EA042856B1 - NEW ANTI-CD3 EPSILON ANTIBODIES - Google Patents

Description

The field to which the invention relates

The present invention generally relates to novel antibodies against human CD3 epsilon.

Prerequisites for the creation of the invention

CD3 (differentiation cluster 3) The T cell coreceptor is a protein complex and consists of four separate chains, a CD3 gamma chain, a CD3 delta chain, and two CD3 epsilon chains. These chains bind to a molecule known as the T cell receptor (TCR) and the zeta chain to generate an activation signal in T lymphocytes. The TCR molecules, zeta chains and CD3 together form a TCR complex in which TCR as a subunit recognizes and binds to an antigen, and CD3 as a subunit carries and transmits antigen stimulation to the signaling pathway and ultimately regulates T cell activity. The CD3 protein is present in almost all T cells.

CD3, together with TCR, forms the CD3-TCR complex, which plays a key role in modulating broad T cell functions in both innate and adoptive immune responses, as well as cellular and humoral immune functions. These include the elimination of pathogenic organisms and the control of tumor growth through a wide range of cytotoxic effects.

Mouse monoclonal antibodies specific for human CD3, such as OKT3 (Kung et al. (1979) Science 206: 347-9), were the first generation of anti-CD3 antibodies for treatment. Although OKT3 has strong immunosuppressive activity, its clinical use has been hampered by serious side effects associated with its immunogenic and mitogenic potential (Chatenoud (2003) Nature Reviews 3:123-132). OKT3 induced an antiglobulin response by promoting its own rapid clearance and neutralization (Chatenoud et al. (1982) Eur. J. Immunol. 137:830-8). In addition, OKT3 induced T cell proliferation and cytokine production in vitro and resulted in large scale cytokine release in vivo (Hirsch et al. (1989) J. Immunol 142: 737-43, 1989). The release of cytokines (also called cytokine storm) in turn leads to an influenza-like syndrome characterized by fever, chills, headache, nausea, vomiting, diarrhea, respiratory distress syndrome, septic meningitis, and hypotension (Chatenoud, 2003). These serious side effects have limited the wider use of OKT3 in transplantation as well as its expansion into other clinical areas such as autoimmune diseases (Id.).

There is a huge need for new anti-CD3 antibodies.

The essence of the invention

The singular and plural are used to refer to one or more than one (i.e., at least one) entity. For example, antibody means one antibody or more than one antibody.

The present invention relates to novel anti-CD3 epsilon monoclonal antibodies, their amino acid and nucleotide sequences and their uses.

In one aspect, the present invention provides isolated antibodies, or antigen-binding fragments thereof, comprising 1, 2, or 3 heavy chain CDR sequences selected 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 group consisting of: SEQ ID NO: 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 some embodiments, the antibodies or antigen-binding fragments thereof comprise 1, 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 with SEQ ID NO: 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 some embodiments, the antibodies or antigen-binding fragments thereof comprise 1, 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 with SEQ ID NO: 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 some embodiments, the antibodies or antigen-binding fragments thereof comprise a heavy chain variable region selected from the group consisting of:

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;

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;

c) a heavy chain variable region comprising 1, 2 or 3 CDR sequences selected from SEQ ID NO: 13, SEQ ID NO: 15 and SEQ ID NO: 17;

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;

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 ID NO: 29;

f) a heavy chain variable region containing 1, 2 or 3 CDR sequences, selected

- 1 042856 those of 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 some embodiments, the antibodies or antigen-binding fragments thereof comprise a kappa light chain variable region selected 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 sequences 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 ID NO: 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 variable region comprising 1, 2 or 3 CDR sequences selected from SEQ ID NO: 44, SEQ ID NO: 46 and SEQ ID NO: 48.

In some embodiments, the antibodies or antigen-binding fragments thereof comprise a heavy chain CDR3 sequence selected from the group consisting of SEQ ID NOS: 5, 11, 17, 23, 29, 35, 41, and 47.

In some embodiments, the antibodies or antigen-binding fragments thereof include:

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; 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 some embodiments, the antibodies or antigen-binding fragments thereof include:

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 some embodiments, the antibodies or antigen-binding fragments thereof include:

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 ID NO: 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 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

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 some embodiments, the antibodies or antigen-binding fragments thereof include:

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 variable region comprising 1, 2 or 3 CDR sequences selected from SEQ ID NO: 13, SEQ ID NO: 15 and SEQ ID NO: 17;

- 2 042856 and a kappa light chain variable region comprising 1, 2 or 3 CDR sequences 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 selected from SEQ ID NO: 20, SEQ ID NO: 22, and SEQ ID NO: 24;

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 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 comprising 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 selected 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 sequences 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 selected from SEQ ID NO: 44, SEQ ID NO: 46, and SEQ ID NO: 48.

In some embodiments, the antibodies or antigen-binding fragments thereof also comprise 1, 2, 3, or 4 heavy chain framework region (FR) sequences selected from the group consisting of: SEQ ID NOs: 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 NOS: 58, 60, 62, 64, 74, 76, 78 and 80.

In some embodiments, the antibodies or antigen-binding fragments thereof also comprise a heavy chain FR1 sequence selected from SEQ ID NOs: 57 and 73; a heavy chain FR2 sequence selected from SEQ ID NOs: 59 and 75; a heavy chain FR3 sequence selected from SEQ ID NOs: 61 and 77; and a heavy chain FR4 sequence selected from SEQ ID NOs: 63 and 79.

In some embodiments, the antibodies or antigen-binding fragments thereof also comprise a light chain FR1 sequence selected from SEQ ID NOs: 58 and 74; a light chain FR2 sequence selected from SEQ ID NOs: 60 and 76; a light chain FR3 sequence selected from SEQ ID NOs: 62 and 78; and a light chain FR4 sequence selected from SEQ ID NOs: 64 and 80.

In some embodiments, the antibodies or antigen-binding fragments thereof comprise a heavy chain variable region 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 ID NO: 113, SEQ ID NO: 117 and a sequence homologous to them having at least 80% (e.g., 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 comprise a light chain variable region 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 and a sequence homologous to them having at least 80% (e.g., 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 comprise all or part of a heavy chain variable region sequence selected from the group consisting of: SEQ ID NOs: 81, 85, 89, 93, 97, 101, 105, 109, 113, and 117; and/or all or part of a light chain variable region sequence selected from the group consisting of: SEQ ID NOS: 83, 87, 91, 95, 99, 103, 107, 111, 115, and 119. In one embodiment, the antibodies or their antigen-binding fragments are a single-domain antibody that consists of all or part of a heavy chain variable region selected from the group consisting of: SEQ ID NOS: 81, 85, 89, 93, 97, 101, 105, 109, 113 and 117.

In some embodiments, the antibodies or antigen-binding fragments thereof include:

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 variable region comprising SEQ ID NO: 93 and a variable region of a cap-

- 3 042856 pa-light chain, including 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 variable 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 variable region comprising SEQ ID NO: 119.

In some embodiments, the antibody or antigen-binding fragment thereof comprises one or more amino acid residue substitutions but retains a specific CD3 epsilon binding affinity.

In some embodiments, the substitution is present in one or more CDR sequences, or in one or more FR sequences, or in one or both variable region sequences, or in an Fc region. In some embodiments, at least one (or all) of the substitutions in the CDR sequences, FR sequences, variable region sequences, or Fc region is a conservative substitution.

In some 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 NOs: 1- 48. In some 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 some embodiments, the antibody or antigen-binding fragment thereof comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitutions in total in the CDR and/or FR sequences of the heavy chain variable region sequences selected of SEQ ID NOs: 81, 85, 89, 93, 97, 101, 105, 109, 113, and 117. In some embodiments, an 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 light chain variable region FR sequences selected from SEQ ID NOs: 83, 87, 91, 95, 99, 103, 107, 111, 115 and 119.

In some embodiments, the substitution imparts one or more of the desired properties selected from the following: a) improving binding affinity for CD3 epsilon, b) introducing or removing a glycosylation site, c) introducing a free cysteine residue, d) increasing or decreasing ADCC or CDC, e) increase in serum half-life; and f) increasing FcRn binding.

In some embodiments, the antibody, or antigen-binding fragment thereof, also includes an immunoglobulin constant region. In some embodiments, the antibody, or antigen-binding fragment thereof, comprises an IgG constant region. In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a human IgG1 constant region.

In some embodiments, the antibodies, or antigen-binding fragments thereof, are a mouse antibody or a humanized antibody.

In some embodiments, the antibodies or antigen-binding fragments thereof are camelized single domain antibody, diabody, scFv, scFv dimer, BsFv, dsFv, (dsFv) ₂ , dsFv-dsFv', Fv fragment, Fab, Fab', F(ab') ₂ , a bispecific antibody, a ds diabody, a nanobody, a domain antibody, or a bivalent domain antibody.

In some embodiments, the antibodies or antigen-binding fragments thereof are bispecific. In some embodiments, the antibody or antigen-binding fragment thereof has a first antigenic specificity for CD3 epsilon and a second antigenic specificity. In some embodiments, the second antigenicity is directed against a second antigen other than CD3 epsilon, where the presence of the second antigen in the vicinity of CD3 epsilon expressing T cells is desirable in order for the second antigen to be recognized by the immune system. In some embodiments, the first antigenic specificity is directed against CD3epsilon and the second antigenic specificity is directed against a tumor-associated antigen.

In some embodiments, the antibodies or antigen-binding fragments thereof are linked to one or more conjugates. In some embodiments, the conjugate may be a chemotherapeutic agent, a toxin, a radioactive isotope, a lanthanide, a fluorescent label, a fluorescent label, or an enzyme-substrate label.

In some embodiments, the antibody, or antigen-binding fragment thereof, is capable of specifically binding to CD3 epsilon. In some embodiments, the CD3 epsilon is from a mouse, rat, monkey, or human. In some embodiments, the CD3epsilon is a recombinant CD3epsilon or cell surface expressed CD3epsilon.

In some embodiments, the antibodies or antigen-binding fragments thereof are capable of specifically binding to human CD3 epsilon expressed on the cell surface at a KD value of not more than 5x10 ^-9 M, not more than 4x10 ^-9 M, not more than 3x10 ^-9 M, no more than 2x10 ^-9 M, no more than 10 ^-9 M, no more than 5x10 ^-10 M, no more than 4x10 ^-10 M, no more than 3x10 ^-10 M, no more than 2x10 ^-10 M, no more than 10 ^-10 M, not more than 5x10 ^-11 M, not more than 4x10 ^-11 M, not more than 3x10 ^-11 M, not more than 2x10 ^-11 M, or not more than 10 ^-11 M, as measured by the flow method cytometry. In some embodiments, the antibodies, or antigen-binding fragments thereof, are capable of specifically binding to human CD3 epsilon expressed on cell surfaces at an EC ₅₀ of not more than 0.50 nM or not more than 1.10 nM, as determined by flow cytometry.

In some embodiments, the antibodies or antigen-binding fragments thereof are capable of specifically binding to recombinant cynomolgus monkey CD3 epsilon with an EC ₅₀ of not more than 0.001 nM, not more than 0.005 nM, not more than 0.01 nM, not more than 0.02 nM, not more than 0.03 nM, not more than 0.04 nM, or not more than 0.05 nM as measured by ELISA.

In some embodiments, the antibodies or antigen-binding fragments thereof are capable of specifically binding to recombinant human CD3 epsilon at an EC ₅₀ of not more than 0.01 nM, not more than 0.02 nM, not more than 0.03 nM, not more than 0, 04 nM, not more than 0.05 nM, not more than 0.06 nM, not more than 0.07 nM, or not more than 0.08 nM, as measured by ELISA.

In some embodiments, the antibodies, or antigen-binding fragments thereof, are capable of specifically binding to human CD3 epsilon expressed on the surface of CD3-expressing cells at an EC ₅₀ of not more than 0.5 nM, not more than 0.6 nM, not more than 0.7 nM , not more than 0.8 nM, not more than 0.9 nM, not more than 1 nM, not more than 2 nM, not more than 3 nM, not more than 4 nM, not more than 5 nM, not more than 6 nM, not more than 7 nM, not more than 8 nM, not more than 9 nM, or not more than 10 nM, as measured by flow cytometry.

In some embodiments, the antibodies, or antigen-binding fragments thereof, are a humanized antibody that is capable of specifically binding to human CD3 epsilon expressed on the surface of CD4-expressing cells at an EC ₅₀ of not more than 0.50 nM or not more than 1.10 nM , as measured by flow cytometry.

In one aspect, the present invention provides an antibody or antigen-binding fragment that competes for the same epitope with the antibody or antigen-binding fragment presented in this application.

In one aspect, the present invention also provides a pharmaceutical composition comprising an antibody or antigen-binding fragment presented in this application, and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition also includes a second agent that is capable of enhancing the therapeutic effect of the antibody or antigen-binding fragment thereof and/or is capable of reducing the side effect of the antibody or antigen-binding fragment thereof.

In one aspect, the present invention also provides an isolated polynucleotide encoding the antibody or antigen-binding fragment presented in this application. In some embodiments, the isolated polynucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 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 invention also provides a vector comprising said isolated polynucleotide.

In one aspect, the present invention also provides a host cell comprising the specified vector.

In one aspect, the present invention also provides a method for expressing an antibody or antigen-binding fragment of the present invention, comprising culturing said host cell under conditions under which said polynucleotide is expressed.

In one aspect, the present invention also provides a method of treating a disease or condition in an individual that may benefit from modulation of CD3 epsilon activity, comprising administering to the individual a therapeutically effective amount of an antibody or antigen-binding fragment of the present invention, or a pharmaceutical composition provided herein. In some embodiments, said individual is a human. In some embodiments, said disease or condition is a CD3 related disease or condition. In some embodiments, said disease or condition is cancer, an autoimmune disease, an inflammatory disease, or an infectious disease.

In one aspect, the present invention also provides a method for activating CD3-epsilon-5 042856 expressing T cells in vivo or in vitro, comprising contacting CD3-epsilon-expressing T cells with an antibody or antigen-binding fragment provided herein.

In one aspect, the present invention also provides a method for modulating CD3 activity in a CD3 epsilon expressing cell, comprising exposing the CD3 epsilon expressing cell to an antibody or antigen-binding fragment thereof of the present invention.

In one aspect, the present invention also provides a method for promoting in vivo or in vitro processing of a second antigen by CD3 epsilon-expressing T cells, comprising contacting CD3 epsilon-expressing T cells with a bispecific antibody or antigen-binding fragment provided herein, wherein the bispecific antibody or antigen-binding fragment is capable of specifically binding to both CD3-epsilon-expressing T cells and the second antigen, thus bringing them into close proximity.

In one aspect, the present invention also provides a method for detecting the presence or amount of CD3 epsilon in a sample, comprising contacting the sample with an antibody or antigen-binding fragment provided herein and determining the presence or amount of CD3 epsilon in the sample.

In one aspect, the present invention also provides a method for diagnosing a CD3-associated disease or condition in an individual, comprising: a) obtaining a sample from the individual; b) contacting the sample with antibodies or antigen-binding fragments provided in this application; c) determining the presence or amount of CD3 epsilon in the sample; and d) relating the presence or amount of CD3 epsilon to a disease or condition in an individual.

In one aspect, the present invention also provides the use of an antibody or antigen-binding fragment of the present invention for the manufacture of a medicament for the treatment of a CD3-associated disease or condition in an individual.

In one aspect, the present invention also provides the use of an antibody or antigen-binding fragment thereof of the present invention to provide a diagnostic reagent for diagnosing a CD3-associated disease or condition.

In one aspect, the present invention also provides a kit comprising the antibody or antigen-binding fragment provided herein useful for detecting CD3epsilon. In some embodiments, the kit includes antibodies or antigen-binding fragments thereof useful for detecting recombinant CD3 epsilon, cell surface expressed CD3 epsilon, or CD3 epsilon expressing cells.

Brief description of the drawings

Fig. 1 shows the binding of monoclonal antibodies WBP3311_2.166.48-uIgG1K, WBP3311_2.306.4-uIgG1K and WBP3311_2.166.48 to recombinant cynomolgus monkey CD3 epsilon protein as measured by ELISA.

Fig. 2 shows the binding of monoclonal antibodies WBP3311_2.166.48-uIgG1K, WBP3311_2.306.4-uIgG1K, WBP3311_2.166.48 and WBP3311_2.306.4 to human CD4 T cells as measured by flow cytometry.

Fig. 3 shows the binding affinity of eight mouse antibodies (W3311-2.166.48, W33112.306.4, W3311-2.383.47, W3311-2.400.5, W3311-2.482.5, W3311-2.488.33, W3311-2.615.8 and W33112. 844.8) with cells expressing human CD3 (Jurkat cells) as measured by flow cytometry.

Fig. 4A shows the binding affinity of the humanized WBP3311_2.166.48-z1uIgG1K antibody to cells expressing human CD3 (Jurkat cells) as measured by flow cytometry.

Fig. 4B shows the binding affinity result of the humanized WBP3311_2.306.4-z1-uIgG1K antibody to cells expressing human CD3 (Jurkat cells) as measured by flow cytometry.

Fig. 4C shows the result of the binding affinity of the OKT3 positive control to cells expressing human CD3 (Jurkat cells) as measured by flow cytometry.

Fig. 5 shows the percentage of cellular internalization of eight mouse antibodies (W3311-2.166.48, W3311-2.306.4, W3311-2.383.47, W3311-2.400.5, W3311-2.482.5, W3311-2.488.33, W3311-2.615.8 and W3311-2.844.8) into cells expressing human CD3 (Jurkat cells) as measured by flow cytometry.

Fig. 6 shows the result of activation of human T cells with eight mouse antibodies (W3311-2.166.48, W3311-2.306.4, W3311-2.383.47, W3311-2.400.5, W3311-2.482.5, W3311-2.488.33, W3311- 2.615.8 and W3311-2.844.8) as measured by intracellular staining of the cytokines TNFalpha and IFN-gamma.

Fig. 7 shows the result of human T cell activation with two humanized antibodies (WBP3311_2.166.48-z1-uIgG1K and WBP3311_2.306.4-z1-uIgG1K) as measured by TNF-alpha and IFN-gamma cytokine intracellular staining.

- 6 042856

Fig. 8 shows the result of epitope binding for seven mouse antibodies (W3311-2.166.48,

W3311-2.306.4, W3311-2.400.5, W3311-2.482.5, W3311-2.488.33, W3311-2.615.8 and W3311-2.844.8) against the WBP3311_2.383.47 clone.

Detailed description of the invention

The following description of the invention is only intended to illustrate various embodiments of the invention. As such, the specific modifications discussed should not be construed as limiting the scope of the invention. Those skilled in the art will appreciate that various equivalents, changes, and modifications are possible without departing from the scope of the invention, and it should be understood that such equivalent embodiments are to be included in the present disclosure. All referenced documents cited in this application, including publications, patents and patent applications, are incorporated into this application by reference in their entirety.

Definitions

The term antibody as used herein includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multispecific antibody, or bispecific antibody that binds to a specific antigen. A native intact antibody includes two heavy (H) chains and two light (L) chains. In mammals, heavy chains are classified as alpha, delta, epsilon, gamma, and mu, each heavy chain consisting of a variable region (VH) and a first, second, and third constant region (C _H1 , C _H2 , CH3, respectively); light chains in mammals are classified as either λ or k, with each light chain consisting of a variable region (VL for λ light chain or V _K for k light chain, respectively) and a constant region (C _L for λ light chain or C _K for k light chain, respectively). The antibody has a Y shape, where the Y leg consists of the second and third constant regions of two heavy chains linked together by disulfide linkage. Each Y arm comprises a single heavy chain variable region and a first constant region associated with a single light chain variable and constant region. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in both chains typically contain three highly variable loops called complementarity determining regions (CDRs) (light chain CDRs including LCDR1, LCDR2 and LCDR3, heavy chain CDRs including HCDR1, HCDR2, HCDR3). CDR boundaries for antibodies and antigen-binding fragments disclosed in this application can be determined or identified by the methods of Kabat, IMGT, Chothia or Al-Lazikani (AlLazikani, B., Chothia, C., Lesk, AM, 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, AM, J. Mol. Biol ., 196,901 (1987); Chothia, C. et al., Nature. Dec 21-28;342(6252):877-83 (1989); Kabat EA et al., National Institutes of Health, Bethesda, Md. ( 1991)). The three CDRs are located between flanking regions known as framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold to support hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding but exhibit different effector functions. Antibodies are classified into classes based on the amino acid sequence of their heavy chain constant region. There are five major classes or isotypes of antibodies, IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of alpha, delta, epsilon, gamma, and mu heavy chains, respectively. Some of the major classes of antibodies are further subclassified, such as IgG1 (gamma1 heavy chain), IgG2 (gamma2 heavy chain), IgG3 (gamma3 heavy chain), IgG4 (gamma4 heavy chain), IgA1 (alpha1 heavy chain), or IgA2 (alpha2 heavy chain). chain).

The term bivalent in the context of this application refers to an antibody or antigen-binding fragment having two antigen-binding sites; the term monovalent refers to an antibody or antigen-binding fragment having only one single antigen-binding site; and the term multivalent refers to an antibody or antigen-binding fragment having multiple antigen-binding sites. In some embodiments, the antibody, or antigen-binding fragment thereof, is bivalent.

In the context of the present application, a bispecific antibody refers to an artificial antibody that has fragments derived from two different monoclonal antibodies and is capable of binding to two different epitopes. The two epitopes may be present on the same antigen, or they may be present on two different antigens.

The term antigen-binding fragment in the context of the present application 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 include the intact structure of a native antibody. Examples of an antigen binding fragment include, without limitation, diabody, Fab, Fab', F(ab') ₂ , Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv) ₂ , bispecific dsFv (dsFvdsFv'), disulfide-stabilized diabody (ds diabody), single chain antibody molecule (scFv), scFv dimer (bivalent diabody), bispecific antibody, multispecific antibody, camelized single domain antibody, nanobody, domain antibody, and bivalent domain antibody. The antigen-binding fragment is capable of binding to the same antigen that the parent antibody binds to.

Fab, in relation to an antibody, refers to that part of an antibody that consists of (both variable and constant regions) one light chain linked to the variable region and the first constant region of one heavy chain by a disulfide bond.

Fab' refers to a Fab fragment that includes part of the hinge region.

F(ab') ₂ refers to the Fab' dimer. Fv, in relation to an antibody, refers to the smallest fragment of an antibody that can contain a complete antigen-binding site. The Fv fragment consists of a single light chain variable region linked to a single heavy chain variable region.

dsFv refers to a disulfide-stabilized Fv fragment, wherein the bond between the variable region of one light chain and the variable region of one heavy chain is a disulfide bond. In some embodiments, the (dsFv)2 or (dsFv-dsFv') comprises three peptide chains: two VH fragments linked by a peptide linker (eg, a long flexible linker) and linked to two V _L fragments, respectively, via disulfide bridges. In some embodiments, the implementation of dsFv-dsFv' is bispecific, where each disulfide-paired heavy and light chain has a different antigen specificity.

Single chain Fv antibody or scFv refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region linked to each other directly or via a peptide linker sequence (Huston JS et al. Proc Natl Acad Sci USA, 85:5879(1988) ).

Fc, in relation to an antibody, refers to that portion of an antibody which consists of the second and third constant regions of the first heavy chain linked to the second and third constant regions of the second heavy chain via a disulfide bond. The Fc portion of an antibody is responsible for various effector functions such as antibody dependent cell mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) but not antigen binding function.

Single chain Fv-Fc antibody or scFv-Fc refers to an engineered antibody consisting of scFv linked to the Fc region of the antibody.

A 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 10;231(1-2):25-38 ( 1999); Muyldermans S., J Biotechnol. Jun; 74(4):277-302 (2001); WO 94/04678; WO 94/25591; US Patent No. 6,005,079). Heavy chain antibodies were originally isolated from Camelidae (camels, dromedaries and llamas). Although they lack 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. et al. Heavy-chain antibodies in Camelidae; a case of evolutionary innovation, Immunogenetics. Apr;54(1):39-47 (2002); Nguyen VK. et al. Immunology. May; 109(1):93-101 (2003)). The variable domain of a heavy chain antibody (VHH domain) is the smallest known antigen-binding unit generated by adaptive immune responses (Koch-Nolte F. et al., FASEB J. Nov; 21(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 comprise small fragments of antibodies with two antigen-binding sites, wherein the fragments comprise a V _H domain linked to a V _L domain in the same polypeptide chain (V _H -V _L or V _L -V _H ) (see, for example, Holliger P. et al., Proc Natl Acad Sci USA Jul 15;90(14):6444-8 (1993); EP404097; WO93/11161). By using a linker that is too short to allow pairing between two domains on the same strand, the domains are forced to pair with complementary domains on the other strand, thus creating two antigen-binding sites. Antigen binding sites may target the same or different antigens (or epitopes). In some embodiments, a bispecific ds diabody is a diabody that targets two different antigens (or epitopes). In some embodiments, the scFv dimer is a bivalent diabody or bivalent ScFv (BsFv) comprising V _H -V _L (linked by a peptide linker) dimerized to another V _H -V _L fragment, such that the V _{H of} one fragment are coordinated to the V _L regions of another fragment and form two binding sites that can target the same antigens (or epitopes) or different antigens (or epitopes). In other embodiments, the scFv dimer is a bispecific diabody comprising V _H1 V _L2 (linked by a peptide linker) linked to V _l1 -Vh ₂ (also linked by a peptide linker) such that VH1 and VL1 are coordinated and VH2 and VL2 are coordinated, and that each coordinated pair have a different antigen specificity.

A domain antibody refers to an antibody fragment containing only a heavy chain variable region or a light chain variable region. In some cases, two or more VH domains are covalently linked by a peptide linker to form a bivalent or multivalent domain antibody. The two VH domains of a bivalent domain antibody may target the same or different antigens.

The term chimeric in the context of this application means an antibody or antigen-binding

- 8 042856 fragment having part of the heavy and/or light chain originating from one species, and the remainder of the heavy and/or light chain originating from another species. In an illustrative example, a chimeric antibody may comprise a human-derived constant region and a non-human variable region, such as a mouse. In some embodiments, the non-human animal is a mammal, such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster.

The term humanized in the context of the present application means that the antibody or antigen binding fragment includes CDRs derived from non-human animals, FR regions derived from a human, and, when applicable, constant regions derived from a human.

CD3, as used herein, refers to a cassette 3 protein derived from any vertebrate species, including mammals such as primates (eg, humans, monkeys) and rodents (eg, mice and rats). In mammals, the CD3 molecule is a multiprotein complex of six chains, including: CD3-gamma chain, CD3-delta chain, two CD3epsilon chains and a homodimer of CD3-zeta chains, where CD3zeta chain is the intracellular tail of the CD3 molecule, and CD3-gamma, CD3 delta and CD3 epsilon chains all contain an extracellular domain (ECD) expressed on the surface of T cells. Illustrative human CD3 sequence includes human CD3 epsilon protein (NCBI Ref Seq No. NP_000724), human CD3 delta protein (NCBI Ref Seq No. NP_000723), and human CD3 gamma protein (NCBI Ref Seq No. NP_000064). Illustrative non-human CD3 sequence includes Macaca fascicularis (monkey) CD3-epsilon protein (NCBI Ref Seq No. NP_001270544), Macaca fascicularis (monkey) CD3-delta protein (NCBI Ref Seq No. NP_001274617), Macaca fascicularis CD3-gamma protein ( monkeys) (NCBI Ref Seq No. NP_001270839); mouse CD3 epsilon protein (NCBI Ref Seq No. NP_031674), mouse CD3 delta protein (NCBI Ref Seq No. NP_038515), mouse CD3 gamma protein (NCBI Ref Seq No. AAA37400); Rattus norvegicus (rat) CD3-epsilon protein (NCBI Ref Seq No. NP_001101610), Rattus norvegicus (rat) CD3-delta protein (NCBI Ref Seq No. NP_037301), Rattus norvegicus (rat) CD3-gamma protein (NCBI Ref Seq No NP_001071114). In some embodiments, the CD3 used in the present invention may also be recombinant CD3, for example, including recombinant CD3 epsilon protein, recombinant CD3 delta protein, and recombinant CD3 gamma protein, which may optionally be expressed as a recombinant CD3 complex . The recombinant CD3 complex may be expressed on the cell surface or, alternatively, may be expressed in a soluble form that is not bound to the cell surface.

The term CD3 epsilon as used herein is intended to cover any form of CD3 epsilon, for example 1) native full-length CD3 epsilon, full-length CD3 epsilon chain, or naturally occurring variants of CD3 epsilon, including, for example, splice variants or allelic variants; 2) any form of CD3-epsilon, which is the result of processing in the cell; or 3) a full-length, fragment (eg, truncated form, extracellular/transmembrane domain) or modified form (eg, mutant form, glycosylated/PEGylated, His-tagged/immunofluorescently-tagged form) of the recombinantly produced CD3 epsilon subunit.

The term anti-CD3 epsilon antibody refers to an antibody that is capable of specifically binding to CD3 epsilon (eg human or monkey CD3 epsilon).

The term specific binding or specific binding in the context of the present application refers to a non-random binding reaction between two molecules, for example between an antibody and an antigen. In some embodiments, the antibodies or antigen-binding fragments provided herein specifically bind to human CD3 epsilon with a binding affinity (KD) of <^10'6M (e.g., <^5x10'7M , <^2x10'7M , <10 ' ⁷ M, <5x10'8 M, <2x10'8 M, <^10'8 M, <5x10'9 M, <4x10'9 M, <3x10'9 M, <2x10'9 M or <10'9 M). KD, as used herein, refers to the ratio of dissociation rate to association rate (k _off /k _on ), which can be determined using any conventional method known in the art, including, but not limited to, the surface plasmon resonance method. , a microscale thermophoresis method, an HPLC-MS method, and a flow cytometry method (such as FACS). In some embodiments, the KD value can be appropriately determined by flow cytometry.

The ability to block binding or compete for the same epitope as used herein refers to the ability of an antibody or antigen-binding fragment to inhibit the binding interaction between two molecules (e.g., a human CD3 epsilon and an anti-CD3 epsilon antibody) to any measurable degree. In some 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 some embodiments, this inhibition may be greater than 85% or greater than 90%.

The term epitope in the context of this application refers to a specific group of atoms or

- 9042856 amino acids on the antigen to which the antibody binds. Two antibodies can bind to the same or a closely related epitope on an antigen if they exhibit competitive binding for that antigen. For example, if an antibody or antigen-binding fragment blocks binding of a reference antibody to an antigen (e.g., recombinant human/simian CD3 epsilon or cell-surface-expressed CD3 epsilon in the present invention) by at least 85% or at least 90%, the antibody or antigen-binding fragment can then be considered to bind to the same/closely related epitope as the reference antibody.

Those skilled in the art will appreciate that it is possible to determine, without undue experimentation, whether a human monoclonal antibody binds to the same epitope as an antibody of the present invention (e.g., mouse monoclonal antibodies 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, WBP3311_2.844.8 and humanized antibodies

WBP3311_2.166.48-z1 and WBP3311_2.306.4-z1) by establishing whether or not the former prevents the latter from binding to the CD3-epsilon antigen polypeptide. If the test antibody competes with an antibody of the present invention, as indicated by reduced binding of the antibody of the present invention to the CD3 epsilon antigen polypeptide, then the two antibodies bind to the same or a closely related epitope. Or, if the binding of the test antibody to the CD3-epsilon antigen polypeptide is inhibited by the antibody of the present invention, then the two antibodies bind to the same or a closely related epitope.

The different symbols used in the names of the antibodies presented in this application have different designations: mIgG2 refers to an antibody with a mouse constant region of the IgG2 isotype; uIgG1 refers to an antibody with a human constant region of the IgG1 isotype; K or L refers to an antibody using a kappa or lambda light chain.

Conservative substitution, in terms of amino acid sequence, refers to the replacement of an amino acid residue with another amino acid residue having a side chain with similar physicochemical properties. For example, conservative substitutions can be between amino acid residues with hydrophobic side chains (e.g., Met, Ala, Val, Leu, and Ile), between residues with neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gln), between residues with acidic side chains (eg Asp, Glu), between amino acids with basic side chains (eg His, Lys and Arg) or between residues with aromatic side chains (eg Trp, Tyr and Phe). As is known in the art, a conservative substitution usually does not result in a significant change in the conformational structure of the protein and therefore may preserve the biological activity of the protein.

The terms homologue and homologous are used interchangeably in the context of this application and refer to nucleic acid sequences (or their complementary strands) or amino acid sequences that share at least 80% sequence identity (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) with other sequences when they are optimally aligned.

Percent (%) sequence identity with respect to an amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleotide) residues in a candidate sequence that are identical to amino acid (or nucleotide) residues in a reference sequence, after alignment of the sequences and, if necessary, , the introduction of gaps, to achieve the maximum number of identical amino acids (or nucleotides). A conservative substitution of amino acid residues may or may not be considered identical residues. Alignment to determine percent amino acid (or nucleotide) sequence identity can be achieved, for example, using publicly available tools such as BLASTN, BLASTp (available from the US National Center for Biotechnology Information (NCBI) website, see also Altschul S.F. et al , J. Mol. Biol., 215:403410 (1990), Stephen F. et al, Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available from the European Bioinformatics Institute website, see also , Higgins D. G. et al, Methods in Enzymology, 266:383402 (1996); Larkin M. A. et al, Bioinformatics (Oxford, England), 23(21): 2947-8 (2007)), and ALIGN or Megalign (DNASTAR) program . Those skilled in the art may use the default parameters provided by the tool, or may adjust the parameters according to the alignment, for example by selecting an appropriate algorithm.

Effector functions in the context of this application refer to biological activities caused by binding of the Fc region of an antibody to its effectors such as the C1 complex and the Fc receptor. Exemplary effector functions include: complement-dependent cytotoxicity (CDC) induced by the interaction of antibodies and C1q on the C1 complex; antibody-dependent cell-mediated cytotoxicity (ADCC) induced by binding of the Fc region of an antibody to an Fc receptor on an effector cell; and phagocytosis.

To treat or treat a condition, in the context of this application, includes preventing or alleviating a condition, slowing the onset or rate of progress of a condition, reducing

- 10 042856 risk of developing the condition, preventing or delaying the development of symptoms associated with the condition, reducing or stopping the symptoms associated with the condition, inducing complete or partial regression of the condition, treating the condition, or some combination of the above.

The isolated substance is altered by human hand from its natural state. If an isolated composition or substance occurs naturally, it has been altered or isolated from its original environment, or both. For example, a polynucleotide or polypeptide naturally present in a living animal in an animal is not isolated, but the same polynucleotide or polypeptide is isolated if it has been sufficiently separated from the coexisting substances of its natural state to exist in an essentially pure state. An isolated nucleic acid sequence refers to the sequence of an isolated nucleic acid molecule. In some embodiments, an isolated antibody or antigen-binding fragment thereof refers to an 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 in the context of the present application refers to a carrier into which a polynucleotide that encodes a protein can be operably inserted to cause expression of that protein. The vector can be used to transform, transduce, or transfect a host cell to cause expression of the genetic element it carries in the host cell. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC), bacteriophages 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, herpes virus (eg, herpes simplex virus), poxvirus, baculovirus, papillomavirus, and papovavirus (eg, SV40). The vector may contain a variety of expression control elements, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. In addition, the vector may contain an origin of replication. The vector may also include substances that facilitate its entry into the cell, including, but not limited to, a viral particle, a liposome, or a protein coat. The vector may be an expression vector or a cloning vector. The present invention provides vectors (e.g., expression vectors) containing the nucleic acid sequence provided herein, encoding an antibody or antigen-binding fragment thereof, at least one promoter (e.g., SV40, CMV, EF-1a) operably linked to the nucleic acid sequence , and at least one selectable marker. Examples of vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus), poxvirus, baculovirus, papillomavirus, papovavirus (e.g., SV40), lambda phage, and M13 phage, plasmid pcDNA3,3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO , pMAL, pMOHO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1/amp, pcDNA3.1, pRc/RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, etc.

The phrase host cell in the context of the present application refers to a cell into which an exogenous polynucleotide and/or vector has been inserted.

A CD3-associated disease or condition, as used herein, refers to any disease or condition that is caused, exacerbated, or otherwise associated with increased or decreased CD3 expression or activity. In some embodiments, the CD3-related condition is an immune disorder such as, for example, cancer, an autoimmune disease, an inflammatory disease, or an infectious disease.

Cancer as used herein refers to any medical condition characterized by malignant cell growth or neoplasm, abnormal proliferation, infiltration or metastasis, and includes both solid tumors and non-solid cancers (hematological malignancies) such as leukemia. The term solid tumor in the context of the present application refers to a solid mass of tumor and/or malignant cells.

The term pharmaceutically acceptable means that said carrier, excipient, diluent, excipient(s) and/or salt are generally chemically and/or physically compatible with the other ingredients included in the composition and physiologically compatible with the recipient.

Anti-CD3 epsilon antibody

The present invention provides anti-CD3 epsilon antibodies and antigen-binding fragments thereof comprising one or more (e.g., 1, 2, 3, 4, 5, or 6) CDR sequences of an anti-CD3 epsilon antibody WBP3311_2.166.48, WBP3311_2.306.4, WBP3311_2.383.47 , WBP3311_2.400.5,

- 11 042856

WBP3311_2.482.5, WBP331_2.488.33, WBP3311_2.615.8 or WBP3311_2.844.8. Throughout the present disclosure, the term WBP3311 referring to antibody names is used interchangeably with W3311. For example, the antibody WBP3311_2.166.48 is also listed as W3311_2.166.48 and these names refer to the same antibody.

WBP3311_2.166.48 as used herein refers to a murine monoclonal antibody having the heavy chain variable region of SEQ ID NO: 81 and the kappal light chain variable region of SEQ ID NO: 83.

WBP3311_2.306.4 as used herein refers to a murine monoclonal antibody having the heavy chain variable region of SEQ ID NO: 85 and the kappal light chain variable region of SEQ ID NO: 87.

WBP3311_2.383.47 as used herein refers to a murine monoclonal antibody having the heavy chain variable region of SEQ ID NO: 89 and the kappal light chain variable region of SEQ ID NO: 91.

WBP3311_2.400.5 as used herein refers to a murine monoclonal antibody having the heavy chain variable region of SEQ ID NO: 93 and the kappal light chain variable region of SEQ ID NO: 95.

WBP3311_2.482.5 as used herein refers to a murine monoclonal antibody having the heavy chain variable region of SEQ ID NO: 97 and the kappal light chain variable region of SEQ ID NO: 99.

WBP331_2.488.33 as used herein refers to a murine monoclonal antibody having the heavy chain variable region of SEQ ID NO: 101 and the kappal light chain variable region of SEQ ID NO: 103.

WBP3311_2.615.8 as used herein refers to a murine monoclonal antibody having the heavy chain variable region of SEQ ID NO: 105 and the kappal light chain variable region of SEQ ID NO: 107.

WBP3311_2.844.8 as used herein refers to a murine monoclonal antibody having the heavy chain variable region of SEQ ID NO: 109 and the kappal light chain variable region of SEQ ID NO: 111.

Tab. 1 shows the CDR sequences of these 8 anti-CD3 epsilon antibodies. The sequences of the heavy chain and light chain variable regions are also shown below.

Table 1

Antibody ID: CDR1 CDR2 CDR3 WBP3311_2.166.48 vh SEQ ID NO: 1 SEQ ID NO: 3 SEQ ID NO: 5 GYSFTTYYIH WIFPGNDNIKYS EKFKG DSVSIYYFDY WBP3311_2.166.48 VK SEQ ID NO: 2 SEQ ID NO: 4 SEQ ID NO: 6 KSSQSLLNSRT RKNYLA WASTRKS TQSFILRT WBP3311 2.306.4 vh SEQ ID NO: 7 SEQ ID NO: 9 SEQ ID NO: 11 GFAFTDYYIH WISPGNVNTKY NENFKG DGYSLYYFDY WBP3311 2.306.4 VK SEQ ID NO: 8 SEQ ID NO: 10 SEQ ID NO: 12 KSSQSLLNSRT RKNYLA WASTRQS TQSHTLRT WBP3311 2.383.47 vh SEQ ID NO: 13 SEQ ID NO: 15 SEQ ID NO: 17 GFTFTNYYIH WISPENGNTKY NENFQD DGYSLYYFDY WBP3311 2.383.47 VK SEQ ID NO: 14 SEQ ID NO: 16 SEQ ID NO: 18 KSSQSLLNSRT RKNYLA WAS IRVS TQSHTLRT WBP3311 2.400.5 vh SEQ ID NO: 19 SEQ ID NO: 21 SEQ ID NO: 23 GYSFTNYYLH WIFPESDNTKY NEKLKG DSVGNYFFDF WBP3311 2.400.5 SEQ ID NO: 20 SEQ ID NO: 22 SEQ ID NO: 24

- 12 042856

VK KSSQSLVNNRT RKNYLA WASTRES AQSFILRT WBP3311 2.482.5 vh SEQ ID NO: 25 SEQ ID NO: 27 SEQ ID NO: 29 GYTFTTYYIH WIFPGSDNIKYN ENFKD DSVSRYYFDY WBP3311 2.482.5 VK SEQ ID NO: 26 SEQ ID NO: 28 SEQ ID NO: 30 KSSQSLVNDRT RKNYLA WASTRES AQSFILRT WBP331 2.488.33 SEQ ID NO: 31 SEQ ID NO: 33 SEQ ID NO: 35 vh GFSFTNYYIH WIFPGTVNTKY NEKFKG DSVGIYYFDF WBP331 2.488.33 VK SEQ ID NO: 32 SEQ ID NO: 34 SEQ ID NO: 36 KSSQSLLNNRT RKNYLA WASTRES TQSFILRT WBP3311 2.615.8 vh SEQ ID NO: 37 SEQ ID NO: 39 SEQ ID NO: 41 GYSFDFYTH WIFPGSDNIKYN EKFKG DSVSVYYFDY WBP3311 2.615.8 VK SEQ ID NO: 38 SEQ ID NO: 40 SEQ ID NO: 42 KSSQSLLNIRTR KNYLA WASTRDS TQSFILRT WBP3311 2.844.8 vh SEQ ID NO: 43 SEQ ID NO: 45 SEQ ID NO: 47 GFAFTDYYIH WISPGNVNTKY NENFKG DGYSLYYFDY WBP3311 2.844.8 SEQ ID NO: 44 SEQ ID NO: 46 SEQ ID NO: 48 VK KSSQSLLNSRT RKNYLA WASTRES TQSHTLRT

Последовательности вариабельных областей тяжелых или каппа-легких цепей 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 и WBP3311_2.844.8 и гуманизированных WBP3311_2.166.48 и WBP3311_2.306.4 антител are presented below.

WBP3311_2.166.48-VH

Amino acid sequence (SEQ ID NO: 81):

OVOLOOSGPELVKPGASVKIACKASGYSFTTYYIHWVKORPGOGLEWIGWIFPG

NDNIKYSEKFKGKATLTADTSSSTAYMQLSSLTSEDSAVYFCAIDSVSIYYFDYWGQGT TLTVSS

Nucleic acid sequence (SEQ ID NO: 82):

- 13 042856

CAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAACCTGGGGCTTCAG TGAAGATTGCCTGCAAGGCTTCTGGCTACAGCTTCACAACCTACTATATACACTGGG TGAAGCAGAGGCCTGGACAGGGACTTGAGTGGATTGGATGGATTTTTCCTGGAAAT GATAATATTAAGTACAGTGAGAAGTTCAAGGGCAAGGCCACACTGACGGCAGACAC TTCCTCCAGTACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCTGT CTATTTCTGTGCTATAGACTCCGTTAGTATCTACTACTTTGACTATTGGGGCCAAGGC ACCACTCTCACAGTCTCCTCA

WBP3311_2.166.48-VK

Amino acid sequence (SEQ ID NO: 83):

DIVMSOSPSSLAVSAGEKVTMSCKSSOSLLNSRTRKNYLAWYOOKPGOSPKLLI YWASTRKSGVPDRFTGSGSGTDFTLTINSVQAEDLAVYYCTQSFILRTFGGGTKLEIK

Nucleic acid sequence (SEQ ID NO: 84):

GACATTGTGATGTCACAGTCTCCATCCTCCCTGGCTGTGTCAGCAGGAGAGA AGGTCACTATGAGCTGCAAATCCAGTCAGAGTCTGCTCAACAGTAGAACCCGAAAG AACTACTTGGCTTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTAC TGGGCATCCACTAGGAAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGG ACAGATTTCACTCTCACCATCAACAGTGTGCAGGCTGAAGACCTGGCAGTTTATTAC TGCACGCAATCTTTTATTCTTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA

WBP3311_2.306.4-VH

Amino acid sequence (SEQ ID NO: 85):

OVQLQQSGPELVKPGASVRISCKASGFAFTDYYIHWVKQRPGQGLEWIGWISPG NVNTKYNENFKGRATLTADLSSSTAYMQLSSLTSEDSAVYFCARDGYSLYYFDYWGQG TTLTVSS

Nucleic acid sequence (SEQ ID NO: 86):

CAGGTCCAGCTGCAGCAGTCTGGACCTGAATTGGTGAAGCCTGGGGCTTCCG TGAGGATATCCTGCAAGGCTTCTGGCTTCGCCTTCACAGACTACTATATACACTGGG TGAAGCAGAGGCCTGGACAGGGTCTTGAGTGGATTGGATGGATTTCTCCTGGAAAT GTTAATACTAAATACAATGAAAACTTCAAGGGCAGGGCCACACTGACTGCAGACCT ATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACCTCTGAGGACTCTGCGGT CTATTTCTGTGCAAGAGATGGATATTCCCTGTATTACTTTGACTACTGGGGCCAAGG CACCACTCTCACAGTCTCCTCA

WBP3311_2.306.4-VK

Amino acid sequence (SEQ ID NO: 87):

DIVMSOSPSSLTVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYOQKPGOSPKLLIY WASTROSGVPDRFTGSGSGTAFTLTISGVQAEDLAVYFCTQSHTLRTFGGGTKLEIK

Nucleic acid sequence (SEQ ID NO: 88):

GACATTGTGATGTCACAGTCTCCATCCTCCCTGACTGTGTCAGCAGGAGAGA AGGTCACTATGAGCTGCAAATCCAGTCAGAGTCTGCTCAACAGTAGAACCCGAAAG AACTACTTGGCTTGGTACCAGCAGAAGCCAGGGCAGTCTCCTAAACTACTAATCTAC TGGGCATCCACTAGGCAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGG

- 14 042856

ACAGCTTTCACTCTCACCATCAGCGGTGTGTCAGGCTGAAGACCTGGCAGTTTATTTC TGCACGCAATCTCATACTCTTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAA A

WBP3311_2.383.47-VH

Amino acid sequence (SEQ ID NO: 89):

OVQLOQSGPELVKPGASVRISCKTSGFTFTNYYIHWVIQRPGQGLEWIGWISPEN GNTKYNENFQDKATLTADISSSTAYMHLSSLTSEDSAVYFCARDGYSLYYFDYWGOGT TLTVSS

Nucleic acid sequence (SEQ ID NO: 90):

CAGGTCCAGCTGCAGCAGTCTGGACCTGAATTGGTGAAGCCTGGGGCTTCAG TGAGGATATCCTGCAAGACTTCTGGCTTCACCTTCACAAACTACTATATACACTGGG TGATACAGAGGCCTGGACAGGGACTTGAGTGGATTGGTTGGATTTCTCCTGAAAATG GTAATACTAAATACAATGAAAACTTCCAGGACAAGGCCACACTGACTGCAGACATA TCGTCCAGCACAGCCTACATGCACCTCAGCAGCCTGACCTCTGAGGACTCTGCGGTC TATTTCTGTGCAAGAGATGGGTATTCCCTTTACTACTTTGACTACTGGGGCCAAGGC ACCACTCTCACAGTCTCCTCA

WBP3311_2.383.47-VK

Amino acid sequence (SEQ ID NO: 91):

DIVMSOSPSSLTVSAGEKVTMSCKSSOSLLNSRTRKNYLAWYOQKPGOSPKLLIY WASIRVSGVPDRFTGSGSGTTFTLTISGVQAEDLAVYYCTQSHTLRTFGGGTKLEIK

Nucleic acid sequence (SEQ ID NO: 92):

GACATTGTGATGTCACAGTCTCCATCCTCCCTGACTGTGTCAGCAGGAGAGA AGGTCACTATGAGCTGCAAATCCAGTCAGAGTCTGCTCAACAGTAGAACCCGAAAG AACTACTTGGCTTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAGCTACTGATCTAC TGGGCATCCATTAGGGTATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGG ACAACTTTCACTCTCACCATCAGCGGTGTGCAGGCTGAAGACCTGGCAGTTTATTAT TGCACGCAATCTCATACTCTTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAA A

WBP3311_2.400.5-VH

Amino acid sequence (SEQ ID NO: 93):

OVQLOQSGPELVNPGASVKISCKASGYSFTNYYLHWVKQRPGQGLEWIGWIFPE SDNTKYNEKLKGKATLTADTSSDTAYMHLSSLTFEDSAVYFCARDSVGNYFFDFWGQG TTLTVSS

Nucleic acid sequence (SEQ ID NO: 94):

CAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAATCCTGGGGCTTCAG TGAAGATATCCTGCAAGGCTTCTGGCTACAGTTTCACAAACTACTATTTACACTGGG TGAAACAGAGGCCTGGACAGGGACTTGAGTGGATTGGATGGATTTTTCCTGAAAGT GATAATACCAAGTACAATGAGAAATTGAAGGGCAAGGCCACACTGACGGCAGACA CATCCTCCGATACAGCCTACATGCACCTCAGCAGCCTGACATTTGAGGACTCTGCAG TCTATTTCTGTGCAAGAGACTCCGTTGGAAACTACTTCTTTGACTTCTGGGGCCAAG

- 15 042856

GCACCACTCTCACAGTCTCCTCA

WBP3311_2.400.5-VK

Amino acid sequence (SEQ ID NO: 95):

DIVMSOSSPSSLAVSAGEKVTMRCKSSOSLVNNRTRKNYLAWYOOKPGOPPKLLI YWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCAQSFILRTFGGGTKLEIK

Nucleic acid sequence (SEQ ID NO: 96):

GACATTGTGATGTCACAGTCTCCATCCTCCCTGGCTGTGTCAGCGGGAGAGA AGGTCACTATGAGGTGCAAATCCAGTCAGAGTCTGGTCAACAATAGAACCCGAAAG AACTACTTGGCATGGTACCAGCAGAAACCAGGGCAGCCTCCTAAACTATTGATCTAC TGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGG ACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTAC TGCGCGCAATCTTTTATTCTTCGGACGTTCGGTGGAGGCACCAAACTGGAAATCAAA WBP3311_2.482.5-VH

Amino acid sequence (SEQ ID NO: 97):

OVQLQQSGPELVKPGSSVKISCKPSGYTFTTYYIHWVKQRPGQGLEWIGWIFPGS DNIKYNENFKDKATLTADTSSSTAYMQLSSLTSEDSAVYFCARDSVSRYYFDYWGQGTI LTVSS

Nucleic acid sequence (SEQ ID NO: 98):

CAGGTTCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAACCTGGGTCTTCAG TGAAGATATCCTGCAAACCTTCTGGCTACACCTTCACAACTTACTATATACATTGGG TGAAGCAGAGGCCTGGACAGGGACTTGAGTGGATTGGATGGATTTTTCCTGGAAGT GATAATATTAAATACAATGAGAATTTCAAGGACAAGGCCACACTGACGGCAGACAC ATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAAGACTCTGCAGT CTATTTCTGTGCAAGAGACTCCGTCAGTAGGTACTACTTTGACTACTGGGGCCAAGG CACCATTCTCACAGTTTCTTCA

WBP3311_2.482.5-VK

Amino acid sequence (SEQ ID NO: 99):

DIVMSQSPSSLAVSAGEKVTMSCKSSOSLVNDRTRKNYLAWYQQKPGLSPKLLI YWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCAQSFILRTFGGGTKLEIK

Nucleic acid sequence (SEQ ID NO: 100):

GACATTGTGATGTCACAGTCTCCATCCTCCCTGGCTGTGTCAGCAGGAGAGA AGGTCACTATGAGCTGCAAATCCAGTCAGAGTCTGGTCAATGATAGAACCCGAAAA AACTACTTGGCTTGGTACCAGCAGAAACCAGGGCTGTCTCCTAAACTGCTGATCTAC TGGGCTTCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGG ACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCTGTTTATTAC TGCGCGCAATCTTTTATTCTTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA

WBP331_2.488.33-VH

Amino acid sequence (SEQ ID NO: 101):

OVQLQQSGPELVKPGTSVKISCKASGFSFTNYYIHWVKQRPGQGPEWIGWIFPGT VNTKYNEKFKGKATLTADTSSNTAFMQLSSLTSADSAVYFCARDSVGIYYFDFWGLGT

- 16 042856

TLTVSS

Nucleic acid sequence (SEQ ID NO: 102):

CAGGTCCAGCTGCAACAGTCTGGACCTGAACTGGTGAAACCTGGGACTTCAG TGAAGATATCCTGCAAGGCTTCTGGCTTCAGCTTCACAAACTACTATATACACTGGG TGAAGCAGAGGCCTGGACAGGGACCTGAGTGGATTGGATGGATTTTTCCTGGAACT GTTAATACTAAGTACAATGAGAAGTTCAAGGGTAAGGCCACACTGACGGCAGACAC ATCCTCCAATACAGCCTTCATGCAGCTCAGCAGCCTGACTTCTGCGGACTCTGCAGT CTATTTCTGTGCAAGAGACTCCGTTGGTATCTACTACTTTGACTTCTGGGGCCTAGGC ACCACTCTCACAGTCTCCTCA

WBP331_2.488.33-VK

Amino acid sequence (SEQ ID NO: 103):

DIVMSQSPSSLAVSAGEKVTVSCKSSOSLLNNRTRKNYLAWYQQKPGQSPKLLI YWASTRESGVPDRFTGSGSGTDFTLTISSVOAEDLAVYYCTQSFILRTFGGGTKLEIK

Nucleic acid sequence (SEQ ID NO: 104):

GACATTGTGATGTCACAGTCTCCATCCTCCCTGGCTGTGTCAGCAGGAGAGA AGGTCACTGTGAGTTGCAAATCCAGTCAGAGTCTGCTCAACAATAGAACCCGAAAA AACTACTTGGCTTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTACTAATCTAC TGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGT ACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTAC TGCACGCAATCTTTTATTCTTCGGACGTTCGGTGGAGGCACCAAGCTGGAGATCAAA WBP3311_2.615.8-VH

Amino acid sequence (SEQ ID NO: 105):

OVQLQQSGPELVKPGTSMKISCKASGYSFTDFYTHWVRQRPGQGLEWIGWIFPG SDNIKYNEKFKGKATLTADTSSSTAYMQLSSLTSEDSAVYFCARDSVSVYYFDYWGOG TTLTVSS

Nucleic acid sequence (SEQ ID NO: 106):

CAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTAAAACCTGGGACTTCAA TGAAAATATCCTGCAAGGCTTCTGGCTACAGTTTCACAGACTTCTATACACACTGGG TGAGGCAGAGGCCTGGACAGGGACTTGAGTGGATTGGATGGATTTTTCCTGGAAGT GATAATATTAAATACAATGAGAAGTTCAAGGGCAAGGCCACACTGACGGCAGACAC ATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCAGT CTATTTCTGTGCAAGAGACTCCGTTAGTGTCTACTACTTTGACTATTGGGGCCAAGG CACCACTCTCACAGTCTCCTCA

WBP3311_2.615.8-VK

Amino acid sequence (SEQ ID NO: 107):

DIVMSQSPSSLAVTAGEKVTMSCKSSQSLLNIRTRKNYLAWYQOKPGQSPKLLIY WASTRDSGVPDRFTGSGSGTDFTLTISSVOAEDLAVYYCTQSFILRTFGGGTKLEIK

Nucleic acid sequence (SEQ ID NO: 108):

GACATCGTGATGTCACAGTCTCCATCCTCCCTGGCTGTGACAGCAGGAGAGA AGGTCACTATGAGCTGCAAATCCAGTCAGTCTGCTCAACATTAGAACCCGAAAG

- 17 042856

AACTACTTGGCTTGGTACCAACAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTAC TGGGCATCCACTAGGGACTCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGG ACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTAC TGCACGCAATCTTTTATTCTTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA_ WBP.33

Amino acid sequence (SEQ ID NO: 109):

OVQLQQSGPELVKPGASVRISCKASGFAFTDYYIHWVKQRPGQGLEWIGWISPG NVNTKYNENFKGRATLTADLSSSTAYMQLSSLTSEDSAVYFCARDGYSLYYFDYWGOG TTLTVSS

Nucleic acid sequence (SEQ ID NO: 110):

CAGGTCCAGCTGCAGCAGTCTGGACCTGAATTGGTGAAGCCTGGGGCTTCCG TGAGGATATCCTGCAAGGCTTCTGGCTTCGCCTTCACAGACTACTATATACACTGGG TGAAGCAGAGGCCTGGACAGGGTCTTGAGTGGATTGGATGGATTTCTCCTGGAAAT GTTAATACTAAATACAATGAAAACTTCAAGGGCAGGGCCACACTGACTGCAGACCT ATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACCTCTGAGGACTCTGCGGT CTATTTCTGTGCAAGAGATGGATATTCCCTGTATTACTTTGACTACTGGGGCCAAGG CACCACTCTCACAGTCTCCTCA

WBP3311_2.844.8-VK

Amino acid sequence (SEQ ID NO: 111):

DIVMSQSPSSLTVSAGEKVTMSCKSSOSLLNSRTRKNYLAWYQQKPGQSPKLLIY WASTRESGVPDRFTGSGSGTAFTLTISGVQAEDLAVYFCTQSHTLRTFGGGTKLEIK

Nucleic acid sequence (SEQ ID NO: 112):

GACATTGTGATGTCACAGTCTCCATCCTCCCTGACTGTGTCAGCAGGAGAGA AGGTCACTATGAGCTGCAAATCCAGTCAGAGTCTGCTCAACAGTAGAACCCGAAAG AACTACTTGGCTTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAGCTACTAATCTAC TGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGG ACAGCTTTCACTCTCACCATCAGCGGTGTGCAGGCTGAAGACCTGGCAGTTTATTTC TGCACGCAATCTCATACTCTTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAA A

It is known that CDRs are responsible for antigen binding, however, it has been found that not all of the 6 CDRs are essential or unchangeable. More specifically, one or more of the CDRs in the anti-CD3 epsilon antibody WBP3311_2.166.48, WBP3311_2.306.4, WBP3311_2.383.47, WBP3311_2.400.5, WBP3311_2.482.5, WBP331_2.483.33, or WBP_BP3311_2. 844.8 while essentially retaining specific binding affinity for CD3-epsilon.

In some embodiments, the anti-CD3 epsilon antibodies and antigen-binding fragments thereof provided herein comprise the heavy chain CDR3 sequence of one of the anti-CD3 epsilon antibodies 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. In some embodiments, the anti-CD3 epsilon antibodies and antigen-binding fragments 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. The CDR3 regions of the heavy chain are located in the center of the antigen-binding site and are therefore thought to have the most contact with the antigen and provide the most free energy for antibody-antigen affinity. It is also believed that the heavy chain CDR3 is by far the most diverse antigen-binding site CDR in terms of length, amino acid composition and conformation through multiple diversification mechanisms (Tonegawa S. Nature. 302: 575-81). Heavy chain CDR3 diversity is sufficient to obtain most antibody specificities (Xu JL, Davis MM. Immunity. 13:37-45) as well as the desired antigen-binding affinity (SchierR, etc. J Mol Biol. 263:551-67) .

In some embodiments, the antibodies and antigen-binding fragments provided herein include suitable framework region (FR) sequences, provided that the antibodies and antigen-binding fragments thereof can specifically bind to CD3epsilon. Sequence CDR presented in table. 1 are derived from mouse antibodies, but can be grafted onto any suitable FR sequences from any suitable species, such as mouse, human, rat, rabbit, among others, using suitable methods known in the art, such as recombinant methods.

In some embodiments, the antibodies and antigen-binding fragments provided herein are humanized. A humanized antibody or antigen-binding fragment is desirable because of its reduced immunogenicity in humans. A humanized antibody is chimeric in its variable regions because non-human CDR sequences are grafted onto human or substantially human FR sequences. Humanization of an antibody or antigen-binding fragment can essentially be accomplished by replacing non-human (such as murine) CDR genes with the corresponding human CDR genes in the 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: 1534-1536).

To achieve this goal, suitable human heavy chain and light chain variable domains can be selected using methods known in the art. In an illustrative example, a best fit approach can be used where the variable domain sequence of a non-human antibody (e.g., a rodent) is screened or BLASTed against a database of known human variable domain sequences, and the human sequence closest to the non-human target sequence is identified and used. as a human scaffold for grafting non-human CDR sequences (see, for example, Sims et al. (1993) J. Immunol. 151:2296; Chothia et al. (1987) J. Mot. Biol. 196:901). Alternatively, a scaffold derived from the consensus sequence of all human antibodies can be used to graft non-human CDRs (see, e.g., Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; Presta et al. ( 1993) J. Immunol., 151: 2623).

In some embodiments, the humanized antibodies or antigen-binding fragments provided herein consist of essentially all human sequences except for CDR sequences that are not human. In some embodiments, the variable region FRs and constant regions, if present, consist wholly or essentially of human immunoglobulin sequences. Human FR sequences and human constant region sequences can be derived from different human immunoglobulin genes, for example, FR sequences derived from one human antibody and the constant region from another human antibody. In some embodiments, the humanized antibody or antigen-binding fragment includes human FR1-4 and human JH and Jk.

In some embodiments, the humanized antibodies and antigen-binding fragments provided herein comprise one or more WBP3311_2.166.48-z1 or WBP3311_2.306.4-z1 FR sequences. Tab. 2 below shows the FR sequences WBP3311_2.166.48-z1 or WBP3311_2.306.4-z1. Native mouse FR sequences are also shown in Table 1. 2. The sequences of the variable regions of the heavy chains and light chains are also shown below.

WBP3311_2.166.48-z1 as used herein refers to a humanized antibody based on WBP3311_2.166.48 which comprises the heavy chain variable region of SEQ ID NO: 113 and the kappa light chain variable region of SEQ ID NO: 115. WBP3311_2.166.48-z1 has a comparable affinity for the antigen compared to its parent antibody WBP3311_2.166.48.

WBP3311_2.306.4-z1 in the context of the present application refers to a humanized antibody based on WBP3311_2.306.4, which includes the heavy chain variable region of SEQ ID NO: 117 and the kappa light chain variable region of SEQ ID NO: 119. WBP3311_2.306.4-z1 has a comparable affinity for the antigen compared to its parent antibody WBP3311_2.306.4.

table 2

FR1 FR2 FR3 FR4 WBP3311 2.1 SEQ ID NO: 49 SEQ GO NO: 51 SEQ ID NO: 53 SEQID NO:

- 19 042856

66.48-VH 55 QVQLQQSGPE LVKPGASVKI ACKAS WVKQRPGQGLE WIG KATLTADTSSST AYMQLSSLTSED SAVYFCAI WGQGTTLT vss WBP3311 2.1 66.48-zl-VH SEQ ID NO: 57 SEQ ID NO: 59 SEQ ID NO: 61 SEQID NO: 63 QVQLVQSGA EVKKPGSSVK VSCKAS WVRQAPGQGL EWMG RVTITADKSTST AYMELSSLRSE DTAVYYCAI WGQGTLVT VSS WBP3311 2.1 66.48-VK SEQ ID NO: 50 SEQ ID NO: 52 SEQ ID NO: 54 SEQID NO: 56 DIVMSQSPSS LAVSAGEKVT MSc WYQQKPGQSPK LLIY GVPDRFTGSGS GTDFFTLTINSVQ AEDLAVYYC FGGGTKLEI TO WBP3311 2.1 66.48-zl-VK SEQ ID NO: 58 SEQ ID NO: 60 SEQ ID NO: 62 SEQID NO: 64 DIVMTQSPDS LAVSLGERAT INC WYQQKPGQPPK LLIY GVPDRFSGSGS GTDFFTLTISSLQ AEDVAVYYC FGGGTKVEI TO WBP3311 2.3 06.4-VH SEQ ID NO: 65 SEQ W NO: 67 SEQ ID NO: 69 SEQ ID NO: 71 QVQLQQSGPE LVKPGASVRL SCKAS WMKQRPGQGL EWIG RATVTADLSSST AYMQLSSLTSED SAVYFCAR WGQGTTLT VSS WBP3311 2.3 06.4-zl-VH SEQ W NO: 73 SEQ W NO: 75 SEQ ID NO: 77 SEQID NO: 79 QVQLVQSGA EVKKPGSSVK VSCKAS WVRQAPGQGL EWMG RVTITADKSTST AYMELSSLRSE DTAVYYCAR WGQGTLVT VSS WBP3311 2.3 06.4-VK SEQ ID NO: 66 SEQ ID NO: 68 SEQ W NO: 70 SEQID NO: 72 DIVMSQSPSSL TVSAGEKVT MSc WYQQKPGQSPK LLIY GVPDRFTGSGS GTAFTLTISGVQ AEDLAVYFC FGGGTKLEI TO WBP3311 2.3 06.4-zl-VK SEQ W NO: 74 SEQ W NO: 76 SEQ W NO: 78 SEQ ID NO: 80 DIVMTQSPDS LAVSLGERAT INC WYQQKPGQPPK LLIY GVPDRFSGSGS GTDFFTLTISSLQ AEDVAVYYC FGGGTKVEI TO

- 20 042856

WBP3311_2.166.48-zl-VH

Amino acid sequence (SEQ ID NO: 113):

QVOLVOSGAEVKKPGSSVKVSCKASGYSFTTYYIHWVROAPGOGLEWMGWIFP

GNDNIKYSEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAIDSVSIYYFDYWGOG

TLVTVSS

Nucleic acid sequence (SEQ ID NO: 114):

CAGGTGCAACTCGTGCAGTCTGGAGCTGAAGTGAAGAAGCCTGGGTCTTCAG TCAAGGTCAGTTGCAAGGCCAGTGGGTATTCCTTCACTACCTACTACATCCACTGGG TGCGGCAGGCACCAGGACAGGGGCTTGAGTGGATGGGCTGGATCTTTCCCGGCAAC GATAATATTAAGTACAGCGAGAAGTTCAAAGGGAGGGTCACCATTACCGCCGACAA ATCCACTTCCACAGCCTACATGGAGTTGAGCAGCCTGAGATCCGAGGATACAGCCG TGTACTACTGTGCCATTGACAGCGTGTCCATCTACTACTTTGACTACTGGGGCCAGG GCACACTGGTCACAGTGAGCAGC

WBP3311_2.166.48-zl-VK

Amino acid sequence (SEQ ID NO: 115):

DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY WASTRKSGVPDRFSGSGSGTDFTLTISSLOAEDVAVYYCTQSFILRTFGGGTKVEIK

Nucleic acid sequence (SEQ ID NO: 116):

GACATCGTCATGACCCAGTCCCCAGACTCTTTGGCAGTGTCTCTCGGGGAAA GAGCTACCATCAACTGCAAGAGCAGCCAGTCCCTTCTGAACAGCAGGACCAGGAAG AATTACCTCGCCTGGTACCAACAGAAGCCCGGACAGCCTCCTAAGCTCCTGATCTAC TGGGCCTCAACCCGGAAGAGTGGAGTGCCCGATCGCTTTAGCGGGAGCGGCTCCGG GACAGATTTCACACTGACAATTTCCTCCCTGCAGGCCGAGGACGTCGCCGTGTATTA CTGTACTCAGAGCTTCATTCTGCGGACATTTGGCGGCGGGACTAAAGTGGAGATTAA G

WBP3311_2.306.4-zl-VH

Amino acid sequence (SEQ ID NO: 117):

OVOLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVROAPGOGLEWMGWIS PGNVNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG QGTLVTVSS

Nucleic acid sequence (SEQ ID NO: 118):

CAGGTGCAGCTTGTGCAGTCTGGGGCAGAAGTGAAGAAGCCTGGGTCTAGTG TCAAGGTGTCATGCAAGGCTAGCGGGTTCGCCTTTACTGACTACTACATCCACTGGG TGCGGCAGGCTCCCGGACAAGGGTTGGAGTGGATGGGATGGATCTCCCCAGGCAAT GTCAACACAAAGTACAACGAGAACTTCAAAGGCCGCGTCACCATTACCGCCGACAA GAGCACCTCCACAGCCTACATGGAGCTGTCCAGCCTCAGAAGCGAGGACACTGCCG TCTACTACTGTGCCAGGGATGGGTACTCCCTGTATTACTTTGATTACTGGGGCCAGG GCACACTGGTGACAGTGAGCTCC

WBP3311_2.306.4-zl-VK

Amino acid sequence (SEQ ID NO: 119):

DIVMTOSPDSLAVSLGERATINCKSSOSLLNSRTRKNYLAWYOQKPGOPPKLLIY

WASTROSGVPDRFSGSGSGTDFTLTISSLOAEDVAVYYCTQSHTLRTF GGGTKVEIK

Nucleic acid sequence (SEQ ID NO: 120):

GATATCGTGATGACCCAGAGCCCAGACTCCCTTGCTGTCTCCCTCGGCGAAA GAGCAACCATCAACTGCAAGAGCTCCCAAAGCCTGCTGAACTCCAGGACCAGGAAG AATTACCTGGCCTGGTATCAGCAGAAGCCCGGCCAGCCTCCTAAGCTGCTCATCTAC TGGGCCTCCACCCGGCAGTCTGGGGTGCCCGATCGGTTTAGTGGATCTGGGAGCGG GACAGACTTCACATTGACAATTAGCTCACTGCAGGCCGAGGACGTGGCCGTCTACTA CTGTACTCAGAGCCACACTCTCCGCACATTCGGCGGAGGGACTAAAGTGGAGATTA AG

Two exemplary humanized anti-CD3 epsilon antibodies, WBP3311_2.166.48-z1 or WBP3311_2.306.4-z1, both retained specific binding affinity for a CD3-expressing cell (e.g., a CD4 T cell), and in this aspect are at least comparable to the original mouse antibodies or even better. These two exemplary humanized antibodies both retained their functional interaction with the CD3 expressing cell in that both could activate human T cells and trigger the release of the cytokines TNF-alpha and IFN-gamma.

In some embodiments, FR regions of human origin may include

- 21 042856 the same amino acid sequence as the human immunoglobulin from which they originate. In some embodiments, one or more amino acid residues of the human FR are replaced with the corresponding residues from the original non-human antibody. In some embodiments, it may be desirable to obtain a humanized antibody, or fragment thereof, that closely resembles the structure of the original non-human antibody. In some embodiments, a 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 substitutions in each of the human FR sequences, or no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue substitutions in all heavy or light chain variable domain FRs. In some embodiments, such a change in amino acid residues may be present only in the FR regions of the heavy chain, only in the FR regions of the light chain, or in both chains.

In some embodiments, the antibodies and antigen-binding fragments 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 ID NO: 113, SEQ ID NO: 117. In some embodiments, the antibodies and antigen-binding fragments thereof provided in herein, include 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, anti-CD3 epsilon antibodies and antigen-binding fragments provided herein comprise all or part of the heavy chain variable domain and/or all or part of the light chain variable domain. In one embodiment, the anti-CD3 epsilon antibody and antigen-binding fragments provided herein is a single domain antibody that consists of all or part of the heavy chain variable domain provided herein. More detailed information about such a single domain antibody can be found in the prior art (see, for example, US patent No. 6248516).

In some embodiments, the anti-CD3 epsilon antibodies and fragments provided herein also include an immunoglobulin constant region. In some embodiments, the immunoglobulin constant region includes a heavy chain and/or light chain constant region. The heavy chain constant region includes CH1, hinge and/or CH2-CH3 regions. In some embodiments, the heavy chain constant region includes an Fc region. In some embodiments, the light chain constant region comprises Ck.

In some embodiments, anti-CD3 epsilon antibodies and antigen-binding fragments thereof have an IgG1 or IgG2a isotype constant region that has a reduced or depleted effector function, such as ADCC or CDC, that can be determined using various assays, such as an Fc binding assay. receptor, dq binding assay and cell lysis assay.

The binding affinity of an antibody and an antigen-binding fragment described herein can be represented by a KD value, which is the ratio of the rate of dissociation to the rate of association (k _off /k _on ) when the binding between the antigen and the antigen-binding molecule reaches equilibrium. Antigen binding affinity (eg, KD) can be appropriately determined using suitable methods known in the art, including, for example, flow cytometry. In some embodiments, the binding of an antibody to an antigen at various concentrations can be determined by flow cytometry, the determined mean fluorescence intensity (MFI) can first be plotted against the antibody concentration, the KD value can then be calculated by fitting the specific binding fluorescence intensity (Y) versus the concentration antibodies (X) into a single center saturation equation: Y=B _max *X/(K _D +X) using Prism version 5 (GraphPad Software, San Diego, CA) where B _max refers to the maximum specific binding of the test antibody to the antigen.

In some embodiments, the anti-CD3 epsilon antibodies and antigen-binding fragments provided herein are capable of specifically binding to cell surface expressed human CD3 epsilon or recombinant human CD3 epsilon. CD3 epsilon is a receptor expressed on the cell. Recombinant CD3 epsilon is a soluble CD3 epsilon that is recombinantly expressed and is not bound to the cell membrane. Recombinant CD3 epsilon can be obtained by various recombinant methods. In one example, a CD3 epsilon DNA sequence encoding the extracellular domain of human CD3 epsilon (NP_000724,1) (Met1-Asp126) can be fused to a polyhistidine tag at the C-terminus in an expression vector, followed by transfection and expression in 293E cells and purification by Niaffin chromatography. .

In some embodiments, the anti-CD3 epsilon antibodies and antigen-binding fragments provided herein are capable of specifically binding to human CD3 epsilon expressed on cell surfaces with a binding affinity (K _D ) of not greater than

- 22 042856

5x10 ^-9 M, no more than 4x10 ^-9 M, no more than 3x10 ^-9 M, no more than 2x10 ^-9 M, no more than 10 ^-9 M, no more than 5x10 ^-10 M, no more than 4x10 ^{- 10} M, no more than 3x10 ^-10 M, no more than 2x10 ^-10 M, no more than

10 ^-10 M, no more than 5x10 ^-11 M or no more than 4x10 ^-11 M, no more than 3x10 ^-11 M or no more than

2x10 ^-11 M, or not more than 10 ^-11 M, as measured by flow cytometry.

In some embodiments, the anti-CD3 epsilon antibodies and antigen-binding fragments provided herein cross-react with cynomolgus monkey CD3 epsilon, e.g., cell surface expressed cynomolgus CD3 epsilon, or soluble recombinant cynomolgus monkey CD3 epsilon.

The binding of antibodies to recombinant CD3 epsilon or CD3 epsilon expressed on the cell surface can also be represented as a half maximum effective concentration (EC ₅₀ ) value, which refers to the concentration of an antibody when 50% of its maximum effect (e.g., binding or inhibition) is observed. etc.). The EC ₅₀ value can be measured by methods known in the art, for example using sandwich assays such as ELISA, Western blotting, flow cytometry, and other binding assays. In some embodiments, the antibodies and fragments provided herein specifically bind to recombinant human CD3 epsilon at an EC ₅₀ (i.e. concentration causing 50% binding) of not more than 0.01 nM, not more than 0, 02 nM, 0.03 nM or less, 0.04 nM or less, 0.05 nM or less, 0.06 nM or less, 0.07 nM or less, or 0.08 nM or less , as determined by the ELISA method. In some embodiments, the antibodies and fragments provided herein specifically bind to human CD3 epsilon expressed on cell surfaces with an EC ₅₀ of not more than 0.5 nM, not more than 0.6 nM, not more than 0 .7 nM, not more than 0.8 nM, not more than 0.9 nM, not more than 1 nM, not more than 2 nM, not more than 3 nM, not more than 4 nM, not more than 5 nM, not more than 6 nM, not more than 7 nM, not more than 8 nM, not more than 9 nM, or not more than 10 nM, as determined by flow cytometry.

In some embodiments, the antibodies and antigen-binding fragments thereof bind to cynomolgus monkey CD3 epsilon with the same binding affinity as human CD3 epsilon. Например, связывание иллюстративных антител WBP3311_2.166.48, WBP3311_2.306.4, WBP3311_2.383.47, WBP3311_2.400.5, WBP3311_2.482.5, WBP3311_2.488.33, WBP3311_2.615.8, WBP3311_2.844.8 с CD3эпсилон яванского макака происходит с такой же аффинностью или при таком же значении EC ₅₀ as with human CD3 epsilon.

In some embodiments, the antibodies and fragments provided herein specifically bind to recombinant cynomolgus monkey CD3 epsilon with an EC ₅₀ of not more than 0.001 nM, not more than 0.005 nM, not more than 0.01 nM, not more than 0 .02 nM, not more than 0.03 nM, not more than 0.04 nM, or not more than 0.05 nM, as determined by ELISA.

In some embodiments, the antibodies and fragments provided herein have a specific binding affinity for human CD3 epsilon that is sufficient to provide diagnostic and/or therapeutic use. A number of therapeutic strategies modulate T-cell immunity by targeting TCR signaling, especially with anti-human CD3 monoclonal antibodies that have clinical applications.

The antibodies or antigen-binding fragments thereof provided herein may be a monoclonal antibody, a polyclonal antibody, a humanized antibody, a chimeric antibody, a recombinant antibody, a bispecific antibody, a labeled antibody, a bivalent antibody, or an anti-idiotypic antibody. A recombinant antibody is an antibody produced in vitro using recombinant methods, and not in animals.

Antibody variants

The present invention also covers various variants of antibodies and their antigennegative fragments presented in this application. In some embodiments, the present invention encompasses various types of variants of an exemplary antibody of the present invention, i.e. 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.

In some embodiments, the antibody variants include one or more modifications or substitutions in one or more of the CDR sequences shown in Table 1. 1, one or more FR sequences presented in table. 2, heavy or light chain variable region sequences provided herein and/or constant region (eg, Fc region). Such variants retain the specific binding affinity of their parent antibodies for CD3 epsilon but have one or more desirable properties acquired through modification(s) or substitution(s). For example, antibody variants may have improved antigen binding affinity, improved glycosylation pattern, reduced risk of glycosylation, reduced deamination, reduced or impoverished effector function(s), improved FcRn receptor binding, increased pharmacokinetic half-life, pH sensitivity, and/or compatibility with conjugation (for example, one or more

- 23 042856 built-in cysteine residues).

The parent antibody sequence can be screened to identify suitable or preferred residues to be modified or substituted using methods known in the art, such as alanine scanning mutagenesis (see, for example, Cunningham and Wells (1989) Science, 244 :1081-1085). Briefly, target residues (eg, charged residues such as Arg, Asp, His, Lys, and Glu) can be identified and replaced with a neutral or negatively charged amino acid (eg, alanine or polyalanine) and modified antibodies produced and screened for the property of interest. If a substitution at a particular amino acid position demonstrates a functional change of interest, then that position can be identified as a potential residue for modification or substitution. Potential residues can be further evaluated by substitution with another type of residue (e.g. cysteine residue, positively charged residue, etc.)

Affinity variant

The affinity variant may contain modifications or substitutions in one or more of the CDR sequences shown in Table 1. 1, one or more FR sequences presented in table. 2, the heavy or light chain variable region sequences provided herein. Affinity variants retain the specific binding affinity of the parent antibody for CD3 epsilon or even have improved specific binding affinity for CD3 epsilon compared to the parent antibody. In some embodiments, at least one (or all) of the substitutions in the CDR sequences, FR sequences, or variable region sequences comprises a conservative substitution.

Specialists in this field should be clear that in the CDR sequences and FR sequences presented in table. 1 and table. 2, one or more amino acid residues can be changed, but the resulting antibody or antigen-binding fragment will still retain binding affinity for CD3 epsilon, or even have improved binding affinity. Various methods known in the art can be used to achieve this goal. For example, a library of antibody variants (such as Fab or scFv variants) can be generated and expressed using phage display technology and then screened for binding affinity to human CD3epsilon. As another example, a computer program can be used to virtually simulate antibody binding to human CD3epilon and identify amino acid residues on antibodies that form a binding interface. Such residues can either be avoided in the substitution to prevent loss of binding affinity, or targeted for substitution to provide stronger binding.

In some embodiments, a humanized antibody or antigen-binding fragment provided herein comprises one or more amino acid residue substitutions in one or more CDR sequences and/or one or more FR sequences. In some embodiments, the affinity 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 some embodiments, anti-CD3 epsilon antibodies and antigen-binding fragments thereof comprise 1, 2, or 3 CDR sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with the sequence (or sequences) shown in table. 1, and at the same time retain the binding affinity for CD3-epsilon at the same level as that of the original antibody, or even higher.

In some embodiments, 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 with the sequence (or sequences) shown in table. 2, and at the same time maintain the binding affinity for CD3-epsilon at the same level as that of the original antibody, or even higher.

In some embodiments, anti-CD3 epsilon antibodies and antigen-binding fragments thereof comprise one or more variable region sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with the sequence (or sequences) shown 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 at the same time retain the binding affinity for CD3epsilon at the same level as that of the original antibody, or even higher. In some embodiments, a total of 1-10 amino acids have been changed, 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,

- 24 042856

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, and SEQ ID NO: 119. In some embodiments, substitutions, insertions, or deletions occur in regions outside of the CDRs (ie, FR regions).

Glycosylation variant

Anti-CD3 epsilon antibodies and antigen-binding fragments provided herein also encompass a glycosylation variant that can be made to either increase or decrease the degree of glycosylation of an antibody or antigen-binding fragment.

An antibody or antigen-binding fragment thereof may include one or more amino acid residues with a side chain to which a carbohydrate moiety (eg, an oligosaccharide structure) may be attached. Glycosylation of antibodies is usually either N-linked or Bound. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an aspartic residue, such as an aspartic residue in a tripeptide sequence such as asparagine-X-serine and asparagine-X-threonine, where X is any amino acid other than proline. Linked glycosylation refers to the addition of one of the sugars N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid, most typically serine or threonine. Deletion of the native glycosylation site is conveniently accomplished, for example, by changing the amino acid sequence so that one of the tripeptide sequences described above (for N-linked glycosylation sites) or serine or threonine residues (for linked glycosylation sites) present in the sequence are replaced. A new glycosylation site can be similarly created by inserting such a tripeptide sequence or a serine or threonine residue.

Cysteine-engineered variant

Anti-CD3 epsilon antibodies and antigen binding fragments provided herein also encompass a cysteine engineered variant that includes one or more inserted free cysteine amino acid residues.

A free cysteine residue is a residue that is not part of a disulfide bridge. A cysteine-engineered variant is useful for conjugation, for example, with a cytotoxic and/or imaging compound, label or radioisotope, among others, at the site of the engineered cysteine, for example, via maleimide or haloacetyl. Methods for constructing antibodies or antigen-binding fragments for insertion of free cysteine residues are known in the art, see, for example, WO 2006/034488.

Fc option

Anti-CD3 epsilon antibodies and antigen binding fragments provided herein also encompass an Fc variant that includes one or more modifications or substitutions of amino acid residues in the Fc region and/or the hinge region.

In some embodiments, the anti-CD3 epsilon antibodies or antigen-binding fragments comprise one or more amino acid substitutions that improve pH-dependent binding to the neonatal Fc receptor (FcRn). Such a variant may have an extended pharmacokinetic half-life because it binds to FcRn at acidic pH, allowing it to avoid degradation in the lysosome, followed by translocation and release from the cell. Methods for constructing an antibody and its antigen-binding fragment to improve FcRn binding affinity 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, Chapter 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 some embodiments, the anti-CD3 epsilon antibodies or antigen-binding fragments comprise one or more amino acid substitutions that alter antibody-dependent cellular cytotoxicity (ADCC). Some amino acid residues in the CH2 domain of the Fc region can also be changed to provide increased ADCC activity. Alternatively or additionally, the carbohydrate structures on the antibody may be substituted to enhance ADCC activity. The use of antibody engineering techniques to modify ADCC activity is known 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 Immunol. 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 some embodiments, the anti-CD3 epsilon antibodies or antigen binding fragments comprise one or more amino acid substitutions that alter complement dependent cytotoxicity (CDC), e.g. Duncan & Winter Nature 322:738-40 (1988), US Pat. No. 5,648,260; US Pat. No. 5,624,821); and WO 94/29351 for other examples of Fc region variants.

In some embodiments, anti-CD3 epsilon antibodies or antigen-binding

- 25 042856 fragments include one or more amino acid substitutions at the border of the Fc region to facilitate and/or promote heterodimerization. These modifications include the introduction of a protrusion into the first Fc polypeptide and a cavity into the second Fc polypeptide, wherein the protrusion may be positioned in the cavity so as to facilitate the interaction of the first and second Fc polypeptides to form a heterodimer or complex. Methods for generating antibodies with these modifications are known in the art, for example, as described in US Pat. No. 5,731,168.

Antigen binding fragments

The present application also provides anti-CD3 epsilon antigen-binding fragments. Various types of antigen-binding fragments are known in the art and can be designed based on the anti-CD3 epsilon antibody of the present invention, including, for example, illustrative antibodies whose CDR and FR sequences are shown in Table. 1 and 2 and their various variants (such as affinity variants, glycosylation variants, Fc variants, cysteine engineered variants, etc.).

In some embodiments, the anti-CD3 epsilon antigen binding fragment provided herein is a camelized single domain antibody, diabody, single chain Fv fragment (scFv), scFv dimer, BsFv, dsFv, (dsFv) ₂ , dsFv-dsFv', Fv fragment, Fab, Fab', F(ab') ₂ , bispecific antibody, ds diabody, nanobody, domain antibody, single domain antibody, or bivalent domain antibody.

Various methods can be used to obtain such antigen-binding fragments. Illustrative methods include enzymatic cleavage of intact antibodies (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)), recombinant expression by host cells such as E. coli (eg, for Fab, Fv, and ScFv antibody fragments), screening from a phage display library as discussed above (eg, for ScFv), and chemical linkage of the two Fab'-SH fragments to form F(ab') ₂ fragments (Carter et al., Bio/Technology 10:163-167 (1992)). Other methods for obtaining antibody fragments will be apparent to the skilled artisan.

In some embodiments, the antigen-binding fragment is a scFv. The production of scFv is described, for example, in WO 93/16185; U.S. Patent Nos. 5,571,894; and 5,587,458. scFv can be fused to an effector protein at the amino or carboxy terminus to form a fusion protein (see, for example, Antibody Engineering, ed. Borrebaeck).

Bispecific antibodies, multivalent antibodies

In some embodiments, the antibodies and antigen-binding fragments provided herein are bivalent, tetravalent, hexavalent, or multivalent. In some embodiments, the antibodies and antigen-binding fragments provided herein are monospecific or bispecific.

The term valent in the context of this application refers to the presence of a certain number of antigen binding sites in a given molecule. Thus, the terms bivalent, tetravalent and hexavalent mean the presence of two binding sites, four binding sites and six binding sites, respectively, in an antigen-binding molecule. A bivalent molecule may be monospecific if two binding sites are both specific for binding the same antigen or the same epitope. Similarly, a trivalent molecule may be bispecific, eg, where two binding sites are monospecific for a first antigen (or epitope) and a third binding site is specific for a second antigen (or epitope).

In some embodiments, the antibodies and antigen-binding fragments provided herein may be monospecific, but bivalent, trivalent, or tetravalent, with at least two binding sites specific for the same antigen or epitope. This, in some embodiments, represents stronger binding to the antigen or epitope than the monovalent counterpart. In some embodiments, in a bivalent antigen-binding fragment, the first binding site valency and the second binding site valence are structurally identical (i.e., have the same sequences) or structurally different (i.e., have different sequences, although with the same specificity).

In some embodiments, the antibodies and antigen-binding fragments provided herein are bispecific. In some embodiments, the bispecific antibodies and antigen-binding fragments provided herein have a first specificity for CD3 epsilon and a second specificity. In some embodiments, the second specificity is for CD3 epsilon but different epitopes. In some embodiments, the second specificity is for a second antigen other than CD3 epsilon and whose presence close to CD3 epsilon expressing T cells is desirable in order for the second antigen to be recognized by the immune system. For example, by bringing CD3epsilon-expressing T cells into close proximity to a tumor or pathogenic antigen, and thus promoting recognition or elimination of such an antigen.

- 26 042856 gene by the immune system.

In some embodiments, the second specificity is for the tumor associated antigen or epitope thereof. The term tumor-associated antigen refers to an antigen that is present or can be presented on the surface of tumor cells, and that is located on or in tumor cells. In some embodiments, tumor-associated antigens can only be presented by tumor cells and not by normal, ie, non-tumor cells. In some other embodiments, the tumor-associated antigens may be expressed exclusively on tumor cells, or may represent a tumor-specific mutation compared to non-tumor cells. In some other embodiments, tumor-associated antigens may be present in both tumor cells and non-tumor cells, but are overexpressed on tumor cells compared to non-tumor cells, or are available for antibody binding in tumor cells due to the less compact structure of tumor tissue along compared to non-tumor tissue. In some embodiments, the implementation of the tumor-associated antigen is in the vasculature of the tumor.

Illustrative examples of a tumor-associated antigen are CD10, CD19, CD20, CD21, CD22, CD25, CD30, CD33, CD34, CD37, CD44v6, CD45, CD133, Fms-like tyrosine kinase 3 (FLT-3, CD135), chondroitin sulfate proteoglycan 4 ( CSPG4, melanoma-associated chondroitin sulfate proteoglycan), epidermal growth factor receptor (EGFR), Her2, Her3, IGFR, IL3R, fibroblast-activating protein (FAP), CDCP1, Derlin1, Tenascin, burn-related protein 1-10, VEGFR2 vascular antigens (KDR/FLK1), VEGFR3 (FLT4, CD309), PDGFR-alpha (CD140α), PDGFR-beta (CD140b), Endoglin, CLEC14, Tem1-8 and Tie2. Other examples may include A33, CAMPATH-1 (CDw52), carcinoembryonic antigen (CEA), carbonic anhydrase IX (MN/CA IX), de2-7, EGFR, EGFRvIII, EpCAM, Ep-CAM, folate binding protein, G250, Fms -like tyrosine kinase 3 (FLT-3, CD135), cKit (CD117), CSF1R (CD115), HLA-DR, IGFR, IL-2 receptor, IL3R, MCSP (melanoma-associated cell surface chondroitin sulfate proteoglycan), Muc-1 , prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), prostate-specific antigen (PSA), and TAG-72.

Bispecific antibodies and antigennegative fragments presented in this application can be obtained by any suitable methods known in the art. The traditional approach involves the co-expression of two immunoglobulin heavy chain-light chain pairs having different antigen specificities in a host cell to generate bispecific antibodies in a recombinant way (see, e.g., Milstein and Cuello, Nature, 305:537 (1983)), with subsequent purification by affinity chromatography.

A recombinant approach can also be used where sequences encoding antibody heavy chain variable domains for two specificities are respectively fused to immunoglobulin constant domain sequences, followed by insertion into an expression vector that is co-transfected with the expression vector for light chain sequences into an appropriate host cell for recombinant expression of a bispecific 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, for example, Gruber et al., J. Immunol., 152:5368 (1994)).

In another way, the leucine zipper peptides from the Fos and Jun proteins can be linked to the Fab' portions of two different antibodies by gene fusion. Bound antibodies are reduced in the hinge region to four half antibodies (ie, monomers) and then re-oxidized to form heterodimers (Kostelny et al., J. Immunol., 148 (5): 1547-1553 (1992)).

The two antigen-binding domains can also be conjugated or fused to form a bispecific antibody or antigen-binding fragment. For example, one antibody may be bound to biotin and another antibody to avidin, and a strong association between biotin and avidin may complex the two antibodies to form a bispecific antibody (see, for example, US Pat. No. 4,676,980; WO 91/00360, WO 92/00373 and EP 03089). In another example, two antibodies or antigen-binding fragments can be linked by conventional methods known in the art, for example, as described in US Pat. No. 4,676,980.

Bispecific antigen binding fragments can be obtained from a bispecific antibody, for example, by proteolytic cleavage or by chemical linkage. For example, an antigen-binding fragment (e.g., Fab') of an antibody can be prepared and converted to a Fab'thiol derivative, and then mixed and reacted with another converted Fab'-derivative having a different antigen specificity to form a bispecific antigen-binding fragment (see ., for example, Brennan et al., Science, 229:81 (1985)).

In some embodiments, the bispecific antibody or antigen-binding fragments thereof can be designed to form a ridge-to-trough association at the interface to promote heterodimerization of two different antigen-binding sites.

- 27 042856

Ridge-in-trough in the context of this application refers to an interaction between two polypeptides (such as a CH3 domain) where one polypeptide has a bulge (i.e. a ridge) due to the presence of an amino acid residue having a bulky side chain (e.g. tyrosine or tryptophan) , and the other polypeptide has a cavity (i.e., a trough) where a small side chain amino acid residue (eg, alanine or threonine) is located, and a bulge is located in the cavity to facilitate the interaction of two polypeptides to form a heterodimer or complex. Methods for producing ridge-to-gallow polypeptides are known in the art, for example, as described in US Pat. No. 5,731,168.

Conjugates

In some embodiments, anti-CD3 epsilon antibodies and antigen-binding fragments thereof also comprise a conjugate. The conjugate may be associated with antibodies and their antigen-binding fragments. A conjugate is a non-protein fragment that can be attached to an antibody or antigen-binding fragment thereof. It is contemplated that various conjugates may be linked to the antibodies or antigen-binding fragments provided 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 can be linked to antibodies or antigen-binding moieties via covalent linkage, affinity linkage, intercalation, coordinating linkage, complexation, association, mixing, or attachment, among other methods.

In some embodiments, the antibodies and antigen-binding fragments disclosed herein can be designed to contain specific sites outside the epitope-binding portion that can be used to bind 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 attachment to the conjugate.

In some embodiments, the antibodies may be linked to the conjugate indirectly or via another conjugate. For example, the antibody or antigen binding fragments can be conjugated to biotin, then indirectly conjugated to a second conjugate that is conjugated to avidin. The conjugate can be a toxin (eg, a chemotherapeutic agent), a detectable label (eg, a radioactive isotope, a lanthanide, a fluorescent label, a fluorescent label, or an enzyme substrate label).

A toxin can be any substance that is harmful to cells or that can damage or kill cells. Examples of a toxin include, without limitation, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids , procaine, tetracaine, lidocaine, propranolol, puromycin and their analogs, antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, decarbazine), alkylating agents (eg, mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine ( BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamine platinum(II) (DDP), cisplatin), anthracyclines (eg daunorubicin, (formerly daunomycin) and doxorubicin), antibiotics ( for example, dactinomycin (formerly actinomycin), bleomycin, mithramycin and anthramycin (AMC)) and antimitotic agents (eg vincristine and vinblastine).

Examples of a detectable label may include fluorescent labels (eg, fluorescein, rhodamine, dansyl, phycoerythrin, or Texas red), label enzyme substrate (eg, horseradish peroxidase, alkaline phosphatase, luciferases, glucoamylase, lysozyme, saccharide oxidase, or β-D-galactosidase), radioisotopes ( for example, ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I, ³⁵ S, ³ H, ⁿ¹ In, ¹¹² In, ¹⁴ C, ⁶⁴ Cu, ⁶⁷ Cu, ⁸⁶ Y, ⁸⁸ Y, ⁹⁰ Y, ¹⁷⁷ Lu, ²¹¹ At, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁵³ Sm, ²¹² Bi and ³² P, other lanthanides, luminescent labels), chromophore group, digoxigenin, biotin/avidin, DNA molecule or gold for detection.

In some embodiments, the implementation of the conjugate may be a pharmacokinetic-modifying fragment that increases the half-life of the antibody. An illustrative example includes water-soluble polymers such as PEG, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, ethylene glycol/propylene glycol copolymers, and the like. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to an antibody may vary, and if more than one polymer is attached, they may be the same or different molecules.

In some embodiments, the conjugate may be a cleaning agent such as a magnetic bead.

In some embodiments, the antibodies and antigen-binding fragments provided herein are used as the basis for a conjugate.

Polynucleotides and recombinant methods

The present invention provides isolated polynucleotides that encode anti-CD3

- 28 042856 epsilon antibodies and their antigen-binding fragments. In some embodiments, isolated polynucleotides comprise one or more of the nucleotide sequences shown in SEQ at NOs: 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112 , 114, 116, 118 and/or 120, which encode the variable region of the exemplary antibodies presented in this application. DNA encoding a monoclonal antibody is readily isolated and sequenced using conventional procedures (eg, using oligonucleotide probes that are capable of specifically binding to the genes encoding the heavy and light chains of the antibody). Coding DNA can also be obtained by synthetic methods.

An isolated polynucleotide that encodes anti-CD3 epsilon antibodies and antigen-binding fragments thereof (e.g., comprising the sequences shown in SEQ IN NOs: 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 (DNA amplification) or for expression using recombinant techniques known in the art. There are many known vectors. Vector components typically 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 (eg, SV40, CMV, EF-1a), and a transcription termination sequence.

In some embodiments, the vector system includes mammalian, bacterial, yeast systems, and so on. and includes 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, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS420, pLexA, pACT2.2, etc., and other laboratory and commercially available vectors. Suitable vectors may include plasmid or viral vectors (eg, replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

Vectors containing a polynucleotide sequence encoding an antibody or antigen-binding fragment can be introduced into a host cell for gene cloning or expression. Suitable host cells for cloning or expression of DNA in the vectors of the present invention are prokaryotic, yeast or higher eukaryotic cells as described above. Suitable prokaryotes for this purpose include eubacteria such as Gram-negative or Gram-positive organisms, e.g. Enterobacteriaceae, such as Escherichia, e.g. E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g. Salmonella typhimurium, Serratia, e.g. Serratia marcescans and Shigella as well as Bacilli such as B. subtilis and B. licheniformis, Pseudomonas such as P. aeruginosa and Streptomyces.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeasts are suitable hosts for cloning or expression of vectors encoding anti-CD3epsilon antibodies. Of the lower eukaryotic host microorganisms, Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used. However, a number of other genera, species and strains are commonly available and useful in the present invention, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as, for example, K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906 ) K. thermotolerans and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183070); Candida Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as Neurospora, Penicillium, Tolypocladium and Aspergillus hosts such as A. nidulans and A. niger.

Suitable host cells for the expression of the glycosylated antibodies or antigen fragment presented in this application come from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains and variants and corresponding host cells have been identified, such as permissive insect cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori . A variety of viral strains for transfection are commonly available, such as Autographa californica NPV variant L-1 and Bombyx mori NPV strain Bm-5, and such viruses can be used as a virus in accordance with the present invention, in particular for transfection of Spodoptera frugiperda cells. Plant cells from crops such as cotton, corn, potatoes, soybeans, petunia, tomatoes, and tobacco can also be used as hosts.

However, vertebrate cells are of greatest interest, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian cell lines as hosts include the SV40-transformed monkey kidney CV1 cell line (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); newborn hamster kidney cell line (BHK, ATCC CCL 10); Chinese hamster ovary cells/-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,

- 29 042856

ATCC CCL 2); dog kidney cells (MDCK, ATCC CCL 34); gray 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 N. Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma cell line (Hep G2). In some preferred embodiments, the host cell is a 293F cell.

Host cells are transformed with the expression or cloning vectors described above to produce anti-CD3 epsilon antibodies and cultured in conventional nutrient media suitably modified to induce promoters, select transformants, or amplify genes encoding the desired sequences. In another embodiment, the antibody can be obtained by homologous recombination, known in this field.

The host cells used to produce the antibodies or antigen-binding fragments provided herein can be cultured in a variety of media. Commercially available media such as F10 Ham's Medium (Sigma), Minimal Culture Medium (MEM), (Sigma), RPMI1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM, Sigma) are suitable for culturing host cells. In addition, any of the carriers described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), US Pat. Nos. 4,767,704; 4657866; 4927762; 4560655; or 5122469; W090/03430; WO 87/00195; or U.S. Pat. Re. 30,985 can be used as a culture medium for host cells. Any of these media can optionally be supplemented with hormones and/or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium and phosphate), buffers (such as HEPES), nucleotides ( such as adenosine and thymidine), antibiotics (such as the drug GENTAMYCIN™), micronutrients (defined as inorganic compounds typically present in final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary additives may also be included at appropriate concentrations, which should be known to those skilled in the art. Culture conditions such as temperature, pH, and the like are those previously used for the host cell selected for expression and will be apparent to one skilled in the art.

When using recombinant methods, the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the environment. If the antibody is produced intracellularly, as a first step, particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10:163-167 (1992) describe a procedure for isolating antibodies that are secreted into the periplasmic space of E. coli. Briefly, the cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for about 30 minutes. Cellular debris can be removed by centrifugation. When the antibody is secreted into the medium, supernatants from such expression systems are typically first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the above steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of incidental contaminants.

Anti-CD3 epsilon antibodies and their antigen-binding fragments derived from cells can be purified, for example, using hydroxylapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out and affinity chromatography, at In this respect, affinity chromatography is the preferred purification method.

In some embodiments, protein A immobilized on the solid phase is used for immunoaffinity purification of an antibody and its antigen-binding fragment. The suitability of Protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies based on gamma1, gamma2 or gamma4 human heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13 (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 affinity ligand is attached is most often agarose, but other matrices exist. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene provide higher flow rates and shorter processing times than can be achieved with agarose. When an antibody includes a CH3 domain, Bakerbond ABX.TM resin is useful for purification. (J.T. Baker, Phillipsburg, N.J.). Other protein purification methods such as ion exchange column fractionation, ethanol precipitation, reverse phase HPLC, silica gel chromatography, heparin-SEFAROSE™ chromatography on an anionic or cation exchange resin (such as a polyaspartic acid column), focus chromatography, SDSPAGE, and sulfate precipitation ammonium are also available depending on the antibody you need.

- 30 042856 share.

Following any prepurification step(s), the mixture comprising the antibody of interest and impurities can be subjected to low pH hydrophobic interaction chromatography using an elution buffer at about pH 2.5-4.5, preferably performed at low salt concentrations (e.g. , about 0-0.25 M salt).

pharmaceutical composition

The present invention also provides pharmaceutical compositions comprising anti-CD3 epsilon antibodies or antigen-binding fragments thereof and one or more pharmaceutically acceptable carriers.

Pharmaceutically acceptable carriers for use in the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous carriers, non-aqueous carriers, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending/dispensing agents , sequestrants or chelating agents, diluents, adjuvants, excipients or non-toxic excipients, other components known in the art, or various combinations of the above.

Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavors, thickeners, colorants, emulsifiers, or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxanisole, butylated hydroxytoluene, and/or propyl gallate. As disclosed in this application, the inclusion of one or more antioxidants, such as methionine, in a composition containing an antibody or antigen-binding fragment and conjugates, as provided in this application, reduces the oxidation of the antibody or antigen-binding fragment. This reduction in oxidation prevents or reduces the loss of binding affinity, thus improving antibody stability and maximizing shelf life. Therefore, in some embodiments, compositions are provided that contain one or more antibodies or antigen-binding fragments as described herein and one or more antioxidants such as methionine. In addition, methods are provided to prevent oxidation, prolong shelf life, and/or increase the potency of an antibody or antigen-binding fragment provided herein by mixing the antibody or antigen-binding fragment with one or more antioxidants, such as methionine.

To further illustrate, pharmaceutically acceptable carriers may include, for example, aqueous vehicles such as sodium chloride solution for injection, Ringer's solution for injection, isotonic dextrose solution for injection, sterile water for injection or Ringer's dextrose and lactate solution for injection, non-aqueous vehicles, such as fixed vegetable oils, cottonseed oil, corn oil, sesame oil or peanut oil, antimicrobial agents at bacteriostatic or fungistatic concentrations, isotonic agents such as sodium chloride or dextrose, buffers such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropylmethylcellulose or polyvinylpyrrolidone, emulsifiers such as polysorbate 80 (TWEEN-80), sequestering or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethyl alcohol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Antimicrobial agents useful as carriers may be added to pharmaceutical compositions in multi-dose containers which include phenols or cresols, mercury compounds, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride. Suitable excipients may include, for example, water, saline, dextrose, glycerol or ethanol. Suitable non-toxic excipients may include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or substances such as sodium acetate, sorbitan monolaurate, triethanolaminoleate, or cyclodextrin.

Pharmaceutical compositions may be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained release formulation, or powder. Compositions for oral administration may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, magnesium carbonate, and the like.

In some embodiments, the pharmaceutical compositions are formulated as an injectable composition. Pharmaceutical compositions for injection may be prepared in any conventional form such as, for example, liquid solution, suspension, emulsion, or solid forms, as appropriate.

- 31 042856 for obtaining a liquid solution, suspension or emulsion. Formulations for injection may include sterile and/or pyrogen-free solutions ready for injection, sterile dry soluble products such as lyophilized powders ready to be combined with a solvent immediately before use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a carrier immediately prior to use, and sterile and/or pyrogen-free emulsions. Solutions may be aqueous or non-aqueous.

In some embodiments, unit dose parenteral preparations are packaged in an ampoule, vial, or syringe with a needle. All preparations for parenteral administration must be sterile, non-pyrogenic, as is known and practiced in the art.

In some embodiments, a sterile lyophilized powder is prepared by dissolving an antibody or antigen-binding fragment described herein in a suitable solvent. The solvent may contain an excipient that improves stability or other pharmacological components of the powder or reconstituted solution prepared from the powder. Excipients that can be used include, but are not limited to, water, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agent. The solvent may contain a buffer such as citrate, sodium or potassium phosphate, or other such buffer known to those skilled in the art, in one embodiment, at approximately neutral pH. Subsequent sterile filtration of the solution, followed by lyophilization under standard conditions known to those skilled in the art, yields the desired composition. In one embodiment, the resulting solution is dispensed into lyophilization vials. Each vial may contain a single dose or multiple doses of the anti-CD3 epsilon antibody or antigen-binding fragment or composition thereof. Overfilling the vials just above the required dose or multiple doses (eg, about 10%) is acceptable to facilitate accurate sampling and accurate dosing. The lyophilized powder can be stored under suitable conditions, for example at about 4° C. to room temperature.

Reconstitution of the lyophilized powder with water for injection is a composition that can be used for parenteral administration. In one embodiment, sterile and/or pyrogen-free water or other suitable liquid carrier is added to the lyophilized powder for reconstitution. The exact amount depends on the chosen therapy and can be determined empirically.

Methods of application

The present invention also provides therapeutic methods comprising: administering a therapeutically effective amount of an antibody or antigen-binding fragment of the present invention to a patient, thereby treating or preventing a CD3-related condition or disorder. In some embodiments, the CD3-related condition or disorder is a cancer, an autoimmune disease, an inflammatory disease, or an infectious disease.

Examples of a cancer include, but are not limited to, non-small cell lung cancer (squamous/nonsquamous), small cell lung cancer, renal cell carcinoma, colorectal cancer, colon cancer, ovarian cancer, breast cancer (including breast basal carcinoma, ductal carcinoma and lobular breast carcinoma), pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymus carcinoma, melanoma , myelomas, fungoid mycoses, Merkel cell carcinoma, hepatocellular carcinoma (HCC), fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, lymphoid malignancy, basal cell carcinoma, adenocarcinoma , sebaceous carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas, sebaceous carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms tumor, cervical cancer, testicular cancer, seminoma, classic Hodgkin's lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell/histiocyte-rich B-cell lymphoma, acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia, chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, polycythelmia vera, mast cell tumors, EBV-positive and -negative PTLD and diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T-cell lymphoma, nasopharyngeal carcinoma, HHV8-associated primary effusion lymphoma, non-Hodgkin's lymphoma, 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, medulloblastoma, craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma and retinoblastoma.

Autoimmune diseases include, but are not limited to, acquired immune deficiency syndrome (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 (ALPS), autoimmune thrombocytopenic purpura (ATP), Behçet's disease, cardiomyopathy, celiac disease, hepetiform dermatitis; chronic fatigue syndrome and immune dysfunction (CFIDS), chronic inflammatory demyelinating polyneuropathy (CIPD), cicatricial pemphigus, cold agglutination syndrome, CREST syndrome, Crohn's disease, Degos disease, juvenile dermatomyositis, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, disease Graves, Guillain-Barré syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, insulin-dependent diabetes mellitus, juvenile chronic arthritis (Still's disease), juvenile rheumatoid arthritis, Meniere's disease, mixed connective tissue disease, disseminated sclerosis, myasthenia gravis, pernicious anemia, polyarteritis nodosum, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's syndrome, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sclerosarcomia arthritis (progressive systemic sclerosis (PSS), also known as systemic sclerosis (SS)), Sjögren's syndrome, rigidity syndrome, systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis/giant cell arteritis, ulcerative colitis, uveitis, vitiligo, and Wegener's granulomatosis. Inflammatory disorders include, for example, chronic and acute inflammatory disorders. Examples of inflammatory disorders include Alzheimer's disease, asthma, atopic allergy, allergy, atherosclerosis, bronchial asthma, eczema, glomerulonephritis, graft-versus-host disease, hemolytic anemias, osteoarthritis, sepsis, stroke, tissue and organ transplantation, vasculitis, diabetic retinopathy, and ventilator-associated lung damage. In some embodiments, the CD3-associated conditions are inflammatory diseases such as systemic lupus erythematosus (SLE), intestinal mucosal inflammation, wasting disease associated with colitis, multiple sclerosis, viral infections, rheumatoid arthritis, osteoarthritis, Cohn's disease, and inflammatory disease. intestines, psoriasis, systemic scleroderma, autoimmune diabetes, etc.

Infectious diseases include, but are not limited to, fungal infection, parasitic/protozoal infection, or chronic viral infection, e.g., malaria, coccidioidomycosis immitis, histoplasmosis, onychomycosis, aspergillosis, blastomycosis, thrush, paracoccidioidomycosis, microsporidiosis, acanthamoeba keratitis, amoebiasis, ascariasis, babesiosis , balantidiasis, baylisascariasis, Chagas disease, clonorchiasis, cochlimiasis, cryptosporidiosis, diphyllobothriasis, dracunculiasis, echinococcosis, elephantiasis, enterobiasis, fascioliasis, fasciolopsidosis, filariasis, giardiasis, gnathostomiasis, hymenolepiasis, isosporiasis, Katayama fever, lyshmaniasis, leishmaniasis, leishmaniasis onchocerciasis, pediculosis, scabies, schistosomiasis, sleeping sickness, strongyloidiasis, teniasis, toxocariasis, toxoplasmosis, trichinosis, trichuriasis, trypanosomiasis, helminth infection, hepatitis B (HBV), hepatitis C (HCV) infection, herpes virus, Epstein-Barr virus, HIV , cytomegalovirus, herpes simplex virus type I, herpes simplex virus type II, human papillomavirus, adenovirus, human immunodeficiency virus I, human immunodeficiency virus II, herpes virus associated with Kaposi's sarcoma, thin ring virus (Torquetenovirus), T-lymphotropic virus human I, human T-lymphotropic virus II, varicella, JC virus or BK virus.

In another aspect, methods are provided for treating a condition in an individual that may benefit from activation of an immune response, comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment of the present invention to a patient.

The therapeutically effective amount of an antibody or antigen-binding fragment of the present invention depends on various factors known in the art, such as, for example, body weight, age, previous medical history, medication being taken, the individual's health status and the potential for cross-reactions, allergies, sensitivity and adverse side effects, as well as the route of administration and the degree of development of the disease. The person skilled in the art (for example, the attending physician or veterinarian) can proportionally reduce or increase the doses in accordance with the circumstances and requirements.

In some embodiments, an antibody or antigen-binding fragment provided herein can be administered at a therapeutically effective dose 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 so

- 33 042856

100 mg/kg). In some of these embodiments, the antibody or antigen binding fragment is administered at a dose of about 50 mg/kg or less, and in some of these embodiments, the dose 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 some embodiments, the implementation of the administered dose may vary during treatment. For example, in some embodiments, the initial dose administered may be higher than subsequent doses administered. In some embodiments, the implementation of the administered dose may vary during treatment depending on the response of the individual.

Administration schedules may be adjusted to provide the optimal desired response (eg, therapeutic response). For example, you can enter a single dose or multiple sub-doses over a period of time.

The antibodies and antigen-binding fragments disclosed herein can be administered by any route known in the art, such as, for example, parenteral (e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular or intradermal injection) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal or topical) routes.

In some embodiments, the antibodies or antigen-binding fragments disclosed herein can be administered alone or in combination with one or more additional therapeutic agents or agents. For example, the antibodies or antigen-binding fragments described herein may be administered in combination with another therapeutic agent, such as a chemotherapeutic agent or an anticancer drug.

In some of these embodiments, the antibody or antigen binding fragment disclosed herein, which is administered in combination with one or more additional therapeutic agents, can be administered simultaneously with one or more additional therapeutic agents, and in some of these embodiments, the antibody or antigen binding fragment and additional therapeutic agent(s) may be administered as part of the same pharmaceutical composition. However, an antibody or antigen-binding fragment administered in combination with another therapeutic agent is optionally administered simultaneously or in the same composition with such agent. An antibody or antigen-binding fragment administered before or after another agent is considered to be administered in combination with that agent, as this phrase is used herein, even if the antibody or antigen-binding fragment and the second agent are administered in different ways. Where possible, additional therapeutic agents administered in combination with the antibodies or antigen-binding fragments disclosed herein are administered according to the schedule indicated in the information leaflet for the additional therapeutic agent, or in accordance with the 2003 Physicians' Handbook (Physicians' Desk Reference, 57th Ed; Medical Economics Company; ISBN: 1563634457; 57th edition (November 2002)) or protocols well known in the art.

The present invention also provides methods for using anti-CD3 epsilon antibodies or antigen-binding fragments thereof. In some embodiments, the present invention provides methods for activating CD3 epsilon expressing T cells in vivo or in vitro, comprising: contacting CD3 epsilon expressing T cells with an antibody or antigen-binding fragment provided herein. In some embodiments, the present invention provides methods for modulating CD3 activity in a CD3 epsilon expressing cell, comprising exposing the CD3 epsilon expressing cell to an antibody or antigen-binding fragment thereof of the present invention.

In some embodiments, the present invention provides methods for promoting in vivo or in vitro processing of a second antigen by a CD3 epsilon-expressing T cell, comprising contacting the CD3 epsilon-expressing T cell with a bespecifically antibody or antigen-binding fragment provided herein, wherein the bispecific antibody or antigen-binding fragment is capable of specifically binding to both CD3 epsilon-expressing T cells and the second antigen, thus bringing them into close proximity.

In some embodiments, the present invention provides methods for detecting the presence or amount of CD3 epsilon in a sample, comprising contacting the sample with an antibody or antigen-binding fragment thereof and determining the presence or amount of CD3 epsilon in the sample.

In some embodiments, the present invention provides methods for diagnosing a CD3-associated disease or condition in an individual, comprising: a) obtaining a sample from the individual; b) contacting a sample obtained from an individual with an antibody or antigen-binding fragment provided in this application; c) determining the presence or amount of CD3 epsilon in the sample; and d) relating the existence of a CD3 epsilon to a CD3-related disease or condition in the individual.

In some embodiments, the present invention also provides the use of an antibody or antigen-binding fragment thereof of the present invention, for the manufacture of a medicament for the treatment of a CD3-associated disease or condition in an individual, for the manufacture of a diagnostic reagent for diagnosing a CD3-associated disease or condition.

The following examples are given to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. All specific compositions, materials and methods described below, in whole or in part, are included in the scope of the present invention. These specific compositions, materials and methods are not intended to limit the invention, but merely to illustrate specific embodiments covered by the scope of the invention. A person skilled in the art can develop equivalent compositions, materials and methods without using inventive possibilities and without departing from the scope of the invention. It should be clear that in the procedures described in this application, many changes can be made without going beyond the scope of the present invention. The inventors assume that such options are included in the scope of the invention.

Example 1 Obtaining antibodies using hybridoma technology.

1.1 Immunization of animals.

Recombinant extracellular domain (ECD) human CD3 proteins, including His-tagged human CD3 epsilon (CD3-epsilon) (cat. No.: 10977-H08H; Sino Biological Inc. Beijing, China), human CD3 Gamma (CD3-gamma ) (cat. No.: abl40563; Abcam Shanghai China) and His-tagged human CD3 delta (CD3 delta) (cat. No.: 10977-H08H; Sino Biological Inc. Beijing, China) were used as immunogens for animal immunization. Balb/c mice were purchased from Shanghai SLAC laboratory animal Co, Ltd. and housed in an IACUC-approved vivarium. Mice were immunized with an ECD protein mixture comprising CD3 epsilon, CD3 gamma, and CD3 delta, or immunized with 7x106 freshly isolated human T cells for each mouse, mixed with Titermax adjuvant, subcutaneous injection, and in the footpad given one week later.

Blood was collected from mice before and after immunization and serum titers against target proteins were monitored by ELISA.

1.2 Obtaining hybridomas.

The mouse with the highest serum titer was selected for cell fusion. B cells from mouse spleen and lymph nodes were fused with SP2/0 myeloma cells by electrofusion according to general electrofusion procedures. After cell fusion, cells were seeded in 96-well DMEM plates supplemented with 20% FBS and 1% HAT selective reagents.

Antibodies present in the supernatant of the hybridoma medium were screened using CB3-expressing Jurkat cells by the flow cytometer method (FACS) and back-screened using CD3-negative MOLT-4 T cells by the FACS method. Hybridoma cells with binding activity to Jurkat cells but not cross-linking to MOLT4 cells were collected as positive hybridoma cell lines and then proceeded to the subcloning step using semi-solid medium.

Individual colonies were seeded in 96-well plates with DMEM supplemented with 10% FBS for 2~3 days and again screened against the CD3 target using CD3-expressing Jurkat cells using a flow cytometer (FACS) method and back-screened with using CD3-negative MOLT-4 T cells by FACS.

1.3 Sequencing using hybridomas.

RNA was extracted from hybridoma cells and cDNA was amplified using the 5'-RACE kit followed by PCR amplification using 3' degenerate primers. The PCR products were then cloned into the pMD18-T vector, transformed, amplified and sequenced.

Received eight mouse monoclonal antibodies, the sequence of the CDR which are shown in table. 1 above.

Example 2. Obtaining a humanized antibody.

2.1 Conversion and humanization of IgG.

Generation of Chimeric Antibody from Mouse mAbs: WBP3311_2.166.48 and WBP3311_2.306.4 and WBP3312_3,179,16 VH and VL genes were reamplified with cloning primers containing appropriate restriction sites and cloned into an expression vector patented by WuXi Biologics to generate the corresponding chimeric antibody clones with constant region of human IgG1.

Humanization and synthesis of humanized V genes: A best fit approach was used to humanize the light and heavy chains of WBP3311_2.166.48 and WBP3311_2.306.4. For light chain amino acid sequences, the respective V genes were subjected to a BLAST study against a proprietary database of human germline V genes. The sequence of the humanized VL gene was obtained by replacing human CDR sequences in the best copy with murine CDR sequences using Kabat's CDR determination. Frameworks were defined using the extended CDR, where the Kabat CDR1 was extended by 5 amino acids at the N-terminus. Several humanized sequences were generated for each heavy chain and light chain by BLASTing mouse scaffolds against an immunoglobulin database.

- 35 042856 human germline. Various FR combinations were generated and analyzed for binding affinity, the top three copies were used to generate humanized VH gene sequences. Humanized genes were reverse translated, codon optimized for mammalian expression, and synthesized using GeneArt Costum Gene Synthesis (Life Technologies). The synthetic genes were re-cloned into an IgG expression vector, expressed and purified. The FRs of the two humanized antibodies and their parental mouse antibodies are shown in Table 1. 2 above.

Transient Expression and Purification of Chimeric and Humanized Antibodies: The chimeric and humanized antibodies described above were inserted into an expression vector patented by WuXi Biologics and expressed from 293F cells. The culture supernatant containing the appropriate antibodies was collected and purified using protein A chromatography.

Example 3 In vitro characterization study.

3.1 Antibody binding determined by ELISA and FACS 3.

Table 3

Antigens, antibodies and cells used in ELISA and FACS

Material Company catalog no. Human protein CD3 epsilon (COZepsilon) (His tag) Sino Biological Inc. 10977-H08H Human protein CD3 delta (CD3 delta) (His tag) Sino Biological Inc. 10981-H08H Human protein CD3 gamma (CD3 gamma) Abeam ab140563 The cynomolgus macaque COZepsilon protein ACRO CDE-C5226 Cyno PBMCs Pharma Legacy Laboratories (Shanghai) Co. Mouse monoclonal antibody against human COZepsilon Sino Biological Inc. 10977-MM03 (Clone No.: 1A7E5G5) Mouse monoclonal antibody against human CD3 delta Sino Biological Inc. 10981-MM08 Mouse monoclonal antibody against human CD3 delta CD3 gamma SANTA CRUZ BIOTECHNOLOGY, INC. sc-55563 Reference antibody OKT3 Abeam ab86883 Jurkat cells ATCC TIB-152 HUT78 cells ATCC ECACC-880401901 MOLT-4 cells ATCC CRL-1582 semi-solid medium Stemcells 03814

Antibody protein binding determined using ELISA and EC ₅₀ : Human CD3 epsilon, CD3 delta and CD3 gamma proteins, respectively, were pre-plated in 96-well plates. The binding affinity of eight mAbs at various concentrations to the extracellular domain of these three different CD3 proteins was determined using the respective HRP labeled second antibodies. Binding EC50 (concentration of test antibody at which half-maximal binding is achieved) was analyzed using the GraphPad Prism equation: Nonlinear regression (curve fitting)-log (agonist) vs. The answer is Variable Slope.

Specific antibody binding to human CD3 epsilon protein as determined by ELISA: Eight mouse mAbs showed highly specific binding activity to human CD3 epsilon without binding to CD3 gamma and CD3 delta, and the data are shown in Table 1. 5.

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Table 5

Specific binding for the human CD3 epsilon subunit

mAb ELISA (A450) Human SVZepsilon (PC: 1.6; NC: 0.05) Human CD3 gamma (PC: 1.21; NC: 0.05) Human CD3 delta (PC: 1.9; NC: 0.05) W3311-2.166.48 1.64 0.05 0.05 W3311-2.306.4 1.46 0.06 0.05 W3311-2.383.47 1.61 0.05 0.05 W3311-2.400.5 1.57 0.05 0.05 W3311-2.482.5 1.33 0.06 0.05 W3311-2.488.33 1.70 0.06 0.05 W3311-2.615.8 1.55 0.05 0.05 W3311-2.844.8 1.54 0.07 0.06

Cellular antibody binding determined using FACS and EC50: Various concentrations of test mAbs were added to Jurkat cells and then the binding activity of the mAb on the cell surface was determined using 2nd antibody-FITC. OKT3 was used as a positive control. Stained cells were analyzed using the BD Biosciences FACSCanto II instrument and Flow Jo Version software. Binding EC50 value was calculated using the GraphPad Prism equation: Nonlinear regression (fitting)-log (agonist) vs. The answer is Variable Slope.

Specific binding to the human CD3 receptor on the surface of T cells: Eight mAbs showed highly specific binding activity to cells expressing human CD3 (Jurkat cells) without binding to CD3-negative cells (MOTL-4 cells and 293F cells), as shown in Table 1. . 6.

Table 6 Specific binding to the human CD3 receptor in various cell lines, determined using FACS

mAb MFI (FACS) (JurkatBlO: 3145-4045) MFI (FACS) (MOLT-4: 22.7-27.3) MFI (FACS) (293F:22.6-24.6) W3311-2.166.48 2139 42.5 24.9 W3311-2.306.4 3365 53.5 29.1 W3311-2.383.47 2132 44.5 29.8 W3311-2.400.5 2741 54.5 25.1 W3311-2.482.5 2390 54.8 25.0 W3311-2.488.33 2266 58.4 30.2 W3311-2.615.8 2215 57.2 25.7 W3311-2.844.8 984 42.5 22.7

3.2 Binding of antibodies to a target protein from various species, determined by ELISA and FACS.

The binding affinities of test antibodies to CD3 from different species were analyzed. The homology of human, simian, rat and mouse CD3 reference sequences is shown below.

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Table 4

Sequence Homology of Human, Monkey, Rat and Mouse CD3 Domain

View SOZepsilon (%) CODelta (%) CD3 gamma (%) Human 100 (NP_000724) 100 (NP_000723) 100 (NP_000064) Masasa fascicularis (monkey) 83 (ΝΡ_001270544) 94 (ΝΡ_001274617) 81 (NP_00127083 9) Masasa mulatta (monkey) 84 (ХР_001097204) 94 (ΝΡ_0010973 02) 82 (NP_001253854) Mouse 59 (NP_031674) 64 (NP_038515) 70 (AAA37400) Rattus norvegicus (rat) 57 (NP_001101610) 69 (NP_037301) 71 (NP_001071114)

Cross-linking of antibodies to cynomolgus CD3 epsilon and mouse CDS epsilon determined by ELISA: Various concentrations of test antibodies, positive and negative controls were added to 96-well plates that had been pre-coated with cynomolgus CD3 epsilon and mouse CD3 epsilon. Antibody binding to cynomolgus CD3 epsilon and mouse CD3 epsilon was determined using the corresponding HRP-labeled ^2nd antibody (BETHYL, A90-231P). EC ₅₀ was calculated using GraphPad Prism software.

As the data show, all eight mAbs showed strong cross-linking activity with cynomolgus CD3 epsilon, but showed no binding with mouse CD3 epsilon. The positive control OKT3 showed no cross-linking activity with either cynomolgus CD3 epsilon or murine CD3 epsilon, Table. 7.

Table 7

Cross-linking versus cynomolgus CD3 epsilon and mouse CD3 epsilon

mAb ELISA A450 SOZepsilon human SOZepsilon of the cynomolgus macaque SOZepsilon mouse Negative control 0.046 0.046 0.179 OKTZ 0.048 0.052 0.24 W3311-2.166.48 2.761 2.808 0.446 W3311-2.306.4 2.909 2.822 0.711 W3311-2.383.47 2.786 2.756 0.666 W3311-2.400.5 2.828 2.794 0.709 W3311-2.482.5 2.953 2.874 0.61 W3311-2.488.33 2.914 2.831 0.607 W3311-2.615.8 2.824 2.713 0.471 W3311-2.844.8 2.923 2.75 0.6

Antibody cross-linking to cynomolgus CD3 epsilon as determined by FACS: Cynomolgus monkey PBMCs were isolated from whole blood of healthy monkeys using Ficoll-Paque PLUS density gradient centrifugation and 100g centrifugation steps to remove platelets. PBMCs were cultured in complete RPMI-1640 medium until ready for use. Various concentrations of test antibodies were added to cynomolgus monkey PBMCs and then the antibody binding activity on the cell surface was determined using 2nd antibody-FITC (Jackson, 115095-008). Stained cells were analyzed using BD Biosciences FACSCanto II and FlowJo Version software. The binding EC ₅₀ value was calculated using the GraphPad Prism equation: Nonlinear regression (curve fitting).

Epitote binding by antibodies determined by FACS method: Different concentrations of test antibodies were mixed with some amount of biotinylated W3311-2.383.47 antibody, respectively. The mixture was then added to CD3-expressing cells in 96-well plates and incubated at 4°C for 1 hour. Binding of the target antibody on CD3-expressing cells

- 38 042856 was determined using PE-conjugated anti-biotin Ab. The samples were tested by flow cytometry and the data was analyzed using the FlowJo software.

Binding to human cynomolgus CD3 epsilon and CD3 epsilon and EC50 calculation by ELISA: Eight mAbs showed strong binding activity to human CD3 epsilon and cross-binding to cynomolgus CD3 epsilon and showed comparable EC50 values in the low nM range for Human CD3 epsilon and cynomolgus monkey CD3 epsilon, as shown in Table 1. 8.

Table 8

EC50 of eight mAbs for human CD3 epsilon and cynomolgus CD3 epsilon

4.3 Detection of cross-species binding of humanized antibodies.

Binding of two humanized mAbs to cynomolgus CD3 epsilon and EC50 value: All data showed that both humanized mAbs retained high binding activity to cynomolgus CD3 epsilon (see Figure 1) with an EC50 value of 0.043 nM for both humanized antibodies, as shown in table. 9.

Table 9

EC50 of two humanized mAbs for cynomolgus CD3 epsilon

Antibody affinity determined by FACS: 5x104 Jurkat cells/well were plated in 96-well plates, followed by the addition of purified test leader antibody at various concentrations as 1st antibody for 1 hour at 4°C. ^2nd goat anti-mouse IgG Fc-FITC was added over 30 min at 4°C and then FITC-stained cells were detected by FACS. KD value was calculated in accordance with the method described above.

Two humanized mAbs were tested for binding activity on human CD4 T cells: Data showed that both humanized antibodies retained high binding activity to CD4 T cells (see Figure 2) with low EC ₅₀ values of 1.01 and 0.46 nM for WBP33112 .166.48-z1-uIgG1k and WBP3311-2.306.4-z1-uIgG1k, respectively, which were 0.5-1-fold lower than the corresponding parental antibody values, as shown in Table 1. 10.

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Table 10

Binding of two humanized mAbs to hCD4 T cells determined using FACS and EC ₅₀

wal EU ₅₀ (nM) W3311-2.166.48-zl-uIgGlK 1.01 W3311-2.166.48-mIgG2aK 3.514 W3311-2.306.4-zl-uIgGlK 0.253 W3311-2.306.4-mIgG2bK 0.461 octz 0.202 Isotype control N/A

4.5 Affinity and EC ₅₀ of antibodies determined by FACS.

Eight mAbs were tested for their EC ₅₀ by FACS and tested for affinity against cells expressing human CD3 (Jurkat cells).

The EC ₅₀ value for the eight mAbs ranged from 0.57 nM to 6.91 nM (see Table 11) and the affinity ranged from 1.3 x 10' ⁹ to 9.3 x 10' ¹⁰ M as shown in table. 12.

Table 11

EC ₅₀ value measured in Jurkat cells by FACS method

K _D value for eight mAbs determined in Jurkat cells by FACS

Sample W331 1- 2.166. 48 W331 1- 2.306. 4 W331 1- 2.383. 47 W33 eleven- 2.40 0.5 W331 1- 2.482. 5 W33 eleven- 2.48 8.33 W33 eleven- 2.61 5.8 W33 eleven- 2.84 4.8 WBP33 Oct 11 3- average value Best match- Wmax 1.26E -10 1.17E -10 1D2E -10 1.13 E-10 1.30E -10 1.30 E-10 1.20 E-10 1.19 E-10 9.41E- eleven The best 1.67E 1.91E 4,OSE 9.34 2.29E 1.32 2.23 2.52 1.29E- conformity-K _o -09 -10 -10 E-10 -09 E-09 E-09 E-10 10 Standard 1.66E 1D9E 1.62E 1.76 2.35E 2.51 2.18 1.00 1.95E- error-vshah -12 -12 -12 E-12 -12 E-12 E-12 E-12 12 Standard 5.62E 8.32E 2D5E 4.38 9.63E 6.98 9.47 8.64 1.27E- error-kts -eleven -12 -eleven E-11 -eleven E-11 E-11 E-12 eleven

4.6 Measurement of K _D affinity and kinetics of two humanized mAbs by FACS.

The two humanized mAbs were tested for affinity in Jurkat cells by FACS. The data showed that both humanized mAbs retained high affinity on Jurkat cells with the same KD values as their respective parent mAbs, as shown in FIG. 4 and in table. 13.

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Table 13

Measurement of KD kinetics of two humanized mAbs on Jurkat cells by FACS

Sample WBP3311-2.166.48-zlulgGlk WBP3311-2.306.4-zlulgGlk octz Best match 7.32E-12 6.83E-12 6.06Е-12 Best match- KD 2.01E-10 5,ZOE-11 2.77E-11 Standard error-Vshah 3.82E-13 3.49E-13 2D9E-13 Standard error-KD 4.38E-11 1D4E-11 5DZE-12

3.3 Cellular functional assays.

Cellular antibody internalization as determined by FACS: Jurkat cells were seeded at 1 x 10 ⁵ /well in 96-well plates. Test antibodies (1 μg/ml) were added to the cells and incubated for 1 hour at 4°C. After incubation, unbound antibodies were removed by washing and cells were then incubated for 3 h at 4°C or 37°C with 5% CO2. After incubation, the presence of antibodies on the cell surface was determined using the 2nd antibody (Abcam, ab98742). Stained cells were analyzed using BD Biosciences FACSCanto II and FlowJo Version software. The percentage of internalization was calculated as shown below:

[(MFI0 hours - MFI3 hours)/MFIo hours] X100%

4.7 Determination of the percentage of cellular internalization.

The percentage of internalization was determined using Jurkat cells by the FACS method. The data showed that all 8 mAbs showed a high percentage of internalization ranging from 86 to 94% of antibodies internalized after the 3 hour test period, while OKT3 showed approximately 72% internalization, FIG. 5.

T-cell activation by antibodies using intracellular cytokine staining method: Intracellular cytokine staining is a flow cytometry-based assay that can detect cytokine production by immune cells in combination with cell surface markers. Human PBMCs were isolated from a healthy donor. Briefly, Ficoll-Paque PLUS density gradient centrifugation was used followed by 100g centrifugation steps to remove platelets. PBMCs were cultured in complete RPMI-1640 medium. PBMCs were resuspended in cell culture medium supplemented with Golgi Stop and dispensed at 2x10 ⁵ /well into 96-well plates. Various concentrations of test antibodies, positive and negative controls were added and then incubated with cells for 4.5 hours at 37°C. After incubation, cells were washed twice with 1% BSA and then stained for their surface markers using anti-human CD4-FITC antibody (BD, 550628) and anti-human CD8-PE antibody (BD, 560959), followed by fixation and permeabilization using Human Regulatory T Cell Staining Kit (eBioscience, 88-8999). After fixation/permeabilization, cells were assessed for cytokine production using an anti-human TNF-APC antibody (BD, 554514) and an anti-human IFN-PerCP-Cy5.5 antibody (eBioscience, 45-7319-42). Stained cells were analyzed using BD Biosciences FACSCanto II and FlowJo Version software.

T cell activation: T cell activation was assessed using human PBMC by intracellular cytokine staining and the cytokines TNF-alpha and INF-gamma were monitored. The data showed that of the 8 mAbs tested, 2.306.4 and 2.844.8 did not show a significant T-cell activation event as no significant release of the cytokines TNF-alpha and INF-gamma was detected, while all 6 other mAbs tested showed T-cell activation in such the same degree as OKT3, except for clone 2.383.47, which showed weaker T cell activation compared to OKT3, the data are shown in FIG. 6.

Activation of human T cells using two humanized mAbs: Two humanized mAbs were tested for T cell activation on human PBMCs by intracellular cytokine staining and monitored for the cytokines TNF-alpha and INF-gamma. The data showed that both humanized antibodies showed the same degree of T cell activation as the OKT3 positive control on CD8+ T cells, with both humanized mAbs showing

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Claims (15)

less activation on CD4+ T cells compared to OKT3. It is also noted that the original mouse antibody clone 2.306.4 consistently showed no T-cell activation. The data is shown in FIG. 7. 3.4 Antibody-epitope binding as determined by FACS. Epitope Binding: Seven of the 8 mAbs clustered against clone 2.383.47 and the result showed that all 8 mAbs shared an epitope group with them, as shown in FIG. 8. CLAIM 1. An isolated anti-CD3 antibody or antigen-binding fragment containing a) a heavy chain variable region containing CDR1 containing the amino acid sequence of SEQ ID NO: 7, a CDR2 containing the amino acid sequence of SEQ ID NO: 9, and CDR3 containing the amino acid sequence of SEQ ID NO: 11; and a kappa light chain variable region containing CDR1 containing the amino acid sequence of SEQ ID NO: 8, a CDR2 containing the amino acid sequence of SEQ ID NO: 10, and CDR3 containing the amino acid sequence of SEQ ID NO: 12; or b) a heavy chain variable region containing CDR containing the amino acid sequence of SEQ ID NO: 43, a CDR2 containing the amino acid sequence of SEQ ID NO: 45, and CDR3 containing the amino acid sequence of SEQ ID NO: 47; and a kappa light chain containing CDR1 containing the amino acid sequence of SEQ ID NO: 44, a CDR2 containing the amino acid sequence of SEQ ID NO: 46, and CDR3 containing the amino acid sequence of SEQ ID NO: 48. 2. The antibody or its antigen-binding fragment according to claim 1, containing a) a heavy chain variable region comprising SEQ ID NO: 117 and a kappa light chain variable region comprising SEQ ID NO: 119; b) a heavy chain variable region comprising SEQ ID NO: 85 and a kappa light chain variable region comprising SEQ ID NO: 87; or c) a heavy chain variable region comprising SEQ ID NO: 109 and a kappa light chain variable region comprising SEQ ID NO: 111. 3. An antibody or antigen-binding fragment thereof according to claim 1, also comprising an immunoglobulin constant region. 4. An antibody or antigen-binding fragment thereof according to claim 1, which is a humanized antibody. 5. An antibody or antigen-binding fragment thereof according to claim 1, which is a camelized single-domain antibody, diabody, scFv, scFv dimer, BsFv, dsFv, (dsFv) ₂ , dsFv-dsFv', Fv fragment, Fab, Fab', F( ab') ₂ , ds diabody, nanobody, domain antibody or bivalent domain antibody. 6. An antibody or antigen-binding fragment thereof according to claim 1, capable of specifically binding to CD3 epsilon. 7. A bispecific antibody or antigen-binding fragment thereof, characterized in that it contains the antigen-binding domain of the anti-CD3 antibody of claim 1 and a second antigen-binding domain. 8. The bispecific antibody or antigen-binding fragment thereof according to claim 7, wherein the bispecific antibody or antigen-binding fragment thereof is specific for CD3 epsilon. 9. The bispecific antibody or antigen-binding fragment thereof according to claim 8, wherein the second specificity is directed to a second antigen other than CD3 epsilon, wherein the presence of the second antigen in the vicinity of CD3 epsilon expressing T cells is desirable for the second antigen to be recognized by the immune system. 10. A conjugate containing an antibody or antigen-binding fragment thereof according to any of the preceding claims, associated with one or more chemotherapeutic agents, toxins, radioactive isotopes, lanthanides, fluorescent labels, fluorescent labels or enzyme-substrate labels. 11. Pharmaceutical composition containing the antibody or antigen-binding fragment according to claim 1 and a pharmaceutically acceptable carrier. 12. An isolated polynucleotide encoding an antibody or antigen-binding fragment thereof according to claim 1. 13. An expression vector comprising the isolated polynucleotide of claim 12. 14. A host cell comprising the vector of claim 13. 15. A method for expressing an antibody or its antigen-binding fragment according to claim 1, including culturing a host cell comprising a vector containing an isolated polynucleotide encoding an antibody or antigen-binding fragment according to claim 1 under conditions under which -