDR3 sequence of one of the anti- CD3epsilon dies WBP3311_2.166.48, WBP3311_2.306.4, WBP33 l l_2.383 .47, WBP3311_2.400.5, WBP3311_2.482.5, WBP331_2.488.33, WBP3311_2.615.8, and 1_2.844.8. In certain embodiments, the anti-CD3epsilon antibodies and the antigen- binding nts provided herein comprise a heavy chain CDR3 sequence selected from the group consisting of SEQ ID NOs: 5, 11, 17, 23, 29, 35, 41, and 47. Heavy chain CDR3 regions are located at the center of the antigen-binding site, and therefore are believed to make the most contact with antigen and provide the most free energy to the af?nity of antibody to antigen. It is also believed that the heavy chain CDR3 is by far the most e CDR of the n- binding site in terms of length, amino acid composition and conformation by multiple diversi?cation mechanisms (Tonegawa S. Nature. 302:575-81). The diversity in the heavy chain CDR3 is suf?cient to produce most antibody speci?cities (Xu JL, Davis MM. Immunity. 13:37—45) as well as desirable antigen-binding af?nity (Schier R, etc. J Mol Biol. 263 :551—67).
In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein comprise suitable framework region (FR) sequences, as long as the dies and antigen-binding fragments thereof can speci?cally bind to CD3epsilon. The CDR sequences provided in Table 1 are obtained from mouse antibodies, but they can be grafted to any suitable FR sequences of any suitable species such as mouse, human, rat, , among others, using le s known in the art such as recombinant techniques.
In certain embodiments, the dies and anti nding fragments thereof provided herein are humanized. A humanized antibody or antigen-binding fragment is desirable in its reduced immunogenicity in human. A humanized antibody is chimeric in its variable regions, as non-human CDR sequences are d to human or substantially human FR ces. Humanization of an antibody or antigen-binding fragment can be essentially performed by substituting the non-human (such as murine) CDR genes for the corresponding human CDR genes in a human immunoglobulin gene (see, for example, Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323—327; Verhoeyen et al. (1988) Science 239: 536).
Suitable human heavy chain and light chain variable domains can be selected to achieve this purpose using methods known in the art. In an illustrative example, "best-?t" approach can be used, where a non-human (e.g. rodent) antibody variable domain sequence is ed or BLASTed against a database of known human variable domain sequences, and the human sequence closest to the non-human query sequence is identi?ed and used as the human scaffold for grafting the non-human CDR sequences (see, for e, Sims et al, (1993) J.
Immunol. 96; Chothia et al. (1987) J. Mot. Biol. 1). Alternatively, a framework derived from the consensus sequence of all human antibodies may be used for the grafting of the non-human CDRs (see, for example, Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 894285, Presta et al. (1993) J. Immunol.,151:2623).
In certain embodiments, the humanized antibodies or antigen-binding fragments provided herein are composed of substantially all human sequences except for the CDR ces which are non-human. In some embodiments, the variable region FRs, and constant regions if present, are entirely or substantially from human immunoglobulin sequences. The human FR sequences and human constant region sequences may be derived different human immunoglobulin genes, for e, FR sequences d from one human dy and constant region from another human antibody. In some embodiments, the humanized antibody or antigen-binding fragment se human FRl-4 and human JH and JK.
In certain embodiments, the humanized antibodies and antigen-binding fragment f provided herein se one or more FR sequences of WBP3311_2.166.48-zl or WBP3311_2.306.4—zl. Table 2 below shows the FR sequences of WBP3311_2.166.48—zl or WBP3311_2.306.4-z1. The native mouse FR sequences are also listed in Table 2. The heavy chain and light chain variable region sequences are also provided below.
"WBP3311_2.166.48—zl" as used herein refers to a humanized antibody based on WBP33 l l_2. 166.48 that comprises a heavy chain variable region of SEQ ID NO: 113, and a kappa light chain variable region of SEQ ID NO: 115. WBP3311_2.166.48—z1 has comparable af?nity to the antigen as compared with its parent antibody WBP3311_2.166.48. 7] "WBP3311_2.306.4-zl" as used herein refers to a humanized antibody based on WBP3311_2.306.4 that comprises a heavy chain variable region of SEQ ID NO: 117, and a kappa light chain variable region of SEQ ID NO: 119. 1_2.306.4-zl has comparable af?nity to the n as compared with its parent antibody WBP3311_2.306.4.
Table 2.
————— SEQ ID NO: 49 SEQ ID NO: 51 SEQ ID NO: 53 SEQ ID NO: 55 WBP3311 QVQLQQSGPEL KATLTADTSSSTA _2.166.48- VKPGASVKIAC ‘LYEV?gRPGQG YMQLSSLTSEDS ‘SNSGQGTTLTV VH KAS AVYFCAI SEQ ID NO 57 SEQ ID NO 59 SEQ ID NO 61 SEQ ID NO 63 WBP3311 QVQLVQSGAE RVTITADKSTST —f‘{f§‘4s' VKKPGSSVKVS AYMELSSLRSED z ' ?‘??éPGQG ‘SNSGQGTLVTV CKAS TAVWCAI ————SEQ ID NO50 SEQ ID NO 52 SEQ ID NO 54 SEQ 11) NO 56 WBP3311 DIVMSQSPSSL GVPDRFTGSGSG {721.366.48- AVSAGEKVTM gingngQs TDFTLTINSVQA FGGGTKLEIK EDLAVYYC SEQ ID NO: 58 SEQ ID NO: 60 SEQ ID NO: 62 SEQ ID NO: 64 WBP3311 DIVMTQSPDSL GVPDRFSGSGSG _2.166.48- AVSLGERATIN QP TDFTLTISSLQAE FGGGTKVEIK zl-VK C DVAVYYC SEQ ID NO: 65 SEQ ID NO: 67 SEQ ID NO: 69 SEQ ID NO: 71 WBP3311 QVQLQQSGPEL RATVTADLSSST _2.306.4- VKPGASVRLSC ‘L‘ggigRPGQG AmQLSSLTSED ‘SVSGQGTTLTV VH KAS SAVYFCAR SEQ ID NO: 73 SEQ ID NO: 75 SEQ ID NO: 77 —SEQ ID NO: 79 WBP3311 QVQLVQSGAE DKSTST —f:3"?§°4' VKKPGSSVKV ?‘??éPGQG AYMELSSLRSED z GTLVTV SCKAS TAVYYCAR SEQ ID NO 66 SEQ ID NO 68 SEQ ID NO 70 —SEQ ID NO: 72 WBP3311 DIVMSQSPSSLT GVPDRFTGSGSG 4' VSAGEKVTMS EEEC?ggPGQS TAFTLTISGVQAE FGGGTKLEIK c DLAVYFC SEQ ID NO: 74 SEQ ID NO: 76 SEQ ID NO: 78 —SEQ ID NO: 80 WBP3311 DIVMTQSPDSL GVPDRFSGSGSG _2.306.4- AVSLGERATIN giggprQP ISSLQAE FGGGTKVElK zl-VK c C WBP3311_2.166.48-zl-VH Amino acid sequence (SEQ ID NO: 113): QVQLVQSGAEVKKPGSSVKVSCKASGYSFTTYYIHWVRQAPGQGLEWMGWIFPGN DNIKYSEKFKGRVTITADKSTSTAWELSSLRSEDTAVYYCAIDSVSIYYFDYWGQG TLVTVSS Nucleic acid sequence (SEQ ID NO: 114): CAGGTGCAACTCGTGCAGTCTGGAGCTGAAGTGAAGAAGCCTGGGTCTTCAGTC AAGGTCAGTTGCAAGGCCAGTGGGTATTCCTTCACTACCTACTACATCCACTGGG TGCGGCAGGCACCAGGACAGGGGCTTGAGTGGATGGGCTGGATCTTTCCCGGCA ACGATAATATTAAGTACAGCGAGAAGTTCAAAGGGAGGGTCACCATTACCGCCG ACAAATCCACTTCCACAGCCTACATGGAGTTGAGCAGCCTGAGATCCGAGGATA TGTACTACTGTGCCATTGACAGCGTGTCCATCTACTACTTTGACTACTG GGGCCAGGGCACACTGGTCACAGTGAGCAGC WBP3311_2.166.48—zl-VK Amino acid sequence (SEQ ID NO: 115): DIVMTQSPDSLAVSLGERATINCKSsg 2SLLNSRTRKNYLAWYQQKPGQPPKLLIYW_A STRKSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTS QSFILRTFGGGTKVEIK Nucleic acid sequence (SEQ ID NO: 116): GACATCGTCATGACCCAGTCCCCAGACTCTTTGGCAGTGTCTCTCGGGGAAAGAG CTACCATCAACTGCAAGAGCAGCCAGTCCCTTCTGAACAGCAGGACCAGGAAGA ATTACCTCGCCTGGTACCAACAGAAGCCCGGACAGCCTCCTAAGCTCCTGATCTA CTGGGCCTCAACCCGGAAGAGTGGAGTGCCCGATCGCTTTAGCGGGAGCGGCTC CGGGACAGATTTCACACTGACAATTTCCTCCCTGCAGGCCGAGGACGTCGCCGTG TATTACTGTACTCAGAGCTTCATTCTGCGGACATTTGGCGGCGGGACTAAAGTGG AGATTAAG WBP3311_2.306.4-zl-VH Amino acid sequence (SEQ ID NO: 117): QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWGQ GTLVTVSS Nucleic acid sequence (SEQ ID NO: 118): CAGGTGCAGCTTGTGCAGTCTGGGGCAGAAGTGAAGAAGCCTGGGTCTAGTGTC AAGGTGTCATGCAAGGCTAGCGGGTTCGCCTTTACTGACTACTACATCCACTGGG TGCGGCAGGCTCCCGGACAAGGGTTGGAGTGGATGGGATGGATCTCCCCAGGCA ATGTCAACACAAAGTACAACGAGAACTTCAAAGGCCGCGTCACCATTACCGCCG ACAAGAGCACCTCCACAGCCTACATGGAGCTGTCCAGCCTCAGAAGCGAGGACA CTGCCGTCTACTACTGTGCCAGGGATGGGTACTCCCTGTATTACTTTGATTACTGG GGCCAGGGCACACTGGTGACAGTGAGCTCC WBP3311_2.306.4-z1-VK Amino acid sequence (SEQ ID NO: 119): DIVMTQSPDSLAVSLGERATINCKSSS 2SLLNSRTRKNYLAWYQQKPGQPPKLLIYW_A STRS 2SGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTS TFGGGTKVEIK Nucleic acid sequence (SEQ ID NO: 120): GATATCGTGATGACCCAGAGCCCAGACTCCCTTGCTGTCTCCCTCGGCGAAAGAG TCAACTGCAAGAGCTCCCAAAGCCTGCTGAACTCCAGGACCAGGAAGA TGGCCTGGTATCAGCAGAAGCCCGGCCAGCCTCCTAAGCTGCTCATCTA CTGGGCCTCCACCCGGCAGTCTGGGGTGCCCGATCGGTTTAGTGGATCTGGGAGC GGGACAGACTTCACATTGACAATTAGCTCACTGCAGGCCGAGGACGTGGCCGTC TACTACTGTACTCAGAGCCACACTCTCCGCACATTCGGCGGAGGGACTAAAGTGG AGATTAAG The two ary humanized anti-CD3epsilon antibodies WBP3311_2.166.48-zl or WBP3311_2.306.4-zl both retained the speci?c binding af?nity to CD3 -expressing cell (e. g.
CD4 T cell), and are at least comparable to, or even better than, the parent mouse dies in that aspect. The two exemplary humanized antibodies both retained their functional interaction with CD3-expressing cell, in that both can activate human T cells and trigger cytokine release of ha and IFNgamma.
In some embodiments, the FR regions derived from human may comprise the same amino acid sequence as the human immunoglobulin from which it is derived. In some embodiments, one or more amino acid residues of the human FR are substituted with the corresponding residues from the parent non-human antibody. This may be desirable in certain ments to make the humanized dy or its fragment closely approximate the non- human parent antibody structure. In certain embodiments, the humanized antibody or antigen- binding fragment provided herein comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue tutions in each of the human FR ces, or no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue substitutions in all the FRs of a heavy or a light chain variable domain. In some embodiments, such change in amino acid residue could be present in heavy chain FR regions only, in light chain FR regions only, or in both chains.
In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein comprise a heavy chain variable domain sequence selected from the group consisting of SEQ ID NO: 81, SEQ ID NO: 85, SEQ ID NO: 89, SEQ ID NO: 93, SEQ ID NO: 97, SEQ ID NO: 101, SEQ ID NO: 105, SEQ ID NO: 109, SEQ IDNO: 113, SEQ ID NO: 117.
In certain embodiments, the antibodies and antigen-binding nts thereof provided herein se a light chain variable domain sequence selected from the group consisting of SEQ ID NO: 83, SEQ ID NO: 87, SEQ ID NO: 91, SEQ ID NO: 95, SEQ ID NO: 99, SEQ ID NO: 103, SEQ ID NO: 107, SEQ ID NO: 111, SEQ ID NO: 115, SEQ ID NO: 119.
In some embodiments, the anti-CD3epsilon antibodies and the antigen-binding fragments provided herein comprise all or a portion of the heavy chain variable domain and/or all or a portion of the light chain variable domain. In one embodiment, the anti-CD3epsilon antibodies and the antigen-binding fragments provided herein is a single domain dy which consists of all or a portion of the heavy chain variable domain provided . More information of such a single domain antibody is available in the art (see, e.g., US. Pat. No. 6,248,516).
In certain embodiments, the anti-CD3epsilon antibodies and the fragments thereof provided herein further comprise an immunoglobulin constant . In some embodiments, an immunoglobulin constant region comprises a heavy chain and/or a light chain constant region. The heavy chain constant region comprises CH1, hinge, and/or CH2-CH3 regions. In certain embodiments, the heavy chain constant region comprises an Fc . In n embodiments, the light chain constant region comprises CK.
In some embodiments, the anti-CD3epsilon dies and antigen-binding fragments thereof have a constant region of IgG1 or IgG2a e, which has reduced or depleted effector function such as ADCC or CDC, which can be evaluated using various assays such as Fc receptor g assay, Clq binding assay, and cell lysis assay.
Binding af?nity of the antibody and antigen-binding fragment provided herein can be represented by K1) value, which represents the ratio of dissociation rate to association rate (keg/ken) when the binding between the antigen and antigen-binding molecule reaches equilibrium. The antigen-binding af?nity (eg. KD) can be appropriately determined using suitable methods known in the art, including, for e, flow cytometry assay. In some embodiments, binding of the antibody to the antigen at different concentrations can be determined by ?ow cytometry, the determined mean cence ity (MFI) can be ?rstly plotted against antibody concentration, KD value can then be calculated by ?tting the dependence of speci?c binding ?uorescence intensity (Y) and the concentration of antibodies (X) into the one site saturation on: Y=Bmax*X/(KD + X) using Prism version 5 Pad Software, San Diego, CA), wherein Bmax refers to the maximum speci?c binding of the tested antibody to the antigen. 0] In certain embodiments, the anti-CD3epsilon antibodies and antigen-binding nts thereof provided herein are capable of speci?cally g to human ilon expressed on a cell surface, or a recombinant human CD3epsilon. CD3epsilon is a receptor expressed on cell. A recombinant CD3epsilon is soluble CD3epsilon which is recombinantly expressed and is not associated with a cell membrane. A recombinant CD3epsilon can be prepared by various recombinant technologies. In one example, the CD3 epsilon DNA sequence encoding the extracellular domain of human CD3 epsilon 0724.1) (Metl- Asp126) can be fused with a polyhistidine tag at the C-terminus in an expression vector, and then transfected and expressed in 293E cells and puri?ed by Ni-Af?nity chromatography.
In some embodiments, the anti-CD3epsilon antibodies and antigen-binding fragments thereof ed herein are capable of speci?cally binding to human CD3epsilon expressed on surface of cells with a binding af?nity (KD) of no more than 5x10'9M, no more than 4xlO'9M, no more than 3x10'9M, no more than M, no more than 10'9M, no more than 5xlO'10M, no more than 4xlO'10M, no more than 3xlO'10M, no more than 2xlO'10M, no more than lO‘lOM, no more than 5x10‘11 M, or no more than 4xlO'11 M, no more than 3xlO'11 M, or no more than 2xlO'11 M, or no more than 10'11 M as measured by ?ow cytometry assay.
In certain embodiments, the anti-CD3epsilon antibodies and antigen-binding fragments thereof provided herein cross-react with Cynomolgus monkey CD3epsilon, for example, Cynomolgus monkey CD3epsilon expressed on a cell surface, or a soluble recombinant Cynomolgus monkey CD3 n.
Binding of the antibodies to recombinant CD3epsilon or CD3epsilon expressed on surface of cells can also be represented by "halfmaximal effective concentration" (ECso) value, which refers to the concentration of an antibody where 50% of its maximal effect (e.g., binding or inhibition etc.) is observed. The ECso value can be measured by methods known in the art, for example, sandwich assay such as ELISA, Western Blot, ?ow cytometry assay, and other binding assay. In certain embodiments, the antibodies and the fragments thereof ed herein speci?cally bind to recombinant human CD3epsilon at an ECso (i.e. 50% binding tration) of no more than 0.01 nM, no more than 0.02 nM, no more than 0.03 nM, no more than 0.04 nM, no more than 0.05 nM, no more than 0.06 nM, no more than 0.07 nM or no more than 0.08 nM by ELISA. In certain embodiments, the antibodies and the fragments thereof provided herein speci?cally bind to human CD3epsilon expressed on surface of cells at an ECso of no more than 0.5 nM, no more than 0.6 nM, no more than 0.7 nM, no more than 0.8 nM, no more than 0.9 nM, no more than 1 nM, no more than 2 nM no more than 3 nM , , no more than 4 nM no more than 5 nM no more than 6 nM, no more than 7 nM, no more , , than 8 nM, no more than 9 nM or no more than 10 nM by ?ow cytometry assay.
In certain embodiments, the dies and antigen-binding nts thereof bind to Cynomolgus monkey CD3epsilon with a binding af?nity similar to that of human CD3epsilon. For example, binding of the exemplary dies WBP33ll_2.l66.48, WBP33ll_2.306.4, l_2.383.47, WBP33ll_2.400.5, WBP33ll_2.482.5, WBP33ll_2.488.33, WBP33ll_2.615.8, WBP33ll_2.844.8 to Cynomolgus monkey CD3epsilon is at a similar y or ECso value to that of human CD3epsilon.
In certain embodiments, the antibodies and the fragments thereof provided herein speci?cally bind to recombinant lgus monkey CD3epsilon with an EC50 of no more than 0.001 nM, no more than 0.005 nM, no more than 0.01 nM, no more than 0.02 nM, no more than 0.03 nM, no more than 0.04 nM, or no more than 0.05 nM by ELISA.
In certain ments, the antibodies and the fragments f ed herein have a speci?c binding af?nity to human CD3epsilon which is suf?cient to provide for diagnostic and/or therapeutic use. A number of therapeutic strategies modulate T cell immunity by targeting TCR signaling, ularly by anti-human CD3 monoclonal antibodies that are clinically used.
The antibodies or antigen-binding fragments thereof provided herein can be a monoclonal antibody, polyclonal antibody, humanized antibody, chimeric antibody, recombinant antibody, bispeci?c antibody, labeled antibody, bivalent antibody, or anti- idiotypic antibody. A recombinant antibody is an antibody prepared in vitro using recombinant s rather than in animals.
Antibody Variants The present disclosure also encompasses various variants of the antibodies and antigen—binding fragments thereof provided herein. In certain embodiments, the present disclosure encompasses various types of variants of an exemplary antibody provided , i.e., WBP33 l l_2. 166.48, WBP3311_2.306.4, WBP3311_2.383.47, WBP3311_2.400.5, WBP33 l l_2.482.5, WBP33 l_2.488.33, WBP33 l l_2.615.8, and WBP33 l l_2.844.8.
In certain ments, the dy variants comprise one or more modi?cations or substitutions in one or more CDR sequences as provided in Table 1, one or more FR sequences provided in Table 2, the heavy or light chain variable region sequences provided herein, and/or the constant region (e. g. Fc region). Such variants retain speci?c binding af?nity to CD3 epsilon of their parent antibodies, but have one or more desirable properties conferred by the modi?cation(s) or substitution(s). For e, the antibody variants may have improved antigen-binding af?nity, ed glycosylation pattern, d risk of glycosylation, reduced deamination, reduced or depleted effector function(s), improved FcRn receptor binding, increased pharrnacokinetic ife, pH sensitivity, and/or compatibility to conjugation (e.g. one or more introduced cysteine residues).
The parent antibody sequence may be screened to identify suitable or preferred residues to be d or tuted, using methods known in the art, for example "alanine scanning mutagenesis" (see, for example, Cunningham and Wells (1989) Science, 244:1081- 1085). , target es (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) can be identi?ed and ed by a neutral or negatively charged amino acid (e.g., alanine or polyalanine), and the modi?ed antibodies are produced and screened for the interested ty.
If tution at a particular amino acid location demonstrates an sted functional change, then the position can be identi?ed as a potential e for modi?cation or substitution. The potential residues may be further assessed by substituting with a different type of residue (e. g. cysteine residue, positively d residue, etc).
Affinity variant Af?nity variant may contain modi?cations or substitutions in one or more CDR sequences as provided in Table 1, one or more FR sequences provided in Table 2, or the heavy or light chain variable region sequences provided herein. The af?nity variants retain speci?c binding af?nity to CD3epsilon of the parent antibody, or even have improved CD3epsilon speci?c g af?nity over the parent antibody. In certain embodiments, at least one (or all) of the substitution(s) in the CDR sequences, FR sequences, or variable region sequences ses a conservative substitution.
A skilled n will understand that in the CDR ces and FR sequences provided in Table 1 and Table 2, one or more amino acid residues may be substituted yet the ing antibody or antigen-binding fragment still retain the binding af?nity to CD3 epsilon, or even have an improved binding af?nity. Various methods known in the art can be used to achieve this purpose. For example, a library of antibody variants (such as Fab or scFv variants) can be generated and expressed with phage display logy, and then screened for the binding af?nity to human CD3epsilon. For another e, computer re can be used to virtually te the binding of the antibodies to human CD3 epsilon, and identify the amino acid residues on the antibodies which form the binding ace. Such residues may be either avoided in the substitution so as to prevent ion in binding af?nity, or targeted for substitution to provide for a stronger binding.
In certain embodiments, the humanized antibody or antigen-binding fragment ed herein comprises one or more amino acid residue substitutions in one or more CDR sequences, and/or one or more FR sequences. In certain embodiments, an af?nity variant comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitutions in the CDR sequences and/or FR sequences in total.
In n embodiments, the anti-CD3epsilon antibodies and antigen-binding fragments thereof comprise 1, 2, or 3 CDR sequences having at least 80% (eg. at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to that (or those) listed in Table 1, and in the meantime retain the binding af?nity to CD3 epsilon at a level similar to or even higher than its parent antibody.
In certain embodiments, the anti-CD3 epsilon antibodies and antigen-binding fragments thereof comprise one or more FR sequences having at least 80% (e. g. at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to that (or those) listed in Table 2, and in the meantime retain the binding af?nity to CD3 epsilon at a level similar to or even higher than its parent antibody.
In certain embodiments, the anti-CD3epsilon antibodies and antigen-binding fragments thereof comprise one or more le region sequences having at least 80% (e. g. at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to that (or those) listed in SEQ ID NO: 81, SEQ ID NO: 85, SEQ ID NO: 89, SEQ ID NO: 93, SEQ ID NO: 97, SEQ ID NO: 101, SEQ ID NO: 105, SEQ ID NO: 109, SEQ ID NO: 113, SEQ ID NO: 117, SEQ ID NO: 83, SEQ ID NO: 87, SEQ ID NO: 91, SEQ ID NO: 95, SEQ ID NO: 99, SEQ ID NO: 103, SEQ ID NO: 107, SEQ ID NO: 111, SEQ ID NO: 115, SEQ ID NO: 119, and in the meantime retain the binding af?nity to CD3epsilon at a level similar to or even higher than its parent antibody. In some embodiments, a total of 1 to 10 amino acids have been substituted, inserted, or deleted in a sequence selected from SEQ ID NO: 81, SEQ ID NO: 85, SEQ ID NO: 89, SEQ ID NO: 93, SEQ ID NO: 97, SEQ ID NO: 101, SEQ ID NO: 105, SEQ ID NO: 109, SEQ ID NO: 113, SEQ ID NO: 117, SEQ ID NO: 83, SEQ IDNO: 87, SEQ ID NO: 91, SEQ ID NO: 95, SEQ ID NO: 99, SEQ ID NO: 103, SEQ ID NO: 107, SEQ ID NO: 111, SEQ ID NO: 115, and SEQ ID NO: 119. In some embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRS (i.e., in the FRS).
Glycosylation variant 0] The anti-CD3 epsilon antibodies and antigen-binding fragments provided herein also encompass a glycosylation variant, which can be ed to either increase or decrease the extent of glycosylation of the antibody or antigen g fragment.
The antibody or antigen g nt thereof may comprise one or more amino acid residues with a side chain to which a carbohydrate moiety (e. g. an accharide structure) can be attached. Glycosylation of antibodies is typically either ed or ed.
N—linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue, for example, an asparagine residue in a tripeptide sequence such as gine-X- serine and asparagine—X-threonine, where X is any amino acid except proline. O-linked glycosylation refers to the attachment of one of the sugars N—aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly to serine or threonine. Removal of a native ylation site can be conveniently accomplished, for example, by altering the amino acid sequence such that one of the above-described tripeptide sequences (for N—linked glycosylation sites) or serine or threonine residues (for O-linked glycosylation sites) present in the sequence is substituted. A new glycosylation site can be created in a similar way by introducing such a tripeptide sequence or serine or threonine residue.
Cysteine-engineered variant The anti-CD3 epsilon dies and antigen-binding fragments ed herein also encompass a cysteine—engineered variant, which comprises one or more introduced free cysteine amino acid residues.
A free cysteine residue is one which is not part of a disul?de bridge. A cysteine- ered variant is useful for conjugation with for example, a cytotoxic and/or imaging compound, a label, or a radioisoptype among others, at the site of the ered cysteine, through for example a maleimide or haloacetyl. Methods for engineering antibodies or antigen- g fragments to introduce free ne residues are known in the art, see, for example, W02006/034488.
Fc Variant The anti-CD3 epsilon antibodies and antigen-binding nts provided herein also encompass an Fc variant, which comprises one or more amino acid residue modi?cations or substitutions at its Fc region and/or hinge .
In certain embodiments, the anti—CD3epsilon antibodies or antigen-binding fragments comprise one or more amino acid substitution(s) that improves pH-dependent binding to neonatal Fc receptor (FcRn). Such a variant can have an extended pharmacokinetic half-life, as it binds to FcRn at acidic pH which allows it to escape from degradation in the lysosome and then be translocated and released out of the cell. s of ering an antibody and antigen-binding fragment thereof to improve binding af?nity with FcRn are well- known in the art, see, for example, Vaughn, D. et al, Structure, 6(1): 63-73, 1998; Kontermann, R. et al, Antibody Engineering, Volume 1, r 27: Engineering of the Fc region for improved PK, published by Springer, 2010; Yeung, Y. et al, Cancer Research, 70: 3269-3277 (2010); and Hinton, P. et al, J. Immunology, 176:346-356 (2006).
In certain ments, the anti-CD3epsilon antibodies or antigen-binding fragments comprise one or more amino acid substitution(s) that alters the antibody-dependent cellular cytotoxicity (ADCC). Certain amino acid residues at CH2 domain of the Fc region can be tuted to provide for ed ADCC activity. Alternatively or additionally, carbohydrate structures on the antibody can be changed to enhance ADCC activity. s of altering ADCC activity by antibody ering have been described in the art, see for example, Shields RL. et al., J Biol Chem. 2001. 276(9): 6591-604; Idusogie EE. et al., J Immunol. 2000.164(8):4178-84; Steurer W. et al., J Immunol. 1995, 155(3): 1165— 74; Idusogie EE. et al., J l. 2001, 166(4): 2571-5; Lazar GA. et al., PNAS, 2006, 103(11): 4005- 4010; Ryan MC. et al., Mol. Cancer Ther., 2007, 6: 3009—3018; Richards JO,. et al., Mol Cancer Ther. 2008, 7(8): 2517-27; Shields R. L. et al, J. Biol. Chem, 2002, 277: 26733-26740; Shinkawa T. et al, J. Biol. Chem, 2003, 278: 3466-3473.
In certain embodiments, the anti—CD3 epsilon antibodies or antigen-binding fragments se one or more amino acid substitution(s) that alters Complement Dependent Cytotoxicity (CDC), for example, by improving or diminishing Clq binding and/or CDC (see, for example, WO99/51642; Duncan & Winter Nature 322:738-40 (1988); US. Pat. No. ,648,260; US. Pat. No. 5,624,821); and WO94/29351 concerning other examples och region variants. 0] In certain ments, the anti-CD3 n antibodies or antigen—binding fragments comprise one or more amino acid substitution(s) in the interface of the Fc region to facilitate and/or promote heterodimerization. These modifications se introduction of a protuberance into a ?rst Fc polypeptide and a cavity into a second Fc polypeptide, wherein the protuberance can be positioned in the cavity so as to promote interaction of the first and second Fc polypeptides to form a heterodimer or a complex, Methods of ting antibodies with these modifications are known in the art, e.g., as bed in US. Pat. No. 5,731,168.
Antigen-binding fragments Provided herein are also anti-CD3 epsilon antigen-binding fragments. Various types of antigen-binding fragments are known in the art and can be developed based on the anti- ilon antibodies provided herein, including for example, the exemplary antibodies whose CDR and FR sequences are shown in Tables 1 and 2, and their different variants (such as y variants, glycosylation variants, Fc variants, cysteine-engineered variants and so on).
In certain embodiments, an anti-CD3 epsilon antigen-binding fragment ed herein is a camelized single domain antibody, a diabody, a single chain Fv fragment (scFv), an scFv dimer, a Bst, a dst, a (dst)2, a dst-dst', an Fv fragment, a Fab, a Fab', a F(ab')2, a bispeci?c antibody, a ds diabody, a nanobody, a domain dy, a single domain dy, or a bivalent domain dy.
Various techniques can be used for the production of such antigen-binding fragments.
Illustrative methods include, enzymatic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)), inant expression by host cells such as E. Coli (e.g. for Fab, Fv and ScFv dy fragments), screening from a phage display library as discussed above (e. g. for ScFv), and chemical coupling of two H fragments to form F(ab')2 fragments (Carter et al., Bio/Technology 10:163-167 (1992)). Other techniques for the production of antibody fragments will be nt to a skilled practitioner.
In certain embodiments, the antigen-binding nt is a scFv. Generation of scFv is described in, for example, WO 93/16185; US. Pat. Nos. 5,571,894; and 5,587,458. scFv may be fused to an effector protein at either the amino or the carboxy terminus to provide for a fusion n (see, for example, Antibody Engineering, ed. Borrebaeck).
Bispecific Antibodies= Multivalent Antibodies In certain embodiments, the antibodies and n-binding fragments thereof provided herein are bivalent, tetravalent, hexavalent, or multivalent, In certain embodiments, the dies and n-binding fragments thereof provided herein are monospeci?c, or bispeci?c.
The term "valent" as used herein refers to the presence of a speci?ed number of antigen g sites in a given molecule. As such, the terms "bivalent37 CL 7 tetravalent", and "hexavalent" denote the presence of two binding site, four g sites, and six binding sites, respectively, in an n-binding molecule. A bivalent molecule can be monospeci?c if the two binding sites are both for speci?c binding of the same antigen or the same e.
Similarly, a trivalent molecule can be bispeci?c, for example, when two binding sites are monospeci?c for a ?rst antigen (or epitope) and the third binding site is speci?c for a second antigen (or epitope).
In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein can be monospeci?c but bivalent, trivalent, or tetravalent, with at least two binding sites c for the same antigen or epitope. This, in certain embodiments, provides for stronger binding to the antigen or the epitope than a monovalent counterpart. In certain embodiments, in a bivalent antigen-binding moiety, the ?rst valent of binding site and the second valent of binding site are structurally identical (i.e. having the same sequences), or structurally different (i.e. having different sequences albeit with the same speci?city).
In certain embodiments, the dies and antigen-binding fragments f ed herein are bispeci?c. In some ments, the bispeci?c antibodies and antigen- g fragments thereof provided herein has a ?rst speci?city for CD3epsilon, and a second speci?city. In some embodiments, the second speci?city is for CD3epsilon but to different epitopes. In some embodiments, the second speci?city is for a second antigen different from CD3epsilon and whose presence in proximity to CD3 epsilon-expressing T cells is desirable for the second antigen to be recognized by immune system. For example, bringing CD3epsilon- expressing T cells in close proximity to a tumor antigen or a pathogen antigen and hence ing recognition or elimination of such an antigen by the immune system.
In certain ments, the second city is for a tumor associated antigen or an epitope thereof. The term "tumor associated antigen" refers to an antigen that is or can be presented on a tumor cell surface and that is located on or within tumor cells. In some embodiments, the tumor associated antigens can be presented only by tumor cells and not by normal, i.e. non-tumor cells. In some other embodiments, the tumor associated ns can be exclusively expressed on tumor cells or may represent a tumor speci?c mutation compared to non-tumor cells. In some other embodiments, the tumor associated antigens can be found in both tumor cells and non-tumor cells, but is overexpressed on tumor cells when compared to mor cells or are accessible for antibody binding in tumor cells due to the less compact structure of the tumor tissue compared to non-tumor tissue. In some embodiments, the tumor associated antigen is located on the ature of a tumor. 2] Illustrative examples of a tumor associated n are CD10, CD19, CD20, CD21, CD22, CD25, CD30, CD33, CD34, CD37, CD44v6, CD45, CD133, Fms-like tyrosine kinase 3 (FLT-3, CD135), chondroitin sulfate glycan 4 (CSPG4, melanoma-associated chondroitin sulfate glycan), Epidermal growth factor or (EGFR), Her2, Her3, IGFR, IL3R, ?broblast activating protein (FAP), CDCPl, Derlinl, Tenascin, frizzled 1—10, the vascular antigens VEGFR2 (KDR/FLKl), VEGFR3 (FLT4, CD309), PDGFR-alpha (CD140a), PDGFR-beta (CDl40b), Endoglin, CLECl4, Tem l -8, and Tie2. Further examples may include A33, CAIVH’ATH-l (CDw52), Carcinoembryonic antigen (CEA), Carboanhydrase IX (MN/CA IX), de2-7, EGFR, EGFRvIII, EpCAM, Ep-CAM, Folate-binding protein, G250, Fms-like tyrosine kinase 3 (FLT—3, CD135), c-Kit (CD1 17), CSFlR (CD1 1 5), , IGFR, IL-2 receptor, IL3R, MCSP (Melanoma-associated cell surface chondroitin sulphate proteoglycane), Muc-l, te-speci?c membrane antigen , Prostate stem cell antigen (PSCA), Prostate speci?c antigen (PSA), and TAG-72.
The bispeci?c dies and antigen-binding fragments provided herein can be made with any le methods known in the art. In a conventional approach, two immunoglobulin heavy chain-light chain pairs having different antigenic speci?cities can be co-expressed in a host cell to produce bispeci?c dies in a recombinant way (see, for example, Milstein and Cuello, Nature, 305: 537 (1983)), followed by puri?cation by af?nity chromatography.
Recombinant approach may also be used, where sequences ng the antibody heavy chain variable domains for the two speci?cities are respectively fused to immunoglobulin constant domain sequences, followed by insertion to an expression vector which is co-transfected with an expression vector for the light chain sequences to a suitable host cell for recombinant expression ofthe bispeci?c antibody (see, for example, WO 94/04690, Suresh et al., Methods in Enzymology, 121:210 (1986)). Similarly, scFv dimers can also be recombinantly constructed and expressed from a host cell (see, e. g. Gruber et al., J. l., 152:5368 (1994).) In another method, leucine zipper es from the Fos and Jun proteins can be linked to the Fab' portions of two different antibodies by gene fusion. The linked antibodies are d at the hinge region to four half antibodies (i.e. monomers) and then re-oxidized to form heterodimers (Kostelny et al., J. Immunol., 148(5): 1547-1553 (1992)). 6] The two antigen-binding domains may also be conjugated or cross—linked to form a bispeci?c antibody or antigen-binding fragment. For example, one antibody can be coupled to biotin while the other antibody to avidin, and the strong association between biotin and avidin would complex the two antibodies together to form a i?c antibody (see, for example, US. Pat. No. 4,676,980; WO 60, WO 92/00373, and EP 03089). For another example, the two antibodies or antigen-binding fragments can be cross-linked by conventional methods known in the art, for example, as disclosed in US. Pat. No. 4,676,980.
Bispeci?c antigen-binding nts may be generated from a bispeci?c antibody, for e, by proteolytic cleavage, or by chemical linking. For example, an antigen-binding fragment (e. g. Fab’) of an antibody may be prepared and converted to Fab'-thiol derivative and then mixed and reacted with another converted Fab’ derivative having a ent antigenic speci?city to form a i?c antigen-binding fragment (see, for example, Brennan et al., e, 229: 81 (1985)).
In certain embodiments, the bispeci?c antibody or antigen-binding fragments may be engineered at the interface so that a knob-into-hole association can be formed to promote heterodimerization of the two different antigen—binding sites. "Knob-into—hole" as used herein, refers to an interaction between two polypeptides (such as CH3 ), where one polypeptide has a protuberance (i.e. ) due to ce of an amino acid e having a bulky side chain (e.g. tyrosine or phan), and the other polypeptide has a cavity (i.e. "hole") where a small side chain amino acid residue resides (e. g. alanine or threonine), and the protuberance is positionable in the cavity so as to promote interaction of the two polypeptides to form a heterodimer or a complex. Methods ofgenerating polypeptides with knobs-into—holes are known in the art, e.g., as described in US, Pat. No. 5,731,168.
Conjugates In some embodiments, the anti-CD3 n dies and antigen-binding fragments thereof further comprise a conjugate. The conjugate can be linked to the antibodies and antigen-binding fragments thereof. A conjugate is a oteinaceous moiety that can be ed to the antibody or antigen-binding fragment thereof. It is contemplated that a variety of conjugates may be linked to the antibodies or antigen-binding fragments ed herein (see, for example, "Conjugate Vaccines", Contributions to Microbiology and Immunology, J.
M. Cruse and R. E. Lewis, Jr. (eds.), Carger Press, New York, (1989)). These conjugates may be linked to the antibodies or antigen-binding fragments by covalent binding, af?nity binding, intercalation, coordinate binding, complexation, association, blending, or addition, among other methods.
In certain embodiments, the antibodies and antigen—binding fragments disclosed herein may be engineered to contain speci?c sites e the epitope binding portion that may be utilized for g to one or more conjugates. For example, such a site may include one or more reactive amino acid residues, such as for example cysteine or histidine residues, to facilitate covalent linkage to a conjugate.
In certain embodiments, the antibodies may be linked to a conjugate indirectly, or through another conjugate. For example, the dy or antigen-binding fragments may be conjugated to biotin, then indirectly conjugated to a second conjugate that is conjugated to . The conjugate can be a toxin (e.g., a chemotherapeutic agent), a detectable label (e. g., a ctive isotope, a lanthanide, a luminescent label, a cent label, or an enzyme- substrate label).
A " can be any agent that is detrimental to cells or that can damage or kill cells.
Examples of toxin include, without limitation, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, cin, etoposide, side, Vincristine, stine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and s thereof, tabolites (e.g., methotrexate, 6- mercaptopurine, 6-thioguanine, cytarabine, 5-?uorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin rly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e. g., vincristine and Vinblastine).
Examples of detectable label may include a ?uorescent labels (e.g. ?uorescein, rhodamine, dansyl, phycoerythrin, or Texas Red), enzyme-substrate labels (e.g. horseradish peroxidase, alkaline phosphatase, luceriferases, glucoamylase, lysozyme, saccharide oxidases or B-D-galactosidase), radioisotopes (e.g. 123I, 1241, 125I, 1311,358,3H, 111In, 112In, 14C, 64Cu, 67Cu, 86Y, 88Y, 90Y, 177Lu, 211At, 186Re, 188Re, 153Sm, 212Bi, and 32F, other lanthanides, luminescent labels), chromophoric moiety, digoxigenin, /avidin, a DNA molecule or gold for detection.
In certain embodiments, the ate can be a pharmacokinetic modifying moiety which helps increase half-life of the antibody. Illustrative example include water-soluble polymers, such as PEG, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl idone, copolymers of ethylene glycol/propylene glycol, and the like. The polymer may be of any molecular weight, and may be branched or unbranched. The number of rs attached to the antibody may vary, and if more than one polymer are attached, they can be the same or different molecules.
In certain ments, the conjugate can be a puri?cation moiety such as a magnetic bead.
In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein is used for a base for a conjugate. cleotides and Recombinant Methods The present disclosure provides isolated cleotides that encode the anti- CD3epsilon antibodies and antigen-binding fragments thereof. In certain embodiments, the isolated cleotides comprise one or more nucleotide sequences as shown in SEQ IN NO: 82, 84, 86, 88,90, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, and/or 120, which encodes the variable region ofthe exemplary antibodies provided . DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e. g., by using oligonucleotide probes that are capable of binding speci?cally to genes encoding the heavy and light chains of the antibody). The encoding DNA may also be obtained by synthetic methods.
The isolated polynucleotide that encodes the anti-CD3epsilon dies and antigen- binding fragments thereof (e. g. including the sequences in as shown in SEQ IN NO: 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, and/or 120) can be inserted into a vector for further cloning (ampli?cation of the DNA) or for expression, using recombinant techniques known in the art. Many vectors are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e. g. SV40, CMV, EF-ld), and a ription termination sequence.
In some embodiments, the vector system includes mammalian, ial, yeast systems, etc, and comprises plasmids such as, but not limited to, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pCMV, pEGFP, pEGFT, pSV2, pFUSE, pVITRO,pVIVO, pMAL, pMD18-T, pMONO, pSELECT, pUNO, pDUO, PsgSL, pBABE, pWPXL, pBI, plSTV-L, pProl8, pTD, pRS420, pLexA, 2 etc, and other laboratorial and commercially available vectors. Suitable vectors may include, plasmid, or viral vectors (e. g., replication defective retroviruses, adenoviruses and adeno-associated viruses).
Vectors comprising the polynucleotide sequence encoding the antibody or antigen- binding fragment can be uced to a host cell for g or gene sion. Suitable host cells for cloning or expressing the DNA in the vectors herein are the yote, yeast, or higher eukaryote cells described above. le prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., ella urium, ia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniforrnis, Pseudomonas such as P. aeruginosa, and Streptomyces.
In on to prokaryotes, eukaryotic microbes such as ?lamentous fungi or yeast are suitable cloning or expression hosts for anti-CD3epsilon antibody-encoding vectors.
Saccharomyces cerevisiae, or common baker’s yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, s, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC ), K. drosophilarum (ATCC 36,906), K. therrnotolerans, and K. nus, yarrowia (EP 402,226), Pichia pastoris (EP 183,070), Candida, Trichoderma reesia (EP 4); Neurospora ; Schwanniomyces such as Schwanniomyces occidentalis; and ?lamentous fungi such as, e. g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.
Suitable host cells for the expression of glycosylated antibodies or antigen-fragment provided here are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila gaster (fruif?y), and Bombyx mori have been identi?ed. A variety of viral strains for transfection are publicly available, e. g., the L-l variant of Autographa califomica NPV and the Bm-5 strain ofBombyx mori NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be ed as hosts. r, interest has been greatest in vertebrate cells, and ation of vertebrate cells in culture (tissue culture) has become a e procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS—7, ATCC CRL 1651), human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)), baby hamster kidney cells (BHK, ATCC CCL 10), Chinese hamster ovary -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243 —251 (1980)), monkey kidney cells (CV1 ATCC CCL 70), African green monkey kidney cells (VERO—76, ATCC CRL—1587), human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2). In some able embodiments, the host cell is 293F cell.
Host cells are transformed with the above-described expression or cloning vectors for anti-CD3 epsilon antibody production and cultured in conventional nutrient media modi?ed as appropriate for ng promoters, selecting transforrnants, or amplifying the genes encoding the d sequences. In another embodiment, the antibody may be ed by homologous recombination known in the art.
The host cells used to produce the antibodies or antigen—binding fragments provided herein may be cultured in a variety ofmedia. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's d Eagle's Medium (DMEM), Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), US. Pat. No. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; wo 90/03430; WO 87/00195; or US. Pat. Re. 30,985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth ), salts (such as sodium chloride, calcium, ium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and ine), antibiotics (such as GENTAMYCINTM drug), trace elements (de?ned as inorganic compounds usually present at ?nal trations in the micromolar , and glucose or an lent energy source. Any other necessary supplements may also be included at appropriate trations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
When using recombinant ques, the antibody can be ed intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a ?rst step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultra?ltration. Carter et al., Bio/Technology :163—167 (1992) describe a procedure for isolating antibodies which are secreted to the periplasmic space of E. coli. Brie?y, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and methylsulfonyl?uoride (PMSF) over about 30 min. Cell debris can be removed by centrifugation. Where the antibody is secreted into the medium, supematants from such expression systems are generally ?rst concentrated using a commercially available protein concentration ?lter, for example, an Amicon or Millipore Pellicon ultra?ltration unit. A protease inhibitor such as PMSF may be ed in any of the ing steps to t proteolysis and antibiotics may be included to t the growth of adventitious inants.
The anti-CD3epsilon antibodies and antigen-binding fragments thereof ed from the cells can be puri?ed using, for example, ylapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate itation, salting out, and af?nity chromatography, with af?nity chromatography being the preferred puri?cation technique.
In certain embodiments, Protein A immobilized on a solid phase is used for immunoaf?nity puri?cation of the antibody and n-binding fragment thereof. The suitability of protein A as an af?nity ligand depends on the species and isotype of any globulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human gammal, gamma2, or gamma4 heavy chains (Lindmark et al., J. Irnmunol. Meth. 621-13 (1983)). Protein G is recommended for all mouse isotypes and for human gamma3 (Guss et al., EMBO J. 5:1567 1575 (1986)). The matrix to which the y ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster ?ow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH3 domain, the ond ABXTM. resin (J. T. Baker, psburg, NJ.) is useful for puri?cation. Other techniques for protein puri?cation such as fractionation on an ion-exchange , ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSETM chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.
Following any preliminary puri?cation step(s), the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations (e.g., from about 0-0.25M salt).
Pharmaceutical Composition The present disclosure further provides pharmaceutical compositions comprising the anti-CD3epsilon antibodies or antigen-binding fragments thereof and one or more pharmaceutically acceptable carriers.
Pharmaceutical acceptable carriers for use in the pharmaceutical compositions sed herein may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, nonaqueous vehicles, crobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending/dispending , sequestering or chelating agents, diluents, adjuvants, excipients, or xic auxiliary substances, other components known in the art, or various ations thereof. le components may include, for example, antioxidants, ?llers, binders, disintegrants, buffers, preservatives, lubricants, ?avorings, thickeners, coloring agents, emulsifiers or stabilizers such as sugars and cyclodextiins. le idants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, se, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxanisol, butylated ytoluene, and/or propyl gallate. As disclosed herein, inclusion of one or more antioxidants such as methionine in a ition comprising an antibody or antigen-binding nt and conjugates as provided herein ses oxidation of the antibody or antigenbinding fragment. This reduction in ion prevents or reduces loss of binding af?nity, thereby improving antibody stability and maximizing shelf-life. Therefore, in n embodiments compositions are provided that comprise one or more antibodies or antigen- binding fragments as disclosed herein and one or more idants such as methionine.
Further provided are methods for preventing oxidation of, extending the shelf-life of, and/or improving the ef?cacy of an antibody or antigen-binding fragment as provided herein by mixing the antibody or antigen-binding fragment with one or more antioxidants such as methionine.
To further illustrate, pharmaceutical acceptable carriers may e, for example, aqueous vehicles such as sodium chloride ion, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection, nonaqueous vehicles such as ?xed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil, antimicrobial agents at bacteriostatic or tatic concentrations, isotonic agents such as sodium chloride or dextrose, buffers such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local etics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcelluose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone, emulsifying agents such as rbate 8O (TWEEN—SO), sequestering or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA ene glycol tetraacetic acid), ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. crobial agents utilized as carriers may be added to pharmaceutical compositions in multiple-dose ners that include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl oxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride, Suitable excipients may include, for example, water, saline, dextrose, glycerol, or ethanol. Suitable xic auxiliary substances may include, for example, wetting or emulsifying agents, pH ing agents, stabilizers, lity enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin. 7] The pharmaceutical compositions can be a liquid solution, suspension, emulsion, pill, capsule, , ned release formulation, or powder. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, , magnesium stearate, polyvinyl pyrollidone, sodium saccharine, cellulose, magnesium carbonate, etc.
In n embodiments, the pharmaceutical compositions are formulated into an able composition. The injectable pharmaceutical compositions may be prepared in any conventional form, such as for example liquid solution, sion, emulsion, or solid forms suitable for generating liquid on, suspension, or emulsion, Preparations for injection may include sterile and/or non-pyretic solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just prior to use, and sterile and/or non-pyretic emulsions. The solutions may be either aqueous or nonaqueous.
In certain embodiments, unit-dose parenteral preparations are packaged in an ampoule, a vial or a syringe with a needle. All preparations for parenteral administration should be sterile and not pyretic, as is known and practiced in the art.
In certain embodiments, a sterile, lyophilized powder is prepared by dissolving an antibody or antigen-binding fragment as disclosed herein in a le solvent. The solvent may contain an excipient which improves the stability or other pharmacological components ofthe powder or reconstituted solution, prepared from the powder. Excipients that may be used include, but are not d to, water, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, e or other le agent. The solvent may contain a buffer, such as citrate, sodium or potassium phosphate or other such buffer known to those of skill in the art at, in one embodiment, about neutral pH. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those of skill in the art es a desirable formulation, In one embodiment, the resulting solution will be apportioned into vials for lyophilization. Each vial can contain a single dosage or multiple dosages of the anti- CD3epsilon antibody or antigen—binding nt thereof or composition thereof. Overfilling vials with a small amount above that needed for a dose or set of doses (e.g., about 10%) is acceptable so as to facilitate accurate sample withdrawal and accurate dosing. The lyophilized powder can be stored under riate conditions, such as at about 4 °C to room temperature.
Reconstitution of a lyophilized powder with water for injection provides a formulation for use in parenteral administration. In one embodiment, for reconstitution the sterile and/or non-pyretic water or other liquid suitable carrier is added to lyophilized powder.
The precise amount depends upon the ed therapy being given, and can be empirically ined.
Methods of Use The present sure also provides eutic methods comprising: administering a eutically effective amount of the antibody or antigen-binding fragment as provided herein to a subject in need f, thereby treating or preventing a CD3—related condition or a disorder. In some embodiment, the CD3 -related condition or a disorder is cancer, mune e, in?ammatory disease, or infectious disease, Examples of cancer include but are not limited to, non-small cell lung cancer (squamous/nonsquamous), small cell lung cancer, renal cell cancer, colorectal cancer, colon cancer, ovarian cancer, breast cancer (including basal breast carcinoma, ductal carcinoma and lobular breast carcinoma), pancreatic cancer, gastric carcinoma, bladder cancer, esophageal cancer, mesothelioma, ma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, melanoma, myelomas, mycoses fungoids, merkel cell cancer, hepatocellular carcinoma (HCC), ?brosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic a, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, lymphoid malignancy, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, ary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, classical Hodgkin lymphoma (Cm), primary mediastinal large B-cell lymphoma, T-cell/histiocyte-rich B-cell lymphoma, acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous ia, chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, polycythemia vera, mast cell derived tumors, EBV-positive and -negative PTLD, and diffuse large B—cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T-cell lymphoma, nasopharyngeal oma, HHV8-associated primary effusion lymphoma, non-Hodgkin's ma, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia, primary CNS lymphoma, spinal aXis tumor, brain stem glioma, astrocytoma, oblastoma, craniopharyogioma, moma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma and blastoma.
Autoimmune diseases include, but are not limited to, Acquired Immunode?ciency me (AIDS, which is a viral disease with an autoimmune component), alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALP S), autoimmune ocytopenic purpura (ATP), Behcet's e, cardiomyopathy, celiac sprue-dermatitis hepetiformis; chronic fatigue immune dysfunction syndrome (CFIDS), chronic in?ammatory demyelinating polyneuropathy , cicatricial gold, cold agglutinin disease, crest syndrome, Crohn's disease, Degos' disease, dermatomyositis-juvenile, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-?bromyositis, Graves' disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, idiopathic pulmonary ?brosis, idiopathic thrombocytopenia purpura (ITP), IgA nephropathy, insulin-dependent es mellitus, juvenile c arthritis (Still's disease), le toid arthritis, Meniere’s disease, mixed connective tissue disease, multiple sis, myasthenia gravis, pemacious , polyarteritis nodosa, ondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary globulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomena, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma (progressive ic sclerosis (PSS), also known as systemic sclerosis (SS)), Sjogren's syndrome, stiff-man syndrome, systemic lupus erythematosus, Takayasu arteritis, temporal arteritis/giant cell arteritis, ulcerative colitis, uveitis, vitiligo and Wegener's granulomatosis.
In?ammatory ers, include, for example, chronic and acute in?ammatory disorders.
Examples of in?ammatory disorders include Alzheimer's disease, asthma, atopic allergy, allergy, atherosclerosis, bronchial asthma, eczema, glomerulonephritis, graft vs. host disease, hemolytic anemias, osteoarthritis, sepsis, stroke, transplantation of tissue and organs, vasculitis, diabetic retinopathy and ventilator d lung injury. In some embodiments, the CD3 associated conditions are in?ammatory diseases such as systemic lupus erythematosus (SLE), inal l in?ammation, wasting disease associated with colitis, multiple sclerosis, viral infections, rheumatoid arthritis, osteoarthritis, Cohn's disease, and in?ammatory bowel disease, sis, systemic scleroderma, autoimmune diabetes and the like.
Infectious disease include, but are not limited to, fungus infection, parasite/protozoan ion or chronic viral infection, for example, malaria, coccidioiodmycosis immitis, histoplasmosis, onychomycosis, aspergilosis, blastomycosis, candidiasis albicans, paracoccidioiomycosis, microsporidiosis, Acanthamoeba tis, Amoebiasis, Ascariasis, Babesiosis, Balantidiasis, Baylisascariasis, Chagas e, Clonorchiasis, Cochliomyia, Cryptosporidiosis, lobothriasis, Dracunculiasis, Echinococcosis, Elephantiasis, Enterobiasis, Fascioliasis, lopsiasis, asis, Giardiasis, Gnathostomiasis, Hymenolepiasis, Isosporiasis, Katayama fever, Leishmaniasis, Lyme disease, Metagonimiasis, Myiasis, Onchocerciasis, Pediculosis, Scabies, Schistosomiasis, Sleeping sickness, Strongyloidiasis, Taeniasis, Toxocariasis, Toxoplasmosis, Trichinosis, Trichuriasis, Trypanosomiasis, helminth infection, infection of hepatitis B (HBV), hepatitis C (HCV), herpes virus, Epstein-Barr virus, HIV, cytomegalovirus, herpes simplex virus type I, herpes x virus type II, human papilloma virus, adenovirus, human immunodeficiency virus I, human immunode?ciency virus II, Kaposi West sarcoma associated herpes virus epidemics, thin ring virus (Torquetenovirus), human T lymphotrophic viruse I, human T trophic viruse II, varicella zoster, JC virus or BK virus.
In another aspect, methods are provided to treat a condition in a subject that would benefit from upregulation of immune response, comprising stering a therapeutically effective amount of the dy or antigen-binding fragment as provided herein to a subject in need thereof.
The therapeutically effective amount of an antibody or antigen-binding fragment as provided herein will depend on various factors known in the art, such as for example body weight, age, past medical y, present medications, state of health of the subject and potential for cross-reaction, allergies, sensitivities and adverse side-effects, as well as the administration route and extent of disease development. Dosages may be proportionally reduced or increased by one of ordinary skill in the art (e.g., physician or veterinarian) as ted by these and other circumstances or requirements.
In certain embodiments, an antibody or antigen—binding fragment as provided herein may be administered at a therapeutically effective dosage of about 0.01 mg/kg to about 100 mg/kg (e.g., about 0.01 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, or about 100 . In certain ofthese embodiments, the dy or n—binding fragment is administered at a dosage of about 50 mg/kg or less, and in certain of these embodiments the dosage is 10 mg/kg or less, 5 mg/kg or less, 3 mg/kg or less, 1 mg/kg or less, 0.5 mg/kg or less, or 0.1 mg/kg or less. In certain embodiments, the administration dosage may change over the course of treatment. For example, in certain ments the initial administration dosage may be higher than subsequent administration dosages. In certain ments, the administration dosage may vary over the course of treatment depending on the reaction of the subj ect‘ 0] Dosage regimens may be adjusted to provide the optimum desired response (e. g., a therapeutic response). For example, a single dose may be administered, or several divided doses may be administered over time.
The antibodies and antigen-binding fragments disclosed herein may be administered by any route known in the art, such as for example eral (e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular, or intradermal injection) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal, or topical) In some embodiments, the dies or antigen-binding fragments disclosed herein may be administered alone or in combination with one or more onal eutic means or agents. For example, the antibodies or antigen-binding fragments disclosed herein may be administered in combination with another therapeutic agent, for example, an chemotherapeutic agent or an anti-cancer drug.
In certain of these embodiments, an antibody or antigen—binding fragment as disclosed herein that is administered in combination with one or more additional eutic agents may be administered simultaneously with the one or more additional therapeutic agents, and in certain of these embodiments the antibody or antigen-binding fragment and the additional therapeutic agent(s) may be administered as part of the same pharmaceutical composition. r, an antibody or n—binding fragment administered "in combination" with another therapeutic agent does not have to be administered simultaneously with or in the same ition as the agent. An dy or antigen-binding nt administered prior to or after another agent is considered to be administered "in combination" with that agent as the phrase is used herein, even if the antibody or antigen-binding fragment and second agent are administered via different routes. Where possible, onal therapeutic agents administered in combination with the antibodies or antigen-binding fragments disclosed herein are stered according to the schedule listed in the product information sheet of the additional therapeutic agent, or ing to the Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57th Ed; Medical Economics Company; ISBN: 1563634457; 57th edition (November 2002)) or protocols well known in the art.
The present disclosure further provides s of using the anti-CD3epsilon antibodies or antigen-binding nts thereof. In some embodiments, the present disclosure provides methods of activating CD3 epsilon-expressing T cells in vivo or in vitro, comprising: contacting the CD3 epsilon-expressing T cells with the antibody or antigen-binding fragment thereof provided herein. In some ments, the present disclosure provides methods of modulating CD3 activity in a ilon-expressing cell, comprising exposing the CD3epsilon-expressing cell to the antibody or antigen-binding fragment thereof provided herein In some embodiments, the present disclosure provides methods of promoting in vivo or in vitro processing of a second antigen by CD3epsilon-expressing T cell, comprising contacting the CD3epsilon-expressing T cells with the bispeci?c antibody or antigen-binding fragment thereof provided herein, wherein the bispeciflc dy or antigen-binding fragment is capable of cally binding to both the CD3epsilon-expressing T cells and a second antigen thereby bringing both in close proximity.
In some embodiments, the present disclosure provides methods of ing presence or amount of CD3epsilon in a sample, comprising contacting the sample with the antibody or antigen-binding fragment thereof, and determining the presence or the amount of CD3epsilon in the sample.
In some embodiments, the present disclosure provides methods of diagnosing a CD3 related e or condition in a t, comprising: a) obtaining a sample from the subject; b) contacting the sample obtained from the t with the antibody or antigen-binding fragment thereof provided herein; c) determining presence or amount of CD3 epsilon in the sample; and d) correlating the nce of the CD3epsilon to the CD3 related disease or condition in the subj ect.
In some embodiments, the present disclosure also provides use of the antibody or antigen-binding fragment thereof provided herein in the manufacture of a medicament for treating a CD3 related disease or condition in a subject, in the manufacture of a diagnostic reagent for diagnosing a CD3 related disease or condition.
The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. All speci?c compositions, als, and methods described below, in whole or in part, fall within the scope of the t invention. These specific compositions, materials, and methods are not intended to limit the invention, but merely to illustrate specific embodiments falling within the scope of the ion. One skilled in the art may develop equivalent compositions, materials, and methods without the exercise ofinventive capacity and without departing from the scope ofthe invention, It will be understood that many variations can be made in the procedures herein bed while still remaining within the bounds of the present ion. It is the ion of the inventors that such variations are included within the scope of the invention.
EXAMPLE 1: Generation of Hybridoma Antibody 1.1 Animal zation: 1] Recombinant extracellular domains (ECD) proteins ofhuman CD3, including human CD3 epsilon (CD3epsilon) with His Tag (cat. No.1 10977—H08H; Sino Biological Inc. Beijing, China), human CD3 Gamma (CD3gamma) (cat. No.: abl40563; Abcam Shanghai China) and human CD3 delta (CD3delta) with His Tag (cat. No.1 10977-H08H; Sino Biological Inc.
Beijing, China) were used as gens for animal immunization. Balb/c mice were purchased from Shanghai SLAC laboratory animal Co; Ltd. and were housed in an IACUC approved animal facility. Mice were immunized with the ECD n mixture of CD3 epsilon, CD3gamma and CD3delta or immunized with 7x106 freshly isolated human T-cells for each mouse, mixed with the Titermax nt by subcutaneous and footpad injections every other week.
Blood were collected from mice before and after immunization and serum titers against target proteins were red by ELISA. 1.2 Hybridoma generation The mouse with the highest serum titer was chosen for cell fusion. The B cells from mouse spleen and lymphanodes were fused with SP2/O myeloma cells by o-fusion according to general electro-fusion procedures. After cell fusion, the cells were plated in 96- well plates with DMEM medium supplemented with 20% FBS and 1% HAT selective reagents.
Antibodies presented in the supernatant of hybridoma medium were screened using CD3-expressing Jurkat cells by Flow cytometer assay (FACS), and counter-screened using gative MOLT-4 T cells by FACS. The hybridoma cells with binding activity to Jurkat cells, but not cross binding to MOLT-4 cells were collected as positive hybridoma cell lines, and then proceed to subcloning stage using semi-solid medium approach.
Single es were picked into 96-well plates with DMEM medium supplemented with 10% FBS for 2~3 days, and re-screened against target of CD3 using CD3-expressing Jurkat cells by Flow cytometer assay , and counter-screened using CD3-negative MOLT-4 T cells by FACS assay. 1.3 Hybridoma sequencing RNA was extracted from hybridoma cells and cDNA was ampli?ed by using 5’-RACE kit, followed by PCR ampli?cation using 3’-degenerated s. PCR products were then cloned into pMD18-T vector, transformed, ed and sequenced.
Eight mouse monoclonal antibodies were generated, CDR sequences of which are shown in Table 1 above.
E 2: Generation of Humanized Antibody 2.1 IgG conversion and humanization Generation of Chimeric antibodyfrom murine-derived mAbs: WBP33 1 1_2. 166.48 and WBP3311_2.306.4 and 2_3.179.16 VH and VL genes were re-amplifled with cloning primers containing appropriate restriction sites and cloned into WuXi Biologics’ propIietary expression vector to create corresponding clones of chimeric antibodies with constant region of human IgG].
Humanization and synthesis 0fhumanized s: "Best Fit" approach was used to humanize WBP3311_2.166.48 and l_2.306.4 light and heavy chains. For light chains amino acid sequences of corresponding V-genes were blasted against in—house human germline V-gene database. The sequence of humanized VL-gene was derived by replacing human CDR sequences in the top hit with mouse CDR sequences using Kabat CDR de?nition.
Frameworks were de?ned using extended CDR where Kabat CDRl was extended by 5 amino acids at N—terminus. Multiple humanized sequences were created for each heavy chain and light chain by blasting mouse frameworks against human germline immunoglobulin database.
Different FR combinations were created and analyzed for binding af?nity, top three hits were used to derive sequences of humanized VH-genes. Humanized genes were back-translated, codon-optimized for mammalian expression, and synthesized by GeneArt Costum Gene Synthesis (Life Technologies). Synthetic genes were ned into IgG sion , expressed and puri?ed. FRs of the two humanized antibodies and their parental mouse antibodies were shown in Table 2 above. ent expression andpurification eric and zed antibodies: The chimeric and humanized antibodies described above were constructed into WuXi Biologics’ etary expression vector and sed from 293F cells. The e supernatant containing corresponding antibodies were harvested and puri?ed using Protein A chromatography.
EXAMPLE 3: In vitro Characterization 3.1 Antibody binding by ELISA and FACS Antigens, antibodies and cells used in the ELISA and FACS described below are listed in table 3.
Table 3. Antigens, antibodies and cells used in ELISA and FACS Company Human CD3 epsilon (CD3epsilon) S1no Blological Inc._ . . lO977-H08H Human CD3 delta (CD3delta) protein Sino Biological Inc. lO981-H08H ab 140563 C nomoly gusMonkey CD3epsilon CDE-C5226 protein Cyno PBMCs 10977—MM03 (Clone NO.: 1A7E5G5) SANTA CRUZ IOTECHNOLOGY, INC Benchmark antibody OKT3 —ab86883 Jurkat cells ATCC TTB-152 ECACC- HUT78 cells ATCC 880401901 MOLT—4 cells ATCC CRL-1582 Semi-solid medium 0—814 Binding ofantibodies toprotein by ELISA o: Human CD3 epsilon, CD3delta and CD3gamma proteins were pre-coated in l plates, respectively. The binding af?nity of eight mAbs at various concentrations to these three different CD3 protein extracellular domain was detected by corresponding HRP labeled 2nd antibodies. The binding ECso (concentration of the test antibody when reaching half maximum binding) were analyzed by using GraphPad Prism software equation: Nonlinear regression (curve ?t)—log st) vs. se—Variable slope.
Speci?c binding of antibodies to human CD3epsilon protein by ELISA: Eight mouse mAbs showed highly speci?c binding activity to human CD3 epsilon without binding to CD3gamma and CD3delta and data were shown in Table 5.
Table 5. Speci?c binding against human CD3epsilon sub-unit protein Human CD3epsilon Human CD3gamma Human CD3 delta (PC: 16; NC: 0.05) (PC: 1.21;NC: 005) (PC: 19; NC: 0.05) W3311-2.l66.48 1.64 W3311-2.306.4 1.46 W3311-2.383.47 1.61 W3311-2.400.5 1.57 W3311-2.482.5 1.33 W3311-2.488.33 1.70 W3311-2.615.8 1.55 W3311-2.844.8 1.54 9] Cellular binding ofantibodies by FACS andEC50: Various concentrations of testing mAbs were added to Jurkat cells, and then the binding activity of mAbs onto the surface of cells was detected by 2nd antibody-FITC. OKT3 was used as the positive control. The d cells were analyzed by using a BD Biosciences FACSCanto II instrument and Flow Jo n software. The binding ECso were calculated by using GraphPad Prism software equation: Nonlinear regression (curve fit)—log st) vs. response—Variable slope.
Speci?c binding to human CD3 receptor on T cell surface: Eight mAbs showed highly speci?c binding activity to human CD3 expressing cells (Jurkat cells), without binding to CD3-negative cells (MOTL-4 cells and 293F cells) in Table 6. 1] Table 6. Speci?c binding to human CD3 receptor cross multiple cell lines by FACS MEI (FACS ) MFI (FACS ) WI (FACS ) mAbs (JurkatB 1 0: 4:22_7~27.3) (293F222.6~24.6) 3 145~4045) W33ll-2.6l5.8 2215 57.2 25.7 3.2 Cross species target n binding of dies by ELISA and FACS Binding affinities of the test antibodies to CD3 from different species were analyzed.
Homology of the human, monkey, rat and mouse CD3 reference ces are shown below.
Table 4. Human, monkey, rat and mouse CD3 domain ces homology CD3epsilon (%) CD3delta (%) (:Dsgamma (%) 100 100 100 Human (NP_000724) (NP_000723) (NP_000064) Macaca fascicularis 83 94 81 (monkey) (NP_001270544) 1274617) (NP_001270839) mulatta 84 94 82 (XP_001097204) (NP_001097302) (NP_001253854) 59 (NP_031674) 64 (NP_O38515) 70 400) 57 71 Rattus norvegicus (Rat) 69 (NP_O3 7301) (NP_001101610) (NP_00107lll4) Cross-binding of antibodies to Cynomolgus Monkey CD3epsilon and mouse CD3 epsilon by ELISA: s concentrations of testing antibodies, positive and negative controls were added to the 96-well plates that were pre-coated with Cynomolgus Monkey CD3 epsilon protein and mouse CD3epsilon. The binding of the dies to the Cynomolgus Monkey CD3epsilon protein and mouse CD3epsilon protein was detected by corresponding HRP labeled 211d antibodies (BETHYL, A90-231P). The ECso were calculated by using ad Prism software.
Data showed that all eight mAbs showed potent cross-binding activity to Cynomolgus Monkey CD3 epsilon, but no binding to mouse CD3 epsilon. The positive control OKT3 showed neither cross-binding activity to Cynomolgus Monkey CD3epsilon or mouse CD3epsilon Table 7.
Table 7. Cross-binding against Cynomolgus Monkey CD3epsilon and mouse CD3epsilon mAbs Human Cyno—Monkey Mouse C—D3epsilonCD3epsilon CD3epsilon OKT3 0.048 0.052 W3311-2.166.48 2.761 2.808 0.446 Cross-binding of antibodies to Cynomolgus Monkey CD3epsilon by FACS: Cyno PBMCs were ed from healthy Cyno whole blood by using Ficoll-Paque PLUS gradient fugation and 100g centrifugation steps to remove thrombocytes. PBMC were cultured in complete RPMI—1640 medium until ready to use. Various concentrations of test antibodies were added to Cyno PBMCs, and then the g activity of the antibodies onto the surface ofthe cells were detected by 2nd dy-FITC (Jackson, 115008). The stained cells were analyzed by using a BD Biosciences FACSCanto II and FlowJo Version software. The binding ECso were calculated by using GraphPad Prism software equation: Nonlinear sion (curve fit). e Binning ofantibodies by FACS: Various concentrations of test antibodies were mixed with certain amount of biotinylated antibody of W3311-2.383.47, respectively.
The mixture was then added to CD3-expressing cells in 96-well plates and incubated at 4°C for 1 hour. The binding of the target antibody onto the cells expressing CD3 was detected using PE-conjugated anti-biotin Ab. Samples were tested by ?ow cytometry and data were analyzed by FlowJo. g t human CD3epsilon and Cynomolgus Monkey CD3epsilon protein and Eng calculation by ELISA: Eight mAbs showed strong binding activity to human CD3epsilon and cross binding to Cynomolgus Monkey CD3epsilon protein and showed comparable EC50 in low nM range n human CD3epsilon and Cynomolgus Monkey CD3epsilon in Table 8.
Table 8. Eight mAbs EC50 for human CD3epsilon and lgus Monkey CD3epsilon Human CD3epsilon Cynomolgus Monkey CD3 epsilon ECso (nM) ECso (nM) 7i5TCZ?£?g_____7i?3___________7RE§_________________ W3311-2.306.4 0.012 0.011 W3311-2.383.47 0.074 0.028 W3311-2.400.5 0.053 0.024 W3311-2.482.5 0.069 0.028 2.488.33 0.026 0.019 W3311-2.615.8 0.033 0.025 W33ll-2.844.8 0.011 0.009 WBP33 l-BMKl 0186 NA (UCHTD 4.3 Detection of cross-species binding of humanized antibodies Cynomolgus Monkey CD3epsilon protein binding and Eng value of two humanized mAbs: All data indicated both humanized mAbs retained their high g activity to Cynomolgus Monkey CD3epsilon protein (see Figure l) with ECso value of 0.043 nM for both humanized antibodies in Table 9.
Table 9. Two humanized mAbs EC50 for Cynomolgus Monkey CD3epsilon mAbs ECso (11M) W33ll-2.l66 48 zl-ngGlK 0.043 W331 l—2.306.4—zl-ngGlK 0.043 W33ll-2.l66.48 0.125 Isotype control N/A A?inity of antibodies by FACS: 5x104 Jurkat cells/well were seeded in 96-well , followed by the addition of puri?ed testing lead antibodies at various concentrations as the 1st antibody for 1 hour at 4°C. The 2nd antibody of Goat ouse IgG Fc-FITC was added for 30 min at 4°C, and then stained cells by FITC were ed by FACS. KD value was calculated according to the method described above.
Two humanized mAbs were tested the binding activity on human CD4 T cells: Data indicated that both humanized antibodies retained high binding activity to CD4 T cells (see Figure 2) with low ECso values of 1.01 and 0.46 nM for WBP331l-2.l66.48-zl-ngle and 1-2.306.4-zl-ngle, respectively, which were 0.5-1 fold lower than that of respective parental antibodies in Table 10.
Table 10. hCD4 T cell binding of two humanized mAbs by FACS and ECso W33ll-2.l66.48-zl-ngGlK W3311-2.l66.48-mIgG2aK 3.514 4.5 Af?nity and ECso of antibodies by FACS Eight mAbs were tested for their ECso by FACS and were tested af?nity to human CD3 expressing cells (Jurkat . The ECso of the 8 mAbs ranged from 0.57 nM to 6.91 nM (see Table 11), and the af?nity ranged from 1.3x 10'9 to 9.3x 10'10 M in Table 12.
Table 11‘ ECso measurement on Jurkat cells by FACS W33 1 l—2. 166.48 W331 l-2.306.4 W3311-2.383.47 W3311-2.400.5 W3311-2.482.5 W33ll-2.488.33 W33ll-2.615.8 W33ll-2.844.8 Table 12. Eight mAbs KD value on Jurkat cells by FACS WBP3311.
W3311- W3311- W33ll- W33ll- W3311- W3311- W33ll- W3311- Sample OKT3— 2.16648 2.3064 2383.47 2.4005 2482.5 2.48833 2.6158 2.8448 Average Best ?t- 1.26E-10 10 1.12E-10 1.13E-10 1.30E-10 1.30E-10 1.20E-10 10 9.4lE-ll Best ?t- 1.67E-09 1.9lE-10 4.03E-10 9.34E-10 2.29E-09 09 2.23E-09 2.52E-10 1.29E-10 Std. Error- 12 1.19E-12 1.62E-12 1.76E-12 2.35E-12 2.51E-12 2.18E-12 1.00E-12 1.95E-12 Std. Error- .62E-11 8.32E-12 2.15E-11 4.38E-11 9.63E-11 6.98E-11 9.47E-11 8.64E-12 1.27E-11 4.6 Af?nity and Kinetic KD measurement of two humanized mAb by FACS Two humanized mAbs were tested for Af?nity measurement on Jurkat cells by FACS.
The data indicated that both humanized mAbs retained their high af?nity activity on Jurkat cells with the K13 values similar to their respective parental mAbs in Figure 4 and Table 13.
Table 13. Kinetic KD measurement of two humanized mAbs on Jurkat cells by FACS WBP3311-2.l66.48—z1— WBP3311-2.306.4—zl— 7.32E-12 6.83E-12 6.06E-12 Best ?t-KD 2,01E-lO 5.30E—11 2.77E-11 Std. Error- 3.82E-l3 43 13 Std. KD 4.38E-ll l.l4E—ll 5.13E-12 3.3 Cell based functional assays Cell internalization ofantibodies by FA CS: Jurkat cells were seeded at l X ll in 96—well plates. Testing antibodies (1 ug/mL) were added to the cells, and incubated for 1 hour at 4°C. After incubation, d antibodies were washed away, and the cells were then incubated for 3 hours at 4°C or 37°C with 5% C02. After incubation, the presence of antibodies on the cell surface was detected by 2nd antibody (Abcam, ab98742). The stained cells were analyzed by using a BD Biosciences nto II and FlowJo Version software. The internalization rate was calculated as below: [(MFIo hour - MF13 hours) / MFIo hour] * 100 %. 4.7 Cell internalization rate ement Internalization rate was tested using Jurkat cells by FACS. The data indicated all 8 mAbs showed high internalization rate ranging from 86-94% antibody internalized after 3 hours study period, whereas the OKT3 showed about 72% internalization rate in Figure 5.
T cell activation of antibodies with ellular cytokine staining assay: Intracellular cytokine staining is a ?ow cytometry-based assay that can detect cytokine production by immune cells in combination with cell surface markers. Human PBMCs were isolated from healthy donor. Brie?y, -Paque PLUS gradient centrifugation was used with subsequent 100g centrifugation steps to remove thrombocytes. PBMC were cultured in complete 640 medium. PBMC were resuspended in cell culture medium supplemented with Golgi Stop and dispended at 2x105/well in 96-well . Various concentrations of test dies, ve and negative controls were added and then incubated with the cells for 4.5 hours at 37°C. After incubation, the cells were washed two times with 1% BSA, and then stained for their surface makers by using anti-human CD4-FITC dy (BD, ), and anti-human CD8-PE antibody (BD, 560959), followed by ?xation and permeabilization using the Human Regulatory T Cell Staining Kit (eBioscience, 88-8999). Post ?xation/permeabilization, the cells were detected for cytokine production by using anti-human TNF-APC dy (BD, 554514) and anti-human IFN—PerCP—Cy55 dy (eBioscience, 4542). The stained cells were analyzed by using BD Biosciences FACSCanto II and Flow]0 Version software T cell activation: T cell activation was evaluated using Human PBMCs by Intracellular cytokine staining method and the cytokines of TNFalpha and INFgamma were monitored. The data indicated that among 8 testing mAbs 2306.4 and 2.8448 did not show signi?cant T cell activation event as no signi?cant cytokines of TNFalpha and INFgamma e was monitored, whereas all other 6 test mAbs showed T cell activation to the extent similar to that of OKT3, except for clone 2.3 83.47 which showed a weaker T cell activation as compared to OKT3, data shown in Figure 6.
Human T cell activation of Two Humanized mAbs: Two humanized mAbs were tested the T cell activation activity on Human PBMCs by Intracellular cytokine staining method and the cytokines of TNFalpha and TFNgamma were monitored. The data indicated that both humanized antibodies showed similar extent T cell activation to OKT3 positive l on CD8+ T cells, whereas both humanized mAbs showed lower extent activation on CD4+ T cells ed to OKT3. It is also noticed that the parental mouse antibody of clone 2.3064 repeatedly displayed no T cells activation. Data were shown in Figure 7. 3.4 Epitope binning of antibodies by FACS Epitope Binning: Seven of 8 mAbs are binned against clone 7 and the result showed that all 8 mAbs sharing the same epitope bin in Figure 8.

Claims (20)

1. An isolated antibody or an antigen-binding nt thereof, comprising: a) heavy chain CDR sequences comprising SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 11; and kappa light chain CDR sequences comprising SEQ ID NO: 8, SEQ ID NO: 10, and SEQ ID NO: 12; b) heavy chain CDR sequences comprising SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5; and kappa light chain CDR sequences comprising SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6; c) heavy chain CDR sequences comprising SEQ ID NO: 13, SEQ ID NO: 15, and SEQ ID NO: 17; and kappa light chain CDR sequences comprising SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 18; d) heavy chain CDR sequences sing SEQ ID NO: 19, SEQ ID NO: 21, and SEQ ID NO: 23; and kappa light chain CDR sequences comprising SEQ ID NO: 20, SEQ ID NO: 22, and SEQ ID NO: 24; e) heavy chain CDR sequences comprising SEQ ID NO: 25, SEQ ID NO: 27, and SEQ ID NO: 29; and kappa light chain CDR sequences comprising SEQ ID NO: 26, SEQ ID NO: 28, and SEQ ID NO: 30; f) heavy chain CDR sequences comprising SEQ ID NO: 31, SEQ ID NO: 33, and SEQ ID NO: 35; and kappa light chain CDR sequences comprising SEQ ID NO: 32, SEQ ID NO: 34, and SEQ ID NO: 36; g) heavy chain CDR sequences sing SEQ ID NO: 37, SEQ ID NO: 39, and SEQ ID NO: 41; and kappa light chain CDR sequences comprising SEQ ID NO: 38, SEQ ID NO: 40, and SEQ ID NO: 42; or h) heavy chain CDR sequences comprising SEQ ID NO: 43, SEQ ID NO: 45, and SEQ ID NO: 47; and kappa light chain CDR sequences comprising SEQ ID NO: 44, SEQ ID NO: 46, and SEQ ID NO: 48.
2. The antibody or an antigen-binding fragment thereof of claim 1, comprising: a) a heavy chain variable region comprising SEQ ID NO: 117 and a kappa light chain le region comprising SEQ ID NO: 119. b) a heavy chain variable region comprising SEQ ID NO: 81 and a kappa light chain variable region comprising SEQ ID NO: 83; c) a heavy chain variable region comprising SEQ ID NO: 85 and a kappa light chain variable region comprising SEQ ID NO: 87; d) a heavy chain variable region comprising SEQ ID NO: 89 and a kappa light chain le region comprising SEQ ID NO: 91; e) a heavy chain variable region comprising SEQ ID NO: 93 and a kappa light chain le region comprising SEQ ID NO: 95; f) a heavy chain variable region comprising SEQ ID NO: 97 and a kappa light chain variable region comprising SEQ ID NO: 99; g) a heavy chain variable region comprising SEQ ID NO: 101 and a kappa light chain variable region comprising SEQ ID NO: 103; h) a heavy chain variable region comprising SEQ ID NO: 105 and a kappa light chain variable region comprising SEQ ID NO: 107; i) a heavy chain variable region comprising SEQ ID NO: 109 and a kappa light chain variable region comprising SEQ ID NO: 111; or j) a heavy chain variable region comprising SEQ ID NO: 113 and a kappa light chain variable region sing SEQ ID NO: 115.
3. The antibody or antigen-binding fragment thereof of any of the preceding claims, further comprising an immunoglobulin constant region, optionally a constant region of IgG, optionally a constant region of human IgG1.
4. The antibody or an antigen-binding fragment f of any of the preceding claims, which is a humanized antibody.
5. The dy or antigen-binding fragment thereof of any of the preceding claims, which is a camelized single domain antibody, a diabody, a scFv, an scFv dimer, a BsFv, a dsFv, a (dsFv)2, a dsFv-dsFv', an Fv nt, a Fab, a Fab', a F(ab')2, a bispecific antibody, a ds diabody, a nanobody, a domain antibody, or a bivalent domain antibody.
6. The antibody or an antigen-binding fragment thereof of claim 5, wherein the antibody or an antigen-binding nt thereof is bispecific and has a first specificity for CD3epsilon, and a second specificity.
7. The antibody or an antigen-binding fragment thereof of claim 6, n the second specificity is for a second n different from CD3epsilon wherein presence of the second antigen in proximity to a CD3epsilon-expressing T cells is desirable for the second antigen to be recognized by immune system.
8. The antibody or antigen-binding nt thereof of any of the preceding claims linked to one or more conjugates, wherein the conjugate comprises a chemotherapeutic agent, a toxin, a radioactive isotope, a lanthanide, a luminescent label, a fluorescent label, or an enzymesubstrate label.
9. The antibody or an antigen-binding fragment thereof of any of the preceding claims, capable of specifically binding to CD3epsilon, and optionally n the CD3epsilon are derived from mouse, rat, monkey or human, and optionally wherein the ilon is a recombinant CD3epsilon or a CD3epsilon expressed on a cell surface.
10. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any of the preceding , and a pharmaceutically able carrier.
11. An isolated polynucleotide encoding the antibody or an antigen-binding fragment thereof of any of the preceding claims.
12. A vector comprising the isolated polynucleotide of claim 11.
13. An isolated host cell comprising the vector of claim 12.
14. A method of expressing the antibody or antigen-binding fragment thereof of any one of claims 1 to 9, comprising culturing an isolated host cell comprising a vector comprising an isolated polynucleotide encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 9 under the ion at which the vector is expressed.
15. The use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 9 for the cture of a medicament for the ent of a CD3 related disease or condition.
16. The antibody or antigen-binding fragment thereof of any one of claims 1 to 9 for use in the treatment of a CD3 related disease or ion.
17. The use or the antibody of claim 15 or 16, wherein the antibody or antigen-binding fragment thereof is bispecific and the disease or condition is a cancer.
18. A method of activating ilon-expressing T cells in vivo in a non-human animal or in vitro, sing contacting the CD3epsilon-expressing T cells with the antibody or antigen-binding nt thereof of any one of claims 1 to 9.
19. A method of promoting in vivo in a non-human animal or in vitro processing of a second antigen by CD3epsilon-expressing T cell, comprising contacting the CD3epsilonexpressing T cells with the bispecific antibody or antigen-binding nt thereof of claim 6, wherein the bispecific antibody or antigen-binding fragment is capable of specifically binding to both the CD3epsilon-expressing T cells and a second antigen thereby bringing both in close ity.
20. A kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 9, useful in ing ilon, optionally recombinant CD3epsilon, CD3epsilon expressed on cell surface, or CD3epsilon-expresing cells, or useful in diagnosing a CD3 related disease or condition in a subject. W3311-2.166.48—ZE-0Eg613< ‘F W3311—2.306.4~21-ulgGlK ¢ MBll—ZJSSAS '4'" W3321—1.80.12-uAb.igG4 0.000001 0.0001 0.01 1 100 Ab(nM)
NZ762170A 2018-09-20 Novel anti-cd3epsilon antibodies NZ762170B2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN2017102622 2017-09-21
PCT/CN2018/106618 WO2019057099A1 (en) 2017-09-21 2018-09-20 Novel anti-cd3epsilon antibodies

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NZ762170A NZ762170A (en) 2023-10-27
NZ762170B2 true NZ762170B2 (en) 2024-01-30

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