Novel human antibody that binds to human CD3 epsilon

Abstract

The present disclosure provides novel fully human antibodies and antibody fragments specific for human CD3 epsilon.

Description

[Technical Field]

[0001] This disclosure relates to fully human antibodies that bind to human CD3 epsilon and improved variants thereof. This disclosure also provides multispecific antibody formats comprising such human CD3-specific antibodies and antibody fragments. This disclosure also relates to nucleic acids, vectors, and host cells capable of expressing such antibodies, pharmaceutical compositions comprising such antibodies, and the use of such antibodies and pharmaceutical compositions for the treatment of specific diseases. [Background technology]

[0002] CD3 is a homodimeric or heterodimeric antigen expressed on T cells along with the T cell receptor complex (TCR), and is required for T cell activation. CD3 is formed when two of four different chains—epsilon, zeta, delta, and gamma—associate to form a dimer. CD3 dimers include gamma / epsilon, delta / epsilon, and zeta / zeta.

[0003] Antibodies bound to CD3 have been shown to cluster CD3 on T cells in a manner similar to how MHC molecules carrying peptides engage with T cell receptors (TCRs), thereby inducing T cell activation. CD3-specific antibodies have been recommended as therapies involving T cell activation. Therefore, dual or multispecific antibody formats that simultaneously engage CD3 and one or more cancer-associated antigens (CAAs) have been developed to novelly direct T cells to attack and lyse cancer cells. While the CD3-targeted approach has shown considerable promise, the therapeutic use of such multispecific antibody formats described in the art shares several common drawbacks. First, there is no specific tumor antigen. In fact, cancer-associated antigens that are typically targeted are often shared by tumors and non-malignant cells. Second, a major problem with multispecific antibody formats that rely on the use of CD3-specific antibody fragments (e.g., single-chain variable fragments (scFv)) is the inherent ability of these fragments to activate T cells regardless of their binding to the target antigen (e.g., cancer-associated antigen). In fact, many of the side effects observed with CD3-based antibody therapy appear to be related to abnormal T cell function (such as the accompanying production of cytokines, which may lead to toxic cytokine release syndrome).

[0004] A novel approach to multispecific antibody formats that overcomes the aforementioned weaknesses of antibody-based therapeutics that simultaneously engage CD3 and cancer-related antigens is presented in International Publication No. 2013 / 104804.

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[0005] As part of the above-described approach, two polypeptides are designed, each consisting of a target component (e.g., a single-chain variable fragment (scFv) or an antibody Fab fragment) fused to either the variable light chain domain (VL) or the complementary variable heavy chain (VH) domain of a T cell activating anti-CD3 antibody. Such split or fragmented Fv domains are referred to herein as "fragmented effector domains," whereas the reassembled Fv domain reproduces the "effector domain."

[0006] Unpaired VH or VL domains (e.g., a fragmented CD3 VH domain and a fragmented CD3 VL domain) cannot bind CD3 alone. However, once the two polypeptides described above bind to their target antigens on the surface of a cell via their target components, the complementary VL and VH domains of the CD3-specific antibody become adjacent and interact with each other to reconstitute the native CD3 antibody (e.g., the CD3-specific antibody Fv domain). The trispecific heterodimeric molecule thus formed on the target cell engages and stimulates T cells for cancer cell destruction, like conventional trivalent bispecific or trispecific antibody formats.

[0007] One critical aspect of the approach described above relies on the identification of a suitable CD3-specific antibody. Such an antibody must not only meet the requirements of conventional bispecific antibody formats in terms of cross-species reactivity, affinity, potency, safety (e.g., therapeutic window), and immunogenicity, but also the specific requirements of the CyCAT format.

[0008] As described above, in the CyCAT approach, the CD3-specific Fv domain used must be cleavable or splittable. This feature implicitly indicates several specific requirements for the CD3 antibody.

[0009] First, the VH and VL domains of the split Fv domain must be producible in the CyCAT format (e.g., as unpaired CyCAT polypeptides) with an acceptable purity, an acceptable yield, and an acceptable monomer content. This is particularly troublesome because unpaired VH or VL domains tend to form strong aggregates after production. This aggregation tendency depends, inter alia, on specific antibody sequences and seems to be more severe for polypeptides with unpaired VH domains. In fact, the inventors of the currently claimed new CD3-specific antibodies have discovered that even minor mutations in the CDR sequences of antibody variants derived from one parental antibody represent a large difference in productivity in the CyCAT format.

[0010] Second, the CD3-specific antibody (e.g., Fv domain) must retain its functional activity in terms of activating T cells and / or newly directing T cell-mediated killing of cancer cells once the unpaired CD3-specific VH and VL domains present in the set of CyCAT polypeptides pair up.

[0011] Third, the unpaired CD3-specific VH or VL domain or each unpaired CyCAT polypeptide containing it should not bind to CD3 alone.

[0012] Fourth, the unpaired CD3-specific VH or VL domain or each unpaired CyCAT polypeptide containing it should not activate T cells alone and / or should not mediate newly directed T cell-mediated killing.

[0013] Finally, unpaired CD3-specific VH or VL domains, or each unpaired CyCAT polypeptide containing them, should not pair before the two polypeptides bind to their respective cancer-associated antigens on target cells. Such undesirable pre-pairing (or heteroassociation) may occur in patient serum after the separate administration of the two polypeptides (e.g., as part of two pharmaceutical compositions) or when both polypeptides are part of a single pharmaceutical composition. Undesirable pre-pairing is thought to be, among other things, dependent on concentration and the affinity of the fragmented VH and VL domains to each other.

[0014] Many antibodies that bind to CD3 are described in this art. However, the majority of these antibodies intended for therapeutic use are derived (e.g., humanized) from a small number of rodent anti-CD3 antibodies discovered in the early 1980s and 1990s. Of these, SP34 (Yoshino N. et al., Exp. Anim 49:97-110, 2000; Conrad ML. et al., Cytometry 71A:925-33, 2007) is one of the very few CD3-specific antibodies that cross-react with cynomolgus monkey CD3 (macaque monkey CD3 or cyno CD3), and therefore can be used for preclinical evaluation of its safety, activity, and / or pharmacokinetic profile in primates, as well as as a human drug in the same form, and is therefore perhaps the antibody most likely to be used in a humanization approach. SP34 recognizes the N-terminal 1-27 amino acid residue polypeptide fragment of the extracellular domain of human CD3 epsilon. However, the inventors of this disclosure have found that at least three prior art antibodies, two of which were derived from SP34, were unsuitable for use in the described CyCAT format due to their undesirable production characteristics and / or functional activity.

[0015] Interestingly, the de novo in vitro generation of a fully human antibody that binds to CD3, recognizes the N-terminal extracellular region of human CD3 epsilon, and is thus cross-reactive to macaque CD3 appears to be troublesome and, to the best of our knowledge, has not been reported to date.

[0016] Accordingly, this disclosure provides novel antibodies and antibody fragments that are specific to CD3 epsilon and are superior to CD3-specific antibodies reported in the art.

[0017] In particular, the antibodies or antibody fragments specific to CD3 epsilon provided in this disclosure are fully human antibodies, cross-react with macaque CD3 with favorable affinity, and possess desirable productivity, functionality, and safety that have never been observed before.

[0018] These features make the antibodies and antibody fragments of this disclosure highly desirable for therapeutic use in multispecific antibody formats, particularly in the CyCAT format described herein. [Overview of the Initiative] [Means for solving the problem]

[0019] Isolated human antibodies or antibody fragments, as well as antibody variable domains specific to human CD3 epsilon, and their respective CDRs and CyCAT polypeptides contained herein are summarized in Tables 2-10 and 17-18 of this disclosure.

[0020] The preferred improved variable heavy chains (VH) and their HCDR1-3 according to this disclosure are summarized in Table 4. The preferred improved variable light chains (VL) and their LCDRS1-3 according to this disclosure are summarized in Table 5.

[0021] Table 10 summarizes preferred isolated human antibodies or antibody fragments specific to human CD3 epsilon according to this disclosure. Table 10 summarizes preferred combinations of enhanced variable heavy chain (VH) and enhanced variable light chain (VL) specific to human CD3 epsilon. Table 17 summarizes the most preferred isolated human antibodies or antibody fragments specific to human CD3 epsilon according to this disclosure. Table 17 summarizes the most preferred combinations of enhanced variable heavy chain (VH) and enhanced variable light chain (VL) specific to human CD3 epsilon.

[0022] In one embodiment, the Disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, comprising six CDRs disclosed in Tables 2 and 3 of the Disclosure.

[0023] In one embodiment, the Disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, comprising three HCDRs of variable heavy chain (VH) disclosed in Table 4 of the Disclosure and three LCDRs of antibody light chain (VL) disclosed in Table 5.

[0024] In one embodiment, the Disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, comprising one HCDR of the antibody or variable domain disclosed in Table 4 of the Disclosure and one LCDR of the antibody or variable domain disclosed in Table 5.

[0025] In one embodiment, the Disclosure provides isolated human antibodies or antibody fragments specific to human CD3 epsilon, comprising combinations of variable heavy chains (VH) and variable light chains (VL) as disclosed in Table 2 or Table 3 and Tables 8-10 of the Disclosure.

[0026] In one embodiment, the Disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, comprising a variable heavy chain (VH) and a variable light chain (VL) from any one of the antibodies disclosed in Tables 2, 3, and 8-10 of the Disclosure. In one embodiment, the Disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, comprising a variable heavy chain (VH) disclosed in Table 4 and a variable light chain (VL) disclosed in Table 5 of the Disclosure. In one embodiment, the Disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, comprising a combination of the variable heavy chain (VH) disclosed in Table 4 and the variable light chain (VL) disclosed in Table 5 of the Disclosure. In one embodiment, the Disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, as disclosed in Table 10 of the Disclosure. In one embodiment, the Disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, as disclosed in Table 17 of the Disclosure.

[0027] In one embodiment, the Disclosure provides isolated human antibodies or antibody fragments specific to human CD3 epsilon, comprising combinations of variable heavy chains (VH) and variable light chains (VL) disclosed in Table 10 of the Disclosure.

[0028] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) An HCDR1 region containing the amino acid sequence GFTFX1SX2X3MX4, where X1 is S, K, or R; X2 is Y or H; X3 is W or Y; and X4 is S or T (SEQ ID NO: 90); b) An HCDR2 region containing the amino acid sequence NIX1X2X3X4X5X6X7YYX8X9SVKG, where X1 is K or D; X2 is Q or Y; X3 is D, Q, or E; X4 is S or G; X5 is S, Q, or T; X6 is E, H, or R; X7 is K, A, or T; X8 is V or A; and X9 is D or E (Sequence ID 91); c) HCDR3 region containing the amino acid sequence of GYSAEFAHRSGLDV (SEQ ID NO: 39); d) An LCDR1 region containing the amino acid sequence SGSSSNIGX1X2YVY, where X1 is S, I, A, K, or Q; and X2 is N or T (SEQ ID NO: 92); e) LCDR2 region containing the amino acid sequence of RNX1X2RPS, where X1 is N, K, S, H, T, or Y; and X2 is Q, I, or K (SEQ ID NO: 93); and f) LCDR3 region containing the amino acid sequence AX1WDX2X3X4X5GAV, where X1 is A or G; X2 is H or R; X3 is H or R; X4 is R, S, or L; and X5 is S or H (SEQ ID NO: 94) The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0029] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) HCDR1 region containing the amino acid sequence of GFTFSSYWMS (SEQ ID NO: 40), GFTFKSYYMS (SEQ ID NO: 41), or GFTFRSHYMT (SEQ ID NO: 42); b) HCDR2 region containing the amino acid sequence of NIKQDGSEKYYVDSVKG (SEQ ID NO: 43), NIDYQSQHAYYAESVKG (SEQ ID NO: 44), or NIDYEGTRTYYAESVKG (SEQ ID NO: 45); c) HCDR3 region containing the amino acid sequence of GYSAEFAHRSGLDV (SEQ ID NO: 39); d) LCDR1 region containing the amino acid sequence of SGSSSNIGSNYVY (SEQ ID NO: 46), SGSSSNIGINYVY (SEQ ID NO: 47), SGSSSNIGANYVY (SEQ ID NO: 48), SGSSSNIGQTYVY (SEQ ID NO: 49), or SGSSSNIGKNYVY (SEQ ID NO: 50); e) LCDR2 region containing the amino acid sequences of RNNQRPS (SEQ ID NO: 51), RNNIRPS (SEQ ID NO: 52), RNKRPS (SEQ ID NO: 53), RNKKRPS (SEQ ID NO: 54), RNKQRPS (SEQ ID NO: 55), RNSQRPS (SEQ ID NO: 56), RNHIRPS (SEQ ID NO: 57), RNTQRPS (SEQ ID NO: 58), or RNYQRPS (SEQ ID NO: 59); and f) LCDR3 region containing the amino acid sequence of AAWDHHRSGAV (SEQ ID NO: 60), AAWDRHSHGAV (SEQ ID NO: 61), or AGWDRRLHGAV (SEQ ID NO: 62) The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0030] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

[0031] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable light chain (VL) containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, and 38.

[0032] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) Variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15; and b) A variable light chain (VL) containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, and 38. The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0033] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) VH containing the amino acid sequence of SEQ ID NO: 13 and VL containing the amino acid sequence of SEQ ID NO: 16, b) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 16 c) VH containing the amino acid sequence of SEQ ID NO: 13 and VL containing the amino acid sequence of SEQ ID NO: 17, d) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 17, e) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 18, f) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 19, g) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 20, h) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 21, i) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 22, j) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 23, k) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 24, l) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 25, m) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 26, n) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 27, o) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 28, p) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 29, q) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 30, r) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 31, s) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 32, t) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 33, u) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 34, v) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 35 w) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 36, x) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 37, y) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 38, z) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 17, aa) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 18, bb) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 19, cc) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 20, dd) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 21, ee) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 22, ff) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 23, gg) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 24, hh) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 25, ii) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 26, jj) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 27, kk) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 28, ll) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 29, VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 30, VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 31, oo) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 32, pp) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 33, qq) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 34, rr) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 35, ss) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 36, VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 37, VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 38 The present invention provides an isolated human antibody or antibody fragment comprising a variable heavy chain (VH) and a variable light chain (VH) selected from the group consisting of the above.

[0034] In one embodiment, an isolated human antibody or antibody fragment specific to human CD3 epsilon according to the Disclosure cross-reacts with macaque CD3 epsilon. In one embodiment, an isolated human antibody or antibody fragment specific to human CD3 epsilon according to the Disclosure is cross-reactive with macaque CD3 epsilon.

[0035] In one embodiment, the isolated human antibody or antibody fragment specific to human CD3 epsilon according to the Disclosure is a recombinant antibody or antibody fragment. In one embodiment, the isolated human antibody or antibody fragment specific to human CD3 epsilon according to the Disclosure is a monoclonal antibody or antibody fragment.

[0036] In one embodiment, the isolated human antibody or antibody fragment specific to human CD3 epsilon according to this disclosure is an antibody fragment selected from the group consisting of Fab, Fv, and scFv. In one embodiment, the isolated human antibody or antibody fragment specific to human CD3 epsilon according to this disclosure is a Fab fragment. In one embodiment, the isolated human antibody or antibody fragment specific to human CD3 epsilon according to this disclosure is a single-chain Fv(scFv) fragment. In one embodiment, the isolated antibody or antibody fragment specific to human CD3 epsilon according to this disclosure is an Fv domain. In one embodiment, the isolated human antibody or antibody fragment specific to human CD3 epsilon according to this disclosure is IgG. In one embodiment, the isolated human antibody or antibody fragment specific to human CD3 epsilon according to this disclosure is full-length IgG. In one embodiment, the isolated human antibody or antibody fragment specific to human CD3 epsilon according to this disclosure is a full-length IgG of an isotype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

[0037] In one embodiment, the isolated human antibody or antibody fragment specific to human CD3 epsilon according to this disclosure is an IgG containing an Fc region having reduced effector function compared to the wild-type Fc receptor. As used in this context, “effector function” refers to the biological activity of the antibody’s Fc region, which varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cell-mediated cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0038] In one embodiment, the isolated human antibody or antibody fragment specific to human CD3 epsilon includes an Fc region in which at least five amino acids at positions corresponding to L234, L235, D237, N330, and P331 in the human IgG1 heavy chain are mutated to A, E, A, S, and S, respectively.

[0039] In one embodiment, the Disclosure provides a multispecific antibody comprising an isolated human antibody or antibody fragment specific to human CD3 epsilon as described herein. In embodiments of the Disclosure, the multispecific antibody also specifically binds to cancer-related antigens.

[0040] In one embodiment, the Disclosure provides a multispecific antibody comprising an isolated human antibody or antibody fragment specific to human CD3 epsilon according to the Disclosure and a second antibody or antibody fragment that binds to a different antigen from the human CD3 epsilon-specific antibody or antibody fragment according to the Disclosure. In one embodiment, the second antibody fragment binds to a cell surface target antigen. In one embodiment, the cell surface target antigen is a tumor antigen. In one embodiment, the second antibody fragment binds to a cancer-associated antigen on cancer cells. In one embodiment, the Disclosure provides a multispecific antibody comprising a first antibody fragment of a human antibody or antibody fragment specific to human CD3 epsilon according to the Disclosure and a second antibody fragment that binds to a different antigen from the first antibody fragment. In one embodiment, the multispecific antibody comprises a third antibody fragment that binds to a different antigen from the first and second antibody fragments. In one embodiment, the multispecific antibody comprises a third antibody fragment that binds to the same antigen as the second antibody fragment. In one embodiment, the second and third antibody fragments bind to a cell surface target antigen. In one embodiment, the cell surface target antigen is a tumor antigen. In one embodiment, the second and third antibody fragments bind to cancer-associated antigens on cancer cells.

[0041] In one embodiment, the Disclosure provides a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding a human antibody or antibody fragment specific to human CD3 epsilon according to the Disclosure, or a multispecific antibody according to the Disclosure. In one embodiment, the Disclosure provides a vector or a vector composition comprising a plurality of nucleic acid sequences according to the Disclosure. In one embodiment, the Disclosure provides a host cell comprising a vector composition according to the Disclosure. In one embodiment, the host cell is a mammalian cell. In one embodiment, the host cell is a prokaryotic cell. In another embodiment, the Disclosure provides a method for producing an isolated human antibody or antibody fragment specific to human CD3 epsilon according to the Disclosure.

[0042] In one embodiment, the Disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, or a multispecific antibody, for use as a pharmaceutical. In a further embodiment, the Disclosure provides an isolated human antibody or antibody fragment specific to CD3 epsilon, for use in the treatment of a target requiring such antibody.

[0043] In one embodiment, the Disclosure also provides a method for treating a patient suffering from a disease such as a proliferative disorder by administering to the patient an effective amount of an isolated human antibody or antibody fragment specific to human CD3 epsilon according to the Disclosure. In one embodiment, the Disclosure provides a pharmaceutical composition comprising a human antibody or antibody fragment specific to CD3 epsilon according to the Disclosure, or a multispecific antibody according to the Disclosure, and a pharmaceutically acceptable carrier or excipient.

[0044] In one embodiment, the present disclosure provides a method for novelly directing the cytotoxic activity of T cells against cancer cells, the method comprising contacting the cancer cells with a multispecific antibody according to the present disclosure in the presence of T cells.

[0045] The claimed antibody or antibody fragment is useful. Furthermore, the claimed method for producing such an antibody or antibody fragment is useful. The use of the claimed antibody or antibody fragment is intended to target CD3-expressing T cells to stimulate T cell activation, for example, under circumstances where T cell-mediated death is beneficial or desirable. In particular, the claimed antibody or antibody fragment is intended for therapeutic use, such as in the treatment of cancer. The present invention includes the following embodiments. <1> an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) GFTFX 1 SX 2 X 3 MX 4 The HCDR1 region containing the amino acid sequence, in the formula, X 1 is S, K, or R; X 2 is Y or H; X 3 is W or Y; and X 4 b) NIX is either S or T (Sequence ID 90); 1X 2 X 3 X 4 X 5 X 6 X 7 YYX 8 X 9 The HCDR2 region containing the amino acid sequence of SVKG, in the formula, X 1 is K or D; X 2 is Q or Y; X 3 is D, Q, or E; X 4 is S or G; X 5 is S, Q, or T; X 6 is E, H, or R; X 7 is K, A, or T; X 8 is V or A; and X 9 c) The HCDR3 region containing the amino acid sequence of GYSAEFAHRSGLDV (SEQ ID NO: 39); d) SGSSSNIGX 1 X 2 The LCDR1 region containing the YVY amino acid sequence, in the formula, X 1 is S, I, A, K, or Q; and X 2 is N or T (Sequence ID 92); e)RNX 1 X 2 The LCDR2 region containing the amino acid sequence of RPS, in the formula, X 1 is N, K, S, H, T, or Y; and X 2 is Q, I, or K (Sequence ID 93); and f)AX 1 WDX 2 X 3 X 4 X 5 The LCDR3 region containing the amino acid sequence of GAV, in the formula, X 1 is A or G; X 2 is H or R; X 3 is H or R; X 4 is R, S, or L; and X 5 This refers to an isolated human antibody or antibody fragment containing S or H (Sequence ID 94). <2> The antibody or antibody fragment is a) HCDR1 region containing the amino acid sequence of GFTFSSYWMS (SEQ ID NO: 40), GFTFKSYYMS (SEQ ID NO: 41), or GFTFRSHYMT (SEQ ID NO: 42); b) HCDR2 region containing the amino acid sequence of NIKQDGSEKYYVDSVKG (SEQ ID NO: 43), NIDYQSQHAYYAESVKG (SEQ ID NO: 44), or NIDYEGTRTYYAESVKG (SEQ ID NO: 45); c) HCDR3 region containing the amino acid sequence of GYSAEFAHRSGLDV (SEQ ID NO: 39); d) SGSSSNIGSNYVY (SEQ ID NO: 46), SGSSSNIGINYVY (SEQ ID NO: 47), SGSSSNIGANYVY (SEQ ID NO: 48), SGSSSNIGQTYVY (SEQ ID NO: 49), or LCDR1 region containing the amino acid sequence of SGSSSNIGKNYVY (SEQ ID NO: 50); e) LCDR2 region containing the amino acid sequence of RNNQRPS (SEQ ID NO: 51), RNNIRPS (SEQ ID NO: 52), RNKRPS (SEQ ID NO: 53), RNKKRPS (SEQ ID NO: 54), RNKQRPS (SEQ ID NO: 55), RNSQRPS (SEQ ID NO: 56), RNHIRPS (SEQ ID NO: 57), RNTQRPS (SEQ ID NO: 58), or RNYQRPS (SEQ ID NO: 59); and f) LCDR3 region containing the amino acid sequence of AAWDHHRSGAV (SEQ ID NO: 60), AAWDRHSHGAV (SEQ ID NO: 61), or AGWDRRLHGAV (SEQ ID NO: 62), <1> An isolated human antibody or antibody fragment specific to human CD3 epsilon as described above. <3> The antibody or antibody fragment includes a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. <1> or <2> An isolated human antibody or antibody fragment specific to human CD3 epsilon as described above. <4> The antibody or antibody fragment includes a variable light chain (VL) containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, and 38. <1> ~ <3> An isolated human antibody or antibody fragment specific to human CD3 epsilon as described in any of the above. <5> The antibody or antibody fragment is a) A variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15; and b) A variable light chain (VL) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38. <1> ~ <4> An isolated human antibody or antibody fragment specific to human CD3 epsilon as described in any of the above. <6> The variable heavy chain and variable light chain are, a) VH containing the amino acid sequence of SEQ ID NO: 13 and VL containing the amino acid sequence of SEQ ID NO: 16, b) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 16, c) VH containing the amino acid sequence of SEQ ID NO: 13 and VL containing the amino acid sequence of SEQ ID NO: 17, d) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 17, e) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 18, f) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 19, g) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 20, h) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 21, i) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 22, j) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 23, k) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 24, l) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 25, m) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 26, n) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 27, o) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 28, p) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 29, q) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 30, r) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 31, s) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 32, t) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 33, u) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 34, v) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 35, w) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 36, x) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 37,y) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 38, z) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 17, aa) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 18, bb) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 19, cc) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 20, dd) VH containing the amino acid sequence of SEQ ID NO: 15 and the amino acid sequence of SEQ ID NO: 21 VL containing the amino acid sequence of SEQ ID NO: 15, ee) VH containing the amino acid sequence of SEQ ID NO: 22, ff) VH containing the amino acid sequence of SEQ ID NO: 15, VL containing the amino acid sequence of SEQ ID NO: 23, gg) VH containing the amino acid sequence of SEQ ID NO: 15, VL containing the amino acid sequence of SEQ ID NO: 24, hh) VH containing the amino acid sequence of SEQ ID NO: 15, VL containing the amino acid sequence of SEQ ID NO: 25, ii) VH containing the amino acid sequence of SEQ ID NO: 15, VL containing the amino acid sequence of SEQ ID NO: 26, jj) VH containing the amino acid sequence of SEQ ID NO: 15, and SEQ ID NO: 27 VL containing the amino acid sequence of (kk) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 28, (ll) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 29, (mm) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 30, (nn) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 31, (oo) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 32, (pp) VH containing the amino acid sequence of SEQ ID NO: 15 and Selected from the group consisting of VL containing the amino acid sequence of SEQ ID NO: 33, VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 34, VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 35, VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 36, VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 37, and VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 38.<1> ~ <5> An isolated human antibody or antibody fragment specific to human CD3 epsilon as described in any of the following. <7> The antibody or antibody fragment binds to macaque monkey CD3 epsilon by cross-reaction. <1> ~ <6> An isolated human antibody or antibody fragment specific to human CD3 epsilon as described in any of the above. <8> The antibody or antibody fragment is a recombinant antibody or antibody fragment. <1> ~ <7> An isolated human antibody or antibody fragment specific to human CD3 epsilon as described in any of the above. <9> The antibody or antibody fragment is a monoclonal antibody or antibody fragment. <1> ~ <8> An isolated human antibody or antibody fragment specific to human CD3 epsilon as described in any of the above. <10> The antibody fragment is selected from the group consisting of Fab, Fv, and scFv. <1> ~ <9> An isolated human antibody or antibody fragment specific to human CD3 epsilon as described in any of the above. <11> The aforementioned antibody is IgG. <1> ~ <9> An isolated human antibody or antibody fragment specific to human CD3 epsilon as described in any of the above. <12> <1> ~ <11> A multispecific antibody comprising an isolated human antibody or antibody fragment specific to human CD3 epsilon as described in any of the above. <13> The multispecific antibody also specifically binds to cancer-related antigens. <12> The multispecific antibodies described above. <14> <1> ~ <11> A human antibody or antibody fragment specific to human CD3 epsilon as described in any of the above, or <12> ~ <13> A pharmaceutical composition comprising a multispecific antibody as described in any of the above and a pharmaceutically acceptable carrier or excipient. <15> For use as a medicine, <1> ~ <11> A human antibody or antibody fragment specific to human CD3 epsilon as described in any of the following: <12> ~ <13> A multispecific antibody as described in any of the following, or <13> The pharmaceutical composition described above. [Brief explanation of the drawing]

[0046] [Figure 1] Figure 1 shows the cell binding of mammalian-produced CD3-specific MAB-1 IgG. Figure 1A shows the binding as a function of IgG concentration determined by flow cytometry in human-derived PBMCs expressing human CD3. IgG against chicken egg white lysozyme was included as a negative control. Figure 1B is the same as Figure 1A, except that it shows the binding to cynomolgus monkey-derived PBMCs expressing macaque CD3. [Figure 2] Figure 2 shows a conceptual diagram of the CyCAT format. A set of polypeptides is designed, each consisting of a target component (e.g., antibody Fab fragment) fused to either the variable light chain domain (VL) or the complementary variable heavy chain (VH) domain of a T cell-activating anti-CD3 antibody. Unpaired VH or VL domains cannot bind to CD3. Once the two polypeptides described above bind to their antigens on the cell surface via their target components, the complementary VL and VH domains become adjacent and interact with each other to reconstitute the original CD3 antibody fragment (e.g., the CD3-specific Fv domain). The triplicate heterodimer molecule thus formed on the target cell engages and stimulates T cells for tumor cell destruction, similar to conventional trivalent bivalent or triplicate antibody formats. [Modes for carrying out the invention]

[0047] definition The term "CD3" refers to an antigen expressed on T cells as part of the multimolecular T cell receptor (TCR), consisting of a homodimer or heterodimer formed from the association of two of the four receptor chains: CD3-epsilon, CD3-delta, CD3-zeta, and CD3-gamma.

[0048] Human CD3 epsilon containing the signal sequence (underlined) has the amino acid sequence of UniProt P07766: [ka]

[0049] Macaque monkey CD3 epsilon containing the signal sequence (underlined) has the amino acid sequence of UniProt Q95LI5: [ka]

[0050] As used herein, the terms “antigen” or “target antigen” refer to any molecule of interest to which one of the binding sites present in the antigen-binding molecule according to this disclosure can bind. Typically, an antigen is a peptide, protein, or any other proteinaceous molecule. Alternatively, an antigen may be any other organic or inorganic molecule, such as a carbohydrate, fatty acid, lipid, pigment, or fluorophore.

[0051] As used herein, the term “antigen-binding molecule” refers in its broadest sense to a proteinaceous molecule that specifically binds to at least one antigen. An antigen-binding molecule may consist of one or more polypeptides. Examples of antigen-binding molecules are immunoglobulins and their derivatives and / or fragments. The antigen-binding molecules disclosed herein may be based on conventional immunoglobulins (e.g., IgG), particularly half-IgG molecules. The antigen-binding molecules disclosed herein consist of at least one target component (e.g., an antibody Fab fragment) and an additional VH or VL domain of the antibody Fv domain, neither of which can bind to the antigen alone. Therefore, the antigen-binding molecules disclosed herein incorporate a half-Fv domain (1 / 2Fv domain) or a half-binding site and a complete binding site, and can thus be described as a 1+1 / 2 or 1 1 / 2 antigen-binding molecule.

[0052] As used herein, the term “antibody” refers to a protein comprising at least two heavy (H) chains and two light (L) chains linked to each other by disulfide bonds, which interacts with an antigen. Each heavy chain consists of a heavy chain variable region or domain (hereinafter abbreviated as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region or domain (hereinafter abbreviated as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are dotted with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody may mediate the binding of immunoglobulins to various cells of the immune system (e.g., effector cells) and to host tissues or host factors, including the first component (C1q) of the classical complement system. The term “antibody” includes, for example, monoclonal antibodies, human antibodies, humanized antibodies, camelized antibodies, and chimeric antibodies. Antibodies can be any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. Both the light and heavy chains are divided into structurally and functionally homologous regions.

[0053] As used herein, the term “antibody fragment” refers to one or more portions of an antibody that possess the ability to specifically interact with an antigen (e.g., by stabilizing binding, steric hindrance, or spatial distribution). Examples of antibody fragments include, but are not limited to, Fab fragments, which are monovalent fragments consisting of VL, VH, CL, and CH1 domains; F(ab)2 fragments, which are bivalent fragments containing two Fab fragments linked by disulfide crosslinks at a hinge region; Fd fragments, which consist of VH and CH1 domains; Fv fragments (or Fv domains), which consist of VL and VH domains of one arm of an antibody; dAb fragments, which consist of a VH domain (Ward et al., (1989) Nature 341:544-546); and isolated complementarity-determining regions (CDRs). Furthermore, the two domains of an Fv fragment (or Fv domain), VL and VH, are encoded by separate genes, but they can be linked together using recombination methods by a synthetic linker, which allows them to be constructed as a single protein chain, with the VL and VH regions paired to form a monovalent molecule (known as single-chain Fv (scFv); see, e.g., Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. 85:5879-5883). Such single-chain antibodies are also intended to be included within the term “antibody fragment.” These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antibody fragments can also be incorporated into single-domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, for example, Hollinger and Hudson, (2005) Nature Biotechnology 23:1126-1136).Antibody fragments can be implanted into polypeptide-based scaffolds, such as fibronectin type III (Fn3) (see U.S. Patent No. 6,703,199, which describes fibronectin polypeptide monobodies). Antibody fragments can be incorporated into single-chain molecules containing a pair of tandem Fv segments (VH-CH1-VH-CH1) that form a pair of antigen-binding sites together with a complementary light chain polypeptide (Zapata et al., (1995) Protein Eng. 8:1057-1062; and U.S. Patent No. 5,641,870).

[0054] As used herein, “human antibody” or “human antibody fragment” includes antibodies and antibody fragments having variable regions in which the framework and CDR regions are derived from human-derived sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from such a sequence. Human origin includes, for example, human germline sequences or mutant versions of human germline sequences or antibodies containing consensus framework sequences derived from human framework sequence analysis, as described, for example, in Knappik et al., (2000) J Mol Biol 296:57-86. Thereafter, the human antibody can be obtained from a technological platform that includes antibodies derived from human germline genes produced by PCR amplification of the VHA / L repertoire isolated from B cells or synthesized. The technological platform includes library-based approaches that include human immunoglobulin genes displayed on phages, ribosomes, or yeast. Each display technique is standard in the scientific community. Furthermore, immunization of transgenic mice possessing a human immunoglobulin repertoire is another approach for producing human antibodies against antigens of interest. Antibodies or fragments thereof selected from the MorphoSys HuCAL® concept-based antibody library (Knappik et al., (2000) J Mol Biol 296:57-86) and the MorphoSys Ylanthia® library (Tiller et al. mAbs 5:3,1-26; May / June (2013) and U.S. Patent No. 8,728,981) are considered fully human.

[0055] In this specification, a "humanized antibody" or "humanized antibody fragment" is defined as an antibody or antibody fragment having a constant antibody region derived from a human sequence, where only the variable antibody region, part thereof, or CDR is derived from another species. For example, a CDR can be implanted into a humanized antibody, where the CDR of the variable domain is of non-human origin, but one or more frameworks of the variable domain are of human origin, and the constant domain (if any) is of human origin.

[0056] The structure and location of immunoglobulin variable domains, such as CDRs, may be defined using well-known numbering schemes, such as the Kabat numbering scheme, the Chothia numbering scheme, or a combination of Kabat and Chothia (e.g., Sequences of Proteins of Immunological Interest, USD Department of Health and Human Services (1991), eds. Kabat et al.; Lazikani et al., (1997) J.Mol.Bio.273:927-948); Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th edit., NIH Publication no.91-3242 USD Department of Health and Human Services; Chothia et al., (1987) J.Mol.Bio.196:901-917; Chothia et al., (1989) Nature 342:877-883; and Al-Lazikani et al. See al., (1997) J.Mol.Biol.273:927-948.

[0057] The term "isolated" refers to a compound that can be substantially free of other antibodies or antibody fragments having different antigen specificities, for example. Thus, in some embodiments, the antibody provided is an isolated antibody separated from an antibody having different specificities. The isolated antibody may be a monoclonal antibody. The isolated antibody may be a recombinant monoclonal antibody. An isolated antibody that specifically binds to a target epitope, isoform, or variant may, however, cross-reactive to other relevant antigens from other species (e.g., species homologs).

[0058] As used herein, the term “recombinant antibody” includes all antibodies prepared, expressed, produced, or cleaved by means not found in nature. For example, antibodies isolated from host cells transformed to express the antibody, antibodies selected and isolated from a recombinant combinatorial human antibody library, and antibodies prepared, expressed, produced, or isolated by any other means involving splicing all or part of a human immunoglobulin gene sequence to other DNA sequences, or antibodies isolated from transchromosomal animals (e.g., mice) or hybridomas prepared therefrom that have been genetically modified for human immunoglobulin genes. Preferably, such recombinant antibodies have a variable region in which the framework and CDR region are derived from a human germline immunoglobulin sequence. In one embodiment, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or in vivo somatic mutagenesis if an animal genetically modified for a human Ig sequence is used), and thus the amino acid sequences of the VH and VL regions of the recombinant antibody are derived from and related to human germline VH and VL sequences, but may not originally be present in the in vivo human germline antibody repertoire. The recombinant antibody may also be a monoclonal antibody.

[0059] As used herein, the term “monoclonal antibody” refers to an antibody derived from a single clone, including any eukaryote, prokaryote, or phage clone, and not to a method by which a monoclonal antibody is produced. The monoclonal antibodies disclosed herein may be prepared by the hybridoma method described in Kohler et al.; Nature, 256:495 (1975) or isolated from a phage library using, for example, the techniques described herein. Other methods for preparing clonal cell lines and the monoclonal antibodies expressed thereby are well known in the art (see, for example, Chapter 11 of Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel et al., eds., John Wiley and Sons, New York). Other exemplary methods for producing other monoclonal antibodies are provided in the examples herein.

[0060] As used herein, binding specificity is a relative rather than an absolute characteristic; therefore, if such an antibody can distinguish such an antigen from one or more reference antigens, the antibody is said to "specifically bind to the antigen," "specific to the antigen," "specific to / against the antigen," or "specifically recognize the antigen." For example, a standard ELISA assay can be performed. Scoring may be performed by standard colorimetric development (e.g., horseradish peroxide and secondary antibody, and hydrogen peroxide and tetramethylbenzidine). The reaction in a well is scored by optical density at, for example, 450 nm. A typical background (= negative reaction) may be 0.1 OD; a typical positive reaction may be 1 OD. This means that the positive / negative difference can be more than 10 times. Typically, the determination of binding specificity is performed by using a set of about 3 to 5 unrelated antigens, such as powdered milk, BSA, transferrin, or other similar antigens, rather than a single reference antigen.

[0061] As used herein, “CD3-binding antibody,” “anti-CD3 antibody,” or “CD3-specific antibody” includes antibodies and antibody fragments that specifically recognize one or more CD3 receptor chains (or subunits) (e.g., epsilon, delta, gamma, or zeta) and antibodies and antibody fragments that specifically recognize dimeric complexes of two CD3 receptor chains (e.g., gamma / epsilon, delta / epsilon, and zeta / zeta CD3 dimers). The antibodies and antibody fragments of this disclosure may bind to soluble CD3 and / or cell surface-expressed CD3. Soluble CD3 includes the native CD3 protein and recombinant CD3 protein variants such as monomeric and dimeric CD3 constructs that lack a transmembrane domain or otherwise do not associate with the cell membrane. The use of the terms “CD3-binding antibody,” “anti-CD3 antibody,” or “CD3-specific antibody” herein is intended to include both monospecific antibodies and bispecific or multispecific antibodies.

[0062] "Cell surface-expressed CD3" includes CD3 proteins contained in the cell membrane in association with functional T cell receptors. The phrase "cell surface-expressed CD3" also includes CD3 proteins expressed on the cell surface as part of homodimers or heterodimers (e.g., gamma / epsilon, delta / epsilon, and zeta / zeta CD3 receptor chain dimers). The phrase "cell surface-expressed CD3" also includes CD3 receptor chains (e.g., CD3-epsilon, CD3-delta, or CD3-gamma or CD3-zeta) expressed on the cell surface without having other CD3 chain types. "Cell surface-expressed CD3" may or may consist of CD3 proteins expressed on the surface of cells that normally express CD3 proteins. Alternatively, "cell surface-expressed CD3" may include or consist of CD3 proteins expressed on the surface of cells that do not normally express human CD3 on their surface but have been artificially engineered to express CD3 on their surface.

[0063] As used herein, the term "binding by cross-reaction" or the term "cross-reactive" is used interchangeably and refers to an antibody having the ability to specifically bind to more than one antigen. For example, in the present disclosure, an antibody binds by cross-reaction to cynomolgus CD3 or cynomolgus CD3 epsilon.

[0064] As used herein, the term "affinity" refers to the strength of the interaction between a polypeptide and its target at a single site. Within each site, the binding region of the polypeptide interacts with its target at multiple sites through weak non-covalent forces, and the greater the interaction, the stronger the affinity. The term "K" as used herein D refers to the dissociation constant, which is obtained from the ratio of K a to K d (i.e., K d / K a ) and is expressed as molar concentration (M). For example, the K D value for an antigen-binding component such as a monoclonal antibody can be determined using methods well established in the art. For example, a method for determining the K D of an antigen-binding component such as a monoclonal antibody is surface plasmon resonance using a biosensor system such as the SET (soluble equilibrium titration) or Biacore® system. In the present disclosure, an antibody specific for the human CD3 epsilon polypeptide typically has a K -2 less than 5×10 -2 M, less than 10 -3 M, less than 5×10 -3 M, less than 10 -4 M, less than 5×10 -4 M, less than 10 -5 M, less than 5×10 -5 M, less than 10 -6 M, less than 5×1w0 -6 M, less than 10 -7 M, less than 5×10 -7 M, less than 10 -8 M, less than 5×10 -8 M, less than 10 -9Less than M, 10 -9 Less than M, 5 x 10 -10 Less than M, 10 -10 Less than M, 5 x 10 -11 Less than M, 10 -11 Less than M, 5 x 10 -12 Less than M, 10 -12 Less than M, 5 x 10 -13 Less than M, 10 -13 Less than M, 5 x 10 -14 Less than M, 10 -14 Less than M, 5 x 10 -15 Less than M, or 10 -15 Dissociation rate constant (K) less than or equal to M D )(k off / k on ) has.

[0065] The compositions of this disclosure may be used for therapeutic or prophylactic applications. Therefore, this disclosure includes pharmaceutical compositions comprising antibodies (or functional antibody fragments) disclosed herein and pharmaceutically acceptable carriers or excipients therefor. In a relevant embodiment, this disclosure provides a method for treating cancer. Such a method includes the step of administering an effective amount of a pharmaceutical composition containing an antibody (or functional antibody fragment) described herein to a subject requiring it.

[0066] This disclosure provides a therapeutic approach comprising administering a therapeutically effective dose of a CD3-specific human antibody or antibody fragment disclosed herein to a subject requiring such treatment. As used herein, “therapeutic effective dose” or “effective dose” refers to the amount of CD3-specific antibody required to induce a desired biological response. In accordance with this disclosure, a therapeutically effective dose is the amount of CD3-specific antibody or antibody fragment required to treat and / or prevent a disease.

[0067] The terms "proliferative disorder" and "proliferative disorder" refer to diseases associated with a certain degree of abnormal cell proliferation. In one embodiment, a proliferative disorder is cancer. In one embodiment, a proliferative disorder is a tumor.

[0068] The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals typically characterized by disordered cell growth.

[0069] As used herein, “delaying the progression” of a disease or disease means delaying, preventing, slowing, interfering with, stabilizing, and / or postponing the onset of a disease or disease (e.g., a cytoproliferative disorder, e.g., cancer). The duration of this delay may vary depending on the medical history and / or the individual being treated. As will be apparent to those skilled in the art, a sufficient or significant delay can essentially encompass prevention in that the individual does not develop the disease. For example, terminal cancer, such as the onset of metastasis, may be delayed.

[0070] The term "IC" as used herein 50 " " refers to the intermediate concentration of an inhibitor (e.g., antibody or antibody fragment) that inhibits the response in the assay, between the maximum response and the baseline. It represents the antibody concentration that reduces a given response by 50%.

[0071] The terms “inhibit” or “inhibit,” “reduce” or “reduce,” or “neutralize” or “neutralize” refer to a reduction or cessation of any phenotypic feature (e.g., binding, biological activity, or function) or a reduction or cessation of the incidence, degree, or possibility of such feature. “Inhibition,” “reduce,” or “neutralize” does not need to be complete, as long as it is detectable using an appropriate assay. In some embodiments, “reduce” or “inhibit” means the ability to cause a reduction of 20% or more. In another embodiment, “reduce” or “inhibit” means the ability to cause a reduction of 50% or more. In yet another embodiment, “reduce” or “inhibit” means the ability to cause an overall reduction of 75%, 85%, 90%, 95%, or more.

[0072] "Administered" or "administered" includes, but is not limited to, the delivery of a drug in an injectable form, such as intravenous, intramuscular, intradermal, or via a subcutaneous or mucosal route, for example, as a nasal spray or aerosol for inhalation, or as an ingestible liquid, capsule, or tablet. Preferably, the administration is in an injectable form.

[0073] As used herein, “treatment,” “to treat,” “to treat,” and “to treat” and similar terms refer to a clinical intervention that seeks to alter the natural course of a disease in the subject being treated, and may be performed for prophylactic purposes or during the course of a clinicopathological examination. Desired effects of treatment include, but are not limited to, preventing the onset or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, recovery or remission of the disease state, and remission or improvement of prognosis. In some embodiments, antibodies or antibody fragments according to this disclosure are used to delay the onset of a disease or to slow the progression of a disease.

[0074] The term "multispecificity" means that an antibody can specifically bind to two or more different antigens. Typically, a multispecific antigen-binding molecule contains two or more antigen-binding sites, each of which is specific to a different antigen or epitope.

[0075] The term "bispecificity" means that an antibody can specifically bind to two different antigens. Typically, a bispecific antigen-binding molecule contains two antigen-binding sites, each of which is specific to a different antigen or epitope.

[0076] As used herein, the terms “first” and “second” with respect to the Fab and / or Fv regions or other similar elements are used to distinguish between components of each type when there is more than one. The use of these terms is not intended to give any particular order or direction unless explicitly stated.

[0077] As used herein, “amino acid residue” or “amino acid” is indicated by its full name or by a standard three-letter or one-letter amino acid code. “Natural amino acid” means the following amino acids:

[0078] [Table 1]

[0079] As used herein, the term "polypeptide" refers to a polymer of amino acid residues and not to a product of a specific length. This term applies to both natural and non-natural amino acid polymers. Unless otherwise specified, a particular amino acid sequence of a polypeptide also implicitly includes its conservatively modified variants (e.g., by replacing an amino acid residue with another amino acid residue having similar structural and / or chemical properties). Polypeptides may originate from natural biological sources or be produced by recombinant techniques, but are not necessarily translated from a specified nucleic acid sequence. They may be produced in any manner, including chemical synthesis. Polypeptides may contain one or more disulfide bonds or may be chemically modified.

[0080] As used herein, the term "domain" refers to a linear molecular chain of amino acids containing the amino acid sequence of an entire polypeptide or a portion of a polypeptide.

[0081] With respect to the CyCAT molecule as disclosed herein, the term "functional domain" as used herein is a domain capable of performing a certain function, such as specific binding to a binding partner or antigen, specific activation of a receptor, mediation of toxic effects, or fluorescence upon excitation with light of an appropriate wavelength.

[0082] With respect to the CyCAT molecule as described herein, the terms "domain fragment" or "functional domain fragment" refer to a linear chain of amino acids that corresponds to a portion of a domain but not to the entire domain or a functional domain.

[0083] In relation to the CyCAT molecule as disclosed herein, the term “component” refers to a linear molecular chain of amino acids containing the amino acid sequence of the whole polypeptide or a portion of the polypeptide. “Target component” is used to refer to a component that specifically binds to an antigen. Non-limiting examples of target components include antibodies, antibody fragments, native ligands of antigens, mutaines of such ligands, or mimetic forms of such ligands.

[0084] The term "non-associating" with respect to the CyCAT molecule in this disclosure means that, with respect to the function of the functional domain, it does not functionally associate, that is, the first fragment and the second fragment of the functional domain do not form the functional domain; or, with respect to the function of the Fv domain, that is, the VH and VL do not form a functional Fv domain.

[0085] As used herein, “non-covalent association” refers to molecular interactions that do not involve interatomic bonding. Non-covalent interactions include, for example, ionic bonds, hydrogen bonds, hydrophobic interactions, and van der Waals forces.

[0086] As used herein, “covalent bond” refers to an interatomic bond characterized by the sharing of electrons.

[0087] Embodiment In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) An HCDR1 region containing the amino acid sequence GFTFX1SX2X3MX4, where X1 is S, K, or R; X2 is Y or H; X3 is W or Y; and X4 is S or T (SEQ ID NO: 90); b) An HCDR2 region containing the amino acid sequence NIX1X2X3X4X5X6X7YYX8X9SVKG, where X1 is K or D; X2 is Q or Y; X3 is D, Q, or E; X4 is S or G; X5 is S, Q, or T; X6 is E, H, or R; X7 is K, A, or T; X8 is V or A; and X9 is D or E (Sequence ID 91); c) HCDR3 region containing the amino acid sequence of GYSAEFAHRSGLDV (SEQ ID NO: 39); d) An LCDR1 region containing the amino acid sequence SGSSSNIGX1X2YVY, where X1 is S, I, A, K, or Q; and X2 is N or T (SEQ ID NO: 92); e) LCDR2 region containing the amino acid sequence of RNX1X2RPS, where X1 is N, K, S, H, T, or Y; and X2 is Q, I, or K (SEQ ID NO: 93); and f) LCDR3 region containing the amino acid sequence AX1WDX2X3X4X5GAV, where X1 is A or G; X2 is H or R; X3 is H or R; X4 is R, S, or L; and X5 is S or H (SEQ ID NO: 94) The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0088] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) HCDR1 region containing the amino acid sequence of GFTFSSYWMS (SEQ ID NO: 40), GFTFKSYYMS (SEQ ID NO: 41), or GFTFRSHYMT (SEQ ID NO: 42); b) HCDR2 region containing the amino acid sequence of NIKQDGSEKYYVDSVKG (SEQ ID NO: 43), NIDYQSQHAYYAESVKG (SEQ ID NO: 44), or NIDYEGTRTYYAESVKG (SEQ ID NO: 45); c) HCDR3 region containing the amino acid sequence of GYSAEFAHRSGLDV (SEQ ID NO: 39); d) LCDR1 region containing the amino acid sequence of SGSSSNIGSNYVY (SEQ ID NO: 46), SGSSSNIGINYVY (SEQ ID NO: 47), SGSSSNIGANYVY (SEQ ID NO: 48), SGSSSNIGQTYVY (SEQ ID NO: 49), or SGSSSNIGKNYVY (SEQ ID NO: 50); e) LCDR2 region containing the amino acid sequences of RNNQRPS (SEQ ID NO: 51), RNNIRPS (SEQ ID NO: 52), RNKRPS (SEQ ID NO: 53), RNKKRPS (SEQ ID NO: 54), RNKQRPS (SEQ ID NO: 55), RNSQRPS (SEQ ID NO: 56), RNHIRPS (SEQ ID NO: 57), RNTQRPS (SEQ ID NO: 58), or RNYQRPS (SEQ ID NO: 59); and f) LCDR3 region containing the amino acid sequence of AAWDHHRSGAV (SEQ ID NO: 60), AAWDRHSHGAV (SEQ ID NO: 61), or AGWDRRLHGAV (SEQ ID NO: 62) The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0089] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) The HCDR1 region containing the amino acid sequence of SEQ ID NO: 40; b) The HCDR2 region containing the amino acid sequence of SEQ ID NO: 43; c) HCDR3 region containing the amino acid sequence of SEQ ID NO: 39; d) The LCDR1 region containing the amino acid sequence of SEQ ID NO: 46; c) The LCDR2 region containing the amino acid sequence of SEQ ID NO: 51; and f) LCDR3 region containing the amino acid sequence of SEQ ID NO: 60 The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0090] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) The HCDR1 region containing the amino acid sequence of SEQ ID NO: 41; b) The HCDR2 region containing the amino acid sequence of SEQ ID NO: 44; c) HCDR3 region containing the amino acid sequence of SEQ ID NO: 39; d) The LCDR1 region containing the amino acid sequence of SEQ ID NO: 46; e) The LCDR2 region containing the amino acid sequence of SEQ ID NO: 51; and f) LCDR3 region containing the amino acid sequence of SEQ ID NO: 60 The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0091] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) The HCDR1 region containing the amino acid sequence of SEQ ID NO: 40; b) The HCDR2 region containing the amino acid sequence of SEQ ID NO: 43; c) HCDR3 region containing the amino acid sequence of SEQ ID NO: 39; d) The LCDR1 region containing the amino acid sequence of SEQ ID NO: 46; e) The LCDR2 region containing the amino acid sequence of SEQ ID NO: 51; and f) LCDR3 region containing the amino acid sequence of SEQ ID NO: 61 The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0092] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) The HCDR1 region containing the amino acid sequence of SEQ ID NO: 41; b) The HCDR2 region containing the amino acid sequence of SEQ ID NO: 44; c) HCDR3 region containing the amino acid sequence of SEQ ID NO: 39; d) The LCDR1 region containing the amino acid sequence of SEQ ID NO: 46; e) The LCDR2 region containing the amino acid sequence of SEQ ID NO: 51; and f) LCDR3 region containing the amino acid sequence of SEQ ID NO: 61 The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0093] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) The HCDR1 region containing the amino acid sequence of SEQ ID NO: 41; b) The HCDR2 region containing the amino acid sequence of SEQ ID NO: 44; c) HCDR3 region containing the amino acid sequence of SEQ ID NO: 39; d) The LCDR1 region containing the amino acid sequence of SEQ ID NO: 47; e) The LCDR2 region containing the amino acid sequence of SEQ ID NO: 52; and f) LCDR3 region containing the amino acid sequence of SEQ ID NO: 61 The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0094] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) The HCDR1 region containing the amino acid sequence of SEQ ID NO: 41; b) The HCDR2 region containing the amino acid sequence of SEQ ID NO: 44; c) HCDR3 region containing the amino acid sequence of SEQ ID NO: 39; d) The LCDR1 region containing the amino acid sequence of SEQ ID NO: 47; e) The LCDR2 region containing the amino acid sequence of SEQ ID NO: 53; and f) LCDR3 region containing the amino acid sequence of SEQ ID NO: 61 The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0095] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) The HCDR1 region containing the amino acid sequence of SEQ ID NO: 41; b) The HCDR2 region containing the amino acid sequence of SEQ ID NO: 44; c) HCDR3 region containing the amino acid sequence of SEQ ID NO: 39; d) The LCDR1 region containing the amino acid sequence of SEQ ID NO: 48; e) The LCDR2 region containing the amino acid sequence of SEQ ID NO: 54; and f) LCDR3 region containing the amino acid sequence of SEQ ID NO: 61 The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0096] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) The HCDR1 region containing the amino acid sequence of SEQ ID NO: 41; b) The HCDR2 region containing the amino acid sequence of SEQ ID NO: 44; c) HCDR3 region containing the amino acid sequence of SEQ ID NO: 39; d) The LCDR1 region containing the amino acid sequence of SEQ ID NO: 47; e) The LCDR2 region containing the amino acid sequence of SEQ ID NO: 55; and f) LCDR3 region containing the amino acid sequence of SEQ ID NO: 61 The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0097] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) The HCDR1 region containing the amino acid sequence of SEQ ID NO: 41; b) The HCDR2 region containing the amino acid sequence of SEQ ID NO: 44; c) HCDR3 region containing the amino acid sequence of SEQ ID NO: 39; d) The LCDR1 region containing the amino acid sequence of SEQ ID NO: 49; e) The LCDR2 region containing the amino acid sequence of SEQ ID NO: 56; and f) LCDR3 region containing the amino acid sequence of SEQ ID NO: 61 The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0098] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) The HCDR1 region containing the amino acid sequence of SEQ ID NO: 41; b) The HCDR2 region containing the amino acid sequence of SEQ ID NO: 44; c) HCDR3 region containing the amino acid sequence of SEQ ID NO: 39; d) The LCDR1 region containing the amino acid sequence of SEQ ID NO: 47; e) The LCDR2 region containing the amino acid sequence of SEQ ID NO: 57; and f) LCDR3 region containing the amino acid sequence of SEQ ID NO: 61 The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0099] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) The HCDR1 region containing the amino acid sequence of SEQ ID NO: 41; b) The HCDR2 region containing the amino acid sequence of SEQ ID NO: 44; c) HCDR3 region containing the amino acid sequence of SEQ ID NO: 39; d) The LCDR1 region containing the amino acid sequence of SEQ ID NO: 48; e) The LCDR2 region containing the amino acid sequence of SEQ ID NO: 51; and f) LCDR3 region containing the amino acid sequence of SEQ ID NO: 61 The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0100] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) The HCDR1 region containing the amino acid sequence of SEQ ID NO: 41; b) The HCDR2 region containing the amino acid sequence of SEQ ID NO: 44; c) HCDR3 region containing the amino acid sequence of SEQ ID NO: 39; d) The LCDR1 region containing the amino acid sequence of SEQ ID NO: 47; e) The LCDR2 region containing the amino acid sequence of SEQ ID NO: 51; and f) LCDR3 region containing the amino acid sequence of SEQ ID NO: 61 The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0101] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) The HCDR1 region containing the amino acid sequence of SEQ ID NO: 41; b) The HCDR2 region containing the amino acid sequence of SEQ ID NO: 44; c) HCDR3 region containing the amino acid sequence of SEQ ID NO: 39; d) The LCDR1 region containing the amino acid sequence of SEQ ID NO: 48; e) The LCDR2 region containing the amino acid sequence of SEQ ID NO: 55; and f) LCDR3 region containing the amino acid sequence of SEQ ID NO: 61 The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0102] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) The HCDR1 region containing the amino acid sequence of SEQ ID NO: 41; b) The HCDR2 region containing the amino acid sequence of SEQ ID NO: 44; c) HCDR3 region containing the amino acid sequence of SEQ ID NO: 39; d) The LCDR1 region containing the amino acid sequence of SEQ ID NO: 47; e) The LCDR2 region containing the amino acid sequence of SEQ ID NO: 58; and f) LCDR3 region containing the amino acid sequence of SEQ ID NO: 61 The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0103] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) The HCDR1 region containing the amino acid sequence of SEQ ID NO: 41; b) The HCDR2 region containing the amino acid sequence of SEQ ID NO: 44; c) HCDR3 region containing the amino acid sequence of SEQ ID NO: 39; d) The LCDR1 region containing the amino acid sequence of SEQ ID NO: 50; e) The LCDR2 region containing the amino acid sequence of SEQ ID NO: 59; and f) LCDR3 region containing the amino acid sequence of SEQ ID NO: 61 The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0104] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) The HCDR1 region containing the amino acid sequence of SEQ ID NO: 41; b) The HCDR2 region containing the amino acid sequence of SEQ ID NO: 44; c) HCDR3 region containing the amino acid sequence of SEQ ID NO: 39; d) The LCDR1 region containing the amino acid sequence of SEQ ID NO: 46; e) The LCDR2 region containing the amino acid sequence of SEQ ID NO: 51; and f) LCDR3 region containing the amino acid sequence of SEQ ID NO: 62 The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0105] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) The HCDR1 region containing the amino acid sequence of SEQ ID NO: 41; b) The HCDR2 region containing the amino acid sequence of SEQ ID NO: 44; c) HCDR3 region containing the amino acid sequence of SEQ ID NO: 39; d) The LCDR1 region containing the amino acid sequence of SEQ ID NO: 49; e) The LCDR2 region containing the amino acid sequence of SEQ ID NO: 58; and f) LCDR3 region containing the amino acid sequence of SEQ ID NO: 61 The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0106] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) The HCDR1 region containing the amino acid sequence of SEQ ID NO: 41; b) The HCDR2 region containing the amino acid sequence of SEQ ID NO: 44; c) HCDR3 region containing the amino acid sequence of SEQ ID NO: 39; d) The LCDR1 region containing the amino acid sequence of SEQ ID NO: 47; e) The LCDR2 region containing the amino acid sequence of SEQ ID NO: 53; and f) LCDR3 region containing the amino acid sequence of SEQ ID NO: 62 The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0107] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) The HCDR1 region containing the amino acid sequence of SEQ ID NO: 41; b) The HCDR2 region containing the amino acid sequence of SEQ ID NO: 44; c) HCDR3 region containing the amino acid sequence of SEQ ID NO: 39; d) The LCDR1 region containing the amino acid sequence of SEQ ID NO: 47; e) The LCDR2 region containing the amino acid sequence of SEQ ID NO: 57; and f) LCDR3 region containing the amino acid sequence of SEQ ID NO: 62 The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0108] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) The HCDR1 region containing the amino acid sequence of SEQ ID NO: 41; b) The HCDR2 region containing the amino acid sequence of SEQ ID NO: 44; c) HCDR3 region containing the amino acid sequence of SEQ ID NO: 39; d) The LCDR1 region containing the amino acid sequence of SEQ ID NO: 48; e) The LCDR2 region containing the amino acid sequence of SEQ ID NO: 51; and f) LCDR3 region containing the amino acid sequence of SEQ ID NO: 62 The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0109] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) The HCDR1 region containing the amino acid sequence of SEQ ID NO: 41; b) The HCDR2 region containing the amino acid sequence of SEQ ID NO: 44; c) HCDR3 region containing the amino acid sequence of SEQ ID NO: 39; d) The LCDR1 region containing the amino acid sequence of SEQ ID NO: 47; e) The LCDR2 region containing the amino acid sequence of SEQ ID NO: 51; and f) LCDR3 region containing the amino acid sequence of SEQ ID NO: 62 The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0110] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) The HCDR1 region containing the amino acid sequence of SEQ ID NO: 41; b) The HCDR2 region containing the amino acid sequence of SEQ ID NO: 44; c) HCDR3 region containing the amino acid sequence of SEQ ID NO: 39; d) The LCDR1 region containing the amino acid sequence of SEQ ID NO: 48; e) The LCDR2 region containing the amino acid sequence of SEQ ID NO: 55; and f) LCDR3 region containing the amino acid sequence of SEQ ID NO: 62 The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0111] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) The HCDR1 region containing the amino acid sequence of SEQ ID NO: 41; b) The HCDR2 region containing the amino acid sequence of SEQ ID NO: 44; c) HCDR3 region containing the amino acid sequence of SEQ ID NO: 39; d) The LCDR1 region containing the amino acid sequence of SEQ ID NO: 47; e) The LCDR2 region containing the amino acid sequence of SEQ ID NO: 58; and f) LCDR3 region containing the amino acid sequence of SEQ ID NO: 62 The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0112] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) The HCDR1 region containing the amino acid sequence of SEQ ID NO: 41; b) The HCDR2 region containing the amino acid sequence of SEQ ID NO: 44; c) HCDR3 region containing the amino acid sequence of SEQ ID NO: 39; d) The LCDR1 region containing the amino acid sequence of SEQ ID NO: 49; e) The LCDR2 region containing the amino acid sequence of SEQ ID NO: 58; and f) LCDR3 region containing the amino acid sequence of SEQ ID NO: 62 The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0113] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) The HCDR1 region containing the amino acid sequence of SEQ ID NO: 41; b) The HCDR2 region containing the amino acid sequence of SEQ ID NO: 44; c) HCDR3 region containing the amino acid sequence of SEQ ID NO: 39; d) The LCDR1 region containing the amino acid sequence of SEQ ID NO: 47; e) The LCDR2 region containing the amino acid sequence of SEQ ID NO: 52; and f) LCDR3 region containing the amino acid sequence of SEQ ID NO: 62 The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0114] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) The HCDR1 region containing the amino acid sequence of SEQ ID NO: 42; b) The HCDR2 region containing the amino acid sequence of SEQ ID NO: 45; c) HCDR3 region containing the amino acid sequence of SEQ ID NO: 39; d) The LCDR1 region containing the amino acid sequence of SEQ ID NO: 46; e) The LCDR2 region containing the amino acid sequence of SEQ ID NO: 51; and f) LCDR3 region containing the amino acid sequence of SEQ ID NO: 61 The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0115] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) The HCDR1 region containing the amino acid sequence of SEQ ID NO: 42; b) The HCDR2 region containing the amino acid sequence of SEQ ID NO: 45; c) HCDR3 region containing the amino acid sequence of SEQ ID NO: 39; d) The LCDR1 region containing the amino acid sequence of SEQ ID NO: 47; e) The LCDR2 region containing the amino acid sequence of SEQ ID NO: 52; and f) LCDR3 region containing the amino acid sequence of SEQ ID NO: 61 The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0116] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) The HCDR1 region containing the amino acid sequence of SEQ ID NO: 42; b) The HCDR2 region containing the amino acid sequence of SEQ ID NO: 45; c) HCDR3 region containing the amino acid sequence of SEQ ID NO: 39; d) The LCDR1 region containing the amino acid sequence of SEQ ID NO: 47; e) The LCDR2 region containing the amino acid sequence of SEQ ID NO: 53; and f) LCDR3 region containing the amino acid sequence of SEQ ID NO: 61 The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0117] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) The HCDR1 region containing the amino acid sequence of SEQ ID NO: 42; b) The HCDR2 region containing the amino acid sequence of SEQ ID NO: 45; c) HCDR3 region containing the amino acid sequence of SEQ ID NO: 39; d) The LCDR1 region containing the amino acid sequence of SEQ ID NO: 48; e) The LCDR2 region containing the amino acid sequence of SEQ ID NO: 54; and f) LCDR3 region containing the amino acid sequence of SEQ ID NO: 61 The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0118] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) The HCDR1 region containing the amino acid sequence of SEQ ID NO: 42; b) The HCDR2 region containing the amino acid sequence of SEQ ID NO: 45; c) HCDR3 region containing the amino acid sequence of SEQ ID NO: 39; d) The LCDR1 region containing the amino acid sequence of SEQ ID NO: 47; e) The LCDR2 region containing the amino acid sequence of SEQ ID NO: 55; and f) LCDR3 region containing the amino acid sequence of SEQ ID NO: 61 The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0119] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) The HCDR1 region containing the amino acid sequence of SEQ ID NO: 42; b) The HCDR2 region containing the amino acid sequence of SEQ ID NO: 45; c) HCDR3 region containing the amino acid sequence of SEQ ID NO: 39; d) The LCDR1 region containing the amino acid sequence of SEQ ID NO: 49; e) The LCDR2 region containing the amino acid sequence of SEQ ID NO: 56; and f) LCDR3 region containing the amino acid sequence of SEQ ID NO: 61 The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0120] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) The HCDR1 region containing the amino acid sequence of SEQ ID NO: 42; b) The HCDR2 region containing the amino acid sequence of SEQ ID NO: 45; c) HCDR3 region containing the amino acid sequence of SEQ ID NO: 39; d) The LCDR1 region containing the amino acid sequence of SEQ ID NO: 47; e) The LCDR2 region containing the amino acid sequence of SEQ ID NO: 57; and f) LCDR3 region containing the amino acid sequence of SEQ ID NO: 61 The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0121] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) The HCDR1 region containing the amino acid sequence of SEQ ID NO: 42; b) The HCDR2 region containing the amino acid sequence of SEQ ID NO: 45; c) HCDR3 region containing the amino acid sequence of SEQ ID NO: 39; d) The LCDR1 region containing the amino acid sequence of SEQ ID NO: 48; e) The LCDR2 region containing the amino acid sequence of SEQ ID NO: 51; and f) LCDR3 region containing the amino acid sequence of SEQ ID NO: 61 The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0122] In one embodiment, the present disclosure provides an isolated human antibody or antibody fragment specific for human CD3 epsilon, wherein the antibody or antibody fragment comprises: a) an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 42; b) an HCDR2 region comprising the amino acid sequence of SEQ ID NO: 45; c) an HCDR3 region comprising the amino acid sequence of SEQ ID NO: 39; d) an LCDR1 region comprising the amino acid sequence of SEQ ID NO: 47; e) an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 51; and f) an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 61. The present disclosure provides an isolated human antibody or antibody fragment.

[0123] In one embodiment, the present disclosure provides an isolated human antibody or antibody fragment specific for human CD3 epsilon, wherein the antibody or antibody fragment comprises: a) an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 42; b) an HCDR2 region comprising the amino acid sequence of SEQ ID NO: 45; c) an HCDR3 region comprising the amino acid sequence of SEQ ID NO: 39; d) an LCDR1 region comprising the amino acid sequence of SEQ ID NO: 48; e) an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 55; and f) an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 61. The present disclosure provides an isolated human antibody or antibody fragment.

[0124] In one embodiment, the present disclosure provides an isolated human antibody or antibody fragment specific for human CD3 epsilon, wherein the antibody or antibody fragment comprises: a) an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 42;​​​​​​ e) The LCDR2 region containing the amino acid sequence of SEQ ID NO: 58; and f) LCDR3 region containing the amino acid sequence of SEQ ID NO: 61 The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0125] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) The HCDR1 region containing the amino acid sequence of SEQ ID NO: 42; b) The HCDR2 region containing the amino acid sequence of SEQ ID NO: 45; c) HCDR3 region containing the amino acid sequence of SEQ ID NO: 39; d) The LCDR1 region containing the amino acid sequence of SEQ ID NO: 50; e) The LCDR2 region containing the amino acid sequence of SEQ ID NO: 59; and f) LCDR3 region containing the amino acid sequence of SEQ ID NO: 61 The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0126] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) The HCDR1 region containing the amino acid sequence of SEQ ID NO: 42; b) The HCDR2 region containing the amino acid sequence of SEQ ID NO: 45; c) HCDR3 region containing the amino acid sequence of SEQ ID NO: 39; d) The LCDR1 region containing the amino acid sequence of SEQ ID NO: 46; e) The LCDR2 region containing the amino acid sequence of SEQ ID NO: 51; and f) LCDR3 region containing the amino acid sequence of SEQ ID NO: 62 The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0127] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) The HCDR1 region containing the amino acid sequence of SEQ ID NO: 42; b) The HCDR2 region containing the amino acid sequence of SEQ ID NO: 45; c) HCDR3 region containing the amino acid sequence of SEQ ID NO: 39; d) The LCDR1 region containing the amino acid sequence of SEQ ID NO: 49; e) The LCDR2 region containing the amino acid sequence of SEQ ID NO: 58; and f) LCDR3 region containing the amino acid sequence of SEQ ID NO: 61 The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0128] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) The HCDR1 region containing the amino acid sequence of SEQ ID NO: 42; b) The HCDR2 region containing the amino acid sequence of SEQ ID NO: 45; c) HCDR3 region containing the amino acid sequence of SEQ ID NO: 39; d) The LCDR1 region containing the amino acid sequence of SEQ ID NO: 47; e) The LCDR2 region containing the amino acid sequence of SEQ ID NO: 53; and f) LCDR3 region containing the amino acid sequence of SEQ ID NO: 62 The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0129] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) The HCDR1 region containing the amino acid sequence of SEQ ID NO: 42; b) The HCDR2 region containing the amino acid sequence of SEQ ID NO: 45; c) HCDR3 region containing the amino acid sequence of SEQ ID NO: 39; d) The LCDR1 region containing the amino acid sequence of SEQ ID NO: 47; e) The LCDR2 region containing the amino acid sequence of SEQ ID NO: 57; and f) LCDR3 region containing the amino acid sequence of SEQ ID NO: 62 Provided is an isolated human antibody or antibody fragment comprising

[0130] In one embodiment, the present disclosure provides an isolated human antibody or antibody fragment specific for human CD3 epsilon, wherein the antibody or antibody fragment a) an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 42; b) an HCDR2 region comprising the amino acid sequence of SEQ ID NO: 45; c) an HCDR3 region comprising the amino acid sequence of SEQ ID NO: 39; d) an LCDR1 region comprising the amino acid sequence of SEQ ID NO: 48; e) an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 51; and f) an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 62 Provided is an isolated human antibody or antibody fragment comprising

[0131] In one embodiment, the present disclosure provides an isolated human antibody or antibody fragment specific for human CD3 epsilon, wherein the antibody or antibody fragment a) an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 42; b) an HCDR2 region comprising the amino acid sequence of SEQ ID NO: 45; c) an HCDR3 region comprising the amino acid sequence of SEQ ID NO: 39; d) an LCDR1 region comprising the amino acid sequence of SEQ ID NO: 47; e) an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 51; and f) an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 62 Provided is an isolated human antibody or antibody fragment comprising

[0132] In one embodiment, the present disclosure provides an isolated human antibody or antibody fragment specific for human CD3 epsilon, wherein the antibody or antibody fragment a) an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 42; b) an HCDR2 region comprising the amino acid sequence of SEQ ID NO: 45; c) an HCDR3 region comprising the amino acid sequence of SEQ ID NO: 39; d) The LCDR1 region containing the amino acid sequence of SEQ ID NO: 48; e) The LCDR2 region containing the amino acid sequence of SEQ ID NO: 55; and f) LCDR3 region containing the amino acid sequence of SEQ ID NO: 62 The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0133] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) The HCDR1 region containing the amino acid sequence of SEQ ID NO: 42; b) The HCDR2 region containing the amino acid sequence of SEQ ID NO: 45; c) HCDR3 region containing the amino acid sequence of SEQ ID NO: 39; d) The LCDR1 region containing the amino acid sequence of SEQ ID NO: 47; e) The LCDR2 region containing the amino acid sequence of SEQ ID NO: 58; and f) LCDR3 region containing the amino acid sequence of SEQ ID NO: 62 The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0134] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) The HCDR1 region containing the amino acid sequence of SEQ ID NO: 42; b) The HCDR2 region containing the amino acid sequence of SEQ ID NO: 45; c) HCDR3 region containing the amino acid sequence of SEQ ID NO: 39; d) The LCDR1 region containing the amino acid sequence of SEQ ID NO: 49; e) The LCDR2 region containing the amino acid sequence of SEQ ID NO: 58; and f) LCDR3 region containing the amino acid sequence of SEQ ID NO: 62 The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0135] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) The HCDR1 region containing the amino acid sequence of SEQ ID NO: 42; b) The HCDR2 region containing the amino acid sequence of SEQ ID NO: 45; c) HCDR3 region containing the amino acid sequence of SEQ ID NO: 39; d) The LCDR1 region containing the amino acid sequence of SEQ ID NO: 47; e) The LCDR2 region containing the amino acid sequence of SEQ ID NO: 52; and f) LCDR3 region containing the amino acid sequence of SEQ ID NO: 62 The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0136] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) having the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

[0137] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) having the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15.

[0138] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) having the amino acid sequence of SEQ ID NO: 14.

[0139] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) having the amino acid sequence of SEQ ID NO: 15.

[0140] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable light chain (VL) containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, and 38.

[0141] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) Variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15; and b) A variable light chain (VL) containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, and 38. The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0142] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) Variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15; and b) A variable light chain (VL) containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, and 38. The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0143] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) Variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15; and b) A variable light chain (VL) containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 18, 24, 25, and 29. The present invention provides isolated human antibodies or antibody fragments containing [the specified substance].

[0144] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) VH containing the amino acid sequence of SEQ ID NO: 13 and VL containing the amino acid sequence of SEQ ID NO: 16, b) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 16 c) VH containing the amino acid sequence of SEQ ID NO: 13 and VL containing the amino acid sequence of SEQ ID NO: 17, d) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 17, e) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 18, f) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 19, g) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 20, h) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 21, i) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 22, j) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 23, k) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 24, l) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 25, m) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 26, n) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 27, o) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 28, p) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 29, q) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 30, r) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 31, s) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 32, t) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 33, u) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 34, v) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 35 w) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 36, x) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 37, y) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 38, z) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 17, aa) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 18, bb) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 19, cc) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 20, dd) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 21, ee) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 22, ff) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 23, gg) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 24, hh) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 25, ii) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 26, jj) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 27, kk) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 28, ll) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 29, VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 30, VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 31, oo) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 32, pp) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 33, qq) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 34, rr) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 35, ss) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 36, VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 37, VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 38 The present invention provides an isolated human antibody or antibody fragment comprising a variable heavy chain (VH) and a variable light chain (VH) selected from the group consisting of the above.

[0145] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 17, b) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 17, c) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 18, d) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 19 e) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 23 f) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 24 g) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 25 h) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 26, i) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 27, j) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 29 k) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 18, l) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 27, m) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 29 The present invention provides an isolated human antibody or antibody fragment comprising a variable heavy chain (VH) and a variable light chain (VH) selected from the group consisting of the above.

[0146] In one embodiment, the present disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is a) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 17, b) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 17, c) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 18, d) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 24 e) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 25, f) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 29 The present invention provides an isolated human antibody or antibody fragment comprising a variable heavy chain (VH) and a variable light chain (VH) selected from the group consisting of the above.

[0147] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 13 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 16.

[0148] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 14 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 16.

[0149] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 13 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 17.

[0150] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 14 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 17.

[0151] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 14 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 18.

[0152] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 14 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 19.

[0153] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 14 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 20.

[0154] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 14 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 21.

[0155] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 14 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 22.

[0156] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 14 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 23.

[0157] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 14 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 24.

[0158] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 14 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 25.

[0159] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 14 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 26.

[0160] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 14 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 27.

[0161] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 14 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 28.

[0162] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 14 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 29.

[0163] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 14 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 30.

[0164] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 14 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 31.

[0165] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 14 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 32.

[0166] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 14 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 33.

[0167] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 14 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 34.

[0168] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 14 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 35.

[0169] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 14 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 36.

[0170] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 14 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 37.

[0171] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 14 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 38.

[0172] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 15 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 17.

[0173] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 15 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 18.

[0174] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 15 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 19.

[0175] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 15 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 20.

[0176] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 15 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 21.

[0177] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 15 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 22.

[0178] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 15 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 23.

[0179] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 15 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 24.

[0180] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 15 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 25.

[0181] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 15 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 26.

[0182] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 15 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 27.

[0183] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 15 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 28.

[0184] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 15 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 29.

[0185] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 15 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 30.

[0186] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 15 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 31.

[0187] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 15 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 32.

[0188] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 15 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 33.

[0189] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 15 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 34.

[0190] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 15 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 35.

[0191] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 15 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 36.

[0192] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 15 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 37.

[0193] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 15 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 38.

[0194] specificity In one embodiment, the Disclosure provides an isolated human antibody or antibody fragment specific to CD3. In one embodiment, the isolated human antibody or antibody fragment specific to CD3 is specific to human CD3. In one embodiment, the isolated human antibody or antibody fragment specific to CD3 is specific to macaque monkey CD3. In one embodiment, the isolated human antibody or antibody fragment specific to CD3 is specific to both human and macaque monkey CD3.

[0195] In one embodiment, the isolated human antibody and antibody fragment specific to CD3 according to the Disclosure is specific to human CD3 epsilon. In one embodiment, the isolated human antibody and antibody fragment specific to CD3 according to the Disclosure is specific to macaque CD3 epsilon. In one embodiment, the isolated human antibody and antibody fragment specific to CD3 according to the Disclosure is specific to both human and macaque CD3 epsilon. In one embodiment, the isolated human antibody and antibody fragment specific to human and macaque CD3 epsilon according to the Disclosure is specific to both human and macaque CD3 epsilon.

[0196] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to human CD3, wherein the antibody or antibody fragment cross-conjugates to macaque CD3. In one embodiment, the disclosure provides an isolated human antibody and antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment cross-conjugates to macaque CD3 epsilon.

[0197] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to CD3, wherein the antibody or antibody fragment specifically binds to human CD3 epsilon. In one embodiment, the isolated human antibody or antibody fragment specific to CD3 specifically binds to human and macaque CD3 epsilon.

[0198] In one embodiment, the disclosure provides an isolated antibody or antibody fragment specific to human CD3 epsilon, wherein the human CD3 epsilon comprises amino acid sequence number 1.

[0199] In one embodiment, the disclosure provides an isolated human antibody or antibody fragment specific to macaque monkey CD3 epsilon, wherein the macaque monkey CD3 epsilon comprises amino acid sequence number 2.

[0200] In one embodiment, a CD3 epsilon-specific isolated human antibody or antibody fragment according to the Disclosure specifically binds to a human CD3 epsilon polypeptide containing the amino acid sequence of SEQ ID NO: 1. In one embodiment, a CD3 epsilon-specific isolated human antibody or antibody fragment according to the Disclosure specifically binds to a human CD3 epsilon polypeptide containing the amino acid sequence of SEQ ID NO: 3. In one embodiment, a CD3 epsilon-specific isolated human antibody or antibody fragment according to the Disclosure specifically binds to a human CD3 epsilon polypeptide containing the amino acid sequence of SEQ ID NO: 5. In one embodiment, a CD3 epsilon-specific isolated human antibody or antibody fragment according to the Disclosure specifically binds to a human CD3 epsilon polypeptide containing the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8. In one embodiment, a CD3 epsilon-specific isolated human antibody or antibody fragment according to the Disclosure specifically binds to a human CD3 epsilon polypeptide containing any of the amino acid sequences selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, and 8.

[0201] In one embodiment, an isolated human antibody or antibody fragment specific to CD3 epsilon according to the Disclosure specifically binds to the extracellular region of human CD3 epsilon. In one embodiment, an isolated human antibody or antibody fragment specific to CD3 epsilon according to the Disclosure specifically binds to the extracellular region of human and macaque monkey CD3 epsilon.

[0202] In one embodiment, an isolated human antibody or antibody fragment specific to CD3 epsilon according to this disclosure specifically binds to a macaque monkey CD3 epsilon polypeptide containing the amino acid sequence of SEQ ID NO: 2. In one embodiment, an isolated human antibody or antibody fragment specific to CD3 epsilon according to this disclosure specifically binds to a macaque monkey CD3 epsilon polypeptide containing the amino acid sequence of SEQ ID NO: 4. In one embodiment, an isolated human antibody or antibody fragment specific to CD3 epsilon according to this disclosure specifically binds to a macaque monkey CD3 epsilon polypeptide containing the amino acid sequence of SEQ ID NO: 6. In one embodiment, an isolated human antibody or antibody fragment specific to CD3 epsilon according to this disclosure specifically binds to a macaque monkey CD3 epsilon polypeptide containing the amino acid sequence of SEQ ID NO: 104. In one embodiment, an isolated human antibody or antibody fragment specific to CD3 epsilon according to this disclosure specifically binds to a macaque monkey CD3 epsilon polypeptide containing any of the amino acid sequences selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 104.

[0203] In one embodiment, the disclosure relates to an isolated human antibody or antibody fragment specific to a polypeptide encoded by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 104, or SEQ ID NO: 8.

[0204] In one embodiment, the disclosure relates to isolated human antibodies or antibody fragments specific to polypeptides encoded by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 104, and SEQ ID NO: 8.

[0205] In one embodiment, the Disclosure relates to isolated human antibodies or antibody fragments according to the Disclosure that specifically bind to polypeptides encoded by SEQ ID NOs: 1, 3, 5, 7, or 8 and polypeptides encoded by SEQ ID NOs: 2, 4, 6, or 104.

[0206] In one embodiment, an isolated human antibody or antibody or antibody fragment specific to human CD3 epsilon according to the Disclosure is cross-reactive to macaque CD3 epsilon. In one embodiment, an isolated human antibody or antibody or antibody fragment specific to human CD3 epsilon according to the Disclosure binds to macaque CD3 epsilon by cross-reactivity.

[0207] In one embodiment, the Disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon according to the Disclosure, wherein the antibody or antibody fragment has a K content of 10 nM or less relative to the human CD3 epsilon peptide comprising SEQ ID NO: 7 or SEQ ID NO: 8, such as 8 nM or less, 7 nM or less, 6 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, 1 nM or less, 0.1 nM or less, 0.2 nM, or 0.1 nM or less. D This relates to an isolated human antibody or antibody fragment having a monovalent affinity.

[0208] In one embodiment, the Disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment has a K between 0.1 nM and 10 nM relative to the human CD3 epsilon peptide comprising SEQ ID NO: 7 or SEQ ID NO: 8. D This relates to an isolated human antibody or antibody fragment having a monovalent affinity.

[0209] In one embodiment, the Disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon according to the Disclosure, wherein the antibody or antibody fragment has a K content of 10 nM or less relative to the macaque monkey CD3 epsilon peptide containing SEQ ID NO: 104, such as 8 nM or less, 7 nM or less, 6 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, 1 nM or less, 0.1 nM or less, 0.2 nM, or 0.1 nM or less.D This relates to an isolated human antibody or antibody fragment having a monovalent affinity.

[0210] In one embodiment, the Disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment has a K between 0.1 nM and 10 nM relative to the macaque monkey CD3 epsilon peptide comprising SEQ ID NO: 104. D This relates to an isolated human antibody or antibody fragment having a monovalent affinity.

[0211] In one embodiment, the Disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment is expressed in a K between 0.1 nM and 10 nM relative to a human CD3 epsilon peptide comprising SEQ ID NO: 7 or SEQ ID NO: 8 and a macaque monkey CD3 epsilon peptide comprising SEQ ID NO: 104. D This relates to an isolated human antibody or antibody fragment having a monovalent affinity.

[0212] In one embodiment, the Disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment has a K between 0.1 nM and 5 nM relative to the human CD3 epsilon peptide comprising SEQ ID NO: 8. D For macaque monkey CD3 epsilon peptides including and SEQ ID NO: 104, K between 0.1 nM and 1 nM was used. D This relates to an isolated human antibody or antibody fragment having a monovalent affinity.

[0213] In one embodiment, the monovalent affinity is determined in antibody Fv or Fab format. In one embodiment, the monovalent affinity is determined as described herein in Example 10 or Example 13.

[0214] In an alternative embodiment, the Disclosure relates to an isolated human antibody or antibody fragment specific to human CD3 epsilon, wherein the antibody or antibody fragment has an EC of between 1 nM and 10 nM, as determined in the FACS assay described herein in Example 14. 50 The present invention relates to isolated human antibodies or antibody fragments that specifically bind to human and / or macaque-derived PBMCs at a given concentration. In one embodiment, the EC 50 The concentration is determined in the Fab format. In one embodiment, the antibody fragment is a Fab.

[0215] In one embodiment, the isolated human antibody or antibody fragment specific to human CD3 epsilon is a) Variable heavy chains (VH) containing the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15; and b) A variable light chain (VL) containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, and 38. Includes.

[0216] Treatment methods In one embodiment, the Disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon for use as a pharmaceutical.

[0217] In one embodiment, the Disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon according to the Disclosure for use in stimulating T cell activation. In one embodiment, the Disclosure provides an isolated human antibody or antibody fragment specific to human CD3 epsilon according to the Disclosure for use in enhancing immune function in subjects having a cell proliferative disorder. In one embodiment, the Disclosure provides a method for treating or delaying the progression of a cell proliferative disorder in a subject in need thereof, comprising administering an effective amount of an isolated human antibody or antibody fragment specific to human CD3 epsilon according to the Disclosure to the subject. In one embodiment, the isolated human antibody or antibody fragment specific to human CD3 epsilon according to the Disclosure may be used for treating or delaying the progression of a cell proliferative disorder in a subject in need thereof. In one embodiment, the cell proliferative disorder is cancer.

[0218] In one embodiment, cancers include esophageal cancer, gastric cancer, small intestine cancer, colorectal cancer, breast cancer, non-small cell lung cancer, non-Hodgkin lymphoma (NHL), B-cell lymphoma, B-cell leukemia, multiple myeloma, kidney cancer, prostate cancer, liver cancer, head and neck cancer, melanoma, ovarian cancer, mesothelioma, glioblastoma, germinal-center B-cell-like (GCB) DLBCL, and activated B-cell-like (activated) cancers. B-cell-like (ABC) DLBCL, follicular lymphoma (FL), mantle cell lymphoma (MCL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), marginal zone lymphoma (MZL), small lymphocytic leukemia (SLL), lymphoplasmacytic lymphoma (LL), Waldenström macroglobulinemia (WM), central nervous system lymphoma (CNSL), Burkitt lymphoma (BL), B-cell prelymphocytic leukemia, splenic marginal zone lymphoma, hairy cell leukemia, unclassifiable splenic lymphoma / leukemia, splenic diffuse red pulp small B-cell lymphoma, hairy cell leukemia variant, Waldenström macroglobulinemia, plasmacytic myeloma, solitary plasmacytoma of bone Plasmacytoma of bone), Extraosseous plasmacytoma, Extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma), Nodal marginal zone lymphoma, Pediatric nodal marginal zone lymphoma, Pediatric follicular lymphoma, Primary cutaneous follicle centre lymphoma, T-cell / histiocyte-rich large B-cell lymphoma, Primary DLBCL (CNS)DLBCL of the CNS, Primary cutaneous DLBCL, leg type, EBV-positive DLBCL of the elderly, DLBCL associated with chronic inflammation, Lymphomatoid granulomatosis, Primary mediastinal (thymic) large B-cell lymphoma), Intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, Plasmablastic lymphoma, Large B-cell lymphoma occurring in HHV8-associated multicentric Castleman disease. Primary coelocellular lymphoma: Selected from a group consisting of unclassifiable B-cell lymphomas with intermediate characteristics between diffuse large B-cell lymphoma and Burkitt lymphoma, and unclassifiable B-cell lymphomas with intermediate characteristics between diffuse large B-cell lymphoma and classical Hodgkin lymphoma.

[0219] In one embodiment, the isolated human antibody or antibody fragment specific to human CD3 epsilon according to this disclosure may be used for pharmaceutical purposes. In one embodiment, this disclosure refers to the isolated human antibody or antibody fragment specific to human CD3 epsilon according to this disclosure for use in medicine. In one embodiment, this disclosure refers to the isolated human antibody or antibody fragment specific to human CD3 epsilon according to this disclosure for use in the preparation of pharmaceuticals. In one embodiment, this disclosure provides the use of the isolated human antibody or antibody fragment specific to human CD3 epsilon according to this disclosure for the manufacture of pharmaceuticals.

[0220] In one embodiment, the Disclosure provides the use of a human CD3 epsilon-specific isolated human antibody or antibody fragment according to the Disclosure for the manufacture of a pharmacopoeia for treating or delaying the progression of a cytoproliferative disorder. In one embodiment, the Disclosure provides the use of a human CD3 epsilon-specific isolated human antibody or antibody fragment according to the Disclosure for the manufacture of a pharmacopoeia for enhancing immune function in a subject having a cytoproliferative disorder.

[0221] In one embodiment, the Disclosure provides a method for administering treatment with an isolated human antibody or antibody fragment specific to human CD3 epsilon in a subject requiring such treatment.

[0222] In one embodiment, an isolated human antibody or antibody fragment specific to human CD3 epsilon according to the Disclosure, or a pharmaceutical composition comprising the antibody or antibody fragment, is administered subcutaneously, intravenously, intramuscularly, topically, orally, percutaneously, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intravenously, or intranasally. In one embodiment, an isolated human antibody or antibody fragment specific to human CD3 epsilon according to the Disclosure, or a pharmaceutical composition according to the Disclosure, is administered intravenously.

[0223] In one embodiment, the isolated human antibody or antibody fragment specific to human CD3 epsilon is a) Variable heavy chains (VH) containing the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15; and b) A variable light chain (VL) containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, and 38. Includes.

[0224] safety In one embodiment, the Disclosure provides an isolated human antibody or antibody fragment that is specific to human CD3 epsilon according to the Disclosure and does not activate T cells. In one embodiment, the Disclosure provides an isolated human antibody or antibody fragment that is specific to human CD3 epsilon according to the Disclosure and does not induce upregulation of CD69 expression on CD4-positive and / or CD8-positive T cells.

[0225] In one embodiment, the isolated human antibody or antibody fragment specific to human CD3 epsilon is a) Variable heavy chains (VH) containing the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15; and b) A variable light chain (VL) containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, and 38. Includes.

[0226] composition In one embodiment, the Disclosure provides a pharmaceutical composition comprising an isolated human antibody or antibody fragment specific to human CD3 epsilon according to the Disclosure and a pharmaceutically acceptable carrier. Such carriers are well known in the Art, and those skilled in the art will be able to find the most suitable formulation and route of administration for treating a subject with the antibody or antibody fragment according to the Disclosure.

[0227] In one embodiment, the disclosure refers to the use of the pharmaceutical composition comprising an isolated human antibody or antibody fragment specific to human CD3 epsilon as described herein in the preparation of a pharmaceutical for the treatment of a disease. In one embodiment, the disclosure refers to the use of the pharmaceutical composition for the treatment of a disease.

[0228] In one embodiment, the Disclosure provides a method for treating a cell proliferation disorder in a subject, comprising administering a pharmaceutical composition comprising a therapeutically effective amount of an isolated human antibody or antibody fragment specific to human CD3 epsilon according to the Disclosure.

[0229] In one embodiment, the Disclosure provides a pharmaceutical composition comprising a combination of an isolated human antibody or antibody fragment specific to human CD3 epsilon according to the Disclosure and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that can be advantageously combined with the human antibody or antibody fragment specific to human CD3 epsilon according to the Disclosure.

[0230] This disclosure provides a therapy for stimulating T cell activation using an isolated human antibody or antibody fragment specific to human CD3 epsilon as described herein, the therapy comprising administering a therapeutically effective amount of a pharmaceutical composition comprising the isolated human antibody or antibody fragment specific to CD3 epsilon as described herein to a subject in need thereof.

[0231] The Disclosure also provides a therapy for newly directing T-cell death of cancerous cells using an isolated human antibody or antibody fragment specific to human CD3 epsilon as disclosed herein, the therapy comprising administering a therapeutically effective amount of a pharmaceutical composition comprising the antibody or antibody fragment as disclosed herein to a subject in need thereof.

[0232] In one embodiment, the isolated human antibody or antibody fragment specific to human CD3 epsilon is a) Variable heavy chains (VH) containing the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15; and b) A variable light chain (VL) containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, and 38. Includes.

[0233] multispecific antibodies The human CD3 epsilon-specific human antibodies or antibody fragments provided herein should be preferredly used in multispecific antibody formats for targeting CD3-expressing T cells and for stimulating T cell activation under circumstances where T cell-mediated death of specific cell types, such as tumor cells, is beneficial or desirable.

[0234] The isolated human antibodies or antibody fragments specific to human CD3 epsilon provided in this disclosure can be conjugated to or co-expressed with another functional molecule, such as another peptide or protein. For example, an antibody or antibody fragment can be functionally conjugated to one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific or multispecific antibody having a second or third binding specificity (e.g., by chemical coupling, gene fusion, non-covalent association, or otherwise). Bispecific or multispecific antibodies capable of binding to two or more antigens are of great interest for therapeutic applications because they enable the simultaneous binding and inactivation of two or more target antigens and thus represent an alternative approach to conventional combination therapies.

[0235] Accordingly, in one embodiment, the present disclosure provides an isolated human antibody or antibody fragment that is specific to human CD3 epsilon, wherein the antibody or antibody fragment is a monospecific, bispecific, or multispecific antibody or antibody fragment.

[0236] A multispecific antibody may contain antibody fragments specific to different epitopes on the same target antigen, or antibody fragments specific to one or more target antigens. With respect to the bispecific or multispecific antibodies of this disclosure, the cell surface target antigen may be a cancer-associated antigen (CAA). Non-limiting examples of cancer-associated antigens include, for example, antigens expressed on the surface of tumor or cancerous cells.

[0237] Exemplary multispecific antibody formats that can be used in connection with this disclosure include, without limitation, scFv-based or diabody bispecific formats, IgG-scFv fusions, bivariable domain (DVD)-Ig, quadromas, knob-into-holes, common light chains (e.g., common light chains with knob-into-holes), CrossMab, CrossFab, (SEED) bodies, leucine zippers, Duobody, IgG1 / IgG2, dual-acting Fab (DAF)-IgG, and Mab2 bispecific formats (see, for example, Klein et al. 2012, mAbs 4:6, 1-11). Preferably, isolated human antibodies or antibody fragments specific to human CD3 epsilon according to this disclosure are used in the formats described in International Publication No. 2020 / 115115. Most preferably, the isolated human antibody or antibody fragment specific to human CD3 epsilon provided herein is used in the CyCAT format as described herein or in International Publication No. 2013 / 104804.

[0238] In one embodiment, the Disclosure provides a bispecific or multispecific antibody comprising a first antibody fragment specific to human CD3 epsilon and a second antibody fragment binding to a different antigen from the first antibody fragment, wherein the first antibody fragment specific to CD3 comprises one of the human CD3 epsilon-specific antibodies or antibody fragments according to the Disclosure. In one embodiment, the first antibody fragment specific to CD3 comprises one of the human CD3 epsilon-specific antibodies or antibody fragments listed in Table 10.

[0239] In one embodiment, the Disclosure provides a multispecific antibody comprising a first antibody fragment specific to human CD3 epsilon and a second antibody fragment binding to a different antigen from the first antibody fragment, wherein the first antibody fragment specific to human CD3 epsilon comprises either a human antibody or antibody fragment specific to human CD3 epsilon according to the Disclosure. In one embodiment, the first antibody fragment specific to CD3 comprises either a human antibody or antibody fragment specific to human CD3 epsilon as listed in Table 10.

[0240] In one embodiment, the second antibody fragment specifically binds to a cell surface antigen. In one embodiment, the first antibody fragment binds to CD3 present on an immune effector cell. In one embodiment, the cell surface target antigen is a cancer-associated antigen. In one embodiment, the immune effector cell is a T cell. In one embodiment, the T cell is a cytotoxic lymphocyte.

[0241] In one embodiment, the Disclosure provides a multispecific antibody comprising a human antibody or antibody fragment specific to human CD3 epsilon as provided for the Disclosure, the multispecific antibody mediating a new directive for the death of a target antigen-expressing cell by a T cell. In one embodiment, the target cell death can be determined by the method described herein in Example 11. In one embodiment, the multispecific antibody as provided for the Disclosure specifically binds to a) CD3 expressed on a T cell and b) a second antigen present on a target cell other than a T cell. In one embodiment, the multispecific antibody activates the T cell after binding to (a) and (b). In some embodiments, the activated T cell can exert a cytotoxic effect and / or an apoptotic effect on the target cell.

[0242] In one embodiment, the Disclosure provides a multispecific antibody disclosed herein, comprising a human antibody or antibody fragment specific to human CD3 epsilon, wherein the multispecific antibody does not induce upregulation of CD69 expression on CD4-positive and CD8-positive T cells in the absence of cell surface antigen-expressing cells. In another embodiment, the Disclosure provides a multispecific antibody comprising a human antibody or antibody fragment specific to human CD3 epsilon, wherein the multispecific antibody induces human T cell proliferation in the presence of cell surface target antigen-expressing cells. In yet another embodiment, the Disclosure provides a multispecific antibody disclosed herein, comprising a human antibody or antibody fragment specific to human CD3 epsilon, wherein the multispecific antibody does not activate T cells in the absence of cell surface antigen-expressing cells.

[0243] In one embodiment, the human antibody or antibody fragment specific to human CD3 epsilon is a) Variable heavy chains (VH) containing the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15; and b) A variable light chain (VL) containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, and 38. Includes.

[0244] CyCAT (registered trademark) The isolated human antibodies or antibody fragments specific to human CD3 epsilon provided herein may be used in the CyCAT approach as described herein or in International Publication No. 2013 / 104804 (which is incorporated herein in its entirety).

[0245] As part of the approach described above, two polypeptides are designed, each consisting of an antibody fragment (e.g., a single-chain variable fragment (scFv) or an antibody Fab fragment) fused to either the variable light chain domain (VL) or the complementary variable heavy chain (VH) domain of a T cell-activating anti-CD3 antibody. The unpaired VH or VL domain cannot bind to CD3 on its own and is thus non-functional. However, once the two CyCAT polypeptides bind to their respective antigens on the cell surface, the complementary VL and VH domains become adjacent and interact with each other to reconstitute the original CD3 antibody fragment (e.g., the Fv domain) and become functional. The triplicate heterodimer thus formed on the target cell engages and stimulates T cells for tumor cell destruction, similar to conventional multispecific antibodies.

[0246] Accordingly, this disclosure provides a CyCAT format comprising a human antibody or antibody fragment specific to human CD3 epsilon as described herein.

[0247] Thus, in one embodiment, this disclosure is, a) A first polypeptide comprising the following: (i) A first target component, wherein the first target component specifically binds to the first antigen, and (ii) The first fragment of the functional domain, Neither the first fragment of the functional domain nor the first polypeptide functions independently with respect to the function of the functional domain; the first polypeptide and b) A second polypeptide comprising the following: (i) A second target component, The second target component specifically binds to the second antigen, and (ii) The second fragment of the functional domain, Neither the second fragment of the functional domain nor the second polypeptide functions independently with respect to the function of the functional domain. A set of polypeptides including, The first polypeptide and the second polypeptide do not associate with each other in the absence of cells having both the first and second antigens on their cell surface. Simultaneously with the dimerization of the second fragment of the second polypeptide and the first fragment of the first polypeptide, the resulting dimer functions with respect to the function of the functional domain. Furthermore, the first fragment of the functional domain comprises VL of a human antibody or antibody fragment specific to human CD3 epsilon according to the Disclosure, and the second fragment of the functional domain comprises VH of the same human antibody or antibody fragment specific to human CD3 epsilon according to the Disclosure; Alternatively, the first fragment of the functional domain comprises VH of a human antibody or antibody fragment specific to human CD3 epsilon according to the Disclosure, and the second fragment of the functional domain comprises VL of the same human antibody or antibody fragment specific to human CD3 epsilon according to the Disclosure; Furthermore, the aforementioned functional domain is or includes an Fv domain. Provides a set of polypeptides.

[0248] In one embodiment, the functional domain is an Fv domain.

[0249] In one embodiment, cells having both the first and second antigens on their cell surface induce dimerization of the first fragment of the functional domain of the first polypeptide with the second fragment of the functional domain of the second polypeptide, whereas cells not having both the first and second antigens on their cell surface do not induce dimerization of the first fragment of the functional domain of the first polypeptide with the second fragment of the functional domain of the second polypeptide.

[0250] In an alternative embodiment, this disclosure is: a) A first polypeptide comprising the following: (i) A first target component, wherein the first target component specifically binds to the first antigen, and (ii) VH or VL of the Fv domain, Neither the VH or VL of the Fv domain nor the first polypeptide functions independently with respect to the function of the Fv domain; the first polypeptide and b) A second polypeptide comprising the following: (i) A second target component, wherein the second target component specifically binds to the second antigen, and (ii) A complementary VH or VL of the Fv domain, Neither the complementary VH or VL of the Fv domain nor the second polypeptide functions independently with respect to the function of the Fv domain. A set of polypeptides including, The first polypeptide and the second polypeptide do not associate with each other in the absence of cells having the first and second antigens on their cell surface. Simultaneously with the dimerization of the VL of the second polypeptide and the VH of the first polypeptide, the resulting dimer functions with respect to the function of the Fv domain or Simultaneously with the dimerization of the VH of the second polypeptide and the VL of the first polypeptide, the resulting dimer functions with respect to the function of the Fv domain. Furthermore, the VL comprises a VL of a human antibody or antibody fragment specific to human CD3 epsilon according to the Disclosure, and the VH domain comprises a VH domain of the same human antibody or antibody fragment specific to human CD3 epsilon according to the Disclosure. Provides a set of polypeptides.

[0251] In one embodiment, cells having both the first and second antigens on their cell surface induce dimerization of the VH of the first polypeptide with the VL of the second polypeptide, whereas cells not having both the first and second antigens on their cell surface do not induce dimerization of the VH of the first polypeptide with the VL of the second polypeptide. Cells that have both the first and second antigens on their cell surface induce dimerization of the VL of the first polypeptide with the VH of the second polypeptide, whereas cells that do not have both the first and second antigens on their cell surface do not induce dimerization of the VL of the first polypeptide with the VH of the second polypeptide.

[0252] In one embodiment, Fv is a) VH containing the amino acid sequence of SEQ ID NO: 13 and VL containing the amino acid sequence of SEQ ID NO: 16, b) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 16 c) VH containing the amino acid sequence of SEQ ID NO: 13 and VL containing the amino acid sequence of SEQ ID NO: 17, d) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 17, e) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 18, f) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 19, g) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 20, h) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 21, i) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 22, j) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 23, k) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 24, l) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 25, m) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 26, n) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 27, o) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 28, p) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 29, q) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 30, r) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 31, s) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 32, t) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 33, u) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 34, v) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 35 w) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 36, x) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 37, y) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 38, z) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 17, aa) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 18, bb) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 19, cc) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 20, dd) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 21, ee) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 22, ff) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 23, gg) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 24, hh) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 25, ii) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 26, jj) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 27, kk) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 28, ll) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 29, VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 30, VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 31, oo) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 32, pp) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 33, qq) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 34, rr) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 35, ss) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 36, VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 37, VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 38 It includes VH and VL selected from the group consisting of the following.

[0253] In one embodiment, VH is selected from the group consisting of SEQ ID NOs: 13, 14, and 15. In one embodiment, VL is selected from the group consisting of SEQ ID NOs: 16 to 38. In one embodiment, VH is selected from the group consisting of SEQ ID NOs: 13, 14, and 15, and VL is selected from the group consisting of SEQ ID NOs: 16 to 38.

[0254] In one embodiment, VH is selected from the group consisting of SEQ ID NO: 14 or SEQ ID NO: 15, and VL is selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 29.

[0255] In one embodiment, the first antigen and / or the second antigen are antigens expressed on the surface of tumor cells or on the surface of tumor precursor cells. In one embodiment, the first antigen and / or the second antigen are cancer-associated antigens expressed on the surface of tumor cells or on the surface of tumor precursor cells.

[0256] In one embodiment, the first antigen and the second antigen are the same antigen. In one embodiment, the first antigen and the antigen are different antigens. In one embodiment, the combination of the first antigen and the second antigen is found only on cancerous cells and not on non-cancerous cells. In one embodiment, the combination of the first antigen and the second antigen is specific to cancerous cells of a certain type of cancer.

[0257] In one embodiment, the first target component and / or the second target component comprises an antibody scFv, Fab, F(ab')2 fragment, or VHH antibody.

[0258] In one embodiment, the set of polypeptides according to this disclosure is intended for use in the treatment of patients with cancer or in the diagnosis of patients with cancer.

[0259] In one embodiment, the Disclosure provides a pharmaceutical composition comprising a set of polypeptides according to the Disclosure, the pharmaceutical composition further comprising a pharmaceutically acceptable carrier. In one embodiment, the Disclosure provides two pharmaceutical compositions, each comprising one of two polypeptides from the set of polypeptides according to the Disclosure, the pharmaceutical compositions further comprising a pharmaceutically acceptable carrier.

[0260] In one embodiment, the Disclosure is a kit comprising a set of polypeptides according to the Disclosure. The set of polypeptides according to the Disclosure may be used for the prevention and treatment of diseases mediated by biological pathways involving a target antigen of interest.

[0261] In one embodiment, the present disclosure provides a method for inducing the lysis of cancer cells, comprising contacting the cancer target cells expressing the first and second antigens with a set of polypeptides according to the present disclosure in the presence of cytotoxic T cells.

[0262] In one embodiment, the present disclosure provides a method for inhibiting the proliferation of cancer cells, comprising contacting the cancer cells expressing the first and second antigens with a set of polypeptides according to the present disclosure in the presence of cytotoxic T cells.

[0263] In one embodiment, the Disclosure provides a method for killing cancer cells, comprising contacting the cancer cells expressing the first and second antigens with a set of polypeptides according to the Disclosure in the presence of cytotoxic T cells.

[0264] In one embodiment, the Disclosure provides a method for inducing a cellular response in cytotoxic T cells, comprising contacting the cytotoxic T cells with a set of polypeptides according to the Disclosure in the presence of cancer cells expressing the first and second antigens. In one embodiment, the cellular response is selected from the group consisting of proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers.

[0265] In one embodiment, the Disclosure provides a method for inducing T cell proliferation in the presence of cancer cells, comprising contacting the cancer cells expressing the first and second antigens with a set of polypeptides according to the Disclosure in the presence of T cells. In one embodiment, the Disclosure provides a method for stimulating a primary T cell response in the presence of cancer cells, comprising contacting the cancer cells expressing the first and second antigens with a set of polypeptides according to the Disclosure in the presence of T cells. In one embodiment, the Disclosure provides a method for newly directing the cytotoxic activity of T cells against cancer cells, comprising contacting the cancer cells expressing the first and second antigens with a set of polypeptides according to the Disclosure in the presence of T cells.

[0266] In one embodiment, the present disclosure relates to the use of a set of polypeptides according to the present disclosure for the treatment of cancer, including cancer cells expressing the first and second cancer-associated antigens in a subject, (a) Selecting subjects who have cancer, (b) Collecting one or more biological samples from the subject, (c) Identifying cancer cells expressing the first and second cancer-associated antigens in one or more samples; and (d) Provides a method comprising administering an effective amount of the set of polypeptides according to this disclosure to a target.

[0267] A combination or set of a first antigen-binding molecule and a second antigen-binding molecule is further provided herein, wherein each of the two antigen-binding molecules comprises a target component specific to a target antigen, fused to an unpaired VL or VH domain of an antibody Fv domain specific to human CD3 epsilon, and the two antigen-binding molecules are not covalently associated.

[0268] In one embodiment, the present disclosure extends from its N-terminus to its C-terminus. a) A target component specific to the first antigen, b) Peptide linker, and c) The VH domain of an antibody Fv domain specific to human CD3 epsilon, selected from the group consisting of SEQ ID NOs: 13, 14, and 15, or the VL domain of an antibody Fv domain specific to human CD3 epsilon, selected from the group consisting of SEQ ID NOs: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, and 38. An antigen-binding molecule consisting of, The target component is fused via a peptide linker to the N-terminus of the VH or VL domain of the antibody Fv domain, which is specific to human CD3 epsilon. Regarding antigen-binding molecules.

[0269] In one embodiment, the target component is fused via a peptide linker to the N-terminus of the VH domain of the antibody Fv domain, which is specific to human CD3 epsilon. In another embodiment, the target component is fused via a peptide linker to the N-terminus of the VL domain of the antibody Fv domain, which is specific to human CD3 epsilon.

[0270] In one embodiment, the target component is an antibody or antibody fragment. In one embodiment, the target component is selected from the group consisting of Fab, scFab, Fab', scFv, dsFv, and VHH. In one embodiment, the target component is Fab.

[0271] In one embodiment of this disclosure, the antigen-binding molecule according to this disclosure consists of two polypeptides. a) The first polypeptide consists of the light chain of Fab, b) The second polypeptide, from its N-terminus to its C-terminus, I.Fab heavy chain II. Peptide linkers, and III. The VH domain of the antibody Fv domain specific to human CD3 epsilon, selected from the group consisting of SEQ ID NOs: 13, 14, and 15, or the VL domain of the antibody Fv domain specific to human CD3 epsilon, selected from the group consisting of SEQ ID NOs: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, and 38. It consists of.

[0272] In one embodiment, the disclosure, a) A first antigen-binding molecule comprising the following from its N-terminus to its C-terminus: i. A first target component specific to the first antigen, ii. The first peptide linker, and iii. The VH domain of the antibody Fv domain specific to human CD3 epsilon, selected from the group consisting of SEQ ID NOs: 13, 14, and 15, or the VL domain of the antibody Fv domain specific to human CD3 epsilon, selected from the group consisting of SEQ ID NOs: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, and 38. The first target component is fused via a first peptide linker to the N-terminus of the VH or VL domain of the antibody Fv domain of the first antigen-binding molecule, which is specific to human CD3 epsilon, and b) A second antigen-binding molecule comprising the following from its N-terminus to its C-terminus: I. A second target component specific to the second antigen, II. The second peptide linker, and III. If the first antigen-binding molecule contains the VH domain of an antibody Fv domain specific to human CD3 epsilon, then the VL domain of an antibody Fv domain specific to human CD3 epsilon selected from the group consisting of SEQ ID NOs: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, and 38, or if the first antigen-binding molecule contains the VL domain of an antibody Fv domain specific to human CD3 epsilon, then the VH domain of an antibody Fv domain specific to human CD3 epsilon selected from the group consisting of SEQ ID NOs: 13, 14, and 15, The second target component is a second antigen-binding molecule, which is fused via a second peptide linker to the N-terminus of the VL or VH domain of the antibody Fv domain, which is specific to human CD3 epsilon. This relates to a set of antigen-binding molecules consisting of the following:

[0273] In one embodiment, the first target component is fused to the N-terminus of the VH domain of the antibody Fv domain specific to human CD3 epsilon of the first antigen-binding molecule via a first peptide linker. In another embodiment, the first target component is fused to the N-terminus of the VL domain of the antibody Fv domain specific to human CD3 epsilon of the first antigen-binding molecule via a first peptide linker.

[0274] In one embodiment, the second target component is fused to the N-terminus of the VH domain of the human CD3 epsilon-specific antibody Fv domain of the second antigen-binding molecule via a second peptide linker. In another embodiment, the second target component is fused to the N-terminus of the VL domain of the human CD3 epsilon-specific antibody Fv domain of the second antigen-binding molecule via a second peptide linker.

[0275] In one embodiment, the target component is an antibody or antibody fragment. In one embodiment, the target component is selected from the group consisting of Fab, scFab, Fab', scFv, dsFv, and VHH. In one embodiment, the target component is Fab.

[0276] In one embodiment, the peptide linker used in the antigen-binding molecule according to this disclosure has a length of 5 to 20 amino acid residues. In one embodiment, the peptide linker used in the antigen-binding molecule according to this disclosure has a length of 5 amino acid residues. In one embodiment, the peptide linker contains the amino acid sequence of GQPSG (SEQ ID NO: 105). In one embodiment, the peptide linker consists of the amino acid sequence of GQPSG (SEQ ID NO: 105).

[0277] In one embodiment of the present disclosure, in the set of antigen-binding molecules according to the present disclosure, the first antigen-binding molecule and the second antigen-binding molecule are not linked by a covalent bond. In one embodiment, the first antigen-binding molecule and the second antigen-binding molecule according to the present disclosure can form a heteromer molecule. In one embodiment, the amount of heteromer molecule formed under conditions in which cells expressing the first and second antigens are present is greater than under conditions in which cells are absent or cells expressing either the first or second antigen are present.

[0278] In embodiments of this disclosure, the VH and VL domains of an antibody Fv domain specific to human CD3 epsilon can non-covalently associate to form an antibody Fv domain specific to human CD3 epsilon. In one embodiment, the non-covalent association of the VH and VL domains specific to human CD3 epsilon results in the formation of an antibody Fv domain specific to human CD3 epsilon. In one embodiment, the non-covalent association of the VH and VL domains specific to human CD3 epsilon dimerizes the first and second antigen-binding molecules. In one embodiment, the non-covalent association dimerizes the first and second antigen-binding molecules, resulting in the formation of a triply specific heteromer antibody. In one embodiment, the formation of the triply specific heteromer antibody occurs on a cell. In one embodiment, the formation of the triply specific heteromer antibody occurs in vivo. In one embodiment, the formation of the triply specific heteromer antibody molecule occurs in vitro. In embodiments of this disclosure, the VH or VL domain of the human CD3 epsilon-specific antibody Fv domain of the first antigen-binding molecule and the complementary human CD3 epsilon-specific VH or VL domain of the second antigen-binding molecule can covalently associate to form a human CD3 epsilon-specific antibody Fv domain. In embodiments of this disclosure, the covalent association of the human CD3 epsilon-specific VH and VL domains preferentially occurs when cells expressing the first and second antigens are present. In one embodiment, the covalent association of the human CD3 epsilon-specific VH and VL domains preferentially occurs when cells expressing the first and second antigens are present and when both antigen-binding molecules bind to those antigens on the cells.

[0279] In one embodiment, the triplicate heterodimer antibody formed on the cells has monovalent binding affinity to a first antigen, monovalent binding affinity to a second antigen, and monovalent binding affinity to human CD3 epsilon. In one embodiment of the present disclosure, the triplicate heterodimer antibody formed on the cells can engage with and stimulate cytotoxic T cells for the destruction of target cells.

[0280] In one embodiment of this disclosure, the first antigen and the second antigen are present on the same cell. In one embodiment, the cell is a tumor cell. In one embodiment, the first antigen and the second antigen are tumor-associated antigens.

[0281] The set of antigen-binding molecules according to this disclosure may be used for the prevention and treatment of diseases mediated by biological pathways involving a target antigen of interest. This may preferably be achieved by recruiting cytotoxic immune cells, such as T cells, to cells expressing the target antigen, preferably CAA. In one embodiment, this disclosure provides a method for inducing the lysis of target cells, such as tumor cells, comprising contacting the cells with the set of antigen-binding molecules according to this disclosure in the presence of cytotoxic T cells.

[0282] In one embodiment, the disclosure provides a method for inhibiting signal transduction in target cells such as tumor cells, comprising contacting the cells with a set of antigen-binding molecules according to the disclosure in the presence of cytotoxic T cells. In one embodiment, the disclosure provides a method for inhibiting the proliferation of target cells such as tumor cells, comprising contacting the cells with a set of antigen-binding molecules according to the disclosure in the presence of cytotoxic T cells. In one embodiment, the disclosure provides a method for inducing a cellular response in cytotoxic T cells, comprising contacting the cytotoxic T cells with a set of antigen-binding molecules according to the disclosure in the presence of target cells such as tumor cells. In one embodiment, the cellular response is selected from the group consisting of proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers.

[0283] In one embodiment, the Disclosure provides a method for inducing human T cell proliferation in the presence of target cells such as tumor cells, comprising contacting the cells with a set of antigen-binding molecules according to the Disclosure in the presence of T cells. In one embodiment, the Disclosure provides a method for stimulating a primary T cell response in the presence of target cells such as tumor cells, comprising contacting the cells with a set of antigen-binding molecules according to the Disclosure in the presence of T cells. In one embodiment, the Disclosure provides a method for newly directing the cytotoxic activity of T cells against target cells such as tumor cells, comprising contacting the cancer cells with a set of antigen-binding molecules according to the Disclosure in the presence of T cells.

[0284] In one embodiment, the present disclosure relates to the use of a set of antigen-binding molecules according to the present disclosure for the treatment of cancer that is positive for at least two cancer-associated antigens (CAAs) in a subject, (a) Selecting subjects who have cancer, (b) Collecting one or more biological samples from the subject, (c) Identifying cancer cells that express at least two tumor-associated antigens in one or more samples, and (d) Provides a use that includes administering an effective amount of the set of antigen-binding molecules described herein to a target.

[0285] In one embodiment, the cancer cells express first and second CAAs. In a preferred embodiment, the first and second CAAs are different. In one embodiment, in the set of antigen-binding molecules according to the Disclosure, the first antigen-binding molecule binds to the first CAA, and the second antigen-binding molecule binds to the second CAA.

[0286] array

[0287] [Table 2]

[0288] [Table 3]

[0289] [Table 4]

[0290] [Table 5]

[0291] [Table 6]

[0292] [Table 7]

[0293] [Table 8]

[0294] Table 9

[0295] Table 10

[0296] Table 11

[0297] Table 12

[0298] Table 13

[0299] Table 14

[0300] Table 15

[0301] Table 16

[0302] Table 17

[0303] Table 18

[0304] [Table 19]

[0305] [Table 20] [Examples]

[0306] Example 1: Antigen generation and quality control The amino acid sequences of human CD3 epsilon and cynomolgus monkey CD3 epsilon were searched and examined using publicly available sources (e.g., Uniprot), and produced in-house or by external service providers.

[0307] antigen Linear peptides covering the N-terminal extracellular regions of human and macaque CD3 epsilon, as well as fusion proteins of human CD3 epsilon and human CD3 delta, were used as antigens for initial panning and screening, and for subsequent binding studies. The peptides were either chemically synthesized or recombinantly produced. Chemically synthesized peptides were synthesized using biotin tags, purified by RP-HPLC, and obtained as lyophilized material. The lyophilized peptides were stored at -80°C.

[0308] Recombinant antigen cell line The HEK293-6E cell line was developed by the National Research Council of Canada (NRC). HEK293-6E cells were maintained in Freestyle F17 medium (Thermo Scientific) in a humidified CO2 incubator at 37°C and 6% CO2. HKB11 (parent clone: ​​U.S. Patent No. 6,136599, J. Biomed. Sci. 2002;9:631-638) is a human hybrid cell line arising from a fusion of HEK293 human fetal kidney cells and 2B8 Burkitt lymphoma cells. HKB11#52 cells were maintained in MAC1.0 medium containing 1% FCS in a humidified CO2 incubator at 37°C and 6% CO2.

[0309] Recombinant antigen production HKB11#52 cells or HEK293-6E cells were transiently transfected one day after inoculation using commercially available transfection reagents according to the manufacturer's instructions. Cells were cultured for 3 days, and the prepared cell culture supernatant was collected by centrifugation followed by sterile filtration (0.22 μm filter). A stable HKB11#52 pool was generated by cell transfection followed by selection with 800 μg / mL G418 (Thermo Scientific). Antigen expression from the stable pool was performed four days after inoculation.

[0310] CD3 FLAG-chLys-Avi tagged antigen DNA encoding the N-terminal extracellular region (ECD) of human or cynomolgus monkey CD3 epsilon containing a natural leader sequence, or a fusion protein of CD3 delta ECD and CD3 epsilon ECD, was cloned in-frame with a C-terminal FLAG-chLys-Avi tag into a modified expression vector, the pMAX expression vector, based on pcDNA3.1 (Thermo Fisher). Antigens were purified by antibody affinity chromatography using a custom-designed MOR003207 column. After binding and washing, proteins were eluted with 0.1 M glycine, pH 3.0. All affinity chromatography steps were performed using an AKTA Express (GE Healthcare) chromatography system. Samples were subsequently neutralized (with 3 M Tris pH 8), buffered to PBS using a PD10 column (GE Healthcare), and filtered sterile (0.2 μm pore size).

[0311] CD3 FC-tagged antigen DNA encoding the N-terminal sequence of human or cynomolgus monkey CD3 epsilon, including the natural leader sequence, was cloned in-frame with a C-terminal human IgG Fc tag into a modified expression vector, the pMAX expression vector, based on pcDNA3.1 (Thermo Fisher). The antigen was purified by protein A chromatography using a HiTrap MabSelect SuRe column (GE Healthcare). After binding and washing, the protein was eluted with 100 mM glycine pH 3.0. All affinity chromatography steps were performed using an AKTA Express (GE Healthcare) chromatography system. The samples were subsequently neutralized (using 3M Tris pH 8), buffered to PBS using a PD10 column (GE Healthcare), and filtered sterile (0.2 μm pore size). Biotinylation of the CD3 antigen was performed using the BirA Kit (Avidity), followed by preparative SEC using a Superdex200 column (GE Healthcare). Protein concentrations were determined by UV spectrophotometry. Sample quality was analyzed by denaturation-reducing or non-reducing SDS-PAGE, Streptavidin-Shift Assay, HP-SEC, and DLS.

[0312] [Table 21]

[0313] [Table 22]

[0314] [Table 23]

[0315] Example 2: Generation of fully human antibodies that bind to human and macaque CD3 epsilon from the MorphoSys Ylanthia® library For antibody generation, the MorphoSys Ylanthia® library was used to select Fab fragments for human and macaque CD3. The MorphoSys Ylanthia® library (Tiller et al. mAbs 5:3,1-26; May / June (2013) and U.S. Patent No. 8,728,981) is a commercially available phagemide library that uses CysDisplay® technology to display Fab on the phage surface (Lohning et al., International Publication No. 2001 / 05950).

[0316] Various panning strategies were performed to identify human / macaque CD3 cross-reactive antibodies. For this purpose, various synthetic and recombinant human and macaque CD3 epsilon antigens corresponding to the N-terminal extracellular region of CD3 epsilon were used. This region coincides with the binding region recognized by the prior art antibody SP34, which exhibits human / macaque CD3 epsilon cross-reactivity. Solution and semi-solution based panning strategies were performed to identify CD3-specific antibodies, and the human and macaque CD3 antigen materials described above were used to select species cross-reactive antibodies. Each panning performed consisted of at least three individual selections.

[0317] Identifying human / macaque CD3 cross-reactive antibodies proved to be extremely challenging, and the parent antibody MAB-1 was the only clone exhibiting specific binding to recombinant human and macaque CD3 epsilon, as well as cell binding to human and macaque PBMCs (see Figure 2). Surprisingly, MAB-1 proved suitable for use in the multispecific CyCAT format described herein.

[0318] To further increase the affinity and specificity of MAB-1 to human and macaque CD3 and to improve its functional activity, MAB-1 was subjected to variable heavy chain (VH) HCDR1+2 affinity maturation and variable light chain (VL) LCDR3 affinity maturation using Jurkat cells having endogenous expression of the soluble CD3 epsilon antigen and / or human CD3 described above.

[0319] Therefore, hundreds of clones were screened for affinity. Functionality was, for example, • Binding of HER2 x CD3 bispecific antibody constructs to human T cells, Jurkat cells with endogenous CD3 expression, and J.RT-T3.5 cells that do not express CD3. • Functional NFAT reporter gene assay using HER2 x CD3 bispecific antibody constructs • Functional cytotoxicity assay as a HER2 x CD3 bispecific construct It was rigorously tested in in vitro assays including [specific assays].

[0320] These studies further characterized the seven most promising affinity-matured variants of MAB-1 in terms of their suitability for the CyCAT format.

[0321] Example 3: Identification of an improved fully human CD3-specific antibody suitable for the CyCAT format. To determine the compatibility of newly identified affinity-matured CD3-specific human antibodies to be used in the CyCAT format, the VH and VL domains of parental MAB-1 or its affinity-matured variants and certain CD3-specific reference prior art antibodies were cloned into the CyCAT format polypeptide set.

[0322] As illustrated herein, each polypeptide in the two polypeptide sets of the CyCAT format consists of an antibody Fab fragment having specificity for the cancer target HER2 and a VL or VH domain of an anti-CD3 antibody of interest. The fusion between the Fab portion and the VL or VH domain is achieved, in each case, by a short peptide linker between the C-terminus of the Fab heavy chain and the N-terminus of the CD3-specific VH or VL domain.

[0323] For HER2 binding, nucleotide sequences encoding the VH and VL domains derived from "trastuzumab" (HERCEPTIN®), as described by Baselga et al. 1998, Cancer Res 58(13): 2825-2831, were used. Trastuzumab and its preparation method are described in U.S. Patent No. 5,821,337.

[0324] Tables 6-7 provide summaries of the protein sequences of CyCAT polypeptides produced with improved CD3-specific VH or VL domains, prepared according to the examples described herein.

[0325] gene synthesis All nucleic acid sequences or desired gene segments were generated in-house or by external providers as linear DNA fragments with appropriate flanking regions (e.g., suitable restriction enzyme recognition sites, linker sequences) using appropriate templates via PCR or gene synthesis. Nucleic acid sequences or gene segments with specific restriction endonuclease cleavage sites located laterally were cloned into their respective mammalian expression vectors using standard molecular biology methods. Where intended for use in mammalian expression vectors, all constructs were designed using 5' end DNA sequences encoding leader peptides that target proteins for secretion in eukaryotic cells. The DNA sequences of the subcloned gene fragments were confirmed by double-strand sequencing.

[0326] Example 4: Recombinant production of CyCAT polypeptide To express the CyCAT polypeptide, exponentially growing eukaryotic HEK293-6E cells were transfected with mammalian expression vectors encoding all components of the CyCAT polypeptide, resulting in 1:1 heterodimers of polypeptides containing the Fab heavy chain and Fab light chain of trastuzumab fused to the VH or VL domain of the CD3 antibody of interest.

[0327] Cell culture supernatant was collected 6 days after transfection and subjected to anti-CH1 affinity chromatography (Capture Select IgG-CH1 or CH1-XL | ThermoFisher Scientific). Buffer exchange with 1×Dulbcecco to PBS (pH 7.2 | Invitrogen) was performed, and the samples were sterile filtered (0.2 μm pore size). Protein concentration was determined by UV spectrophotometry, and construct purity was analyzed under denaturation-reduced and non-reducing conditions using CE-SDS (LabChip GX Touch | Perkin Elmer | USA). UHP-SEC was performed to analyze individual, unpaired CyCAT polypeptide preparations in their native state.

[0328] result: The VH and VL domains of three CD3-specific prior art antibodies (Ref-MAB-1, Ref-MAB-2, and Ref-MAB-3) were produced as CyCAT polypeptides. Ref-MAB-2 and Ref-MAB-3 are SP34-based prior art antibodies and are cross-reactive to macaque CD3. Ref-MAB-2 was first described in International Publication No. 2008 / 119567, and Ref-MAB-3 was first described in International Publication No. 2019 / 034580. Ref-MAB-1 binds to a distinct epitope in CD3 and is not cross-reactive to macaque CD3.

[0329] In addition, the newly identified parental fully human antibody MAB-1 and the VH and VL domains of the seven affinity maturation variable domains of MAB-1 were produced as CyCAT polypeptides.

[0330] Table 12 summarizes the results of quality control of mammalian-produced CyCAT polypeptides. This data reflects the strong dependence of CyCAT polypeptide productivity on CD3-specific antibody variable domains. Overall, polypeptides with unpaired CD3-specific VH domains appeared to be more prone to aggregation compared to polypeptides with (unpaired) CD3-specific VL domains.

[0331] Overall, CyCAT polypeptides possessing the CD3-specific VH domains of prior art reference antibodies (Ref-MAB-1, Ref-MAB-2, Ref-MAB-3) exhibited poor productivity in terms of monomer content and purity. Surprisingly, both variable domains of the newly identified parent CD3-specific human antibody MAB-1 could be produced as unpaired CyCAT polypeptides with acceptable yield (data not shown), monomer content, and purity (see Table 12). However, two productive polypeptides possessing the affinity-matured VH domains of MAB-1 (VH of MAB-2 and VH of MAB-3) failed to produce, while five polypeptides possessing other affinity-matured VH domains of MAB-1 (VH of MAB-4, MAB-5, MAB-6, and MAB-1_25) or the affinity-matured VL domain of MAB-1 (VL of MAB-1_2) could be produced to an acceptable degree.

[0332] These results highlight the difficulty in finding suitable pairs of CD3-specific variable domains that can actually be produced in the CyCAT format.

[0333] [Table 24]

[0334] The produced CyCAT polypeptide having an optimized CD3-specific variable domain derived from MAB-1 was then further characterized for affinity and functional activity in cytotoxicity assays, as described herein in Examples 10 and 11, by combining a CyCAT polypeptide having VH of a CD3-specific antibody with a CyCAT polypeptide having VL of a CD3-specific antibody.

[0335] These studies identified a novel, improved human CD3-specific antibody named MAB-7, which contains the affinity-matured VH domain of MAB-1_25 (MAB-1_25_VH (SEQ ID NO: 14)) and the affinity-matured VL domain of MAB-1_2 (MAB-1_2_VL (SEQ ID NO: 17)).

[0336] Table 15 (column 1) shows an overview of the functional characteristics of the MAB-7 antibody (CyCAT-7), where MAB-7 is 5 nM K D This indicates that when bound to human CD3 epsilon and tested in the bispecific CyCAT format, it efficiently mediated T cell death of HER-2-expressing SW-480 colon cancer cells, with an IC50 value in the single-digit nM range.

[0337] Example 5: Identification of further improved human antibodies specific to CD3 To identify further enhanced human CD3-specific variable domains of MAB-1 and MAB-7 in terms of affinity, potency, and productivity in the CyCAT format, various strategies were pursued: 1. Additional LCDR1+2 affinity maturation of variable light chain (MAB-1_2_VL) of MAB-7 using Jurkat cells expressing human and macaque CD3 antigens and native CD3. 2. Identification of novel LCDR3 variants of the parent antibody MAB-1 by next-generation sequencing (NGS) based on analysis of the product after LCDR3 affinity maturation is complete from the first affinity maturation operation described in Example 2. 3. Identification of a new HCDR1+2 variant of parent MAB-1 based on NGS analysis of affinity-matured products derived from the first affinity-maturation operation described in Example 2.

[0338] From these studies, 81 clones that were most promising, for example in terms of affinity, were produced as unpaired CyCAT polypeptides, as described in Example 4.

[0339] Example 6: Production of unpaired CyCAT polypeptides having improved CD3-specific variable domains compared to Example 5. From 81 clones in Example 5, we successfully produced variable domains of 57 affinity-mature antibodies as unpaired CyCAT polypeptides that were at least as suitable as those possessing the VH domain of MAB-7 (MAB-1_25_VH; (SEQ ID NO: 14) or the VL domain of MAB-7 (MAB-1_2_VL; (SEQ ID NO: 17)). These included six of only eight newly identified affinity-mature VH domains.

[0340] result: Overall, the production data indicated that a higher number of acidic amino acids in the CD3-specific variable domain's CDR was associated with more favorable CyCAT polypeptide productivity. Table 13 outlines the quality control of the produced CyCAT polypeptide having 13 improved CD3-specific VL domains (MAB-1_3_VL~MAB-1_15_VL) and one improved VH domain (MAB-1_24_VH) from the parent MAB-1.

[0341] These polypeptides could be produced in high yield (data not shown) and exhibited excellent monomer content and purity.

[0342] In particular, CyCAT polypeptides possessing the affinity-matured VH domain of MAB-1_24 (MAB-1_24_VH; SEQ ID NO: 15) could be produced with a high monomer content of approximately 90%, a significant increase compared to the previously determined monomer content for its parent antibody MAB-1, which was only in the 80% range (see Table 12).

[0343] MAB-1_24_VH was independently identified using a novel rational design approach based on the sequences of 100 unique affinity screening hits after H-CDR1+2 maturation. The positional amino acid frequencies of the affinity-matured HCDR1+2 sequences were analyzed via next-generation sequencing. For each position, the amino acid residue showing the best enrichment compared to the maturation module design was selected. If two or more amino acids similarly enriched the sequence, an acidic amino acid was selected.

[0344] [Table 25]

[0345] Example 7: Characterization of paired CyCAT polypeptides possessing improved CD3-specific variable domains as described in Examples 5 and 6. The CyCAT polypeptides successfully produced in Example 6 (52) were combined with CyCAT polypeptides having MAB-7 VH (MAB-1_25_VH (SEQ ID NO: 14)) or MAB-7 VL (MAB-1_2_VL (SEQ ID NO: 17)), and subjected to affinity determination as described in Example 10 and functional testing as described in Example 11.

[0346] These studies resulted in the identification of 13 improved and most preferred CD3-specific VL domains and one CD3-specific VH domain derived from MAB-1 or MAB-7 of Example 6 (summarized in Table 13).

[0347] Table 15 outlines the functional characteristics of preferred combinations of CD3-specific VL and VH domains, and the newly identified CD3-specific antibodies in this disclosure have a K of 5 nM or less. D It binds to human CD3 epsilon and mediates efficient T-cell-mediated death of SW-480 colon cancer cells in CyCAT format, IC 50 This indicates that the value was, in most cases, within the range of nM or less.

[0348] From these VH / VL combinations, MAB-7, MAB-8, MAB-9, MAB-12, MAB-13, and MAB-16 were selected as CD3-specific major antibodies for use in the T cell engagement multispecific antibody format.

[0349] Example 8: Production of unpaired CyCAT polypeptides having further improved CD3-specific variable domains of MAB-1 or MAB-7 As the final optimization step, eight cross-clones were generated by combining the LCDR1+2 sequence of the best-in-class LCDR1+2 affinity-matured mutant of MAB-7 (see Table 13) with the LCDR3 sequence of MAB-1_14_VL (SEQ ID NO: 29). MAB-1_14_VL was identified as a consensus sequence based on enrichment of a specific amino acid residue at a certain position in LCDR3 compared to the affinity-matured module design used, as described in Example 5 (Option 2 in the numbered list). The CyCAT polypeptide was then produced as described in Example 4.

[0350] result Table 14 outlines the quality control of eight cross-clones of the produced CyCAT polypeptides possessing the improved CD3-specific VL domain. These produced polypeptides exhibited a less favorable overall monomer content and a higher degree of high molecular weight impurities after production compared to the polypeptides with the improved CD3-specific variable domain shown in Table 15. Therefore, the polypeptides were subjected to size exclusion chromatography (Prep-SEC). Prep-SEC preparation resulted in very high monomer content for the CyCAT polypeptides, as shown in Table 14.

[0351] [Table 26]

[0352] Example 9: Characterization of paired CyCAT polypeptides possessing the improved cross-clonal CD3-specific variable light chain domains from Example 8. The CyCAT polypeptides produced in Example 8 were subjected to affinity determination as described in Example 10 and functional testing as described in Example 11 by combining them with CyCAT polypeptides having the VH domain of MAB-7 (MAB-1_25_VH (SEQ ID NO: 14)) or a newly identified optimized VH domain of MAB-1_24 (MAB-1_24_VH (SEQ ID NO: 15)).

[0353] Table 16 summarizes the functional characteristics of the further improved CD3-specific VL and VH domains. This data is based on the K21 D This indicates binding to human CD3 epsilon. However, further enhanced T cell-mediated death of SW-480 colon cancer cells in CyCAT format could not be observed (data not shown).

[0354] Example 10: Affinity determination of paired CD3 VH / VL domains in CyCAT format for human CD3 epsilon The unpaired CyCAT polypeptides produced, each possessing a VH or VL domain of a CD3-specific antibody according to this disclosure, are combined to enable the formation of a paired, functional CD3-specific Fv domain. Functional complementation of the CD3-specific VH and VL domains occurs once each of the two CyCAT polypeptides binds to its target antigen via its Fab moiety, and the two polypeptides become adjacent to each other.

[0355] method K for paired VH and VL domains D The determination was made using an Octet HTX (ForteBIO, Sartorius AG) instrument at 27°C.

[0356] A streptavidin-coated biosensor (ForteBIO, Sartorius AG) was loaded with purified and biotinylated human HER2_his_Avi_bio (ACROBiosystems, HE2-H82E2) in immobilization buffer (DPBS, 0.05% (v / v) Tween 20) to obtain a high-density human HER2 sensor surface. Subsequently, CyCAT polypeptides were diluted in Octet buffer (DPBS, 0.05% (v / v) Tween-20, 0.1% (w / v) BSA) and then injected into CyCAT_VH_CD3 and CyCAT_VL_CD3, respectively, until a loading threshold of approximately 0.5 nm was reached. Reaction kinetics were measured using three different concentrations of human CD3 epsilon (CD3e(1-118)_FLAG_chLys_AVI-bio(MorphoSys AG)) (SEQ ID NO: 8) (4-fold serial dilutions, 120-7.5 nM in Octet buffer), with association times of 300 seconds and dissociation times of 720 seconds. After each dissociation step, the sensor was regenerated to remove the bound CyCAT molecule (2 × 30 seconds, Gly / HCl, pH 1.5).

[0357] All data were relative to streptavidin-coated biosensors loaded with human HER2_his_Avi_bio incubated in Octet buffer instead of analyte-containing buffer. Sensorgrams were fitted using Octet Data Analysis Software 10.0 (ForteBio, Sartorius AG) to determine the kon and koff rate constants (using a 1:1 binding model), and these were converted to K D It was used to calculate [something].

[0358] result Tables 15 and 16 provide an overview of the affinities determined for preferred VH and VL pairs against human CD3 epsilon, representing the monovalent affinities of the newly identified CD3-specific antibodies in this disclosure against human CD3 epsilon in the range of one order of nanomoles to three orders of picomoles.

[0359] As expected, testing of unpaired, uncombined CyCAT polypeptides below 500 nM concentrations—CyCAT_MAB-1_24_VH, CyCAT_MAB-1_3_VL, CyCAT_MAB-1_14_VL, and CyCAT_MAB-1_16_VL~CyCAT_MAB-1_23_VL—did not show binding to CD3 epsilon due to the absence of functional CD3-specific Fv binding fragment formation.

[0360] Example 11: T cell-mediated death of cancer cells induced by paired CyCAT polypeptides The produced unpaired CyCAT polypeptides were combined to enable the formation of a CyCAT molecule containing a newly formed CD3-specific Fv-binding domain. Functional complementation of the CD3-specific VH and VL domains occurs once each of the two CyCAT polypeptides binds to its target antigen on cancer cells via its Fab moiety, and the two polypeptides are adjacent. The newly directed cancer cell death via immune cells is mediated by the binding of the newly formed CD3-specific antibody Fv domain to T cells.

[0361] method Isolation of human T cells Human whole blood from healthy donors was collected, for example, in a Li-heparin-containing S-Monovette container (Sarstedt). 20 mL of blood was transferred to a 50 mL conical tube and mixed with 1 mL of RosetteSep human CD8+ enrichment cocktail (Stemcell Technologies, #15063), and incubated at room temperature for 20 minutes. The blood-containing RosetteSep human CD8+ enrichment cocktail was diluted with an equal volume of PBS containing 2% fetal bovine serum (Sigma, #F7524) and 2 mM EDTA. The diluted blood was transferred to a SepMate-50 tube (Stemcell Technologies, #85450) containing 15 mL of Lymphoprep density gradient medium (Stemcell Technologies, #07811), and centrifuged at 1200 x g for 20 minutes at room temperature. The supernatant was transferred to a 50 mL conical tube and diluted to 45 mL with PBS containing 2% fetal bovine serum and 2 mM EDTA, and centrifuged at 800 x g for 5 minutes. The supernatant was discarded, and the cell pellet was resuspended in 1 mL of PBS containing 2% fetal bovine serum. The cell suspension was pooled, transferred to a 50 mL tube, and diluted in 30 mL of PBS containing 2% fetal bovine serum. The cells were pelletized by centrifuging at 800 x g for 5 minutes. The cell pellet was resuspended in 2 mL of 1 × Pharm Lyse erythrocyte lysis buffer (BD, #555899) and incubated at 4°C for 10 minutes. PBS containing 2% fetal bovine serum was added to a final volume of 15 mL. The cells were pelleted at 120 x g for 10 minutes, and the supernatant was decanted. Cells were washed twice with PBS containing 2% fetal bovine serum and then counted (CASY TT device, Beckmann Coulter).

[0362] Assay method 75,000 HER-2-expressing SW-480 colon cancer cells (ATCC® CCL-228®) were suspended in a culture medium supplemented with 10% FCS, inoculated into black 96-well assay plates (Corning), and incubated overnight at 37°C with 5% CO2 and humidity.

[0363] CellToxGreen dye (Promega, #G8731), serially diluted CyCAT polypeptide (final concentration: 0.00001-100 nM), and purified human T cells (E:T ratio 1:10) or human PBMCs (E:T ratio 1:30) were all diluted in assay medium containing RPMI1640 w / o phenol red (Gibco, #32404-014), GlutaMAX (Gibco 35050-038), and 10% fetal bovine serum, added to the cells, and incubated for 48 or 72 hours at 37°C with 5% CO2 and humidity.

[0364] Cytotoxic activity was determined by measuring the embedded CellToxGreen fluorescence using a Tecan Infinite F500 device with 485nm excitation and 535nm emission.

[0365] result The experimental results for selected CyCAT polypeptides possessing newly identified variable domains of CD3-specific antibodies as described in this disclosure are summarized in Table 15.

[0366] Co-culture of T cells with combinations of CyCAT polypeptides specific to HER-2 and CD3 induced the death of HER-2-positive SW-480 target cells in a dose-dependent manner. As expected, negative control combinations of CyCAT polypeptides did not induce death activity (data not shown). Co-culture of T cells with unpaired, uncombined CyCAT polypeptides specific to HER-2 and CD3 did not induce the death of HER-2-positive SW480 target cells. In the presence of HER-2-negative cells, none of the HER-2-specific combinations of CyCAT polypeptides tested induced cytotoxic activity (data not shown).

[0367] These results clearly demonstrate that the CD3-specific VH and VL domains contained in each unpaired CyCAT polypeptide, once both CyCAT polypeptides bind to the target antigen on cancer cells via their Fab moieties and the two polypeptides are adjacent, complement the functional CD3-specific antibody Fv domain to become complete. Therefore, the newly identified CD3-specific human antibodies according to this disclosure can be used in a variety of approaches to efficiently direct T cells to kill tumor-expressing cells.

[0368] Summary of the affinity and functional activity of the new CD3-specific antibody as disclosed herein

[0369] [Table 27]

[0370] [Table 28]

[0371] [Table 29]

[0372] Example 12: Reconversion and production of newly identified and optimized CD3-specific VH / VL pairs to conventional Fab and IgG antibody backbones To determine the suitability of newly identified and optimized CD3-specific VH / VL pairs for use in conventional antibody formats, the most preferred VH / VL pairs (see Table 17) were cloned into mammalian IgG1f-AEASS (effector-silent) or FabCys expression vectors for IgG or Fab production, respectively.

[0373] All nucleic acid sequences or desired gene segments were generated in-house or by external providers as linear DNA fragments having appropriate flanking regions for Gibson assembly (e.g., suitable restriction enzyme recognition sites and 21bp duplicate sequences) by PCR using appropriate templates or by gene synthesis. The nucleic acid sequences were cloned into their respective Ylanthia® FabCys mammalian expression vectors.

[0374] For IgG expression, FabCys vectors encoding antibody variable domains were enzymatically digested, and the resulting inserts were ligated with Ylanthia® mammalian expression cassettes and further subcloned into their respective mammalian human silent IgG vectors.

[0375] [Table 30]

[0376] [Table 31]

[0377] [Table 32]

[0378] [Table 33]

[0379] [Table 34]

[0380] Experimental scale production of IgG Eukaryotic HEK293 cells were transfected with mammalian expression vector DNA encoding both the heavy and light chains of IgG. Cell culture supernatant was collected 3 days after transfection and subjected to standard protein A affinity chromatography (MabSelect SURE | GE Healthcare). Unless otherwise specified, buffer exchange with 1×Dulbcecco in PBS (pH 7.2 | Invitrogen) was performed, and samples were filtered sterile (0.2 μm pore size).

[0381] Protein concentration was determined by UV spectrophotometry, and IgG purity was analyzed under denaturation-reduced and non-reducing conditions using CE-SDS (LabChip GXII | Perkin Elmer | USA). HP-SEC was performed to analyze IgG preparations in their natural state.

[0382] Experimental-scale production of Tagless FabCys Eukaryotic HEK293 cells were transfected with mammalian expression vector DNA encoding both the heavy and light chains of disulfide-crosslinked FabCys. Cell culture supernatant was collected 3 days after transfection and subjected to CH1 affinity chromatography (Capture Select CH1-XL | Thermo Scientific). Unless otherwise specified, buffer exchange with 1× Dulbecco PBS (pH 7.2 | Invitrogen) was performed, and the samples were filtered sterile (0.2 μm pore size).

[0383] Protein concentrations were determined by UV spectrophotometry, and the purity of FabCys was analyzed under denaturation-reduced and non-reducing conditions using CE-SDS (LabChip GXII | Perkin Elmer | USA). HP-SEC was performed to analyze FabCys preparations in their natural state.

[0384] Results of production: Tables 19-21 summarize the quality control procedures for mammalian-produced FabCys and human IgG1.

[0385] This data indicates that antibodies could be produced with acceptable yield, monomer content, and purity. For the three IgGs, the monomer content was determined to be in the range of 85% to 90%. All produced IgGs were subjected to size exclusion chromatography (Prep-SEC) for separation. Preparation by Prep-SEC resulted in a monomer content of over 93% for all produced IgGs, as shown in Table 21.

[0386] [Table 35]

[0387] [Table 36]

[0388] [Table 37]

[0389] Example 13: K2 in human and macaque CD3 epsilon by Biolayer Interferometry (BLI) D decision K DFor the determination, monomer fractions of the antibody protein (Fab-Cys) were used (containing at least 90% monomers, as analyzed by analytical SEC). Affinity was determined by determining the kinetic rate constant, as described below, using an Octet HTX (ForteBIO, Sartorius AG) instrument.

[0390] Various Fab-Cys samples diluted in assay buffer (D-PBS, 0.05% (v / v) PS20, 0.1% (w / v) BSA) were captured on a Fab-specific BLI sensor at a load level of approximately 0.5 nm. For analysis, human CD3 epsilon antigen hCD3e(1-118)_F-chLys_avi (SEQ ID NO: 7) and cynomolgus monkey CD3 epsilon antigen cyCD3e(1-109)_F-chLys_avi (SEQ ID NO: 104) were diluted in assay buffer to concentrations ranging from 1.56 nM to 100 nM. Blank samples with assay buffer were included for reference, i.e., to correct for the dissociation of captured antibodies. The association phase was recorded for 300 seconds, followed by the dissociation phase for 900 seconds. After each cycle, the biosensor was regenerated twice with 10 mM glycine HCl pH 1.7 to remove bound ligand / antibody complexes while maintaining the integrity of the capture surface. During the regeneration step, the biosensor was washed with assay buffer for 20 seconds.

[0391] The sensorgram was fitted using Octet Data Analysis Software 10.0 (ForteBio, Sartorius AG) to determine the kon and koff rate constants (using a 1:1 coupled model), and these were then converted to K D It was used to calculate [something].

[0392] K D The results of the determination are summarized in Table 22. The newly identified CD3-specific antibodies have a comparable range of K for binding to recombinant human and cynomolgus monkey CD3 epsilon antigens. D The value was shown.

[0393] [Table 38]

[0394] Example 14: Cell conjugation of CD3-specific antibodies against human and macaque-derived PBMCs Isolation of human and macaque PBMCs Whole human blood from healthy donors (collected in-house) and whole blood from cynomolgus monkeys (collected from LPT Laboratory of Pharmacology and Toxicology, Hamburg, Germany) were collected in Li-heparin-containing S-Monovette containers (Sarstedt). The blood was transferred to a 50 ml conical tube and mixed with an equal volume of PBS containing 2% fetal bovine serum (Sigma, #F7524) and 2 mM EDTA. The diluted blood was transferred to a SepMate-50 tube (StemCell Technologies, #86450) containing 15 ml of Biocoll solution (Biochrom, #L6115) and centrifuged at 1200 xg for 10 minutes. The supernatant was transferred to a 50 ml conical tube, diluted to 45 ml with PBS, and centrifuged at 300 xg for 8 minutes. The supernatant was discarded, the cell pellet was resuspended in 1 ml of PBS, and the cells were counted using a Neubauer chamber.

[0395] Cell binding assay The CD3-specific FabCys molecule described herein and the prior art anti-CD3 antibody UCHT-1 were tested by FACS for their ability to bind to human and macaque-derived PBMCs using in-house prepared Fab-Cys (which are known not to cross-react with cynomolgus monkey CD3).

[0396] 200,000 purified human or macaque monkey PBMCs (pre-blocked with recombinant human Fc2-H and goat anti-human IgG (Jackson Immuno Research, #109-005-097) or recombinant cynomolgus monkey IgG1 (R&D Systems, #9315-HG) and mouse anti-monkey IgG (Southern Biotech, SB108a), respectively, produced in-house) were mixed with antibodies (final concentrations: 0.012 nM to 200 nM) serially diluted in D-PBS (Gibco) containing 3% fetal bovine serum (Sigma, #F7524), and incubated on ice for 1 hour. The bound antibodies were detected using goat anti-human IgG-AF647 (Jackson Immuno Research, #109-606-097).

[0397] Antibody staining was measured using a multi-format Flow Cytometer (NovoCyte, Agilent Technologies) and analyzed using NovoExpress software (version 1.5.0). Lymphocytes were identified by morphological gating using forward and lateral scattering. EC 50 The values ​​were calculated using 4-parameter nonlinear regression analysis in Prism software (GraphPad Software Inc., version 8.4.3).

[0398] The experimental results are shown in Table 23, indicating that the newly identified CD3-specific antibody showed equivalent binding to human and macaque monkey PBMCs. As expected, the UCHT-1 control Fab showed binding to humans but not to cynomolgus monkey PBMCs at a concentration of 200 nM.

[0399] [Table 39]

Claims

1. An isolated human antibody or antibody fragment specific to human CD3 epsilon, The antibody or antibody fragment comprises a variable heavy chain and a variable light chain. The variable heavy chain and variable light chain are, a) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 17, b) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 17 c) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 18, d) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 24 e) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 25 f) VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 29 An isolated human antibody or antibody fragment specific to human CD3 epsilon, selected from the group consisting of the following.

2. The antibody or antibody fragment is an isolated human antibody or antibody fragment specific to human CD3 epsilon according to claim 1, wherein the antibody or antibody fragment binds to macaque CD3 epsilon by cross-reaction.

3. The antibody or antibody fragment is a recombinant antibody or antibody fragment, an isolated human antibody or antibody fragment specific to human CD3 epsilon according to claim 1 or 2.

4. The antibody or antibody fragment is a monoclonal antibody or antibody fragment, an isolated human antibody or antibody fragment specific to human CD3 epsilon according to any one of claims 1 to 3.

5. The antibody fragment is an isolated human antibody or antibody fragment specific to human CD3 epsilon according to any one of claims 1 to 4, selected from the group consisting of Fab, Fv, and scFv.

6. The antibody is IgG, and the isolated human antibody or antibody fragment is specific to human CD3 epsilon as described in any one of claims 1 to 5.

7. A multispecific antibody comprising an isolated human antibody or antibody fragment specific to human CD3 epsilon as described in any one of claims 1 to 6.

8. The multispecific antibody according to claim 7, wherein the multispecific antibody also specifically binds to cancer-related antigens.

9. A pharmaceutical composition comprising an isolated human antibody or antibody fragment specific to human CD3 epsilon as described in any one of claims 1 to 6, or a multispecific antibody as described in claim 7 or 8, and a pharmaceutically acceptable carrier or excipient.

10. A pharmaceutical product comprising an isolated human antibody or antibody fragment specific to human CD3 epsilon as described in any one of claims 1 to 6, a multispecific antibody as described in claim 7 or 8, or the pharmaceutical composition as described in claim 9.

11. The isolated human antibody or antibody fragment specific to human CD3 epsilon according to claim 6, wherein the IgG comprises an Fc region having reduced effector function compared to the wild-type Fc receptor.

12. An isolated human antibody or antibody fragment specific to human CD3 epsilon according to claim 6 or 11, wherein the IgG comprises an Fc region in which at least five amino acids at positions corresponding to L234, L235, G237, N330, and P331 in the human IgG1 heavy chain are mutated to A, E, A, S, and S, respectively.

13. A method for in vitro instructing the cytotoxic activity of T cells against cancer cells, comprising contacting the cancer cells with the multispecific antibody described in claim 7 or 8 in the presence of T cells.

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