Recombinant T cell receptor

Abstract

The present disclosure relates to the field of molecular biology, more particularly to antigen-binding molecule technology. The present disclosure also relates to methods of medical treatment and prophylaxis, particularly methods of cellular immunotherapy.

Description

[Technical Field]

[0001] Technical Field The present disclosure relates to the field of molecular biology, more particularly to antigen-binding molecule technology. The present disclosure also relates to methods of medical treatment and prophylaxis, particularly methods of cellular immunotherapy. [Background technology]

[0002] background Chimeric antigen receptor (CAR)-expressing T cells have demonstrated clinical efficacy in a variety of hematological malignancies. For solid tumor indications, CAR-T cell therapy has yet to provide significant clinical benefits over conventional chemotherapy and immunotherapy. Various strategies have been pursued to overcome the limitations of CAR-T cell therapy, which can be summarized as systemic toxicity and insufficient CAR-T cell-mediated antitumor immune responses. The most widespread CAR format to date is the second-generation CAR, which typically features a costimulatory signaling domain derived from the CD28 or 4-1BB costimulatory receptor and a CD3 zeta T cell receptor (TCR) signaling domain. While this format is widely used, it exhibits well-documented inherent limitations. Expression levels of second-generation CARs are higher than those of native T cell receptors, and some commonly used antibody-derived single-chain fragment variable (scFv) domains may exhibit a tendency to misdiffusion and aggregation on the cell surface under these conditions. This can result in constitutive CAR signaling in the absence of tumor antigen, leading to toxicity and T cell exhaustion. Therefore, research efforts have been focused on generating CAR formats that exhibit more physiological and strictly antigen-dependent CAR signaling.

[0003] One early-described strategy aims to add or replace antigen specificity in the native TCR complex. Various techniques for achieving this have been described. By adding an antibody variable domain to the CD3 epsilon domain of the TCR, a TCR complex with a second antigen specificity can be generated (Nolan et al., Clin. Cancer Res. (1999) 5:3928-3941; Baeuerle et al., Nat. Comms. (2019) 10:2087). This additional specificity can mediate peptide-human leukocyte antigen (pHLA)-independent T cell activation via the TCR complex. Another approach aims to replace the variable alpha and beta domains of the TCR with antibody-derived variable light and heavy domains (Kuwana et al., Biochem. Biophys. Res. Commun. (1987) 149:960-968; Liu et al., Sci. Transl. Med. (2021) 13:1-16; Mansilla-Soto et al., Nat. Med. (2022) 28:345-352). This, combined with enzyme-mediated gene knockout of the genes encoding the endogenous TCR alpha and beta chains, results in the loss of the native T cell specificity and the acquisition of a desired new antigen specificity. Using both the above format and the previously described second-generation CAR format, the final recombinant T cell product will be characterized by a single new antigen specificity unless several different CAR genes are introduced.

[0004] The present inventors have previously described a modular CAR that can bind to predetermined adapter molecules with specificity for various tumor antigens (Darowski et al. MAbs (2019) 11(4):621-631). T cells expressing a chimeric antigen receptor (CAR) construct containing an antigen-binding portion specific for a variant Fc domain are disclosed in International Publication No. 2018 / 177966. The CAR construct is a second-generation CAR that contains a costimulatory sequence in conjunction with the intracellular domain of CD3ζ. Summary of the Invention

[0005] overview In a first aspect, the present disclosure provides a recombinant CD3-TCR complex polypeptide comprising: (i) an antigen-binding portion, or component thereof, that binds to a variant Fc domain having an amino acid sequence that includes at least one amino acid difference relative to a reference Fc domain to which the antigen-binding portion does not bind; and (ii) a CD3-TCR complex-associated domain having an amino acid sequence derived from a CD3-TCR complex polypeptide; The present invention provides a recombinant CD3-TCR complex polypeptide comprising:

[0006] In some embodiments, the recombinant CD3-TCR complex polypeptide is capable of associating with one or more CD3-TCR complex polypeptides via its CD3-TCR complex association domain to form a CD3-TCR complex.

[0007] In some embodiments, the amino acid sequence derived from a CD3-TCR complex polypeptide is derived from CD3ε, TCRα, or TCRβ.

[0008] In some embodiments, the CD3-TCR complex association domain comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity to one of SEQ ID NOs: 30, 52, 1, 53, 5, 54 or 9.

[0009] In some embodiments, the amino acid sequence derived from the CD3-TCR complex polypeptide is derived from CD3ε. In some embodiments, the CD3-TCR complex association domain comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:30.

[0010] In some embodiments, the amino acid sequence derived from the CD3-TCR complex polypeptide is derived from TCR alpha or TCR beta. In some embodiments, the CD3-TCR complex association domain comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity to one of SEQ ID NOs: 52, 1, 53, 5, 54, or 9.

[0011] In some embodiments, the antigen-binding portion that binds to the variant Fc domain comprises the variable heavy (VH) and variable light (VL) regions of an antibody that binds to the variant Fc domain.

[0012] In some embodiments, the antigen-binding portion is or comprises an Fv, scFv, Fab, Fab', Fab'-SH, F(ab')2, crossFab, scFab, or dAb portion. In some embodiments, the antigen-binding portion is or comprises an scFv.

[0013] In some embodiments, the antigen-binding moiety component is or comprises a heavy chain variable (VH) region or a light chain variable (VL) region of an antibody that binds to a variant Fc domain.

[0014] In some embodiments, the antigen-binding portion or component thereof is linked at its C-terminus to the N-terminus of the CD3-TCR complex-associated domain, optionally via a linker sequence.

[0015] In some embodiments, the variant Fc domain binds to an Fc receptor with a lower affinity than the affinity with which the reference Fc domain binds to the Fc receptor, and optionally the Fc receptor is an Fcγ receptor or a neonatal Fc receptor (FcRn).

[0016] In some embodiments, the variant Fc domain comprises a CH2-CH3 region that comprises an amino acid difference at one or more of the following positions relative to the amino acid sequence of the CH2-CH3 region of a reference Fc domain according to EU numbering: L234, L235, I253, N297, S298, H310, P329, E333, K334, or H435.

[0017] In some embodiments, the variant Fc domain comprises a CH2-CH3 region that comprises G329 according to EU numbering.

[0018] In some embodiments, the antigen binding portion comprises the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 69; LC-CDR2 having the amino acid sequence of SEQ ID NO: 70; and LC-CDR3 having the amino acid sequence of SEQ ID NO: 71; It contains a VL region incorporating

[0019] In some embodiments, the antigen-binding moiety is (i) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 56; HC-CDR2 having the amino acid sequence of SEQ ID NO: 64; and HC-CDR3 having the amino acid sequence of SEQ ID NO: 58; or (ii) the following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 56; HC-CDR2 having the amino acid sequence of SEQ ID NO: 57; and HC-CDR3 having the amino acid sequence of SEQ ID NO: 58; It contains a VH region incorporating:

[0020] In some embodiments, the antigen-binding moiety is (a)(i) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 56; HC-CDR2 having the amino acid sequence of SEQ ID NO: 64; and HC-CDR3 having the amino acid sequence of SEQ ID NO: 68; and (ii) a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 69; LC-CDR2 having the amino acid sequence of SEQ ID NO: 70; and LC-CDR3 having the amino acid sequence of SEQ ID NO: 71; or (b)(i) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 56; HC-CDR2 having the amino acid sequence of SEQ ID NO: 57; and HC-CDR3 having the amino acid sequence of SEQ ID NO: 58; and (ii) a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 69; LC-CDR2 having the amino acid sequence of SEQ ID NO: 70; and LC-CDR3 having the amino acid sequence of SEQ ID NO: 71; Includes.

[0021] In some embodiments, the antigen-binding portion comprises a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:68.

[0022] In some embodiments, the antigen-binding portion comprises a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 65, 63, or 55.

[0023] In some embodiments, the antigen-binding moiety is (a)(i) a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 65; and (ii) a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 68; or (b)(i) a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 63; and (ii) a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 68; or (c)(i) a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 55; and (ii) a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 68; Includes.

[0024] In some embodiments, the variant Fc domain comprises a CH2-CH3 region comprising A298, A333 and A334 according to EU numbering.

[0025] In some embodiments, the antigen binding portion comprises the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 77; HC-CDR2 having the amino acid sequence of SEQ ID NO: 78; and HC-CDR3 having the amino acid sequence of SEQ ID NO: 79; It contains a VH region incorporating:

[0026] In some embodiments, the antigen binding portion comprises the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 85; LC-CDR2 having the amino acid sequence of SEQ ID NO: 86; and LC-CDR3 having the amino acid sequence of SEQ ID NO: 87; It contains a VL region incorporating

[0027] In some embodiments, the antigen-binding moiety is (i) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 77; HC-CDR2 having the amino acid sequence of SEQ ID NO: 78; and HC-CDR3 having the amino acid sequence of SEQ ID NO: 79; and (ii) a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 85; LC-CDR2 having the amino acid sequence of SEQ ID NO: 86; and LC-CDR3 having the amino acid sequence of SEQ ID NO: 87; Includes.

[0028] In some embodiments, the antigen-binding portion comprises a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:76.

[0029] In some embodiments, the antigen-binding portion comprises a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:84.

[0030] In some embodiments, the antigen-binding moiety is (i) a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 76; and (ii) a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 84; Includes.

[0031] The present disclosure also provides a recombinant CD3-TCR complex polypeptide, comprising: (i) an antigen-binding moiety, or component thereof, that is an scFv, wherein the antigen-binding moiety binds to a variant Fc domain having an amino acid sequence that includes at least one amino acid difference relative to a reference Fc domain to which the antigen-binding moiety does not bind, and the variant Fc domain includes a CH2-CH3 region that includes G329 according to EU numbering; and (ii) a CD3-TCR complex association domain having an amino acid sequence derived from a CD3-TCR complex polypeptide, wherein the amino acid sequence derived from a CD3-TCR complex polypeptide is derived from CD3ε; The present invention provides a recombinant CD3-TCR complex polypeptide comprising:

[0032] The present disclosure also provides a polypeptide complex comprising: (a) a first recombinant CD3-TCR complex polypeptide comprising: (i) a first component of the antigen-binding portion, wherein the antigen-binding portion binds to a variant Fc domain having an amino acid sequence that includes at least one amino acid difference relative to a reference Fc domain to which the antigen-binding portion does not bind; and (ii) a CD3-TCR complex-associated domain having an amino acid sequence derived from a CD3-TCR complex polypeptide; and (b) a second recombinant CD3-TCR complex polypeptide comprising: (i) a second component of the antigen-binding portion of(a)(i); and (ii) a CD3-TCR complex-associated domain having an amino acid sequence derived from a CD3-TCR complex polypeptide; Includes; The first and second recombinant CD3-TCR complex polypeptides provide a polypeptide complex that is capable of associating via their CD3-TCR complex-associating domains to form an antigen-binding portion.

[0033] In some embodiments, the first component of the antigen-binding portion is or comprises a heavy chain variable (VH) region of an antibody that binds to a variant Fc domain, and the second component of the antigen-binding portion is or comprises a light chain variable (VL) region of an antibody that binds to a variant Fc domain.

[0034] In some embodiments, (i) the CD3-TCR complex-associated domain of the first recombinant CD3-TCR complex polypeptide is derived from TCRα and the CD3-TCR complex-associated domain of the second recombinant CD3-TCR complex polypeptide is derived from TCRβ; or (ii) the CD3-TCR complex-associated domain of the first recombinant CD3-TCR complex polypeptide is derived from TCRβ, and the CD3-TCR complex-associated domain of the second recombinant CD3-TCR complex polypeptide is derived from TCRα;

[0035] In some embodiments, the CD3-TCR complex-associating domain from TCR alpha comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 52 or 1.

[0036] In some embodiments, the CD3-TCR complex association domain from TCR β comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity to one of SEQ ID NOs: 53, 5, 54 or 9.

[0037] The present disclosure also provides a polypeptide complex comprising: (a) a first recombinant CD3-TCR complex polypeptide comprising: (i) a first component of the antigen-binding portion, wherein the antigen-binding portion binds to a variant Fc domain having an amino acid sequence that includes at least one amino acid difference relative to a reference Fc domain to which the antigen-binding portion does not bind, the variant Fc domain including a CH2-CH3 region that includes G329 according to EU numbering; and (ii) a CD3-TCR complex-associated domain having an amino acid sequence derived from a CD3-TCR complex polypeptide; and (b) a second recombinant CD3-TCR complex polypeptide comprising: (i) a second component of the antigen-binding portion of(a)(i); and (ii) a CD3-TCR complex-associated domain having an amino acid sequence derived from a CD3-TCR complex polypeptide; Includes; the first and second recombinant CD3-TCR complex polypeptides are capable of associating via their CD3-TCR complex-associating domains to form an antigen-binding portion; a first component of the antigen-binding portion is or comprises a heavy chain variable (VH) region of an antibody that binds to the variant Fc domain, and a second component of the antigen-binding portion is or comprises a light chain variable (VL) region of an antibody that binds to the variant Fc domain; and (i) the CD3-TCR complex-associated domain of the first recombinant CD3-TCR complex polypeptide is derived from TCRα and the CD3-TCR complex-associated domain of the second recombinant CD3-TCR complex polypeptide is derived from TCRβ; or (ii) providing a polypeptide complex, wherein the CD3-TCR complex association domain of the first recombinant CD3-TCR complex polypeptide is derived from TCRβ and the CD3-TCR complex association domain of the second recombinant CD3-TCR complex polypeptide is derived from TCRα;

[0038] The present disclosure also provides a CD3-TCR polypeptide complex comprising a recombinant CD3-TCR complex polypeptide or polypeptide complex according to the present disclosure.

[0039] The present disclosure also provides a composite polypeptide comprising: (a) an amino acid sequence encoding a first recombinant CD3-TCR complex polypeptide comprising: (i) a first component of the antigen-binding portion, wherein the antigen-binding portion binds to a variant Fc domain having an amino acid sequence that includes at least one amino acid difference relative to a reference Fc domain to which the antigen-binding portion does not bind; and (ii) a CD3-TCR complex-associated domain having an amino acid sequence derived from a CD3-TCR complex polypeptide; and (b) an amino acid sequence encoding a second recombinant CD3-TCR complex polypeptide comprising: (i) a second component of the antigen-binding portion of(a)(i); and (ii) a CD3-TCR complex-associated domain having an amino acid sequence derived from a CD3-TCR complex polypeptide; Includes; the first and second recombinant CD3-TCR complex polypeptides are capable of associating via their CD3-TCR complex association domains to form a CD3-TCR complex comprising an antigen-binding portion; The composite polypeptide further comprises a cleavage site between the amino acid sequences of (a) and (b).

[0040] In some embodiments, the first component of the antigen-binding portion is or comprises a heavy chain variable (VH) region of an antibody that binds to a variant Fc domain, and the second component of the antigen-binding portion is or comprises a light chain variable (VL) region of an antibody that binds to a variant Fc domain.

[0041] In some embodiments, (i) the CD3-TCR complex-associated domain of the first recombinant CD3-TCR complex polypeptide is derived from TCRα and the CD3-TCR complex-associated domain of the second recombinant CD3-TCR complex polypeptide is derived from TCRβ; or (ii) the CD3-TCR complex-associated domain of the first recombinant CD3-TCR complex polypeptide is derived from TCRβ, and the CD3-TCR complex-associated domain of the second recombinant CD3-TCR complex polypeptide is derived from TCRα;

[0042] In some embodiments, the CD3-TCR complex-associating domain from TCR alpha comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 52 or 1.

[0043] In some embodiments, the CD3-TCR complex association domain from TCR β comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity to one of SEQ ID NOs: 53, 5, 54 or 9.

[0044] The present disclosure also provides a nucleic acid or nucleic acids encoding a recombinant CD3-TCR complex polypeptide, polypeptide complex, or complex polypeptide according to the present disclosure.

[0045] The present disclosure also provides a nucleic acid or a plurality of nucleic acids, (a) a first recombinant CD3-TCR complex polypeptide comprising: (i) a first component of the antigen-binding portion, wherein the antigen-binding portion binds to a variant Fc domain having an amino acid sequence that includes at least one amino acid difference relative to a reference Fc domain to which the antigen-binding portion does not bind; and (ii) a CD3-TCR complex-associated domain having an amino acid sequence derived from a CD3-TCR complex polypeptide; and (b) a second recombinant CD3-TCR complex polypeptide comprising: (i) a second component of the antigen-binding portion of(a)(i); and (ii) a CD3-TCR complex-associated domain having an amino acid sequence derived from a CD3-TCR complex polypeptide; Code; The first and second recombinant CD3-TCR complex polypeptides provide a nucleic acid or nucleic acids that can associate via their CD3-TCR complex association domains to form a CD3-TCR complex that includes an antigen-binding portion.

[0046] The present disclosure also provides an expression vector or vectors comprising a nucleic acid or nucleic acids according to the present disclosure.

[0047] The present disclosure also provides a cell comprising a recombinant CD3-TCR complex polypeptide, polypeptide complex, CD3-TCR polypeptide complex, complex polypeptide, nucleic acid or nucleic acids, or expression vector or expression vectors according to the present disclosure.

[0048] The present disclosure also provides a pharmaceutical composition comprising a cell according to the present disclosure.

[0049] The present disclosure also provides a cell or pharmaceutical composition according to the present disclosure for use in a method of medical treatment or prevention.

[0050] The present disclosure also provides a cell or pharmaceutical composition according to the present disclosure, for use in a method for treating or preventing a disease in which cells containing or expressing a target antigen are pathologically involved, the method comprising administering the cells or pharmaceutical composition to a subject to whom the antigen-binding molecule has been administered or will be administered; The antigen-binding molecule comprises (a) an antigen-binding domain that binds to a target antigen, and (b) a variant Fc domain having an amino acid sequence that includes at least one amino acid difference relative to a reference Fc domain; Provided are cells or pharmaceutical compositions in which an antigen-binding portion of a recombinant CD3-TCR complex polypeptide, polypeptide complex, CD3-TCR polypeptide complex or composite polypeptide contained in a cell according to the disclosure or in a cell comprised in a pharmaceutical composition according to the disclosure, or an antigen-binding portion of a CD3-TCR complex polypeptide, polypeptide complex, CD3-TCR polypeptide complex or composite polypeptide encoded by a nucleic acid or nucleic acids or expression vector or nucleic acids contained in a cell according to the disclosure or in a cell comprised in a pharmaceutical composition according to the disclosure, binds to a variant Fc domain.

[0051] The present disclosure also provides a method of depleting or killing cells containing or expressing a target antigen, the method comprising: (i) a cell or pharmaceutical composition according to the present disclosure; and (ii) an antigen-binding molecule comprising: (a) an antigen-binding domain that binds to a target antigen; and (b) a variant Fc domain having an amino acid sequence that includes at least one amino acid difference relative to a reference Fc domain. including contacting the Methods are provided wherein an antigen-binding portion of a recombinant CD3-TCR complex polypeptide, polypeptide complex, CD3-TCR polypeptide complex or composite polypeptide contained in a cell according to the disclosure or in a cell comprised in a pharmaceutical composition according to the disclosure, or an antigen-binding portion of a CD3-TCR complex polypeptide, polypeptide complex, CD3-TCR polypeptide complex or composite polypeptide encoded by a nucleic acid or nucleic acids or expression vector or nucleic acids contained in a cell according to the disclosure or in a cell comprised in a pharmaceutical composition according to the disclosure, binds to a variant Fc domain.

[0052] The present disclosure also provides a kit, comprising: (i) a cell or pharmaceutical composition according to the present disclosure; and (ii) an antigen-binding molecule comprising: (a) an antigen-binding domain that binds to a target antigen; and (b) a variant Fc domain having an amino acid sequence that includes at least one amino acid difference relative to a reference Fc domain. Includes; Kits are provided in which an antigen-binding portion of a recombinant CD3-TCR complex polypeptide, polypeptide complex, CD3-TCR polypeptide complex or composite polypeptide contained in a cell according to the disclosure or in a cell comprised in a pharmaceutical composition according to the disclosure, or an antigen-binding portion of a CD3-TCR complex polypeptide, polypeptide complex, CD3-TCR polypeptide complex or composite polypeptide encoded by a nucleic acid or nucleic acids or expression vector or nucleic acids contained in a cell according to the disclosure or in a cell comprised in a pharmaceutical composition according to the disclosure, binds to a variant Fc domain.

[0053] The present disclosure also provides a kit, comprising: (i) a nucleic acid or nucleic acids according to the present disclosure, or an expression vector or expression vectors according to the present disclosure; and (ii) an antigen-binding molecule comprising: (a) an antigen-binding domain that binds to a target antigen; and (b) a variant Fc domain having an amino acid sequence that includes at least one amino acid difference relative to a reference Fc domain. Includes; Kits are provided in which a CD3-TCR complex polypeptide, polypeptide complex, CD3-TCR polypeptide complex, or antigen-binding portion of a complex polypeptide encoded by a nucleic acid or nucleic acids according to the disclosure or contained in an expression vector or vectors according to the disclosure is bound to a variant Fc domain.

[0054] explanation The present disclosure provides recombinant CD3-TCR complex polypeptides, polypeptide complexes (including CD3-TCR polypeptide complexes) comprising such recombinant CD3-TCR complex polypeptides, nucleic acids and vectors encoding such polypeptides and polypeptide complexes, cells comprising such polypeptides, polypeptide complexes, nucleic acids and vectors, compositions comprising such cells, and uses thereof.

[0055] More specifically, the present disclosure relates to novel recombinant CD3-TCR complex polypeptides and polypeptide complexes comprising the same, which have an antigen-binding moiety specific for a variant Fc domain and include a CD3-TCR complex association domain, such that when expressed by a cell (particularly a T cell), the recombinant CD3-TCR complex polypeptide can associate with the CD3-TCR complex polypeptide to form a CD3-TCR complex that recognizes the variant Fc domain. Binding of the antigen-binding moiety to its cognate variant Fc domain triggers CD3-TCR complex-mediated signaling and activates the CD3-TCR polypeptide complex-expressing cell.

[0056] Unexpectedly, the present disclosure demonstrates that novel recombinant CD3-TCR complex polypeptides that do not contain domains inherently capable of triggering CD3-TCR complex-mediated signaling (e.g., the CD3ζ intracellular domain) can associate with endogenous CD3-TCR complex polypeptides expressed by a cell to form functional CD3-TCR complexes that nevertheless trigger T cell activation in response to a variant Fc domain to which the antigen-binding moiety binds. The present disclosure further demonstrates that this technical effect is achieved across different embodiments, particularly (i) embodiments in which the recombinant CD3-TCR complex polypeptide comprises an scFv that binds to the variant Fc domain and a CD3-TCR complex association domain derived from CD3ε, and (ii) embodiments in which the antigen-binding moiety specific for the variant Fc domain is formed by VH and VL region portions provided on separate recombinant CD3-TCR complex polypeptides that comprise CD3-TCR complex association domains derived from TRAC and TRBC1, respectively.

[0057] Even more unexpectedly, experiments providing a direct comparison of the activation levels of T cells in response to cells presenting variant Fc domains show that T cells expressing the novel CD3-TCR complexes described in (i) and (ii) of the previous paragraph are activated to a similar or greater extent than T cells expressing a CAR construct with the same antigen-binding portion (scFv format) containing the costimulatory and CD3ζ intracellular signaling domains (disclosed in WO 2018 / 177966) - see Figures 6, 7, 10 and 11.

[0058] Recombinant CD3-TCR complex polypeptide Aspects and embodiments of the present disclosure relate to recombinant CD3-TCR complex polypeptides.

[0059] As referred to herein, a "recombinant" polypeptide refers to a polypeptide that does not occur in nature. A recombinant polypeptide is sometimes referred to as a "synthetic" polypeptide. A recombinant polypeptide can comprise or consist of an amino acid sequence that is not encoded by the genome of a naturally occurring organism (e.g., a wild-type organism). That is, a recombinant polypeptide can comprise or consist of an amino acid sequence that is not included in the amino acid sequence of a polypeptide produced by a naturally occurring organism. A recombinant polypeptide can be encoded by a nucleic acid produced using recombinant nucleic acid technology. A recombinant polypeptide can be produced by expression (e.g., by transcription, translation, and any subsequent post-translational processing) from a recombinant nucleic acid encoding the polypeptide. Recombinant nucleic acid technology includes techniques for constructing and manipulating the nucleotide sequence of a nucleic acid, including molecular cloning.

[0060] The CD3-TCR complex (sometimes referred to as the TCR-CD3 complex; see, e.g., Dong et al., Nature (2019) 573(7775):546-552) is a polypeptide complex expressed on the cell surface of T cells that is involved in antigen-specific T cell activation. The structure and function of the CD3-TCR complex are reviewed, for example, in Mariuzza et al., J Biol Chem. 2020 Jan 24; 295(4):914-925, the entire contents of which are incorporated herein by reference.

[0061] In mammals, the CD3-TCR complex comprises multiple TCR polypeptides that together form a heterodimeric TCR (either TCRα and TCRβ, or TCRγ and TCRδ) for antigen recognition, provided in noncovalent association with invariant CD3ε, CD3δ, CD3γ, and CD3ζ polypeptides. Classical octameric CD3-TCR complexes include TCRα and TCRβ heterodimers (i.e., TCRαβ) or TCRγ and TCRδ heterodimers (i.e., TCRγδ), heterodimers comprising CD3ε and CD3δ (i.e., CD3δε), heterodimers comprising CD3ε and CD3γ (i.e., CD3γε), and CD3ζ homodimers (i.e., CD3ζζ). Such TCR-CD3 complexes can be represented as CD3γε / CD3δε / CD3ζζ / TCRαβ and CD3γε / CD3δε / CD3ζζ / TCRγδ, respectively (see, e.g., Zheng et al., Nature (2019) 573(7775):546-552).

[0062] In some embodiments, the CD3-TCR complex is a CD3-TCRα / β complex. In some embodiments, the CD3-TCR complex is a CD3-TCRγ / δ complex. The CD3-TCRα / β complex may comprise TCRα and TCRβ polypeptides, as well as CD3γ, CD3ε, CD3δ, and / or CD3ζ polypeptides. The CD3-TCRα / β complex may comprise or consist of CD3γε / CD3δε / CD3ζζ / TCRαβ. The CD3-TCRγ / δ complex may comprise TCRγ and TCRδ polypeptides, as well as CD3γ, CD3ε, CD3δ, and / or CD3ζ polypeptides. The CD3-TCRγ / δ complex may comprise or consist of CD3γε / CD3δε / CD3ζζ / TCRγ / δ.

[0063] As used herein, "CD3-TCR complex polypeptide" refers to a constituent polypeptide of a CD3-TCR complex. In some embodiments, the CD3-TCR complex polypeptide is selected from TCRα, TCRβ, TCRγ, TCRδ, TRAC, TRBC1, TRBC2, TRGC1, TRGC2, TRDC, CD3ε, CD3δ, CD3γ, CD3ζ, and CD3η. In some embodiments, the CD3-TCR complex polypeptide is a recombinant CD3-TCR complex polypeptide described herein.

[0064] As used herein, "TCRα," "TCRβ," "TCRγ," "TCRδ," "TRAC," "TRBC1," "TRBC2," "TRGC1," "TRGC2," "TRDC," "CD3ε," "CD3δ," "CD3γ," "CD3ζ," and "CD3η" refer to TCRα, TCRβ, TCRγ, TCRδ, TRAC, TRBC1, TRBC2, TRGC1, TRGC2, TRDC, CD3ε, CD3δ, CD3γ, CD3ζ, and CD3η, respectively, from any species, and include isoforms, fragments, variants, or homologs from any species. In some embodiments, the TCRα, TCRβ, TCRγ, TCRδ, TRAC, TRBCl, TRBC2, TRGC1, TRGC2, TRDC, CD3ε, CD3δ, CD3γ, CD3ζ, or CD3η is derived from a mammal (e.g., a eutherian, a placental, an epithelioid, an archaean, a primate (rhesus, cynomolgus, non-human primate, or human)). In some embodiments, the TCRα, TCRβ, TCRγ, TCRδ, TRAC, TRBCl, TRBC2, TRGC1, TRGC2, TRDC, CD3ε, CD3δ, CD3γ, CD3ζ, or CD3η is human.

[0065] As used herein, an isoform, fragment, variant, or homolog of a given reference protein (e.g., TCRα, TCRβ, TCRγ, TCRδ, TRAC, TRBC1, TRBC2, TRGC1, TRGC2, TRDC, CD3ε, CD3δ, CD3γ, CD3ζ, or CD3η) may be characterized as having one of at least 70% sequence identity, preferably ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99% or 100% amino acid sequence identity with the amino acid sequence of the reference protein.

[0066] A "fragment" generally refers to a fraction of a reference protein. A "variant" generally refers to a protein having an amino acid sequence that contains one or more amino acid substitutions, insertions, deletions, or other modifications compared to the amino acid sequence of the reference protein, but that retains a significant degree of sequence identity (e.g., at least 60%) with the amino acid sequence of the reference protein. An "isoform" generally refers to a variant of a reference protein that is expressed by the same species as the species of the reference protein. A "homolog" generally refers to a variant of a reference protein that is produced by a different species compared to the species of the reference protein. Homologs include orthologs.

[0067] An isoform, fragment, variant or homologue of a given reference protein may optionally be characterized as having one of at least 70%, preferably ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99% or 100% amino acid sequence identity with the amino acid sequence of the immature or mature (i.e., after processing to remove the signal peptide) form of a particular isoform of the related protein from a given species, e.g., human.

[0068] In some embodiments, the TCRα comprises an amino acid sequence having at least 70%, preferably ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99% or 100% amino acid sequence identity to SEQ ID NO: 1. In some embodiments, the TRAC comprises or consists of an amino acid sequence having at least 70%, preferably ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99% or 100% amino acid sequence identity to SEQ ID NO: 1.

[0069] In some embodiments, TCRβ comprises an amino acid sequence having at least 70%, preferably ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99% or 100% amino acid sequence identity with SEQ ID NO: 5 or 9. In some embodiments, TRBC1 comprises or consists of an amino acid sequence having at least 70%, preferably ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99% or 100% amino acid sequence identity with SEQ ID NO: 5. In some embodiments, TRBC2 comprises or consists of an amino acid sequence having at least 70%, preferably ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99% or 100% amino acid sequence identity with SEQ ID NO:9.

[0070] In some embodiments, TCRγ comprises an amino acid sequence having at least 70%, preferably ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99%, or 100% amino acid sequence identity to SEQ ID NO: 13 or 17. In some embodiments, TRGC1 comprises or consists of an amino acid sequence having at least 70%, preferably ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99%, or 100% amino acid sequence identity to SEQ ID NO: 13. In some embodiments, TRGC2 comprises or consists of an amino acid sequence having at least 70%, preferably ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99% or 100% amino acid sequence identity to SEQ ID NO: 17.

[0071] In some embodiments, TCRδ comprises an amino acid sequence having at least 70%, preferably ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99% or 100% amino acid sequence identity to SEQ ID NO: 21. In some embodiments, TRDC comprises or consists of an amino acid sequence having at least 70%, preferably ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99% or 100% amino acid sequence identity to SEQ ID NO: 21.

[0072] In some embodiments, CD3ε comprises or consists of an amino acid sequence having at least 70%, preferably ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99% or 100% amino acid sequence identity to SEQ ID NO: 25 or 30.

[0073] In some embodiments, CD3δ comprises or consists of an amino acid sequence having at least 70%, preferably ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99% or 100% amino acid sequence identity to SEQ ID NO: 31 or 36.

[0074] In some embodiments, CD3γ comprises or consists of an amino acid sequence having at least 70%, preferably ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99% or 100% amino acid sequence identity to SEQ ID NO: 37 or 42.

[0075] In some embodiments, CD3ζ comprises or consists of an amino acid sequence having at least 70%, preferably ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99%, or 100% amino acid sequence identity to SEQ ID NO: 43 or 48.

[0076] In some embodiments, CD3η comprises or consists of an amino acid sequence having at least 70%, preferably ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99% or 100% amino acid sequence identity to SEQ ID NO: 49 or 51.

[0077] In some embodiments, the recombinant CD3-TCR complex polypeptide according to the present disclosure comprises: (i) an antigen-binding portion or component thereof described herein; and (ii) a CD3-TCR complex-associated domain as described herein; Includes.

[0078] In some embodiments, the amino acid sequence of the antigen-binding portion / component thereof is N-terminal to the amino acid sequence of the CD3-TCR complex association domain in the amino acid sequence of the recombinant CD3-TCR complex polypeptide, i.e., in some embodiments, the recombinant CD3-TCR complex polypeptide comprises the following structure: N-terminus-[...]-[antigen-binding portion / component thereof]-[CD3-TCR complex association domain]-[...]-C-terminus.

[0079] In some embodiments, a recombinant CD3-TCR complex polypeptide according to the present disclosure does not contain a domain or amino acid sequence containing an immunoreceptor tyrosine-based activation motif (ITAM). An ITAM comprises an amino acid sequence according to YXXL / I (SEQ ID NO: 222), where "X" represents any amino acid. In ITAM-containing proteins, YXXL / I sequences are often separated by 6-8 amino acids (i.e., a sequence of the formula: YXXL / I(X) 6-8 YXXL / I; according to SEQ ID NO: 223). Addition of a phosphate group to the tyrosine residue of an ITAM by a tyrosine kinase initiates a signaling cascade within the cell. ITAM-containing sequences include the intracellular domains of CD3ζ and FcγRI. In some embodiments, a recombinant CD3-TCR complex polypeptide according to the present disclosure does not comprise the amino acid sequence set forth in SEQ ID NO: 47. In some embodiments, a recombinant CD3-TCR complex polypeptide does not comprise the amino acid sequence set forth in SEQ ID NO: 223. In some embodiments, a recombinant CD3-TCR complex polypeptide does not comprise the amino acid sequence set forth in SEQ ID NO: 222.

[0080] In some embodiments, a recombinant CD3-TCR complex polypeptide according to the present disclosure does not comprise a costimulatory sequence. As referred to herein, a "costimulatory sequence" refers to an amino acid sequence that provides costimulation to immune cells expressing a recombinant CD3-TCR complex polypeptide. Costimulation promotes proliferation and survival, and may also promote cytokine production, differentiation, cytotoxic function, and memory formation. The molecular mechanisms of T cell costimulation are reviewed, for example, in Chen and Flies, (2013) Nat Rev Immunol 13(4):227-242. A costimulatory sequence may be or be derived from the intracellular domain of a costimulatory protein. Examples of costimulatory proteins include CD28, 4-1BB, ICOS, CD27, OX40, HVEM, CD2, SLAM, TIM-1, CD30, GITR, DR3, CD226, and LIGHT. In some embodiments, a recombinant CD3-TCR complex polypeptide according to the present disclosure does not comprise the amino acid sequence set forth in SEQ ID NO: 101 (the intracellular domain of human 4-1BB).

[0081] CD3-TCR complex-associated domain The recombinant CD3-TCR complex polypeptides of the present disclosure comprise a CD3-TCR complex association domain, the essential function of which is to provide for the formation of a polypeptide complex comprising a recombinant CD3-TCR complex polypeptide according to the present disclosure and one or more CD3-TCR complex polypeptides.

[0082] A "CD3-TCR complex association domain" refers to a domain through which a polypeptide comprising the domain can associate with a CD3-TCR complex polypeptide (e.g., a CD3-TCR complex polypeptide as described hereinabove). Thus, a CD3-TCR complex association domain according to the present disclosure comprises or consists of an amino acid sequence that confers on a polypeptide comprising the domain the ability to associate with a CD3-TCR complex polypeptide to form a polypeptide complex comprising the CD3-TCR complex polypeptide and a polypeptide having the CD3-TCR complex association domain.

[0083] The association between a CD3-TCR complex association domain / polypeptide comprising a CD3-TCR complex association domain and a CD3-TCR complex polypeptide may be characterized by a non-covalent protein:protein interaction. In some embodiments, the association comprises electrostatic interactions (e.g., ionic bonds, hydrogen bonds) and / or van der Waals forces.

[0084] In some embodiments, a CD3-TCR complex association domain is or is derived from the amino acid sequence of a CD3-TCR complex polypeptide. It will be understood that a CD3-TCR complex association domain can be or can be derived from a region of a CD3-TCR complex polypeptide in which a CD3-TCR complex polypeptide interacts with another CD3-TCR complex polypeptide to form a polypeptide complex. In some embodiments, a CD3-TCR complex association domain is or is derived from the amino acid sequence of a region of a CD3-TCR complex polypeptide that is necessary for association between a CD3-TCR complex polypeptide and another CD3-TCR complex polypeptide to form a polypeptide complex comprising such polypeptide.

[0085] Regions of a CD3-TCR complex polypeptide required for such interactions can be determined by site-directed mutagenesis and / or truncation studies, in which the amino acid sequence of a CD3-TCR complex polypeptide is altered or truncated, and the effect of such alteration / truncation on its ability to associate with other CD3-TCR complex polypeptides is assessed. Suitable techniques for examining such protein:protein interactions include resonance energy transfer techniques, such as fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET), using appropriately labeled interaction partners, as described, for example, in Ciruela, Curr. Opin. Biotechnol. (2008) 19(4):338-43.

[0086] As used herein, polypeptides, domains and amino acid sequences "derived from" a reference polypeptide / domain / amino acid sequence have at least 60%, preferably one of ≥ 70%, ≥ 75%, ≥ 80%, ≥ 85%, ≥ 90%, ≥ 91%, ≥ 92%, ≥ 93%, ≥ 94%, ≥ 95%, ≥ 96%, ≥ 97%, ≥ 98%, ≥ 99% or 100% amino acid sequence identity with the amino acid sequence of the reference polypeptide / domain / amino acid sequence. Polypeptides, domains and amino acid sequences "derived from" a reference polypeptide / domain / amino acid sequence preferably retain functional and / or structural properties of the reference polypeptide / domain / amino acid sequence.

[0087] In some embodiments, the CD3-TCR complex association domain comprises modifications to promote association with CD3-TCR complex polypeptides, hi some embodiments, the CD3-TCR complex association domain comprises modifications to promote heteromerization, i.e., association with non-identical CD3-TCR complex polypeptides.

[0088] As used herein, "modification" refers to a difference relative to a reference amino acid sequence. The reference amino acid sequence may be the amino acid sequence encoded by the most common nucleotide sequence of a gene encoding a related protein. In embodiments herein (and more generally in the art), "modification" may also be referred to as "substitution" or "mutation." Modification typically involves the substitution of an amino acid residue. Substitution of an amino acid residue involves the substitution of an amino acid residue with a non-identical "replacement" amino acid residue. The replacement amino acid residue in the modification according to the present disclosure may be a naturally occurring amino acid residue (i.e., encoded by the genetic code) that is not identical to the amino acid residue at the relevant position in the amino acid sequence prior to modification, selected from alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine ​​(Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val). In some embodiments, the replacement amino acid residue in the modification may be a non-naturally occurring amino acid residue, i.e., an amino acid residue other than those listed in the preceding sentence. Examples of non-naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine, aib, and other amino acid residue analogs such as those described in Ellman, et al., Meth. Enzym. 202 (1991) 301-336.

[0089] By way of example, in embodiments herein, the CD3-TCR complex association domain derived from TRAC includes a modification that replaces the threonine residue at position 47 (numbered relative to SEQ ID NO: 1) with a cysteine ​​residue (SEQ ID NO: 52), and the CD3-TCR complex association domain derived from TCRβ includes a modification that replaces the serine residue at position 56 (numbered relative to SEQ ID NO: 5) with a cysteine ​​residue (SEQ ID NO: 53). The introduction of these cysteine ​​residues promotes heteromerization between the modified domains via the formation of interchain disulfide bridges.

[0090] Embodiments in which the CD3-TCR complex association domain further comprises a modification to promote association with a CD3-TCR complex polypeptide are particularly contemplated in relation to aspects and embodiments of the present disclosure in which a recombinant CD3-TCR complex polypeptide comprising such a CD3-TCR complex association domain is provided for use with another recombinant CD3-TCR complex polypeptide. For example, such a CD3-TCR complex association domain is particularly contemplated for use in a first recombinant CD3-TCR complex polypeptide and / or a second recombinant CD3-TCR complex polypeptide of a polypeptide complex of the present disclosure comprising such a recombinant CD3-TCR complex polypeptide.

[0091] In some embodiments, the CD3-TCR complex association domain is or is derived from the CD3-TCR complex association domain of CD3ε. In some embodiments, the CD3-TCR complex association domain is or is derived from a region of CD3ε necessary for association with CD3γ and / or CD3δ (i.e., to form a CD3ε:CD3γ polypeptide complex or a CD3ε:CD3δ polypeptide complex). In some embodiments, the CD3-TCR complex association domain comprises or consists of an amino acid sequence having at least 60%, preferably ≧70%, ≧75%, ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99%, or 100% amino acid sequence identity to SEQ ID NO:30.

[0092] In some embodiments, the CD3-TCR complex association domain is or is derived from the CD3-TCR complex association domain of TRAC. In some embodiments, the CD3-TCR complex association domain is or is derived from a region of TRAC necessary for association with TCRβ, TRBC1, and / or TRBC2 (i.e., to form a TRAC:TCRβ polypeptide complex, or a TRAC:TRBC1 polypeptide complex, or a TRAC:TRBC2 polypeptide complex). In some embodiments, the CD3-TCR complex association domain comprises or consists of an amino acid sequence having at least 60%, preferably ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to SEQ ID NO:1. In some embodiments, the CD3-TCR complex association domain is derived from TRAC and further comprises modifications to promote association with another CD3-TCR complex polypeptide. In some embodiments, the CD3-TCR complex association domain comprises or consists of an amino acid sequence having at least 60%, preferably >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, or >99% amino acid sequence identity to SEQ ID NO:1, and comprises a cysteine ​​residue at a position corresponding to position 47 numbered according to SEQ ID NO:1. In some embodiments, the CD3-TCR complex association domain comprises or consists of an amino acid sequence having at least 60%, preferably ≧70%, ≧75%, ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99% or 100% amino acid sequence identity to SEQ ID NO:52.

[0093] In some embodiments, the CD3-TCR complex association domain is or is derived from the CD3-TCR complex association domain of TRBC1. In some embodiments, the CD3-TCR complex association domain is or is derived from a region of TRBC1 required for association with TCRα and / or TRAC (i.e., forming a TRBC1:TCRα polypeptide complex or a TRBC1:TRAC polypeptide complex). In some embodiments, the CD3-TCR complex association domain comprises or consists of an amino acid sequence having at least 60%, preferably ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to SEQ ID NO:5. In some embodiments, the CD3-TCR complex association domain is derived from TRBC1 and further comprises modifications to promote association with another CD3-TCR complex polypeptide. In some embodiments, the CD3-TCR complex association domain comprises or consists of an amino acid sequence having at least 60%, preferably ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% amino acid sequence identity to SEQ ID NO: 5, and comprises a cysteine ​​residue at a position corresponding to position 56 numbered according to SEQ ID NO: 5. In some embodiments, the CD3-TCR complex association domain comprises or consists of an amino acid sequence having at least 60%, preferably ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to SEQ ID NO:53.

[0094] In some embodiments, the CD3-TCR complex association domain is or is derived from the CD3-TCR complex association domain of TRBC2. In some embodiments, the CD3-TCR complex association domain is or is derived from a region of TRBC2 necessary for association with TCRα and / or TRAC (i.e., forming a TRBC2:TCRα polypeptide complex or a TRBC2:TRAC polypeptide complex). In some embodiments, the CD3-TCR complex association domain comprises or consists of an amino acid sequence having at least 60%, preferably ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to SEQ ID NO:9. In some embodiments, the CD3-TCR complex association domain is derived from TRBC2 and further comprises modifications to promote association with another CD3-TCR complex polypeptide. In some embodiments, the CD3-TCR complex association domain comprises or consists of an amino acid sequence having at least 60%, preferably ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% amino acid sequence identity to SEQ ID NO: 9, and comprises a cysteine ​​residue at a position corresponding to position 56 numbered according to SEQ ID NO: 9. In some embodiments, the CD3-TCR complex association domain comprises or consists of an amino acid sequence having at least 60%, preferably ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to SEQ ID NO: 54.

[0095] antigen binding part The recombinant CD3-TCR complex polypeptides of the present disclosure comprise an antigen-binding portion or component thereof, the essential function of which is to provide binding to a variant Fc domain, as described herein below.

[0096] As used herein, "antigen-binding portion" refers to a portion that binds to a given target antigen.

[0097] Antigen-binding moieties include antibodies (i.e., immunoglobulins (Ig)), as well as antigen-binding fragments and derivatives thereof. In some embodiments, an antigen-binding moiety according to the present disclosure comprises or consists of a monoclonal antibody, a monospecific antibody, a multispecific (e.g., bispecific, trispecific, etc.) antibody, a variable fragment (Fv) portion, a single-chain Fv (scFv) portion, a fragment antigen-binding (Fab) portion, a single-chain Fab portion (scFab), a crossFab portion, a Fab' portion, a Fab'-SH portion, a F(ab')2 portion, a diabody portion, a triabody portion, a scFv-Fc portion, a minibody portion, a heavy chain-only antibody (HCAb) portion, or a single domain antibody (dAb, VHH) portion.

[0098] Antigen-binding moieties according to the present disclosure also include additional target antigen-binding peptides / polypeptides such as peptide aptamers, thioredoxins, anticalins, Kunitz domains, avimers, knottins, finomers, atrimers, DARPins, affibodies, affilins, armadillo repeat proteins (ArmRPs), OBodys and adnectins (reviewed, e.g., in Reverdatto et al., Curr Top Med Chem. 2015;15(12):1082-1101, which is incorporated herein by reference in its entirety (see also, e.g., Boersma et al., J Biol Chem (2011) 286:41273-85 and Emanuel et al., Mabs (2011) 3:38-48)). Antigen-binding moieties according to the present disclosure also include target antigen-binding nucleic acids, such as nucleic acid aptamers (reviewed, e.g., in Zhou and Rossi Nat Rev Drug Discov. 2017 16(3):181-202). Antigen-binding moieties according to the present disclosure also include target antigen-binding small molecules (e.g., low molecular weight (<1000 daltons, typically about 300-700 daltons) organic compounds).

[0099] An antigen-binding portion of a recombinant CD3-TCR complex polypeptide of the present disclosure is capable of binding to a variant Fc domain according to the present disclosure. An antigen-binding portion capable of binding to a variant Fc domain according to the present disclosure may also be described as an antigen-binding portion that binds to a variant Fc domain according to the present disclosure.

[0100] The antigen-binding moieties described herein preferably exhibit specific binding to a variant Fc domain according to the present disclosure. As used herein, "specific binding" refers to binding that is selective for a target antigen and can be distinguished from non-specific binding to non-target antigens. An antigen-binding moiety that specifically binds to a given target antigen preferably binds to the target antigen with higher affinity and / or longer duration than it binds to other non-target antigens.

[0101] The ability of a given moiety to specifically bind to a given target antigen can be determined by analysis using methods known in the art, such as ELISA, surface plasmon resonance (SPR; see, e.g., Hearty et al., Methods Mol Biol (2012) 907:411-442), biolayer interferometry (BLI; see, e.g., Lad et al., (2015) J Biomol Screen 20(4):498-507), flow cytometry, or radiolabeled antigen binding assay (RIA) enzyme-linked immunosorbent assay. Such analysis can measure and quantify binding to a given target antigen. In some embodiments, the level of binding can be a response detected in a given assay.

[0102] In some embodiments, the antigen-binding moieties described herein have binding affinity in the micromolar range, i.e., K D =9.9×10 -4 ~1×10 -6 M. In some embodiments, the antigen-binding moieties described herein bind to a variant Fc domain according to the present disclosure with an affinity (e.g., as determined by SPR or BLI) of submicromolar, i.e., K D <1×10 -6In some embodiments, the antigen-binding moieties described herein bind to a variant Fc domain according to the present disclosure with an affinity to the Fc domain in the nanomolar range, i.e., K D =9.9×10 -7 ~1×10 -9 In some embodiments, the antigen-binding moieties described herein bind to a variant Fc domain according to the present disclosure with an affinity of sub-nanomolar, i.e., K D <1×10 -9 In some embodiments, the antigen-binding moieties described herein bind to a variant Fc domain according to the present disclosure with an affinity to the Fc domain in the picomolar range, i.e., K D =9.9x10 -10 ~1x10 -12 In some embodiments, the antigen-binding moieties described herein bind to a variant Fc domain according to the present disclosure with an affinity of at least picomolar, i.e., K D <1x10 -12 M binds to a variant Fc domain according to the present disclosure.

[0103] The antigen-binding portion of a recombinant CD3-TCR complex polypeptide according to the present disclosure preferably does not exhibit specific binding to a reference Fc domain according to the present disclosure. In some embodiments, the antigen-binding portion does not bind to, or exhibits substantially no binding to, a reference Fc domain according to the present disclosure.

[0104] An antigen-binding moiety that "does not bind" or "exhibits substantially no binding" to a given antigen exhibits a level of binding to the given antigen that is similar to the level of binding to an antigen that the antigen-binding moiety is known not to bind or not specifically bind, e.g., a non-target antigen. In some embodiments, the level of binding of an antigen-binding moiety that does not bind or exhibits substantially no binding to a given antigen is between 0.5 and 2 times lower, e.g., between 0.75 and 1.5 times lower, between 0.8 and 1.4 times lower, between 0.85 and 1.3 times lower, between 0.9 and 1.2 times lower, or between 0.95 and 1.1 times lower, than the level of binding exhibited by the antigen-binding moiety for an antigen that the antigen-binding moiety is known not to bind or not specifically bind, e.g., a non-target antigen.

[0105] In some embodiments, the level of binding of an antigen-binding moiety to a reference Fc domain according to the present disclosure is 10% or less of the binding of the antigen-binding moiety to a variant Fc domain according to the present disclosure, as determined, for example, by ELISA, SPR, BLI, or RIA. In some embodiments, the antigen-binding moiety has a K D The equilibrium dissociation constant (K D ; e.g., as determined by SPR or BLI) binds to a reference Fc domain according to the present disclosure.

[0106] An antigen-binding portion according to the present disclosure may be or comprise an antigen-binding peptide / polypeptide or an antigen-binding peptide / polypeptide complex. An antigen-binding portion may comprise two or more peptides / polypeptides that together form an antigen-binding domain. The peptides / polypeptides may be covalently or non-covalently associated. In some embodiments, the peptide / polypeptide forms part of a larger polypeptide that comprises the peptide / polypeptide (e.g., in the case of an scFv portion comprising a VH region and a VL region, or in the case of an scFab portion comprising a VH-CH1 and a VL-CL).

[0107] In some embodiments, the antigen-binding portion of the present disclosure comprises an antibody heavy chain variable (VH) region and an antibody light chain variable (VL) region capable of binding to a given target antigen. In some embodiments, the antigen-binding portion comprises or consists of an Fv portion formed by the VH and VL regions of an antibody capable of binding to a given target antigen. In some embodiments, the VH and VL regions may be provided in the same polypeptide and connected by a linker sequence. In some embodiments, the antigen-binding portion comprises or consists of an scFv portion that binds to a given target antigen.

[0108] An antigen-binding portion of the disclosure generally comprises six complementarity-determining region CDRs; three in the heavy chain variable (VH) region: HC-CDR1, HC-CDR2, and HC-CDR3, and three in the light chain variable (VL) region: LC-CDR1, LC-CDR2, and LC-CDR3. The six CDRs together define the paratope of the antigen-binding moiety, which is the part that binds to the target antigen.

[0109] The VH and VL regions each contain framework regions (FRs) on either side of each CDR, which provide a scaffold for the CDR. From the N-terminus to the C-terminus, the VH region comprises the following structure: N-terminus-[HC-FR1]-[HC-CDR1]-[HC-FR2]-[HC-CDR2]-[HC-FR3]-[HC-CDR3]-[HC-FR4]-C-terminus; the VL region comprises the following structure: N-terminus-[LC-FR1]-[LC-CDR1]-[LC-FR2]-[LC-CDR2]-[LC-FR3]-[LC-CDR3]-[LC-FR4]-C-terminus.

[0110] There are several different conventions for defining antibody CDRs and FRs, for example, (i) the Chothia system described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991); (ii) the Chothia system described in Chothia et al., J. Mol. Biol. 196:901-917 (1987); and (iii) the international IMGT (ImMunoGeneTics) information system (LeFranc et al., Nucleic Acids Res. (2015) 43 (Database Issue): D413-22), which uses the IMGT V-DOMAIN numbering convention described in Lefranc et al., Dev. Comp. Immunol. (2003) 27:55-77.

[0111] The CDRs and FRs of the VH and VL regions of the antigen-binding moieties described herein are defined according to the Kabat system.

[0112] In some embodiments, the antigen-binding portion comprises the CDRs of an antigen-binding portion that binds to a variant Fc domain according to the present disclosure. In some embodiments, the antigen-binding portion comprises the FRs of an antigen-binding portion that binds to a variant Fc domain according to the present disclosure. In some embodiments, the antigen-binding portion comprises the CDRs and FRs of an antigen-binding portion that binds to a variant Fc domain according to the present disclosure. That is, in some embodiments, the antigen-binding portion comprises the VH and VL regions of an antigen-binding portion that binds to a variant Fc domain according to the present disclosure.

[0113] Wessels et al., Bioanal. (2017) 9(11):849-59, describe the identification of an antibody that binds to an antibody containing an Fc domain derived from human IgG1 containing P329G, but not to an antibody containing an equivalent Fc domain lacking the P329G substitution. The antibody also binds to an antibody having an hIgG1-derived Fc region containing P329G and further containing L234A and L235A. Darowski et al., Protein Eng. Des. Sel. (2019) 32(5):207-218 and Stock et al., Journal for ImmunoTherapy of Cancer (2022) 10:e005054 provide the structure of an anti-P329G Fab having an Fc containing P329G, L234A, and L235A. The anti-P329G Fab interacts with an Fc containing P329G, L234A, and L235A in a 1:1 stoichiometry. The epitope is disclosed to include positions N325 to P331 (including G329) and further S267 to E272.

[0114] In some embodiments, the antigen-binding portion comprises the CDR, FR and / or VH and / or VL regions of an antigen-binding molecule described herein that binds to a variant Fc domain according to the present disclosure, or comprises the CDR, FR and / or VH and / or VL regions derived from an antigen-binding molecule described herein that binds to a variant Fc domain according to the present disclosure. In some embodiments, the antigen-binding molecule that binds to a variant Fc domain according to the present disclosure is selected from αP329G_VH1 / VL1, αP329G_VH2 / VL1, αP329G_VH3 / VL1, and αAAA.

[0115] In some embodiments, the antigen-binding portion comprises a VH region set forth in (1) or (2) below: (1) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 56 HC-CDR2 having the amino acid sequence of SEQ ID NO: 57 HC-CDR3 having the amino acid sequence of SEQ ID NO: 58; Or a variant thereof in which one, two or three amino acids in HC-CDR1, and / or one, two or three amino acids in HC-CDR2, and / or one, two or three amino acids in HC-CDR3 are replaced with another amino acid. (2) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 56 HC-CDR2 having the amino acid sequence of SEQ ID NO: 64 HC-CDR3 having the amino acid sequence of SEQ ID NO: 58; Or a variant thereof in which one, two or three amino acids in HC-CDR1, and / or one, two or three amino acids in HC-CDR2, and / or one, two or three amino acids in HC-CDR3 are replaced with another amino acid.

[0116] In some embodiments, the antigen-binding portion comprises a VH region set forth in (3) or (4) below: (3) A VH region incorporating the following FR: HC-FR1 having the amino acid sequence of SEQ ID NO: 59 HC-FR2 having the amino acid sequence of SEQ ID NO: 60 HC-FR3 having the amino acid sequence of SEQ ID NO: 61 HC-FR4 having the amino acid sequence of SEQ ID NO: 62; Or a variant thereof in which one, two or three amino acids in HC-FR1, and / or one, two or three amino acids in HC-FR2, and / or one, two or three amino acids in HC-FR3, and / or one, two or three amino acids in HC-FR4 are substituted with another amino acid. (4) A VH region incorporating the following FR: HC-FR1 having the amino acid sequence of SEQ ID NO: 66 HC-FR2 having the amino acid sequence of SEQ ID NO: 60 HC-FR3 having the amino acid sequence of SEQ ID NO: 67 HC-FR4 having the amino acid sequence of SEQ ID NO: 62; Or a variant thereof in which one, two or three amino acids in HC-FR1, and / or one, two or three amino acids in HC-FR2, and / or one, two or three amino acids in HC-FR3, and / or one, two or three amino acids in HC-FR4 are substituted with another amino acid.

[0117] In some embodiments, the antigen-binding portion comprises a VH region comprising the CDRs described in (1) or (2) above and the FRs described in (3) or (4) above. In some embodiments, the antigen-binding portion comprises a VH region set forth in (5) or (6) below: (5) A VH region comprising the CDRs described in (1) and the FRs described in (3). (6) A VH region comprising the CDRs described in (2) and the FRs described in (4). (7) A VH region comprising the CDRs described in (2) and the FRs described in (3).

[0118] In some embodiments, the antigen-binding portion comprises a VH region according to one of the following (8) to (10): (8) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 55. (9) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 63. (10) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 65.

[0119] In some embodiments, the antigen-binding portion comprises a VL region as set forth in (11) below: (11) A VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 69 LC-CDR2 having the amino acid sequence of SEQ ID NO: 70 LC-CDR3 having the amino acid sequence of SEQ ID NO: 71; Or a variant thereof in which one, two or three amino acids in LC-CDR1, and / or one, two or three amino acids in LC-CDR2, and / or one, two or three amino acids in LC-CDR3 are replaced with another amino acid.

[0120] In some embodiments, the antigen-binding portion comprises a VL region as set forth in (12) below: (12) A VL region incorporating the following FR: LC-FR1 having the amino acid sequence of SEQ ID NO: 72 LC-FR2 having the amino acid sequence of SEQ ID NO: 73 LC-FR3 having the amino acid sequence of SEQ ID NO: 74 LC-FR4 having the amino acid sequence of SEQ ID NO: 75; or a variant thereof in which one, two or three amino acids in LC-FR1, and / or one, two or three amino acids in LC-FR2, and / or one, two or three amino acids in LC-FR3, and / or one, two or three amino acids in LC-FR4 are substituted with another amino acid.

[0121] In some embodiments, the antigen-binding portion comprises a VL region as set forth in (13) below: (13) A VL region comprising the CDRs described in (11) and the FRs described in (12).

[0122] In some embodiments, the antigen-binding portion comprises a VL region as set forth in (14) below: (14) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 68.

[0123] In some embodiments, the antigen-binding portion comprises a VH region described in any one of (1) to (10) above and a VL region described in any one of (11) to (14) above.

[0124] In some embodiments, the antigen-binding portion comprises a VH region set forth in (15) below: (15) A VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 77 HC-CDR2 having the amino acid sequence of SEQ ID NO: 78 HC-CDR3 having the amino acid sequence of SEQ ID NO: 79; Or a variant thereof in which one, two or three amino acids in HC-CDR1, and / or one, two or three amino acids in HC-CDR2, and / or one, two or three amino acids in HC-CDR3 are replaced with another amino acid.

[0125] In some embodiments, the antigen-binding portion comprises a VH region set forth in (16) below: (16) A VH region incorporating the following FR: HC-FR1 having the amino acid sequence of SEQ ID NO: 80 HC-FR2 having the amino acid sequence of SEQ ID NO: 81 HC-FR3 having the amino acid sequence of SEQ ID NO: 82 HC-FR4 having the amino acid sequence of SEQ ID NO: 83; Or a variant thereof in which one, two or three amino acids in HC-FR1, and / or one, two or three amino acids in HC-FR2, and / or one, two or three amino acids in HC-FR3, and / or one, two or three amino acids in HC-FR4 are substituted with another amino acid.

[0126] In some embodiments, the antigen-binding portion comprises a VH region set forth in (17) below: (17) A VH region comprising the CDRs described in (15) and the FRs described in (16).

[0127] In some embodiments, the antigen-binding portion comprises a VH region set forth in (18) below: (18) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 76.

[0128] In some embodiments, the antigen-binding portion comprises a VL region set forth in (19) below: (19) A VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 85 LC-CDR2 having the amino acid sequence of SEQ ID NO: 86 LC-CDR3 having the amino acid sequence of SEQ ID NO: 87; Or a variant thereof in which one, two or three amino acids in LC-CDR1, and / or one, two or three amino acids in LC-CDR2, and / or one, two or three amino acids in LC-CDR3 are replaced with another amino acid.

[0129] In some embodiments, the antigen-binding portion comprises a VL region set forth in (20) below: (20) A VL region incorporating the following FR: LC-FR1 having the amino acid sequence of SEQ ID NO: 88 LC-FR2 having the amino acid sequence of SEQ ID NO: 89 LC-FR3 having the amino acid sequence of SEQ ID NO: 90 LC-FR4 having the amino acid sequence of SEQ ID NO: 91; or a variant thereof in which one, two or three amino acids in LC-FR1, and / or one, two or three amino acids in LC-FR2, and / or one, two or three amino acids in LC-FR3, and / or one, two or three amino acids in LC-FR4 are substituted with another amino acid.

[0130] In some embodiments, the antigen-binding portion comprises a VL region set forth in (21) below: (21) A VL region comprising the CDRs described in (19) and the FRs described in (20).

[0131] In some embodiments, the antigen-binding portion comprises a VL region set forth in (22) below: (22) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 84.

[0132] In some embodiments, the antigen-binding portion comprises a VH region described in any one of (15) to (18) above and a VL region described in any one of (19) to (22) above.

[0133] Amino acid substitutions according to the present disclosure can be biochemically conservative. In some embodiments, if the substituted amino acid is provided in one of rows 1-5 of the table below, the replacement amino acid for the substitution is another non-identical amino acid provided in the same row. [table] TIFF2025530146000001.tif42170

[0134] By way of example, in some embodiments where the substitution is for a Met residue, the replacement amino acid may be selected from Ala, Val, Leu, Ile, Trp, Tyr, Phe, and norleucine.

[0135] In some embodiments, the replacement amino acid in the substitution may have the same side chain polarity as the amino acid residue it replaces. In some embodiments, the replacement amino acid in the substitution may have the same side chain charge (at pH 7.4) as the amino acid residue it replaces. [table] TIFF2025530146000002.tif132170

[0136] That is, in some embodiments, a non-polar amino acid is substituted with another non-identical non-polar amino acid; in some embodiments, a polar amino acid is substituted with another non-identical polar amino acid; in some embodiments, an acidic polar amino acid is substituted with another non-identical acidic polar amino acid; in some embodiments, a basic polar amino acid is substituted with another non-identical basic polar amino acid; in some embodiments, a neutral amino acid is substituted with another non-identical neutral amino acid; in some embodiments, a positive amino acid is substituted with another non-identical positive amino acid; in some embodiments, a negative amino acid is substituted with another non-identical negative amino acid.

[0137] In some embodiments, the substitution(s) may be functionally conservative, i.e., in some embodiments, the substitution may not affect (or may not substantially affect) one or more functional properties (e.g., target antigen binding) of the antigen-binding moiety comprising the substitution, compared to the equivalent unsubstituted molecule.

[0138] In some embodiments, an antigen-binding portion of the present disclosure comprises a VH as described herein. In some embodiments, an antigen-binding portion comprises a VL as described herein. In some embodiments, an antigen-binding portion comprises one or more antibody heavy chain constant regions (CH). In some embodiments, an antigen-binding portion comprises one or more antibody light chain constant regions (CL). In some embodiments, an antigen-binding portion comprises a CH1, CH2 and / or CH3 region of an immunoglobulin (Ig). In some embodiments, an antigen-binding portion comprises a linker sequence as described herein.

[0139] In some embodiments, an antigen-binding portion of the disclosure comprises a polypeptide(s) comprising: (i) a VH region comprising HC-CDR1, HC-CDR2, and HC-CDR3 as set forth in column A of Table A; and (ii) a VL region comprising LC-CDR1, LC-CDR2, and LC-CDR3 as set forth in column B of Table A, wherein the sequences in columns A and B are selected from the same row of Table A.

[0140] In some embodiments, the antigen-binding portion of the present disclosure comprises (i) a VH region comprising an HC-CDR1 set forth in SEQ ID NO: 56, an HC-CDR2 set forth in SEQ ID NO: 64, and an HC-CDR3 set forth in SEQ ID NO: 58, and (ii) a VL region comprising an LC-CDR1 set forth in SEQ ID NO: 69, an LC-CDR2 set forth in SEQ ID NO: 70, and an LC-CDR3 set forth in SEQ ID NO: 71.

[0141] In some embodiments, an antigen-binding portion of the disclosure comprises (i) a polypeptide(s) comprising a VH region comprising HC-FR1, HC-FR2, HC-FR3, and HC-FR4 as set forth in column A of Table B, and a VL region comprising LC-FR1, LC-FR2, LC-FR3, and LC-FR4 as set forth in column B of Table B, wherein the sequences in columns A and B are selected from the same row of Table B.

[0142] In some embodiments, an antigen-binding portion of the present disclosure comprises a polypeptide(s) comprising: (i) a VH region comprising HC-FR1 set forth in SEQ ID NO: 66, HC-FR2 set forth in SEQ ID NO: 60, HC-FR3 set forth in SEQ ID NO: 67, and HC-FR4 set forth in SEQ ID NO: 62; and (ii) a VL region comprising LC-FR1 set forth in SEQ ID NO: 72, LC-FR2 set forth in SEQ ID NO: 73, LC-FR3 set forth in SEQ ID NO: 74, and LC-FR4 set forth in SEQ ID NO: 75.

[0143] In some embodiments, an antigen-binding portion of the disclosure comprises a polypeptide(s) comprising: (i) an amino acid sequence having at least 70%, preferably ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99% or 100% amino acid sequence identity to an amino acid sequence set forth in column A of Table C, and (ii) an amino acid sequence having at least 70%, preferably ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99% or 100% amino acid sequence identity to an amino acid sequence set forth in column B of Table C, wherein the sequences in columns A and B are selected from the same row of Table C.

[0144] In some embodiments, an antigen-binding portion of the disclosure comprises amino acids having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to SEQ ID NO: 55, 63, or 65. In some embodiments, an antigen-binding portion of the disclosure comprises amino acids having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to SEQ ID NO: 68.

[0145] In some embodiments, antigen-binding portions of the disclosure comprise amino acids having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to SEQ ID NO: 65. In some embodiments, antigen-binding portions of the disclosure comprise amino acids having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to SEQ ID NO: 68.

[0146] In some embodiments, an antigen-binding portion of the disclosure comprises amino acids having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to SEQ ID NO: 76. In some embodiments, an antigen-binding portion of the disclosure comprises amino acids having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to SEQ ID NO: 84.

[0147] In some embodiments, an antigen-binding portion of the disclosure comprises or consists of amino acids having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to SEQ ID NO: 94. In some embodiments, an antigen-binding portion comprises or consists of amino acids having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to SEQ ID NO: 95. In some embodiments, the antigen-binding portion comprises or consists of amino acids having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to SEQ ID NO: 96. In some embodiments, the antigen-binding portion comprises or consists of amino acids having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to SEQ ID NO:97.

[0148] In aspects and embodiments of the present disclosure, the recombinant CD3-TCR complex polypeptide comprises a component of an antigen-binding portion. This is particularly true when it is intended to use the recombinant CD3-TCR complex polypeptide together with another non-identical, complementary recombinant CD3-TCR complex polypeptide. In such aspects and embodiments, the two non-identical, complementary polypeptides preferably associate with each other to form a polypeptide complex comprising the antigen-binding portion. That is, association between the recombinant CD3-TCR complex polypeptides reconstitutes a functional antigen-binding portion.

[0149] By way of example, in the embodiments described herein, a recombinant CD3-TCR complex polypeptide comprises a VH region of an antigen-binding portion specific for a variant Fc domain and the ECD, TMD, and ICD of TRAC(T47C), and it is contemplated that this recombinant CD3-TCR complex polypeptide be used in combination with a recombinant CD3-TCR complex polypeptide comprising a VL region of an antigen-binding portion specific for a variant Fc domain and the ECD, TMD, and ICD of TRBC1(S56C). When expressed in a cell, the two recombinant CD3-TCR complex polypeptides associate to form a polypeptide complex comprising an Fv specific for the variant Fc domain formed by the VH region from the first polypeptide and the VL region from the second polypeptide.

[0150] In some aspects and embodiments of the present disclosure, a first and second component of the antigen-binding portion is provided. It will be understood that according to such aspects and embodiments, the first and second component of the antigen-binding portion are complementary and can associate to form a (complete, functional) antigen-binding portion.

[0151] In some embodiments according to the present disclosure, a component of the antigen-binding portion may be or comprise a VH region of an antigen-binding portion specific for a variant Fc domain (e.g., as described herein). In some embodiments, a component of the antigen-binding portion may be or comprise a VL region of an antigen-binding portion specific for a variant Fc domain (e.g., as described herein). In preferred embodiments, the VH and VL regions may be derived from the same antigen-binding portion.

[0152] In some embodiments, the antigen-binding portion comprises or consists of a VH as described herein. In some embodiments, the antigen-binding portion comprises or consists of a VL as described herein. In some embodiments, the antigen-binding portion comprises one or more antibody heavy chain constant regions (CH). In some embodiments, the antigen-binding portion comprises one or more antibody light chain constant regions (CL). In some embodiments, the antigen-binding portion comprises an immunoglobulin (Ig) CH1, CH2 and / or CH3 region.

[0153] In some embodiments, a component of the antigen-binding portion comprises or consists of a polypeptide(s) comprising a VH region comprising HC-CDR1, HC-CDR2 and HC-CDR3 as shown in column A of Table A. In some embodiments, a component of the antigen-binding portion comprises or consists of a polypeptide(s) comprising a VL region comprising LC-CDR1, LC-CDR2 and LC-CDR3 as shown in column B of Table A.

[0154] In some embodiments, a component of the antigen-binding portion comprises or consists of a polypeptide(s) comprising a VH region comprising HC-FR1, HC-FR2, HC-FR3 and HC-FR4 as shown in column A of Table B. In some embodiments, a component of the antigen-binding portion comprises or consists of a polypeptide(s) comprising a VL region comprising LC-FR1, LC-FR2, LC-FR3 and LC-FR4 as shown in column B of Table B.

[0155] In some embodiments, a component of the antigen-binding portion comprises or consists of a polypeptide(s) comprising an amino acid sequence having at least 70%, preferably ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99% or 100% amino acid sequence identity to an amino acid sequence set forth in column A of Table C. In some embodiments, a component of the antigen-binding portion comprises or consists of a polypeptide(s) comprising an amino acid sequence having at least 70%, preferably ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99% or 100% amino acid sequence identity to an amino acid sequence set forth in column B of Table C.

[0156] In some embodiments, a component of the antigen-binding portion comprises or consists of amino acids having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to SEQ ID NO: 55, 63 or 65. In some embodiments, a component of the antigen-binding portion comprises or consists of amino acids having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to SEQ ID NO: 68.

[0157] In some embodiments, a component of the antigen-binding portion comprises or consists of amino acids having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to SEQ ID NO: 76. In some embodiments, a component of the antigen-binding portion comprises or consists of amino acids having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to SEQ ID NO: 84.

[0158] It will be understood that where components of an antigen-binding moiety are provided in aspects and embodiments of the present disclosure, it is intended that the provided components are complementary and capable of associating to form a (complete, functional) antigen-binding moiety.

[0159] Variant Fc domains The antigen-binding portion of the recombinant CD3-TCR complex polypeptide of the present disclosure provides binding to a variant Fc domain.

[0160] As used herein, "Fc domain" refers to a polypeptide complex formed by the interaction between two polypeptides, each of which contains the CH2-CH3 region of an immunoglobulin (Ig) heavy chain constant sequence.

[0161] G-type immunoglobulins (i.e., IgG) are glycoproteins of approximately 150 kDa that contain two heavy chains and two light chains. From the N-terminus to the C-terminus, the heavy chain contains a VH followed by a heavy chain constant region containing three constant domains (CH1, CH2, CH3), and similarly, the light chain contains a VL followed by a CL. Depending on the heavy chain, immunoglobulins can be classified as IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM. The light chain can be kappa (κ) or lambda (λ).

[0162] As used herein, the term "CH2 domain" refers to the amino acid sequence corresponding to the CH2 domain of an immunoglobulin (Ig). The CH2 domain is the region of an Ig formed by positions 231-340 of the immunoglobulin constant domain according to the EU numbering system described in Edelman et al., Proc Natl Acad Sci USA (1969) 63(1):78-85. The term "CH3 domain" refers to the amino acid sequence corresponding to the CH3 domain of an immunoglobulin (Ig). The CH3 domain is the region of an Ig formed by positions 341-447 of the immunoglobulin constant domain according to the EU numbering system described in Edelman et al., Proc Natl Acad Sci USA (1969) 63(1):78-85. The term "CH2-CH3 region" refers to the amino acid sequence corresponding to the CH2 and CH3 domains of an immunoglobulin (Ig). The CH2-CH3 region is the region of an Ig formed by positions 231 to 447 of the immunoglobulin constant domain according to the EU numbering system described in Edelman et al., Proc Natl Acad Sci USA (1969) 63(1):78-85.

[0163] In some embodiments, a CH2 domain, a CH3 domain, and / or a CH2-CH3 region according to the present disclosure corresponds to a CH2 domain / CH3 domain / CH2-CH3 region of an IgG (e.g., IgG1, IgG2, IgG3, IgG4), an IgA (e.g., IgA1, IgA2), an IgD, an IgE, or an IgM. In some embodiments, a CH2 domain, a CH3 domain, and / or a CH2-CH3 region corresponds to a CH2 domain / CH3 domain / CH2-CH3 region of a human IgG (e.g., hIgG1, hIgG2, hIgG3, hIgG4), an hIgA (e.g., hIgA1, hIgA2), an hIgD, an hIgE, or an hIgM. In some embodiments, the CH2 domain, CH3 domain and / or CH2-CH3 region correspond to the CH2 domain / CH3 domain / CH2-CH3 region of a human IgG1 allotype (eg, G1m1, G1m2, G1m3 or G1m17).

[0164] It will be understood that an Fc domain according to the present disclosure may form part of a larger molecule comprising an Fc domain, for example, a variant Fc domain according to the present disclosure may be included in an antigen-binding molecule (e.g., an antibody) that comprises an antigen-binding portion specific for a target antigen and a variant Fc domain according to the present disclosure.

[0165] The Fc domain interacts with Fc receptors and other molecules of the immune system to produce functional effects. Fc-mediated effector functions are outlined, for example, in Jefferis et al., Immunol Rev 1998 163:59-76 (incorporated herein by reference in its entirety), and include Fc-mediated recruitment and activation of immune cells (e.g., macrophages, dendritic cells, neutrophils, basophils, eosinophils, platelets, mast cells, NK cells, and T cells) through the interaction between the Fc region and Fc receptors expressed by immune cells, recruitment of complement pathway components through binding of the Fc region to the complement protein C1q, and consequent activation of the complement cascade. Fc-mediated functions include Fc receptor binding, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), formation of the membrane attack complex (MAC), cell degranulation, cytokine and / or chemokine production, and antigen processing and presentation.

[0166] The CH2-CH3 region sequence of human IgG1 G1m1 allotype is shown in SEQ ID NO: 206. The CH2-CH3 region sequence of human IgG1 G1m3 allotype is shown in SEQ ID NO: 207. The CH2-CH3 region sequence of human IgG2 is shown in SEQ ID NO: 208. The CH2-CH3 region sequence of human IgG3 is shown in SEQ ID NO: 209. The CH2-CH3 region sequence of human IgG4 is shown in SEQ ID NO: 210.

[0167] A variant Fc domain according to the present disclosure comprises an amino acid sequence that includes at least one amino acid difference relative to a reference Fc domain.

[0168] In some embodiments, a reference Fc domain according to the present disclosure comprises two polypeptides, each comprising a CH2-CH3 region comprising or consisting of an amino acid sequence having at least 70% sequence identity, more preferably at least ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 206. In some embodiments, a reference Fc domain comprises two polypeptides, each comprising a CH2-CH3 region comprising or consisting of SEQ ID NO: 206.

[0169] In some embodiments, the reference Fc domain comprises two polypeptides, each comprising a CH2-CH3 region comprising or consisting of an amino acid sequence having at least 70% sequence identity, more preferably at least ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 207. In some embodiments, the reference Fc domain comprises two polypeptides, each comprising a CH2-CH3 region comprising or consisting of SEQ ID NO: 207.

[0170] In some embodiments, the reference Fc domain comprises two polypeptides, each comprising a CH2-CH3 region comprising or consisting of an amino acid sequence having at least 70% sequence identity, more preferably at least ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 208. In some embodiments, the reference Fc domain comprises two polypeptides, each comprising a CH2-CH3 region comprising or consisting of SEQ ID NO: 208.

[0171] In some embodiments, the reference Fc domain comprises two polypeptides, each comprising a CH2-CH3 region comprising or consisting of an amino acid sequence having at least 70% sequence identity, more preferably at least ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 209. In some embodiments, the reference Fc domain comprises two polypeptides, each comprising a CH2-CH3 region comprising or consisting of SEQ ID NO: 209.

[0172] In some embodiments, the reference Fc domain comprises two polypeptides, each comprising a CH2-CH3 region comprising or consisting of an amino acid sequence having at least 70% sequence identity, more preferably at least ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 210. In some embodiments, the reference Fc domain comprises two polypeptides, each comprising a CH2-CH3 region comprising or consisting of SEQ ID NO: 210.

[0173] A variant Fc domain according to the present disclosure may comprise amino acid differences relative to the amino acid sequence of one or both of the polypeptides of a reference Fc domain according to the present disclosure.

[0174] In some embodiments, a variant Fc domain according to the disclosure comprises two polypeptides, each polypeptide comprising a CH2-CH3 region, and one or both of the CH2-CH3 regions comprises an amino acid sequence that is not identical to SEQ ID NO: 206. In some embodiments, a variant Fc domain according to the disclosure comprises two polypeptides, each polypeptide comprising a CH2-CH3 region, and one or both of the CH2-CH3 regions comprises an amino acid sequence with one or more (e.g., 1, 2, 3, 4, 5 or more) amino acid differences relative to SEQ ID NO: 206. In some embodiments, each CH2-CH3 region of the variant Fc domain comprises or consists of an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:206, more preferably at least ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98% or ≥99% sequence identity, and one or both of the CH2-CH3 regions comprises an amino acid sequence that is not identical to SEQ ID NO:206. In some embodiments, each CH2-CH3 region of the variant Fc domain comprises or consists of an amino acid sequence having at least 70% sequence identity, more preferably at least ≧75%, ≧80%, ≧85%, ≧86%, ≧87%, ≧88%, ≧89%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, or ≧99% sequence identity, to the amino acid sequence of SEQ ID NO:206, and one or both of the CH2-CH3 regions comprises an amino acid sequence with one or more (e.g., 1, 2, 3, 4, 5 or more) amino acid differences relative to SEQ ID NO:206.

[0175] In some embodiments, a variant Fc domain according to the disclosure comprises two polypeptides, each polypeptide comprising a CH2-CH3 region, and one or both of the CH2-CH3 regions comprises an amino acid sequence that is not identical to SEQ ID NO: 207. In some embodiments, a variant Fc domain according to the disclosure comprises two polypeptides, each polypeptide comprising a CH2-CH3 region, and one or both of the CH2-CH3 regions comprises an amino acid sequence with one or more (e.g., 1, 2, 3, 4, 5 or more) amino acid differences relative to SEQ ID NO: 207. In some embodiments, each CH2-CH3 region of the variant Fc domain comprises or consists of an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 207, more preferably at least ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98% or ≥99% sequence identity, and one or both of the CH2-CH3 regions comprises an amino acid sequence that is not identical to SEQ ID NO: 207. In some embodiments, each CH2-CH3 region of the variant Fc domain comprises or consists of an amino acid sequence having at least 70% sequence identity, more preferably at least ≧75%, ≧80%, ≧85%, ≧86%, ≧87%, ≧88%, ≧89%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, or ≧99% sequence identity, to the amino acid sequence of SEQ ID NO:207, and one or both of the CH2-CH3 regions comprises an amino acid sequence with one or more (e.g., 1, 2, 3, 4, 5 or more) amino acid differences relative to SEQ ID NO:207.

[0176] In some embodiments, a variant Fc domain according to the disclosure comprises two polypeptides, each polypeptide comprising a CH2-CH3 region, and one or both of the CH2-CH3 regions comprises an amino acid sequence that is not identical to SEQ ID NO: 208. In some embodiments, a variant Fc domain according to the disclosure comprises two polypeptides, each polypeptide comprising a CH2-CH3 region, and one or both of the CH2-CH3 regions comprises an amino acid sequence with one or more (e.g., 1, 2, 3, 4, 5 or more) amino acid differences relative to SEQ ID NO: 208. In some embodiments, each CH2-CH3 region of the variant Fc domain comprises or consists of an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:208, more preferably at least ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98% or ≥99% sequence identity, and one or both of the CH2-CH3 regions comprises an amino acid sequence that is not identical to SEQ ID NO:208. In some embodiments, each CH2-CH3 region of the variant Fc domain comprises or consists of an amino acid sequence having at least 70% sequence identity, more preferably at least ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity, to the amino acid sequence of SEQ ID NO:208, and one or both of the CH2-CH3 regions comprises an amino acid sequence with one or more (e.g., 1, 2, 3, 4, 5 or more) amino acid differences relative to SEQ ID NO:208.

[0177] In some embodiments, a variant Fc domain according to the disclosure comprises two polypeptides, each polypeptide comprising a CH2-CH3 region, and one or both of the CH2-CH3 regions comprises an amino acid sequence that is not identical to SEQ ID NO: 209. In some embodiments, a variant Fc domain according to the disclosure comprises two polypeptides, each polypeptide comprising a CH2-CH3 region, and one or both of the CH2-CH3 regions comprises an amino acid sequence with one or more (e.g., 1, 2, 3, 4, 5 or more) amino acid differences relative to SEQ ID NO: 209. In some embodiments, each CH2-CH3 region of the variant Fc domain comprises or consists of an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 209, more preferably at least ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98% or ≥99% sequence identity, and one or both of the CH2-CH3 regions comprises an amino acid sequence that is not identical to SEQ ID NO: 209. In some embodiments, each CH2-CH3 region of the variant Fc domain comprises or consists of an amino acid sequence having at least 70% sequence identity, more preferably at least ≧75%, ≧80%, ≧85%, ≧86%, ≧87%, ≧88%, ≧89%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, or ≧99% sequence identity, to the amino acid sequence of SEQ ID NO: 209, and one or both of the CH2-CH3 regions comprises an amino acid sequence with one or more (e.g., 1, 2, 3, 4, 5 or more) amino acid differences relative to SEQ ID NO: 209.

[0178] In some embodiments, a variant Fc domain according to the disclosure comprises two polypeptides, each polypeptide comprising a CH2-CH3 region, and one or both of the CH2-CH3 regions comprises an amino acid sequence that is not identical to SEQ ID NO: 210. In some embodiments, a variant Fc domain according to the disclosure comprises two polypeptides, each polypeptide comprising a CH2-CH3 region, and one or both of the CH2-CH3 regions comprises an amino acid sequence with one or more (e.g., 1, 2, 3, 4, 5 or more) amino acid differences relative to SEQ ID NO: 210. In some embodiments, each CH2-CH3 region of the variant Fc domain comprises or consists of an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 210, more preferably at least ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98% or ≥99% sequence identity, and one or both of the CH2-CH3 regions comprises an amino acid sequence that is not identical to SEQ ID NO: 210. In some embodiments, each CH2-CH3 region of the variant Fc domain comprises or consists of an amino acid sequence having at least 70% sequence identity, more preferably at least ≧75%, ≧80%, ≧85%, ≧86%, ≧87%, ≧88%, ≧89%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, or ≧99% sequence identity, to the amino acid sequence of SEQ ID NO: 210, and one or both of the CH2-CH3 regions comprises an amino acid sequence with one or more (e.g., 1, 2, 3, 4, 5 or more) amino acid differences relative to SEQ ID NO: 210.

[0179] In some embodiments, each CH2-CH3 region of a variant Fc domain according to the disclosure comprises an amino acid difference relative to a reference Fc domain according to the disclosure, and in some embodiments, the amino acid sequences of the CH2-CH3 regions of the constituent polypeptides of a variant Fc domain according to the disclosure are identical (i.e., they have the same amino acid sequence).

[0180] The amino acid differences of a variant Fc domain according to the present disclosure (relative to a reference Fc domain) may affect Fc-mediated functions.

[0181] Modifications to Fc domains that affect Fc-mediated function are known in the art, such as those described in Wang et al., Protein Cell (2018) 9(1):63-73 and Saunders et al., Front Immunol. (2019) 10:1296, both of which are incorporated herein by reference in their entireties. Exemplary Fc domain modifications known to affect Fc-mediated function are summarized in Table 1 of Wang et al., Protein Cell (2018) 9(1):63-73 and Tables 1, 2, and 3 of Saunders et al., Front Immunol. (2019) 10:1296. In some embodiments, a variant Fc domain of the present disclosure comprises an Fc domain that includes an amino acid difference compared to a reference Fc domain (e.g., a reference Fc domain according to the present disclosure) that increases or decreases an Fc-mediated function.

[0182] In some embodiments, the variant Fc domain comprises an amino acid difference compared to a reference Fc domain that increases an Fc-mediated function. In some embodiments, the variant Fc domain comprises an amino acid difference compared to a reference Fc domain that increases ADCC, ADCP, and / or CDC. Thus, in some embodiments, the variant Fc domain exhibits an increased level of Fc-mediated function compared to the reference Fc domain. In some embodiments, the variant Fc domain exhibits increased ADCC, ADCP, and / or CDC compared to the reference Fc domain.

[0183] In some embodiments, the variant Fc domain comprises an amino acid difference compared to a reference Fc domain that increases binding to an Fc receptor (e.g., an Fcγ receptor, e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa and / or FcγRIIIb). In some embodiments, the variant Fc domain comprises an amino acid difference compared to a reference Fc domain that increases binding to FcRn. In some embodiments, the variant Fc domain comprises an amino acid difference compared to a reference Fc domain that increases binding to a complement protein (e.g., C1q). In some embodiments, the variant Fc domain comprises an amino acid difference compared to a reference Fc domain to increase hexamerization of an antigen-binding molecule comprising the variant Fc domain. In some embodiments, the Fc domain comprises an amino acid difference compared to a reference Fc domain that increases the half-life of an antigen-binding molecule comprising the variant Fc domain. Thus, in some embodiments, the variant Fc domain exhibits increased binding to an Fc receptor (e.g., an Fcγ receptor, e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa and / or FcγRIIIb) compared to a reference Fc domain. In some embodiments, the variant Fc domain exhibits increased binding to FcRn compared to a reference Fc domain. In some embodiments, the variant Fc domain exhibits increased binding to a complement protein (e.g., C1q) compared to a reference Fc domain. In some embodiments, an antigen binding molecule comprising the variant Fc domain exhibits increased hexamerization compared to an antigen binding molecule comprising the reference Fc domain. In some embodiments, an antigen binding molecule comprising the variant Fc domain exhibits increased half-life compared to an antigen binding molecule comprising the reference Fc domain.

[0184] In some embodiments, the variant Fc domain comprises an amino acid difference compared to a reference Fc domain that reduces an Fc-mediated function. In some embodiments, the variant Fc domain comprises an amino acid difference compared to a reference Fc domain that reduces ADCC, ADCP, and / or CDC. Thus, in some embodiments, the variant Fc domain exhibits a reduced level of Fc-mediated function compared to the reference Fc domain. In some embodiments, the variant Fc domain exhibits reduced ADCC, ADCP, and / or CDC compared to the reference Fc domain.

[0185] In some embodiments, the variant Fc domain comprises an amino acid difference compared to a reference Fc domain that reduces binding to an Fc receptor (e.g., an Fcγ receptor, e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa and / or FcγRIIIb). In some embodiments, the variant Fc domain comprises an amino acid difference compared to a reference Fc domain that reduces binding to FcRn. In some embodiments, the variant Fc domain comprises an amino acid difference compared to a reference Fc domain that reduces binding to a complement protein (e.g., C1q). In some embodiments, the variant Fc domain comprises an amino acid difference compared to a reference Fc domain to increase hexamerization of an antigen-binding molecule comprising the variant Fc domain. In some embodiments, the Fc domain comprises an amino acid difference compared to a reference Fc domain that reduces the half-life of an antigen-binding molecule comprising the variant Fc domain. Thus, in some embodiments, the variant Fc domain exhibits reduced binding to an Fc receptor (e.g., an Fcγ receptor, e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa and / or FcγRIIIb) compared to a reference Fc domain. In some embodiments, the variant Fc domain exhibits reduced binding to FcRn compared to a reference Fc domain. In some embodiments, the variant Fc domain exhibits reduced binding to complement proteins (e.g., C1q) compared to a reference Fc domain. In some embodiments, an antigen binding molecule comprising the variant Fc domain exhibits reduced hexamerization compared to an antigen binding molecule comprising the reference Fc domain. In some embodiments, an antigen binding molecule comprising the variant Fc domain exhibits reduced half-life compared to an antigen binding molecule comprising the reference Fc domain.

[0186] In some embodiments, the variant Fc domain comprises a CH2-CH3 region that comprises an amino acid difference at one or more of the following positions relative to the amino acid sequence of the CH2-CH3 region of a reference Fc domain: 233, 234, 235, 253, 297, 298, 310, 329, 331, 333, 334, or 435 (according to the EU numbering system). In some embodiments, the variant Fc domain comprises a CH2-CH3 region that comprises an amino acid difference at one or more of the following positions relative to the amino acid sequence of the CH2-CH3 region of a reference Fc domain: 234, 235, 253, 297, 298, 310, 329, 333, 334, or 435. In some embodiments, the variant Fc domain comprises a CH2-CH3 region that comprises an amino acid difference at position 329 relative to the amino acid sequence of the CH2-CH3 region of a reference Fc domain. In some embodiments, the variant Fc domain comprises a CH2-CH3 region that comprises amino acid differences at positions 234, 235, and 329 relative to the amino acid sequence of the CH2-CH3 region of the reference Fc domain. In some embodiments, the variant Fc domain comprises a CH2-CH3 region that comprises amino acid differences at positions 298, 333, and 334 relative to the amino acid sequence of the CH2-CH3 region of the reference Fc domain.

[0187] In some embodiments, the variant Fc domain comprises a CH2-CH3 region comprising an amino acid difference at one or more of the following positions relative to the amino acid sequence of the CH2-CH3 region of a reference Fc domain: E233, L234, L235, 1253, N297, S298, H310, P329, P331, E333, K334, or H435 (according to the EU numbering system). In some embodiments, the variant Fc domain comprises a CH2-CH3 region comprising an amino acid difference at one or more of the following positions relative to the amino acid sequence of the CH2-CH3 region of a reference Fc domain: L234, L235, 1253, N297, S298, H310, P329, E333, K334, or H435. In some embodiments, the variant Fc domain comprises a CH2-CH3 region comprising an amino acid difference at P329 relative to the amino acid sequence of the CH2-CH3 region of a reference Fc domain. In some embodiments, the variant Fc domain comprises a CH2-CH3 region comprising amino acid differences at positions L234, L235, and P329 relative to the amino acid sequence of the CH2-CH3 region of the reference Fc domain, hi some embodiments, the variant Fc domain comprises a CH2-CH3 region comprising amino acid differences at positions S298, E333, and K334 relative to the amino acid sequence of the CH2-CH3 region of the reference Fc domain.

[0188] In some embodiments, the variant Fc domain comprises a CH2-CH3 region comprising one or more of the following specific amino acid residues: P233, A234, A235, A253, A297, A298, A310, G329, S331, A333, A334, or A435 (according to the EU numbering system). In some embodiments, the variant Fc domain comprises a CH2-CH3 region comprising one or more of the following specific amino acid residues: A234, A235, A253, A297, A298, A310, G329, A333, A334, or A435. In some embodiments, the variant Fc domain comprises a CH2-CH3 region comprising G329. In some embodiments, the variant Fc domain comprises a CH2-CH3 region comprising A234, A235, and G329. In some embodiments, the variant Fc domain comprises a CH2-CH3 region comprising A298, A333 and A334.

[0189] In some embodiments, the variant Fc domain comprises a CH2-CH3 region that comprises one or more of the following amino acid substitutions relative to the amino acid sequence of the CH2-CH3 region of a reference Fc domain: E233P, L234A, L235A, I253A, N297A, S298A, H310A, P329G, P331S, E333A, K334A, or H435A (according to the EU numbering system). In some embodiments, the variant Fc domain comprises a CH2-CH3 region that comprises one or more of the following amino acid substitutions relative to the amino acid sequence of the CH2-CH3 region of a reference Fc domain: L234A, L235A, I253A, N297A, S298A, H310A, P329G, E333A, K334A, or H435A. In some embodiments, the variant Fc domain comprises a CH2-CH3 region that comprises the amino acid substitution P329G relative to the amino acid sequence of the CH2-CH3 region of the reference Fc domain. In some embodiments, the variant Fc domain comprises a CH2-CH3 region that comprises the amino acid substitutions L234A, L235A, and P329G relative to the amino acid sequence of the CH2-CH3 region of the reference Fc domain. In some embodiments, the variant Fc domain comprises a CH2-CH3 region that comprises the amino acid substitutions S298A, E333A, and K334A relative to the amino acid sequence of the CH2-CH3 region of the reference Fc domain.

[0190] In some embodiments, a variant Fc domain according to the disclosure comprises a polypeptide comprising a CH2-CH3 region comprising or consisting of an amino acid sequence having at least 70% sequence identity, more preferably at least ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 211 or 214, wherein the CH2-CH3 region comprises G329. In some embodiments, a variant Fc domain according to the disclosure comprises two polypeptides, each polypeptide comprising a CH2-CH3 region comprising or consisting of an amino acid sequence having at least 70% sequence identity, more preferably at least ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 211 or 214, wherein the CH2-CH3 region comprises G329. In some embodiments, a variant Fc domain according to the disclosure comprises one or more (e.g., two) polypeptides comprising the amino acid sequence of SEQ ID NO: 211 or 214.

[0191] In some embodiments, a variant Fc domain according to the disclosure comprises a polypeptide comprising a CH2-CH3 region comprising or consisting of an amino acid sequence having at least 70% sequence identity, more preferably at least ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 213 or 216, wherein the CH2-CH3 region comprises A234, A235 and G329. In some embodiments, a variant Fc domain according to the disclosure comprises two polypeptides, each polypeptide comprising a CH2-CH3 region comprising or consisting of an amino acid sequence having at least 70% sequence identity, more preferably at least ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 213 or 216, wherein the CH2-CH3 region comprises A234, A235 and G329. In some embodiments, a variant Fc domain according to the disclosure comprises one or more (e.g., two) polypeptides comprising the amino acid sequence of SEQ ID NO: 213 or 216.

[0192] In some embodiments, a variant Fc domain according to the present disclosure comprises a polypeptide comprising a CH2-CH3 region comprising or consisting of an amino acid sequence having at least 70% sequence identity, more preferably at least ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 212 or 215, wherein the CH2-CH3 region comprises A298, A333 and A334. In some embodiments, a variant Fc domain according to the disclosure comprises two polypeptides, each polypeptide comprising a CH2-CH3 region comprising or consisting of an amino acid sequence having at least 70% sequence identity, more preferably at least ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 212 or 215, wherein the CH2-CH3 region comprises A298, A333, and A334. In some embodiments, a variant Fc domain according to the disclosure comprises one or more (e.g., two) polypeptides comprising the amino acid sequence of SEQ ID NO: 212 or 215.

[0193] Complex Polypeptides Aspects and embodiments of the present disclosure relate to composite polypeptides.

[0194] A composite polypeptide of the present disclosure comprises an amino acid sequence encoding at least two recombinant CD3-TCR complex polypeptides according to the present disclosure. The amino acid sequences of the recombinant CD3-TCR complex polypeptides of the composite polypeptide are provided in tandem. A cleavage site may be provided in the composite polypeptide between the amino acid sequences of the recombinant CD3-TCR complex polypeptides, as described herein below.

[0195] In some embodiments, the individual recombinant CD3-TCR complex polypeptides included in a composite polypeptide of the present disclosure are not identical. In some embodiments, the recombinant CD3-TCR complex polypeptides included in a composite polypeptide of the present disclosure are complementary. That is, in some embodiments, the recombinant CD3-TCR complex polypeptides included in a composite polypeptide of the present disclosure can associate with each other (e.g., via non-covalent protein:protein interactions) to form a polypeptide complex (e.g., a polypeptide complex described herein).

[0196] In further preferred embodiments, individual recombinant CD3-TCR complex polypeptides included in a composite polypeptide of the present disclosure are capable of associating with one another to form an antigen-binding portion according to the present disclosure. In some embodiments, recombinant CD3-TCR complex polypeptides included in a composite polypeptide of the present disclosure comprise complementary components of an antigen-binding portion according to the present disclosure. The complementary components of the antigen-binding portion are capable of associating (e.g., via non-covalent protein:protein interactions) to form the antigen-binding portion. In some embodiments, association between recombinant CD3-TCR complex polypeptides included in a composite polypeptide of the present disclosure results in the formation of an antigen-binding portion according to the present disclosure. In some embodiments, a recombinant CD3-TCR complex polypeptide of a composite polypeptide of the present disclosure comprises a VH region of an antigen-binding portion that binds to a variant Fc domain (e.g., as described herein), and another CD3-TCR complex polypeptide of the composite polypeptide comprises a VL region of an antigen-binding portion.

[0197] As an example, in the embodiments described herein, the composite polypeptide comprises (i) a recombinant CD3-TCR complex polypeptide comprising a VH region of an antigen-binding portion specific for a variant Fc domain and the ECD, TMD, and ICD of TRAC(T47C), and (ii) a recombinant CD3-TCR complex polypeptide comprising a VL region of an antigen-binding portion specific for a variant Fc domain and the ECD, TMD, and ICD of TRBC1(S56C), and (iii) a T2A cleavage site between polypeptides (i) and (ii). After cleavage of the composite polypeptide, polypeptides (i) and (ii) associate to form a polypeptide complex comprising an Fv specific for the variant Fc domain formed by the VH from (i) and the VL from (ii).

[0198] Linkers, Labels and Conjugates Polypeptides of the present disclosure (eg, recombinant CD3-TCR complex polypeptides and complex polypeptides) may further comprise additional amino acids or amino acid sequences.

[0199] Polypeptides may contain one or more linker sequences between amino acid sequences. By way of example, a linker sequence may be provided between different domains of a recombinant CD3-TCR complex polypeptide (e.g., between an antigen-binding portion and a CD3-TCR complex-associated domain). As a further example, a linker sequence may be provided between subsequences of domains of a recombinant CD3-TCR complex polypeptide (e.g., between the VH and VL regions of an antigen-binding portion).

[0200] Linker sequences are known to those skilled in the art and are described, for example, in Chen et al., Adv Drug Deliv Rev (2013) 65 (10): 1357-1369, the entire contents of which are incorporated herein by reference. In some embodiments, the linker sequence can be a flexible linker sequence. A flexible linker sequence allows the relative movement of the amino acid sequences connected by the linker sequence. Flexible linkers are known to those skilled in the art, and some are identified in Chen et al., Adv Drug Deliv Rev (2013) 65 (10): 1357-1369. Flexible linker sequences often contain a high proportion of glycine and / or serine residues.

[0201] In some embodiments, the linker sequence comprises at least one glycine residue and / or at least one serine residue. In some embodiments, the linker sequence comprises or consists of glycine and serine residues. In some embodiments, the linker sequence has the following structure: (GxS)n or (GxS)nGm; where G = glycine, S = serine, x = 3 or 4, n = 2, 3, 4, 5, or 6, and m = 0, 1, 2, or 3. In some embodiments, the linker sequence comprises one or more (e.g., 1, 2, 3, 4, 5, or 6) copies (e.g., in tandem) of the sequence motif G4S. In some embodiments, the linker sequence comprises or consists of (G4S)3 or (G4S)4. In some embodiments, the linker sequence has a length of 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 10, 1 to 15, 1 to 20, 1 to 25, or 1 to 30 amino acids.

[0202] In some embodiments, the linker sequence comprises one or more copies of the amino acid sequence according to SEQ ID NO: 92. In some embodiments, the linker sequence comprises at least 1, 2, 3, or 4 copies of the amino acid sequence according to SEQ ID NO:92.

[0203] In some embodiments, the linker sequence comprises or consists of an amino acid sequence having at least 60%, preferably ≧70%, ≧75%, ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99% or 100% amino acid sequence identity to SEQ ID NO:93.

[0204] In some embodiments, the linker sequence comprises a cleavage site, such as a cleavage site described herein below.

[0205] Polypeptides of the present disclosure may comprise an amino acid sequence(s) to facilitate expression, folding, transport, processing, purification, or detection of the antigen-binding molecule / polypeptide. For example, a recombinant CD3-TCR complex polypeptide of the present disclosure may further comprise a sequence of amino acids that forms a detectable moiety, e.g., as described herein below.

[0206] Polypeptides may further contain a signal peptide (also known as a leader sequence or signal sequence). Signal peptides typically consist of a sequence of 5 to 30 hydrophobic amino acids that form a single alpha helix. Secretory proteins and proteins expressed on the cell surface often contain signal peptides. Signal peptides are known for many proteins and are recorded in databases such as GenBank, UniProt, and Ensembl, and / or can be identified / predicted using amino acid sequence analysis tools such as SignalP (Petersen et al., 2011 Nature Methods 8:785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24:2172-2176).

[0207] The signal peptide may be present at the N-terminus of the recombinant CD3-TCR complex polypeptide or in a newly synthesized polypeptide. The signal peptide provides efficient transport of the recombinant CD3-TCR complex polypeptide. The signal peptide is often removed by cleavage and therefore is not included in the mature recombinant CD3-TCR complex polypeptide.

[0208] Signal peptides are known for many proteins and are recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensembl, and InterPro, and / or can be identified / predicted using amino acid sequence analysis tools such as, for example, SignalP (Petersen et al., 2011 Nature Methods 8:785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24:2172-2176).

[0209] In some embodiments, the signal peptide comprises or consists of an amino acid sequence having at least 60%, preferably ≧70%, ≧75%, ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99% or 100% amino acid sequence identity to SEQ ID NO:98.

[0210] In some embodiments, polypeptides of the present disclosure (e.g., recombinant CD3-TCR complex polypeptides and complex polypeptides) comprise a detectable moiety. In some embodiments, the detectable moiety is provided at the N-terminus and / or C-terminus of the polypeptide.

[0211] In some embodiments, the detectable moiety is a fluorescent label, a phosphorescent label, a luminescent label, an immunodetectable label (e.g., an epitope tag), a radioactive label, a chemical label, a nucleic acid label, or an enzymatic label. The recombinant CD3-TCR complex polypeptide may be covalently or non-covalently labeled with a detectable moiety.

[0212] Fluorescent labels include, for example, fluorescein, rhodamine, allophycocyanin, eosin, and NDB, green fluorescent protein (GFP), enhanced GFP (eGFP), rare earth chelates such as europium (Eu), terbium (Tb), and samarium (Sm), tetramethylrhodamine, Texas Red, 4-methylumbelliferone, 7-amino-4-methylcoumarin, Cy3, and Cy5. Radioactive labels include hydrogen 3 ,sulfur 35 ,carbon 14 , Phosphorus 32 , iodine 123 , iodine 125 , iodine 126 , iodine 131 , iodine 133 ,bromine 77 ,technetium 99m ,indium 111 ,indium 113m ,gallium 67 ,gallium 68 ,ruthenium 95 ,ruthenium 97 ,ruthenium 103 ,ruthenium 105 ,mercury 207 ,mercury 203 ,rhenium 99m ,rhenium 101 ,rhenium 105 ,scandium 47 , tellurium 121m , tellurium 122m , tellurium 125m ,thulium 165 ,thulium 167 ,thulium 168 ,copper 67 , fluorine 18 ,yttrium 90 ,palladium 100 , bismuth217 and antimony 211 Examples of luminescent labels include radioactive isotopes such as fluoroisotopes, ...

[0213] In some embodiments, the recombinant CD3-TCR complex polypeptide comprises an epitope tag, such as His, (e.g., 6XHis), FLAG, c-Myc, StrepTag, hemagglutinin, E, calmodulin-binding protein (CBP), glutathione-S-transferase (GST), maltose-binding protein (MBP), thioredoxin, S-peptide, T7 peptide, SH2 domain, avidin, streptavidin, and a hapten (e.g., biotin, digoxigenin, dinitrophenol), optionally at the N-terminus or C-terminus of the recombinant CD3-TCR complex polypeptide.

[0214] In some embodiments, the recombinant CD3-TCR complex polypeptide comprises a moiety having a detectable activity, such as an enzymatic moiety, including, for example, luciferase, glucose oxidase, galactosidase (e.g., beta-galactosidase), glucorinidase, phosphatase (e.g., alkaline phosphatase), peroxidase (e.g., horseradish peroxidase), and cholinesterase.

[0215] In some embodiments, a polypeptide of the present disclosure comprises a fluorescent label. In some embodiments, the polypeptide comprises an eGFP moiety. In some embodiments, the polypeptide comprises an amino acid sequence having at least 60%, preferably ≧70%, ≧75%, ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99%, or 100% amino acid sequence identity to SEQ ID NO: 103.

[0216] Polypeptides of the present disclosure may also comprise one or more cleavage sites, which refers to a sequence of amino acids that acts as a substrate for an enzyme capable of cleaving a peptide bond.

[0217] Many such cleavage sites are known to and can be used by those skilled in the art of molecular biology. In some embodiments, the cleavage sequence comprises a self-cleavage site. Self-cleavage sites include the 2A cleavage sequence from the picornavirus "NPGP," which cleaves at "G / P." Additional self-cleavage sites are described, for example, in Kim et al., PLoS ONE (2011) 6:e18556 (incorporated herein by reference in its entirety), including, for example, T2A, P2A, E2A, and F2A cleavage sites. The amino acid sequences of the T2A, P2A, E2A, and F2A cleavage sites are set forth in SEQ ID NOs: 104, 220, 109, and 221, respectively.

[0218] Cleavage sites can be included in polypeptides according to the present disclosure to provide for removal of a portion or domain. It may be desirable to remove a given portion or domain so that it is not included in the polypeptide complex formed by the polypeptide. For example, in embodiments of recombinant CD3-TCR complex polypeptides according to the present disclosure, a cleavage site (specifically, a T2A cleavage site) is provided upstream of the eGFP portion to provide for its removal from being included in a CD3-TCR complex comprising the CD3-TCR complex polypeptide. Thus, in some embodiments, polypeptides according to the present disclosure include a cleavage site adjacent to a detectable portion according to the present disclosure (i.e., in the amino acid sequence of the polypeptide, e.g., immediately upstream or immediately downstream of the polypeptide).

[0219] Cleavage sites can be included in composite polypeptides according to the present disclosure to provide for separation of recombinant CD3-TCR complex polypeptides contained in the composite polypeptide. For example, in composite polypeptide embodiments of the present disclosure, a cleavage site (e.g., a T2A cleavage site) is provided between amino acid sequences encoding complementary recombinant CD3-TCR complex polypeptides, providing for physical separation of the recombinant CD3-TCR complex polypeptides. Thus, in some embodiments, a polypeptide according to the present disclosure comprises a cleavage site adjacent to the amino acid sequence encoding a recombinant CD3-TCR complex polypeptide according to the present disclosure (i.e., in the amino acid sequence of the polypeptide, e.g., immediately upstream or immediately downstream of the polypeptide).

[0220] In some embodiments, the cleavage site according to the present disclosure is a 2A cleavage site, for example, selected from T2A, P2A, E2A, and F2A cleavage sites. In some embodiments, the cleavage site is a T2A cleavage site.

[0221] In some embodiments, a polypeptide according to the disclosure comprises an amino acid sequence having at least 60%, preferably ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to SEQ ID NO: 104, 220, 109, or 221. In some embodiments, a polypeptide according to the disclosure comprises an amino acid sequence having at least 60%, preferably ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to SEQ ID NO: 104.

[0222] Polypeptide Complex The present disclosure also provides polypeptide complexes. A "polypeptide complex" refers to a molecule formed by non-covalent protein:protein interactions between two or more polypeptides. In some embodiments, the association involves electrostatic interactions (e.g., ionic bonds, hydrogen bonds) and / or van der Waals forces.

[0223] Aspects and embodiments of the present disclosure relate to polypeptide complexes formed by association between non-identical complementary recombinant CD3-TCR complex polypeptides according to the present disclosure.

[0224] In some embodiments, the constituent polypeptides of such polypeptide complexes comprise complementary CD3-TCR complex association domains. In some embodiments, the association between the constituent polypeptides of such polypeptide complexes comprises an interaction between the CD3-TCR complex association domains of the recombinant CD3-TCR complex polypeptides. For example, one of the recombinant CD3-TCR complex polypeptides may comprise a CD3-TCR complex association domain derived from TRAC, and the other may comprise a CD3-TCR complex association domain derived from TRBC1 or TRBC2. In some embodiments, the CD3-TCR complex association domains of the recombinant CD3-TCR complex polypeptides of such complexes comprise complementary modifications to promote their association. In some embodiments, the CD3-TCR complex association domains of the recombinant CD3-TCR complex polypeptides comprise modifications that introduce cysteine ​​residues for interchain disulfide bond formation between the CD3-TCR complex association domains.

[0225] For example, a polypeptide complex according to the present disclosure may include (i) a recombinant CD3-TCR complex polypeptide comprising a CD3-TCR complex association domain derived from TRAC, further comprising the modification T47C, and (ii) a recombinant CD3-TCR complex polypeptide comprising a CD3-TCR complex association domain derived from TRBC1 or TRBC2, further comprising the modification S56C.

[0226] In some embodiments, the constituent polypeptides of such polypeptide complexes comprise complementary components of antigen-binding portions specific for a variant Fc domain (e.g., as described herein). In some embodiments, the association between the constituent polypeptides comprises an interaction between individual components of the antigen-binding portions provided by different recombinant CD3-TCR complex polypeptides. For example, one of the recombinant CD3-TCR complex polypeptides may comprise a VH region of an antigen-binding portion specific for a variant Fc domain, and the other may comprise a VL region of the antigen-binding portion.

[0227] Aspects and embodiments of the present disclosure also relate to CD3-TCR polypeptide complexes.

[0228] A CD3-TCR polypeptide complex according to the present disclosure comprises a recombinant CD3-TCR complex polypeptide according to the present disclosure. In some embodiments, a CD3-TCR polypeptide complex can comprise one or more (e.g., two, three, four or more) recombinant CD3-TCR complex polypeptides according to the present disclosure.

[0229] In some aspects and embodiments where the CD3-TCR polypeptide complex comprises multiple recombinant CD3-TCR complex polypeptides according to the present disclosure, the recombinant CD3-TCR complex polypeptides may be identical.

[0230] In some aspects and embodiments, the CD3-TCR polypeptide complex comprises a non-identical recombinant CD3-TCR complex polypeptide according to the present disclosure. In some aspects and embodiments, the CD3-TCR polypeptide complex comprises a polypeptide complex as described above (i.e., a polypeptide complex formed by the association of complementary recombinant CD3-TCR complex polypeptides according to the present disclosure). That is, the polypeptide complex may be a CD3-TCR polypeptide complex or may form part of a CD3-TCR polypeptide complex.

[0231] It will be understood that in addition to (a) recombinant CD3-TCR complex polypeptide(s), a CD3-TCR polypeptide complex according to the present disclosure may include (b) one or more additional CD3-TCR complex polypeptides selected from, for example, TCRα, TCRβ, TCRγ, TCRδ, TRAC, TRBC1, TRBC2, TRGC1, TRGC2, TRDC, CD3ε, CD3δ, CD3γ, CD3ζ, and CD3η.

[0232] A CD3-TCR polypeptide complex according to the present disclosure may optionally be characterized by its ability to induce one or more of the following by an immune cell (e.g., a T cell) expressing the CD3-TCR polypeptide complex in response to an antigen for which the CD3-TCR polypeptide complex comprises an antigen-binding portion (or in response to a cell containing or expressing the antigen): CD3-TCR complex-mediated signaling, proliferation / population expansion, growth factor (e.g., IL-2) expression, IFNγ expression, CD107a expression, TNFα expression, GM-CSF expression, perforin expression, granzyme expression, granulysin expression, and / or FAS ligand (FASL) expression.

[0233] For example, an immune cell (e.g., a T cell) expressing a CD3-TCR polypeptide complex according to the present disclosure may be able to elicit one or more of the functional properties listed in the previous paragraph in response to a variant Fc domain according to the present disclosure (i.e., a variant Fc domain bound by a recombinant CD3-TCR complex polypeptide / antigen-binding portion of the complex comprised in the CD3-TCR polypeptide complex), or in response to a cell comprising / expressing such a variant Fc domain.

[0234] Certain Exemplary CD3-TCR Complex Polypeptides, Complex Polypeptides, and Polypeptide Complexes In some embodiments, a CD3-TCR complex polypeptide according to the present disclosure comprises or consists of one of the following structures: N-terminus - [signal peptide] - [antigen-binding portion or its component] - [CD3-TCR complex-associated domain] - C-terminus N-terminus - [antigen-binding portion or its component] - [CD3-TCR complex-associated domain] - C-terminus N-terminus - [signal peptide] - [antigen-binding portion or component thereof] - [CD3-TCR complex-associated domain] - [cleavage site] - [detectable portion] - C-terminus N-terminus - [antigen-binding portion or component thereof] - [CD3-TCR complex-associated domain] - [cleavage site] - [detectable portion] - C-terminus

[0235] In some embodiments, a composite polypeptide according to the present disclosure comprises or consists of one of the following structures: N-terminus - [signal peptide] - [antigen-binding component] - [CD3-TCR complex-associated domain] - [cleavage site] - [signal peptide] - [antigen-binding component] - [CD3-TCR complex-associated domain] - C-terminus N-terminus - [signal peptide] - [antigen-binding component] - [CD3-TCR complex-associated domain] - [cleavage site] - [signal peptide] - [antigen-binding component] - [CD3-TCR complex-associated domain] - [cleavage site] - [detectable moiety] - C-terminus

[0236] In some embodiments, a CD3-TCR complex polypeptide according to the present disclosure comprises or consists of (e.g., from N-terminus to C-terminus): (1)(i) an amino acid sequence encoding a signal peptide, e.g., an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to SEQ ID NO: 98; (ii) an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to a sequence selected from column A of Table 1; and (iii) an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity with a sequence selected from column B of Table 1; The sequence selected from column A of Table 1 and the sequence selected from column B of Table 1 are selected from the same row of Table 1. (2)(i) an amino acid sequence having at least 70%, preferably ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99%, or 100% amino acid sequence identity to a sequence selected from column A of Table 1; and (ii) an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity with a sequence selected from column B of Table 1; The sequence selected from column A of Table 1 and the sequence selected from column B of Table 1 are selected from the same row of Table 1. (3)(i) an amino acid sequence encoding a signal peptide, e.g., an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to SEQ ID NO: 98; (ii) an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity with a sequence selected from column A of Table 1; (iii) an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity with a sequence selected from column B of Table 1; (iv) an amino acid sequence encoding a cleavage site, e.g., an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to SEQ ID NO: 104; and (v) an amino acid sequence encoding a detectable moiety, e.g., an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to SEQ ID NO: 103; The sequence selected from column A of Table 1 and the sequence selected from column B of Table 1 are selected from the same row of Table 1. (4)(i) an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to a sequence selected from column A of Table 1; (ii) an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity with a sequence selected from column B of Table 1; (iii) an amino acid sequence encoding a cleavage site, e.g., an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to SEQ ID NO: 104; and (iv) an amino acid sequence encoding a detectable moiety, e.g., an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to SEQ ID NO: 103; The sequence selected from column A of Table 1 and the sequence selected from column B of Table 1 are selected from the same row of Table 1. [Table 1]

[0237] In some embodiments, a CD3-TCR complex polypeptide according to the disclosure comprises or consists of an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to one of SEQ ID NOs: 110-173. In some embodiments, a CD3-TCR complex polypeptide according to the disclosure comprises or consists of an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to SEQ ID NO: 121.

[0238] In some embodiments, a composite polypeptide according to the present disclosure comprises or consists of (e.g., from N-terminus to C-terminus): (1)(i) an amino acid sequence encoding a signal peptide, e.g., an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to SEQ ID NO: 98; (ii) an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to a sequence selected from column A of Table 2; and (iii) an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity with a sequence selected from column B of Table 2; (iv) an amino acid sequence encoding a cleavage site, e.g., an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to SEQ ID NO: 104; (v) an amino acid sequence encoding a signal peptide, e.g., an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to SEQ ID NO: 98; (vi) an amino acid sequence having at least 70%, preferably ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99%, or 100% amino acid sequence identity with a sequence selected from column C of Table 2; and (vii) an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity with a sequence selected from column D of Table 2; The sequence selected from column A of Table 2 and the sequence selected from column B of Table 2, and the sequence selected from column C of Table 2 and the sequence selected from column D of Table 2 are selected from the same row of Table 2. (2)(i) an amino acid sequence encoding a signal peptide, e.g., an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to SEQ ID NO: 98; (ii) an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to a sequence selected from column A of Table 2; and (iii) an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity with a sequence selected from column B of Table 2; (iv) an amino acid sequence encoding a cleavage site, e.g., an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to SEQ ID NO: 104; (v) an amino acid sequence encoding a signal peptide, e.g., an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to SEQ ID NO: 98; (vi) an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity with a sequence selected from column C of Table 2; (vii) an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity with a sequence selected from column D of Table 2; (viii) an amino acid sequence encoding a cleavage site, e.g., an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to SEQ ID NO: 104; and (ix) an amino acid sequence encoding a detectable moiety, e.g., an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to SEQ ID NO: 103; The sequence selected from column A of Table 2 and the sequence selected from column B of Table 2, and the sequence selected from column C of Table 2 and the sequence selected from column D of Table 2 are selected from the same row of Table 2. [Table 2] TIFF2025530146000005.tif37170

[0239] In some embodiments, a composite polypeptide according to the present disclosure comprises or consists of an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to one of SEQ ID NOs: 174-205. In some embodiments, a composite polypeptide according to the present disclosure comprises or consists of an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to SEQ ID NO: 178. In some embodiments, a composite polypeptide according to the present disclosure comprises or consists of an amino acid sequence having at least 70%, preferably ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99% or 100% amino acid sequence identity to SEQ ID NO: 186.

[0240] In some embodiments, a polypeptide complex according to the present disclosure comprises a CD3-TCR complex polypeptide according to an embodiment described herein.

[0241] In some embodiments, a polypeptide complex according to the present disclosure comprises: (a) a polypeptide comprising (e.g., from N-terminus to C-terminus): (i) an amino acid sequence having at least 70%, preferably ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99%, or 100% amino acid sequence identity to a sequence selected from column A of Table 2; and (ii) an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity with a sequence selected from column B of Table 2; and (b) a polypeptide comprising (e.g., from N-terminus to C-terminus): (i) an amino acid sequence having at least 70%, preferably ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99%, or 100% amino acid sequence identity with a sequence selected from column C of Table 2; and (ii) an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity with a sequence selected from column D of Table 2; The sequence selected from column A of Table 2 and the sequence selected from column B of Table 2, and the sequence selected from column C of Table 2 and the sequence selected from column D of Table 2 are selected from the same row of Table 2.

[0242] In some embodiments, a polypeptide complex according to the present disclosure comprises: (1)(i) a polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to SEQ ID NO: 129; and (ii) a polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to SEQ ID NO: 165; or (2)(i) a polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to SEQ ID NO: 133; and (ii) a polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to SEQ ID NO: 165; or (3)(i) a polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to SEQ ID NO: 137; and (ii) a polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to SEQ ID NO: 165; or (4)(i) a polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to SEQ ID NO: 145; and (ii) a polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to SEQ ID NO: 173; or (5)(i) a polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to SEQ ID NO: 141; and (ii) a polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to SEQ ID NO: 153; or (6)(i) a polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to SEQ ID NO: 141; and (ii) a polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to SEQ ID NO: 157; or (7)(i) a polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to SEQ ID NO: 141; and (ii) a polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to SEQ ID NO: 161; or (8)(i) a polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to SEQ ID NO: 149; and (ii) A polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to SEQ ID NO: 169.

[0243] In a preferred embodiment, a polypeptide complex according to the present disclosure comprises: (i) a polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99% or 100% amino acid sequence identity to SEQ ID NO: 137; and (ii) A polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to SEQ ID NO: 165.

[0244] In a preferred embodiment, a polypeptide complex according to the present disclosure comprises: (i) a polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99% or 100% amino acid sequence identity to SEQ ID NO: 141; and (ii) A polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to SEQ ID NO: 161.

[0245] Nucleic acids and vectors The present disclosure provides a nucleic acid or nucleic acids encoding a recombinant CD3-TCR complex polypeptide, complex polypeptide, or polypeptide complex according to the present disclosure. In some embodiments, the nucleic acid(s) comprise or consist of DNA and / or RNA.

[0246] A recombinant CD3-TCR complex polypeptide, composite polypeptide, or polypeptide complex according to the present disclosure can be produced intracellularly by translation of RNA encoding the recombinant CD3-TCR complex polypeptide, composite polypeptide, or polypeptide complex. A recombinant CD3-TCR complex polypeptide, composite polypeptide, or polypeptide complex according to the present disclosure can be produced intracellularly by transcription from a nucleic acid encoding the recombinant CD3-TCR complex polypeptide, composite polypeptide, or polypeptide complex, and subsequent translation of the transcribed RNA.

[0247] In some embodiments, the nucleic acid(s) may be or may be included / contained in a vector, or multiple vectors. As used herein, a "vector" is a nucleic acid molecule used as a vehicle to transfer exogenous nucleic acid into a cell.

[0248] Thus, the present disclosure also provides a vector or vectors comprising a nucleic acid or nucleic acids according to the present disclosure. The vector can facilitate delivery of a nucleic acid(s) encoding a recombinant CD3-TCR complex polypeptide, composite polypeptide, or polypeptide complex according to the present disclosure to a cell. The vector can be an expression vector containing elements necessary for expression of a recombinant CD3-TCR complex polypeptide, composite polypeptide, or polypeptide complex according to the present disclosure. The vector can contain elements that facilitate integration of the nucleic acid(s) into the genomic DNA of a cell into which the vector is introduced.

[0249] The nucleic acids and vectors according to the present disclosure can be provided in purified or isolated form, i.e., from other nucleic acids or naturally occurring biological materials.

[0250] The vector can be a vector for expressing a nucleic acid in a cell (i.e., an expression vector). Such a vector can include a promoter sequence operably linked to a nucleotide sequence encoding a recombinant CD3-TCR complex polypeptide, composite polypeptide, or polypeptide complex according to the present disclosure. The vector can also include a stop codon (i.e., 3' in the nucleotide sequence of the vector to the nucleotide sequence encoding the recombinant CD3-TCR complex polypeptide / composite polypeptide / polypeptide complex) and an expression enhancer. Any suitable vector, promoter, enhancer, and stop codon known in the art can be used to express a peptide or polypeptide from a vector according to the present disclosure.

[0251] The term "operably linked" can include a situation in which a nucleic acid encoding a recombinant CD3-TCR complex polypeptide, composite polypeptide, or polypeptide complex of the present disclosure and a regulatory nucleic acid sequence(s) (e.g., a promoter and / or enhancer) are covalently linked in such a manner that expression of the nucleic acid encoding the recombinant CD3-TCR complex polypeptide, composite polypeptide, or polypeptide complex is under the influence or control of the regulatory nucleic acid sequence(s) (thereby forming an expression cassette). Thus, a regulatory sequence is operably linked to a selected nucleic acid sequence if the regulatory sequence is capable of effecting transcription of the nucleic acid sequence. The resulting transcript(s) can then be translated into the desired polypeptide(s).

[0252] Vectors contemplated in connection with the present disclosure include DNA vectors, RNA vectors, plasmids (e.g., conjugative plasmids (e.g., F plasmids), non-conjugative plasmids, R plasmids, col plasmids, episomes), viral vectors (e.g., retroviral vectors, e.g., gamma retroviral vectors (e.g., murine leukemia virus (MLV)-derived vectors, e.g., SFG vectors), lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, vaccinia viral vectors, and herpes viral vectors), transposon-based vectors, and artificial chromosomes (e.g., yeast artificial chromosomes), as described, for example, in Maus et al., Annu Rev Immunol (2014) 32:189-225 and Morgan and Boyerinas, Biomedicines (2016) 4:9, both of which are incorporated herein by reference in their entireties. In some embodiments, a vector according to the present disclosure is a lentiviral vector.

[0253] In some embodiments, the vector can be a eukaryotic vector, i.e., a vector that contains elements necessary for expression of a protein from the vector in a eukaryotic cell, hi some embodiments, the vector can be a mammalian vector that includes, for example, a cytomegalovirus (CMV) or SV40 promoter driving protein expression.

[0254] In some embodiments, the nucleic acid(s) or vector(s) according to the present disclosure comprise an EF1α promoter.

[0255] In some embodiments, the nucleic acid(s) or vector(s) according to the present disclosure encode a CD3-TCR complex polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to one of SEQ ID NOs: 110-173. In some embodiments, the nucleic acid(s) or vector(s) according to the present disclosure encode a CD3-TCR complex polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to SEQ ID NO: 121.

[0256] In a preferred embodiment, the nucleic acid(s) or vector(s) according to the present disclosure comprise the nucleotide sequence of SEQ ID NO:224, or a codon-degenerate nucleotide sequence thereof that encodes the amino acid sequence encoded by SEQ ID NO:224.

[0257] As used herein, a "codon-degenerate nucleotide sequence" of a reference nucleotide sequence refers to a nucleotide sequence that has a nucleotide sequence that is not identical to the nucleotides of the reference nucleotide sequence, but that, as a result of the degeneracy of the genetic code, encodes the same amino acid sequence as the amino acid sequence encoded by the reference nucleotide sequence.

[0258] The constituent polypeptides of a polypeptide complex according to the present disclosure can be encoded by different nucleic acids of a plurality of nucleic acids according to the present disclosure or by different vectors of a plurality of nucleic acids according to the present disclosure.

[0259] In aspects and embodiments of the disclosure, a nucleic acid or nucleic acids according to the disclosure encode two or more (e.g., two, three, four or more) recombinant CD3-TCR complex polypeptides according to the disclosure. In aspects and embodiments of the disclosure, a vector or vectors according to the disclosure encode two or more (e.g., two, three, four or more) recombinant CD3-TCR complex polypeptides according to the disclosure.

[0260] In some embodiments where the nucleic acid(s) or vector(s) encode two or more (e.g., 2, 3, 4 or more) recombinant CD3-TCR complex polypeptides, the recombinant CD3-TCR complex polypeptides are not identical. In some embodiments, the nucleic acid(s) or vector(s) encode complementary recombinant CD3-TCR complex polypeptides. That is, in some embodiments, the nucleic acid(s) or vector(s) encode CD3-TCR complex polypeptides that can associate with each other (e.g., via non-covalent protein:protein interactions) to form a polypeptide complex (e.g., a polypeptide complex described herein).

[0261] In some embodiments, the nucleic acid(s) or vector(s) encode CD3-TCR complex polypeptides that can associate with each other to form an antigen-binding portion according to the present disclosure. In some embodiments, the nucleic acid(s) or vector(s) encode CD3-TCR complex polypeptides that comprise complementary components of an antigen-binding portion according to the present disclosure (i.e., components of an antigen-binding portion that can associate (e.g., via non-covalent protein:protein interactions) to form the antigen-binding portion).

[0262] By way of illustration, in one embodiment, the nucleic acid(s) or vector(s) encode (i) a recombinant CD3-TCR complex polypeptide comprising a VH region of an antigen-binding portion specific for the variant Fc domain and the ECD, TMD, and ICD of TRAC(T47C), and (ii) a recombinant CD3-TCR complex polypeptide comprising a VL region of an antigen-binding portion specific for the variant Fc domain and the ECD, TMD, and ICD of TRBC1(S56C). After expression from the nucleic acid(s) or vector(s), polypeptides (i) and (ii) associate to form a polypeptide complex comprising an Fv specific for the variant Fc domain formed by the VH from (i) and the VL from (ii).

[0263] In some embodiments, the nucleic acid(s) or vector(s) according to the present disclosure encodes: (1)(i) a polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to SEQ ID NO: 129; and (ii) a polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to SEQ ID NO: 165; or (2)(i) a polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to SEQ ID NO: 133; and (ii) a polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to SEQ ID NO: 165; or (3)(i) a polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to SEQ ID NO: 137; and (ii) a polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to SEQ ID NO: 165; or (4)(i) a polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to SEQ ID NO: 145; and (ii) a polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to SEQ ID NO: 173; or (5)(i) a polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to SEQ ID NO: 141; and (ii) a polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to SEQ ID NO: 153; or (6)(i) a polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to SEQ ID NO: 141; and (ii) a polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to SEQ ID NO: 157; or (7)(i) a polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to SEQ ID NO: 141; and (ii) a polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to SEQ ID NO: 161; or (8)(i) a polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to SEQ ID NO: 149; and (ii) A polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to SEQ ID NO: 169.

[0264] In a preferred embodiment, the nucleic acid(s) or vector(s) according to the present disclosure encode: (i) a polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99% or 100% amino acid sequence identity to SEQ ID NO: 137; and (ii) A polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to SEQ ID NO: 165.

[0265] In a preferred embodiment, the nucleic acid(s) or vector(s) according to the present disclosure encode: (i) a polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99% or 100% amino acid sequence identity to SEQ ID NO: 141; and (ii) A polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to SEQ ID NO: 161.

[0266] In a preferred embodiment, the nucleic acid(s) or vector(s) according to the present disclosure comprise: (i) the nucleotide sequence of SEQ ID NO: 225, or a codon-degenerate nucleotide sequence thereof encoding the amino acid sequence encoded by SEQ ID NO: 225; and (i) The nucleotide sequence of SEQ ID NO: 226, or a codon-degenerate nucleotide sequence thereof that encodes the amino acid sequence encoded by SEQ ID NO: 226.

[0267] In a preferred embodiment, the nucleic acid(s) or vector(s) according to the present disclosure comprise: (i) the nucleotide sequence of SEQ ID NO: 227, or a codon-degenerate nucleotide sequence thereof encoding the amino acid sequence encoded by SEQ ID NO: 227; and (i) The nucleotide sequence of SEQ ID NO: 228, or a codon-degenerate nucleotide sequence thereof that encodes the amino acid sequence encoded by SEQ ID NO: 228.

[0268] In some embodiments, the nucleic acid(s) or vector(s) according to the present disclosure encode a composite polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to one of SEQ ID NOs: 174-205. In some embodiments, the nucleic acid(s) or vector(s) according to the present disclosure encode a composite polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity to SEQ ID NO: 178. In some embodiments, the nucleic acid(s) or vector(s) according to the present disclosure encode a composite polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99% or 100% amino acid sequence identity to SEQ ID NO: 186.

[0269] In some embodiments where the nucleic acid(s) or vector(s) encode two or more (e.g., two, three, four or more) recombinant CD3-TCR complex polypeptides according to the present disclosure, transcription of the nucleic acids encoding the two or more recombinant CD3-TCR complex polypeptides is under the control of the same promoter.

[0270] In some embodiments, the nucleic acid(s) or vector(s) comprises a nucleic acid encoding an internal ribosome entry site (IRES). In some embodiments, an IRES is provided between the nucleotide sequences encoding the recombinant CD3-TCR complex polypeptides. In some embodiments, the nucleic acid(s) or vector(s) comprises a nucleic acid that allows two or more recombinant CD3-TCR complex polypeptides to be translated separately from the same RNA transcript.

[0271] In some embodiments, the two or more recombinant CD3-TCR complex polypeptides are encoded by nucleotide sequences provided in the same reading frame. In some embodiments, the nucleic acid(s) or vector(s) encode a fusion protein comprising two or more recombinant CD3-TCR complex polypeptides. In some embodiments, the fusion protein encoded by the nucleic acid(s) or vector(s) comprises a cleavage site (e.g., a cleavage site described herein) between the amino acid sequences of the recombinant CD3-TCR complex polypeptides. In some embodiments, the nucleic acid(s) or vector(s) encode a composite polypeptide according to the present disclosure.

[0272] In some embodiments, transcription of nucleic acids encoding two or more recombinant CD3-TCR complex polypeptides is under the control of different promoters.

[0273] In some embodiments, the nucleic acid(s) or vector(s) are multicistronic (e.g., bicistronic, tricistronic, etc.). That is, in some embodiments, the nucleic acid(s) or vector(s) comprises nucleotide sequences encoding multiple polypeptides. In some embodiments, nucleic acids encoding two or more recombinant CD3-TCR complex polypeptides are provided in different cistrons.

[0274] Cells containing / expressing the polypeptides, polypeptide complexes and nucleic acids / vectors of the disclosure The present disclosure also provides a cell comprising a recombinant CD3-TCR complex polypeptide, complex polypeptide or polypeptide complex according to the present disclosure, or a nucleic acid(s) or vector(s) according to the present disclosure.

[0275] It will be understood that where a cell is referred to herein in the singular (ie, "a / the cell"), a plurality / population of such cells is also contemplated.

[0276] The cell may be a eukaryotic cell, for example, a mammalian cell. The mammal may be a primate (rhesus monkey, cynomolgus monkey, non-human primate, or human) or a non-human mammal (e.g., rabbit, guinea pig, rat, mouse, or other rodent (including any animal of the order Rodents), cat, dog, pig, sheep, goat, cow (including cows, e.g., dairy cows, or any animal of the order Bovidae), horse (including any animal of the order Equines), donkey, and non-human primate). In a preferred embodiment, the cell is a human cell.

[0277] In some embodiments, the cells are immune cells. The immune cells can be cells of hematopoietic origin, such as neutrophils, eosinophils, basophils, dendritic cells, lymphocytes, or monocytes. The lymphocytes can be, for example, T cells, B cells, NK cells, NKT cells, or innate lymphoid cells (ILCs), or their precursors. The immune cells can express one or more CD3-TCR complex polypeptides, such as TCRα, TCRβ, TCRγ, TCRδ, TRAC, TRBC1, TRBC2, TRGC1, TRGC2, TRDC, CD3ε, CD3δ, CD3γ, CD3ζ, and / or CD3η. The immune cells can express CD27, CD28, CD4, and / or CD8. In some embodiments, the immune cells are T cells, such as CD3+ T cells. In some embodiments, the T cells are CD3+, CD4+ T cells. In some embodiments, the T cells are CD3+, CD8+ T cells. In some embodiments, the T cells are T helper cells (T H In some embodiments, the T cell is a cytotoxic T cell (e.g., a cytotoxic T lymphocyte (CTL)).

[0278] Aspects and embodiments of the present disclosure relate in particular to T cells comprising / expressing a CD3-TCR complex comprising a recombinant CD3-TCR complex polypeptide according to the present disclosure.

[0279] In some aspects and embodiments, cells according to the present disclosure express / display recombinant CD3-TCR complex polypeptides or polypeptide complexes according to the present disclosure on the cell surface. That is, the recombinant CD3-TCR complex polypeptides or polypeptide complexes may be present in or at the cell membrane. Cells can be assessed for surface expression of CD3-TCR complex polypeptides and polypeptide complexes (e.g., after introduction of nucleic acids encoding same into the cells) using, for example, antibody-based methods such as flow cytometry (e.g., as described in Example 1 of the present disclosure).

[0280] In aspects and embodiments of the present disclosure, a cell according to the present disclosure comprises or expresses a recombinant CD3-TCR complex polypeptide, composite polypeptide, or polypeptide complex according to the present disclosure. In some aspects and embodiments, a cell according to the present disclosure comprises a nucleic acid encoding a recombinant CD3-TCR complex polypeptide, composite polypeptide, or polypeptide complex according to the present disclosure. In some aspects and embodiments, a cell according to the present disclosure comprises a nucleic acid(s) or vector(s) according to the present disclosure.

[0281] In aspects and embodiments of the present disclosure, cells according to the present disclosure contain or express a polypeptide complex according to the present disclosure that binds to a variant Fc domain described herein. It will be understood that binding to the variant Fc domain is achieved via the binding moiety of the recombinant CD3-TCR complex polypeptide / polypeptide complex. The cell may express / contain a polypeptide complex according to the present disclosure as a result of expression of a nucleic acid encoding such a polypeptide complex. The cell may also be engineered to contain a nucleic acid encoding such a polypeptide complex.

[0282] In some embodiments, cells according to the present disclosure may comprise a modification that reduces expression of a CD3-TCR complex polypeptide (i.e., compared to the level of expression of a CD3-TCR complex polypeptide by a comparable unmodified cell). In some embodiments, the cells comprise a modification to reduce expression of an endogenous CD3-TCR complex polypeptide, i.e., a CD3-TCR complex polypeptide encoded by the genome of a comparable unmodified cell.

[0283] In some embodiments, the cells comprise a modification that reduces expression of a CD3-TCR complex polypeptide from which the CD3-TCR complex association domain of the recombinant polypeptide CD3-TCR complex polypeptide is derived. By way of example, in embodiments in which the cells comprise or express a recombinant CD3-TCR complex polypeptide (or a composite polypeptide or polypeptide complex comprising such a recombinant CD3-TCR complex polypeptide) comprising a CD3-TCR complex association domain derived from CD3ε, the cells may comprise a modification that reduces expression of CD3ε (i.e., endogenous CD3ε) by the cells. By way of further example, in embodiments in which the cells comprise or express a recombinant CD3-TCR complex polypeptide(s) (or a composite polypeptide or polypeptide complex comprising such a recombinant CD3-TCR complex polypeptide(s)) comprising a CD3-TCR complex association domain(s) derived from TRAC, TRBC1, and / or TRBC2, the cells may comprise a modification that reduces expression of TRAC / TRBC1 / TRBC2 (i.e., endogenous TRAC / TRBC1 / TRBC2) by the cells.

[0284] In some embodiments, the cell comprises a modification to a nucleic acid (e.g., an endogenous nucleic acid) encoding a CD3-TCR complex polypeptide. In some embodiments, one or more alleles of a gene encoding a CD3-TCR complex polypeptide in the cell are modified. In some embodiments, the modification comprises an insertion, substitution, or deletion in the nucleotide sequence of the nucleic acid encoding the CD3-TCR complex polypeptide. In some embodiments, the modification reduces or prevents endogenous expression of the CD3-TCR complex polypeptide from the modified nucleotide sequence. In some embodiments, the modified cell lacks an endogenous nucleic acid encoding a CD3-TCR complex polypeptide. In some embodiments, the modification introduces a premature stop codon in the nucleotide sequence of an RNA transcribed from the endogenous nucleic acid encoding the CD3-TCR complex polypeptide. In some embodiments, the nucleotide sequence of the modified nucleic acid encodes a truncated and / or non-functional form of the CD3-TCR complex polypeptide. In some embodiments, the nucleotide sequence of the modified nucleic acid encodes a misfolded and / or degraded version of the CD3-TCR complex polypeptide. In some embodiments, the nucleotide sequence of the modified nucleic acid encodes a version of a CD3-TCR complex polypeptide that is unable to participate in a functional CD3-TCR polypeptide complex.

[0285] In some embodiments, the cell comprises a modification to a nucleic acid (e.g., an endogenous nucleic acid) encoding CD3ε (e.g., a polypeptide having the sequence of SEQ ID NO: 25). In some embodiments, one or more alleles of CD3E are modified. In some embodiments, the modification comprises an insertion, substitution, or deletion in the nucleotide sequence of CD3E. In some embodiments, the modification reduces or prevents endogenous expression of CD3ε by the cell. In some embodiments, the modified cell lacks an endogenous nucleic acid encoding CD3ε. In some embodiments, the modification introduces a premature stop codon in the nucleotide sequence of RNA transcribed from the endogenous nucleic acid encoding CD3ε. In some embodiments, the nucleotide sequence of the modified nucleic acid encodes a truncated and / or non-functional form of CD3ε. In some embodiments, the nucleotide sequence of the modified nucleic acid encodes a misfolded and / or degraded version of CD3ε. In some embodiments, the nucleotide sequence of the modified nucleic acid encodes a version of CD3ε that is unable to participate in a functional CD3-TCR polypeptide complex.

[0286] In some embodiments, the cells comprise a modification to a nucleic acid (e.g., an endogenous nucleic acid) encoding TRAC (e.g., a polypeptide having the sequence of SEQ ID NO: 1), TRBC1 (e.g., a polypeptide having the sequence of SEQ ID NO: 5), and / or TRBC2 (e.g., a polypeptide having the sequence of SEQ ID NO: 9). In some embodiments, the cells comprise a modification to a nucleic acid (e.g., an endogenous nucleic acid) encoding TRAC and TRBC1. In some embodiments, one or more alleles of TRAC, TRBC1, and / or TRBC2 (e.g., TRAC and TRBC1) are modified. In some embodiments, the modification comprises an insertion, substitution, or deletion in the nucleotide sequence of TRAC, TRBC1, and / or TRBC2 (e.g., TRAC and TRBC1). In some embodiments, the modification reduces or prevents endogenous expression of TRAC, TRBC1, and / or TRBC2 (e.g., TRAC and TRBC1) by the cells. In some embodiments, the modified cells lack endogenous nucleic acids encoding TRAC, TRBC1, and / or TRBC2 (e.g., TRAC and TRBC1). In some embodiments, the modification introduces a premature stop codon into the nucleotide sequence of RNA transcribed from an endogenous nucleic acid encoding TRAC, TRBC1 and / or TRBC2 (e.g., TRAC and TRBC1). In some embodiments, the nucleotide sequence of the modified nucleic acid encodes a truncated and / or non-functional form of TRAC, TRBC1 and / or TRBC2 (e.g., TRAC and TRBC1). In some embodiments, the nucleotide sequence of the modified nucleic acid encodes a misfolded and / or degraded version of TRAC, TRBC1 and / or TRBC2 (e.g., TRAC and TRBC1). In some embodiments, the nucleotide sequence of the modified nucleic acid encodes a version of TRAC, TRBC1 and / or TRBC2 (e.g., TRAC and TRBC1) that is unable to participate in a functional CD3-TCR polypeptide complex.

[0287] Immune cells (e.g., T cells) according to the present disclosure can be characterized by specific functional properties in response to an antigen (or in response to a cell containing or expressing an antigen) for which the CD3-TCR polypeptide complex comprises an antigen-binding portion: CD3-TCR complex-mediated signaling; cell proliferation / population expansion; growth factor (e.g., IL-2) expression; IFNγ expression; CD107a expression; TNFα expression; GM-CSF expression; perforin expression; granzyme expression; granulysin expression; and / or FAS ligand (FASL) expression.

[0288] For example, an immune cell (e.g., a T cell) according to the present disclosure may exhibit one or more of the functional properties listed in the previous paragraph in response to a variant Fc domain according to the present disclosure (i.e., a variant Fc domain bound by an antigen-binding moiety comprised in a CD3-TCR polypeptide complex expressed by the cell), or in response to a cell comprising / expressing such a variant Fc domain.

[0289] CD3-TCR complex-mediated signaling can be investigated by analyzing one or more correlates of CD3-TCR complex-mediated signaling. For example, CD3-TCR complex-mediated signaling can be examined by assessing the phosphorylation of one or more signaling molecules in the CD3-TCR complex signaling pathway. The level of CD3-TCR complex-mediated signaling can be analyzed by detecting and quantifying the phosphorylation level of CD3ζ, ZAP-70, Lck, LAT, and / or SLP-76. The level of CD3-TCR complex-mediated signaling can also be analyzed using reporter-based methods, such as quantifying the activity of transcription factors whose expression / activity is upregulated in response to CD3-TCR complex-mediated signaling, such as NFAT, NF-κB, and / or AP-1, or quantifying the expression of genes whose expression is upregulated by CD3-TCR complex-mediated signaling, such as IL2. For example, CD3-TCR complex-mediated signaling can be examined using reporter cell lines (e.g., GloResponse Jurkat NFAT-RE-luc2P (Promega #CS176501) or T Cell Activation Bioassay TCRαβ-KO CD4+ (Promega #GA1172)) that stably express a luciferase reporter driven by CD3-TCR complex-mediated signaling, as in Example 1 of the present disclosure.

[0290] Cell proliferation / population expansion can be investigated by analyzing cell division or cell number over a period of time. Cell division can be measured, for example, by: 3Analysis can be performed by in vitro analysis of H-thymidine incorporation, or by a CFSE dilution assay, for example, as described in Fulcher and Wong, Immunol Cell Biol (1999) 77(6):559-564, the entire contents of which are incorporated herein by reference. Proliferating cells can also be identified by analysis of 5-ethynyl-2'-deoxyuridine (EdU) incorporation, for example, as described in Buck et al., Biotechniques. 2008 Jun;44(7):927-9, and Sali and Mitchison, PNAS USA 2008 Feb 19;105(7):2415-2420, the entire contents of which are incorporated herein by reference.

[0291] As used herein, "expression" can refer to gene or protein expression. Gene expression encompasses transcription from DNA to RNA and can be measured by various means known to those skilled in the art, for example, by measuring mRNA levels by quantitative real-time PCR (qRT-PCR) or by using reporter-based methods. Similarly, protein expression can be measured by various methods well known in the art, for example, antibody-based methods, such as Western blot, immunohistochemistry, immunocytochemistry, flow cytometry, ELISA, ELISPOT, or reporter-based methods.

[0292] Immune cells (e.g., T cells) according to the present disclosure may be cytotoxic to cells containing / expressing a variant Fc domain according to the present disclosure, i.e., immune cells (e.g., T cells) according to the present disclosure may have the ability to kill cells containing / expressing a variant Fc domain according to the present disclosure.

[0293] A cell comprising a variant Fc domain according to the present disclosure can do so as a result of an antigen binding molecule comprising the variant Fc domain binding to an antigen expressed by the cell (e.g., on the surface of the cell, i.e., in or at the cell membrane). In some embodiments, a cell comprising a variant Fc domain according to the present disclosure comprises (e.g., on the surface of the cell) a polypeptide complex comprising (i) an antigen binding molecule comprising the variant Fc domain, and (ii) a target antigen for the antigen binding molecule.

[0294] Cytotoxicity and cell killing can be investigated using, for example, any of the methods outlined in Zaritskaya et al., Expert Rev Vaccines (2011), 9(6):601-616, which is incorporated herein by reference in its entirety. Examples of in vitro cytotoxicity / cell killing assays include release assays, e.g., 51 Examples of assays for cell killing include Cr release assay, lactate dehydrogenase (LDH) release assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) release assay, and calcein-acetoxymethyl (calcein-AM) release assay. These assays measure cell killing based on the detection of factors released from lysed cells. Cell killing by a given test cell type (e.g., immune cells (e.g., T cells) according to the present disclosure) can be analyzed, for example, by co-culturing the test cells with a given target cell type (e.g., cells containing a variant Fc domain according to the present disclosure) and measuring the number / proportion of viable (i.e., unlysed) / dead (e.g., lysed) target cells after an appropriate period of time. Other suitable assays include the xCELLigence real-time cytolytic in vitro potency assay (described in Cerignoli et al., PLoS One. (2018) 13(3):e0193498, which is incorporated herein by reference in its entirety), and the Incucyte immune cell killing assay used in the experimental examples of the present disclosure.

[0295] Immune cells (e.g., T cells) according to the present disclosure may have one or more novel, similar, or improved functional properties compared to a CAR that includes the same antigen-binding portion (i.e., the antigen-binding portion of the recombinant CD3-TCR complex polypeptide / polypeptide complex expressed by the cell). In some embodiments, immune cells according to the present disclosure may have one or more novel, similar, or improved functional properties compared to a CAR-expressing cell described in WO 2018 / 177966, which is incorporated herein by reference in its entirety.

[0296] In some embodiments, an immune cell (e.g., a T cell) according to the present disclosure, in response to an antigen for which a CD3-TCR polypeptide complex comprises an antigen-binding portion, or in response to a cell containing or expressing an antigen (e.g., in response to a variant Fc domain according to the present disclosure (i.e., a variant Fc domain bound by an antigen-binding portion included in a CD3-TCR polypeptide complex expressed by a cell), or in response to a cell containing / expressing such a variant Fc domain), may exhibit a level of CD3-TCR complex-mediated signaling that is similar to or greater than the level of CD3-TCR complex-mediated signaling exhibited by a CAR comprising the same antigen-binding portion.

[0297] A level of CD3-TCR complex mediated signaling that is "similar" to a reference level of CD3-TCR complex mediated signaling can be one of: 0.5 to 2 times the reference level of CD3-TCR complex mediated signaling, for example, 0.55 to 1.9 times, 0.6 to 1.8 times, 0.65 to 1.7 times, 0.7 to 1.6 times, 0.75 to 1.5 times, 0.8 to 1.4 times, 0.85 to 1.3 times, 0.9 to 1.2 times, or 0.95 to 1.1 times the reference level of CD3-TCR complex mediated signaling. In some embodiments, a level of CD3-TCR complex-mediated signaling that is "greater than" a reference level of CD3-TCR complex-mediated signaling can be more than 1-fold the reference level of CD3-TCR complex-mediated signaling, for example, one of 1.01-fold or more, 1.02-fold or more, 1.03-fold or more, 1.04-fold or more, 1.05-fold or more, 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.5-fold or more, 1.6-fold or more, 1.7-fold or more, 1.8-fold or more, 1.9-fold or more, 2-fold or more, 3-fold or more, 4-fold or more, or 5-fold or more.

[0298] In some embodiments, T cells expressing a recombinant CD3-TCR complex polypeptide / polypeptide complex according to the present disclosure comprising an antigen binding portion comprising a VH region according to SEQ ID NO: 65 and a VL region according to SEQ ID NO: 68 may be assessed in an assay comprising: (i) contacting a T cell expressing a recombinant CD3-TCR complex polypeptide / polypeptide complex of the present disclosure, the polypeptide complex comprising an antigen-binding portion comprising a VH region set forth in SEQ ID NO: 65 and a VL region set forth in SEQ ID NO: 68, with an antigen-presenting cell expressing a given target antigen, the antigen-presenting cell comprising an Fc domain having a CH2-CH3 region set forth in SEQ ID NO: 213, in contact with an antigen-binding molecule that binds to the given target antigen, and subsequently analyzing the level of CD3-TCR complex-mediated signaling by the T cell; (ii) contacting T cells expressing a CAR according to SEQ ID NO: 108 (e.g., equivalent T cells, i.e., derived from the same source as the T cells of (i)) with antigen-presenting cells as defined in (i), followed by analyzing the level of CD3-TCR complex-mediated signaling by the T cells; and (iii) To compare the level of CD3-TCR complex-mediated signaling by T cells in (i) with T cells in (ii).

[0299] In some embodiments, in an assay performed as described in the previous paragraph, the T cells described in (i) exhibit a level of CD3-TCR complex-mediated signaling that is 0.5 to 2 times, e.g., one of 0.55 to 1.9 times, 0.6 to 1.8 times, 0.65 to 1.7 times, 0.7 to 1.6 times, 0.75 to 1.5 times, 0.8 to 1.4 times, 0.85 to 1.3 times, 0.9 to 1.2 times, or 0.95 to 1.1 times, the level of CD3-TCR complex-mediated signaling exhibited by the T cells described in (ii). In some embodiments, in an assay performed as described in the previous paragraph, the T cells described in (i) exhibit one of the following levels of CD3-TCR complex-mediated signaling that are more than 1-fold, e.g., 1.01-fold or more, 1.02-fold or more, 1.03-fold or more, 1.04-fold or more, 1.05-fold or more, 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.5-fold or more, 1.6-fold or more, 1.7-fold or more, 1.8-fold or more, 1.9-fold or more, 2-fold or more, 3-fold or more, 4-fold or more, or 5-fold or more, the level of CD3-TCR complex-mediated signaling exhibited by the T cells described in (ii).

[0300] The present disclosure also provides methods for producing cells according to the present disclosure, and cells obtained or obtainable by such methods.

[0301] Methods for producing cells containing / expressing a polypeptide / polypeptide complex of interest are well known to those skilled in the art and generally involve introducing into the cell nucleic acid(s) / vector(s) encoding the polypeptide(s) of interest.

[0302] Such methods may include nucleic acid transfer for permanent (i.e., stable) or transient expression of the transferred nucleic acid. In some embodiments, after introduction into a cell, the nucleic acid(s) encoding the polypeptide(s) of interest may be integrated into or form part of the genomic DNA of the cell. In some embodiments, after introduction into a cell, the nucleic acid(s) encoding the polypeptide(s) of interest may be maintained extrachromosomally.

[0303] Any suitable genetic engineering platform may be used, including gammaretroviral vectors, lentiviral vectors, adenoviral vectors, DNA transfection, transposon-based gene delivery, and RNA transfection, as described, for example, in Maus et al., Annu Rev Immunol (2014) 32:189-225, the entire contents of which are incorporated herein by reference. Methods also include, for example, those described in Wang and Riviere Mol Ther Oncolytics. (2016) 3:16015, the entire contents of which are incorporated herein by reference. Suitable methods for introducing nucleic acid(s) / vector(s) into cells include transduction, transfection, and electroporation.

[0304] Methods for in vitro / ex vivo generation / expansion of a population of cells containing / expressing a polypeptide(s) of interest are well known to those skilled in the art. Appropriate culture conditions (i.e., cell culture medium, additives, stimuli, temperature, gas atmosphere), cell number, culture period, and method for introducing nucleic acid(s) / vector(s) encoding a polypeptide(s) of interest into cells can be determined, for example, by reference to WO 2018 / 177966. In some embodiments, the cells / cell populations according to the present disclosure are prepared under GMP (Good Manufacturing Practice; for example, the Good Manufacturing Practice guidelines published by the European Commission (Volume 4 of "The rules governing medicinal products in the European Union" contains guidance for the interpretation of the principles and guidelines of Good Manufacturing Practice for medicinal products for human and veterinary use set forth in European Commission Directive 91 / 356 / EEC, as amended by Directives 2003 / 94 / EC and 91 / 412 / EEC, respectively).

[0305] Conveniently, cell cultures according to the present disclosure can be maintained at 37°C in a humidified atmosphere containing 5% CO2. Cells of the cell culture can be established and / or maintained at any suitable density, as can be easily determined by one skilled in the art. Culturing can be carried out in any container suitable for the volume of the culture, such as a well of a cell culture plate, a cell culture flask, a bioreactor, etc. In some embodiments, cells are cultured in a bioreactor, such as the bioreactor described in Somerville and Dudley, Oncoimmunology (2012) 1(8):1435-1437, the entire contents of which are incorporated herein by reference. Immune cells (e.g., T cells) can be activated prior to the introduction of nucleic acid(s) encoding the polypeptide(s) of interest. For example, T cells within a population of PBMCs can be nonspecifically activated by in vitro stimulation with agonistic anti-CD3 and anti-CD28 antibodies in the presence of IL-2.

[0306] Introducing nucleic acid(s) into cells can include transduction, for example, lentiviral transduction. Transduction of immune cells by viral vectors is described, for example, in Simmons and Alberola-Ila, Methods Mol Biol. (2016) 1323:99-108, the entire contents of which are incorporated herein by reference.

[0307] Drugs can be used to enhance transduction efficiency. Hexadimethrine bromide (polybrene) is a cationic polymer commonly used to improve transduction by neutralizing the charge repulsion between virions and sialic acid residues expressed on the cell surface. Other drugs commonly used to enhance transduction include, for example, LentiBOOST (Sirion Biotech), Retronectin (Takara), Vectofusin (Miltenyi Biotech), and poloxamer-based drugs such as SureENTRY (Qiagen) and ViraDuctin (Cell Biolabs). In some embodiments, the method involves centrifuging cells into which it is desired to introduce nucleic acid encoding a polypeptide(s) of interest in the presence of cell culture medium containing a viral vector containing the nucleic acid (referred to in the art as "spinfection").

[0308] The methods generally involve introducing nucleic acids encoding a polypeptide(s) of interest into cells and culturing the cells under conditions suitable for expression of the polypeptide(s) of interest by the cells. In some embodiments, the methods involve culturing the immune cells into which nucleic acids encoding the polypeptide(s) of interest have been introduced to expand their numbers.

[0309] In some embodiments, the method includes analyzing the cells to confirm successful introduction of the nucleic acid into the cells, hi some embodiments, the method includes analyzing the cells to confirm expression of the polypeptide(s) of interest by the cells (e.g., via evaluation of a detectable entity).

[0310] In some embodiments, the method further comprises, for example, cells that express a polypeptide(s) of interest from other cells (e.g., cells that do not express the polypeptide(s) of interest). Methods for purifying / isolating immune cells from heterogeneous cell populations are well known in the art and may use, for example, FACS or MACS-based methods to sort cell populations based on the expression of immune cell markers. In some embodiments, the method purifies / isolates a specific type of cell, for example, CD8+ T cells or CTLs that express a polypeptide(s) of interest.

[0311] Methods of producing cells according to the present disclosure can include modifying the cells to reduce expression of a CD3-TCR complex polypeptide. In some embodiments, the methods include modifying a nucleic acid (e.g., an endogenous nucleic acid) encoding the CD3-TCR complex polypeptide.

[0312] Modification of a given target nucleic acid can be achieved in a variety of ways known to those skilled in the art, including modifying the target nucleic acid by homologous recombination and target nucleic acid editing using site-specific nucleases (SSNs).

[0313] Suitable methods may use targeting by homologous recombination, as outlined in, for example, Mortensen Curr Protoc Neurosci. (2007) Chapter 4: Unit 4.29 and Vasquez et al., PNAS 2001, 98(15): 8403-8410 (both of which are incorporated herein by reference in their entirety). Targeting by homologous recombination involves the exchange of nucleic acid sequences through crossover events guided by homologous sequences. Other suitable techniques include nucleic acid editing using SSNs. Gene editing using SSNs is outlined in, for example, Eid and Mahfouz, Exp Mol Med. 2016 Oct; 48(10): e265, which is incorporated herein by reference in its entirety. Enzymes capable of generating site-specific double-strand breaks (DSBs) can be engineered to introduce DSBs into the target nucleic acid sequence(s) of interest. DSBs can be repaired either by error-prone non-homologous end joining (NHEJ), in which the two ends of the break are rejoined, often with the insertion or deletion of nucleotides. Alternatively, DSBs can be repaired by homology-directed repair (HDR), a high-fidelity mechanism in which a DNA template with ends homologous to the break is provided and introduced at the site of the DSB.

[0314] SSNs that can be engineered to generate target nucleic acid sequence-specific DSBs include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced palindromic repeats / CRISPR-associated 9 (CRISPR / Cas9) systems. ZFN systems are generally described, for example, in Umov et al., Nat Rev Genet. (2010) 11(9):636-46, the entire contents of which are incorporated herein by reference. ZFNs contain a programmable zinc finger DNA-binding domain and a DNA-cleavage domain (e.g., a FokI endonuclease domain). DNA-binding domains can be identified by screening zinc finger arrays capable of binding to target nucleic acid sequences. The TALEN system is generally described, for example, in Mahfouz et al., Plant Biotechnol J. (2014) 12(8):1006-14, the entire contents of which are incorporated herein by reference. TALENs contain a programmable DNA-binding TALE domain and a DNA-cleavage domain (e.g., a FokI endonuclease domain). TALEs contain a repeat domain consisting of 33-39 amino acid repeats that are identical except for two residues at positions 12 and 13 of each repeat, the repeat variable di-residues (RVDs). Each RVD determines the binding of the repeat to a nucleotide in the target DNA sequence according to the following relationship: "HD" binds to C, "NI" binds to A, "NG" binds to T, and "NN" or "NK" binds to G (Moscou and Bogdanove, Science (2009) 326(5959):1501). CRISPR / Cas9 and related systems, such as CRISPR / Cpf1, CRISPR / C2c1, CRISPR / C2c2, and CRISPR / C2c3, are reviewed, for example, in Nakade et al., Bioengineered (2017) 8(3):265-273, which is incorporated herein by reference in its entirety.These systems include an endonuclease (e.g., Cas9, Cpf1, etc.) and a single guide RNA (sgRNA) molecule. The sgRNA can be engineered to target the endonuclease activity to a nucleic acid sequence of interest.

[0315] In some embodiments, modifying a nucleic acid (e.g., an endogenous nucleic acid) encoding a CD3-TCR complex polypeptide according to the present disclosure uses a site-specific nuclease (SSN) system that targets the nucleic acid encoding the CD3-TCR complex polypeptide. The SSN system can be a ZFN system, a TALEN system, a CRISPR / Cas9 system, a CRISPR / Cpf1 system, a CRISPR / C2c1 system, a CRISPR / C2c2 system, or a CRISPR / C2c3 system.

[0316] In some embodiments, a method for producing a cell according to the present disclosure comprises introducing into a cell nucleic acid(s) encoding a CD3E-targeting CRISPR / Cas9 system. In some embodiments, the nucleic acid(s) encode a CRISPR RNA (crRNA) that targets CD3E (e.g., an exon of CD3E, e.g., exon 7 of CD3E) and a trans-activating crRNA (tracrRNA) for processing the crRNA into its mature form.

[0317] In some embodiments, a method for producing a cell according to the present disclosure comprises introducing into a cell nucleic acid(s) encoding a CRISPR / Cas9 system(s) targeting TRAC, TRBCl and / or TRBC2 (e.g., TRAC and TRBCl). In some embodiments, the nucleic acid(s) encode a CRISPR RNA (crRNA) targeting TRAC, TRBCl and / or TRBC2 (e.g., TRAC and TRBCl; e.g., an exon of TRAC, TRBCl and / or TRBC2 (e.g., TRAC and TRBCl)) and a trans-activating crRNA (tracrRNA) for processing the crRNA into its mature form.

[0318] composition The present disclosure also provides compositions comprising the polypeptides, polypeptide complexes, nucleic acids, expression vectors and cells described herein.

[0319] The polypeptides, polypeptide complexes, nucleic acids, expression vectors, and cells described herein (particularly the nucleic acids, expression vectors, and cells described herein) can be formulated as pharmaceutical compositions or medicaments for clinical use and may include pharmaceutically acceptable carriers, diluents, excipients, or adjuvants. In preferred aspects and embodiments, the present disclosure provides pharmaceutical compositions or medicaments comprising cells according to the present disclosure. Accordingly, the present disclosure also provides pharmaceutical compositions / medicines comprising the polypeptides, polypeptide complexes, nucleic acid(s), expression vector(s), or cells described herein. In preferred embodiments, the pharmaceutical compositions / medicines according to the present disclosure comprise nucleic acid(s), expression vector(s), or cells described herein.

[0320] The pharmaceutical compositions / medicaments of the present disclosure may contain one or more pharmaceutically acceptable carriers (e.g., liposomes, micelles, microspheres, nanoparticles), diluents / excipients (e.g., starch, cellulose, cellulose derivatives, polyols, dextrose, maltodextrin, magnesium stearate), adjuvants, fillers, buffering agents, preservatives (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium citrate, methylparaben, propylparaben), antioxidants, or the like. They may contain oxidizing agents (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium), lubricants (e.g., magnesium stearate, talc, silica, stearic acid, vegetable stearin), binders (e.g., sucrose, lactose, starch, cellulose, gelatin, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), xylitol, sorbitol, mannitol), stabilizers, solubilizers, surfactants (e.g., wetting agents), masking agents, or colorants (e.g., titanium oxide).

[0321] As used herein, the term "pharmaceutically acceptable" refers to compounds, ingredients, materials, compositions, dosage forms, etc., which, within the scope of sound medical judgment, are suitable for use in contact with the tissues of the subject in question (e.g., a human subject) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each carrier, diluent, excipient, adjuvant, filler, buffer, preservative, antioxidant, lubricant, binder, stabilizer, solubilizer, surfactant, masking agent, colorant, flavoring, or sweetener of a composition according to the present disclosure must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, binders, stabilizers, solubilizers, surfactants, masking agents, colorants, flavorings, or sweetening agents can be found in standard pharmaceutical texts, such as Remington's "The Science and Practice of Pharmacy" (Ed. A. Adejare), 23rd Edition (2020), Academic Press.

[0322] The pharmaceutical compositions and medicaments of the present disclosure can be formulated for topical, parenteral, systemic, intracavitary, intravenous, intraarterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, intrathecal, oral, or transdermal administration. In some embodiments, the pharmaceutical compositions / medicaments can be formulated for administration by injection or infusion, or by ingestion.

[0323] Suitable formulations may include cells provided in sterile or isotonic medium. Medicaments and pharmaceutical compositions may be formulated in fluid forms, including gels. Fluid formulations may be formulated for administration by injection or infusion (e.g., via a catheter) into selected areas of the human or animal body.

[0324] In some embodiments, the pharmaceutical composition / medicament is formulated for injection or infusion, for example, into a blood vessel, tissue / organ, or tumor of interest.

[0325] The present disclosure also provides methods for producing pharmaceutically useful compositions, which may comprise one or more steps selected from the following: Producing the cells described herein; Isolating / purifying the cells described herein; and / or The cells described herein are mixed with a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.

[0326] For example, a further aspect of the present disclosure relates to a method of formulating or producing a medicament or pharmaceutical composition for use in treating a disease / condition (e.g., a disease / condition described herein), comprising formulating the pharmaceutical composition or medicament by mixing a cell described herein with a pharmaceutically acceptable carrier, adjuvant, excipient, or diluent.

[0327] Therapeutic and prophylactic uses The articles of the present disclosure find use in therapeutic and prophylactic methods. In particular, cells according to the present disclosure, e.g., cells comprising / expressing a CD3-TCR polypeptide complex, including a recombinant CD3-TCR complex polypeptide according to the present disclosure, find use in therapeutic and prophylactic methods. Similarly, compositions according to the present disclosure, e.g., pharmaceutical compositions comprising cells according to the present disclosure, e.g., cells comprising / expressing a CD3-TCR polypeptide complex, including a recombinant CD3-TCR complex polypeptide according to the present disclosure, find use in such methods.

[0328] Accordingly, the present disclosure provides cells or compositions described herein for use in methods of medical treatment or prevention. Also provided are cells or compositions described herein for use in methods of treating or preventing a disease or condition described herein. Also provided is the use of cells or compositions described herein in the manufacture of a medicament for treating or preventing a disease or condition described herein. Also provided is a method of treating or preventing a disease or condition described herein, comprising administering a therapeutically or prophylactically effective amount of a cell or composition described herein to a subject.

[0329] The interventions described in the previous paragraphs may be effective for reducing the onset or progression of a disease / condition, alleviating the symptoms of a disease / condition, or reducing the pathology of a disease / condition. The intervention may be effective for preventing the progression of a disease / condition, for example, for preventing the progression of a disease / condition from worsening or for slowing the rate of onset of a disease / condition. In some embodiments, the intervention may result in an improvement of a disease / condition, for example, a reduction in the symptoms of a disease / condition or a reduction in some other correlate of the severity / activity of a disease / condition. In some embodiments, the intervention may prevent the progression / onset of a disease / condition at a later stage (e.g., a chronic stage or metastasis).

[0330] Therapeutic or prophylactic intervention according to the present disclosure generally involves administering a cell or pharmaceutical composition according to the present disclosure to a subject who has been or will be administered an antigen binding molecule comprising (a) an antigen binding domain that binds to a target antigen, and (b) a variant Fc domain according to the present disclosure.

[0331] It will be understood that according to such intervention, the cell (or cells of the composition) comprises / expresses a CD3-TCR complex comprising an antigen-binding portion that binds to the variant Fc domain of the antigen-binding molecule. Illustratively, the intervention may comprise administering T cells expressing a CD3-TCR polypeptide complex comprising a recombinant CD3-TCR complex polypeptide set forth in SEQ ID NO: 121 to a subject who has been administered or will be administered an antibody comprising an Fc domain comprising a CH2-CH3 region set forth in SEQ ID NO: 213. Illustratively, the intervention may comprise administering T cells expressing a CD3-TCR polypeptide complex comprising (i) a recombinant CD3-TCR complex polypeptide set forth in SEQ ID NO: 137 and (ii) a recombinant CD3-TCR complex polypeptide set forth in SEQ ID NO: 165 to a subject who has been administered or will be administered an antibody comprising an Fc domain comprising a CH2-CH3 region set forth in SEQ ID NO: 213. By way of further example, the intervention may include administering T cells expressing a CD3-TCR polypeptide complex comprising a recombinant CD3-TCR complex polypeptide set forth in SEQ ID NO: 125 to a subject who has been or will be administered an antibody comprising an Fc domain comprising a CH2-CH3 region set forth in SEQ ID NO: 212.

[0332] In the therapeutic / prophylactic interventions of the present disclosure, antigen-binding molecules having variant Fc domains act as adapter molecules, directing the activity of cells according to the present disclosure against the antigen to which the antigen-binding molecule binds. That is, in embodiments in which the cells are immune cells (e.g., T cells), antigen-binding molecules having variant Fc domains direct a cell-mediated immune response (e.g., a T-cell-mediated immune response) against cells expressing the antigen to which the antigen-binding molecule binds.

[0333] By way of example, in examples of the present disclosure, T cells expressing a CD3-TCR polypeptide complex comprising a recombinant CD3-TCR complex polypeptide according to SEQ ID NO: 121 are used in conjunction with an anti-FolR1 antibody comprising an Fc domain comprising P329G to direct the T cells to FolR1-expressing cells. By way of further example, in examples of the present disclosure, T cells expressing a CD3-TCR polypeptide complex comprising a recombinant CD3-TCR complex polypeptide according to SEQ ID NO: 121 are used in conjunction with an anti-CD19 antibody comprising an Fc domain comprising P329G to direct the T cells to CD19-expressing cells. By way of further example, in examples of the present disclosure, T cells expressing a CD3-TCR polypeptide complex comprising (i) a recombinant CD3-TCR complex polypeptide set forth in SEQ ID NO: 137 and (ii) a recombinant CD3-TCR complex polypeptide set forth in SEQ ID NO: 165 are used in conjunction with an anti-FolR1 antibody comprising an Fc domain comprising P329G to direct the T cells to FolR1-expressing cells. By way of further illustration, in examples of the present disclosure, T cells expressing a CD3-TCR polypeptide complex comprising (i) a recombinant CD3-TCR complex polypeptide set forth in SEQ ID NO: 137 and (ii) a recombinant CD3-TCR complex polypeptide set forth in SEQ ID NO: 165 are used in conjunction with an anti-CD19 antibody comprising an Fc domain comprising P329G to direct the T cells against CD19-expressing cells.

[0334] The antigen binding molecules with variant Fc domains used with the cells or compositions according to the present disclosure may bind to any given target antigen.

[0335] The target antigen can be any target antigen expressed by cells that are desired to be killed / depleted to achieve a therapeutic / prophylactic effect. In some embodiments, the target antigen is an antigen whose expression / activity, or its upregulated expression / activity, is positively associated with a disease / symptom (e.g., cancer, infectious disease, or autoimmune disease). The target antigen is preferably expressed on the cell surface of the cells that express the target antigen.

[0336] In some embodiments, the target antigen may be a cancer cell antigen. A cancer cell antigen is an antigen expressed or overexpressed by cancer cells. A cancer cell antigen may be any peptide / polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof. Expression of a cancer cell antigen may be associated with cancer. A cancer cell antigen may be aberrantly expressed by cancer cells (e.g., the cancer cell antigen may be expressed with abnormal localization) or may be expressed in an abnormal structure by cancer cells. A cancer cell antigen may be capable of eliciting an immune response. In some embodiments, the antigen is expressed on the cell surface of cancer cells (i.e., the cancer cell antigen is a cancer cell surface antigen). In some embodiments, a portion of the antigen bound by the antigen binding molecules described herein is presented on the outer surface (i.e., extracellularly) of cancer cells. A cancer cell antigen may be a cancer-associated antigen. In some embodiments, a cancer cell antigen is an antigen whose expression is associated with the onset, progression, or severity of cancer symptoms. A cancer-associated antigen may be associated with the cause or pathology of cancer or may be aberrantly expressed as a result of cancer. In some embodiments, a cancer cell antigen is an antigen whose expression is upregulated (e.g., at the RNA and / or protein level) by cancer cells, e.g., compared to the expression level by comparable non-cancerous cells (e.g., non-cancerous cells derived from the same tissue / cell type). In some embodiments, a cancer-associated antigen may be preferentially expressed by cancerous cells and not expressed by comparable non-cancerous cells (e.g., non-cancerous cells derived from the same tissue / cell type). In some embodiments, a cancer-associated antigen may be the product of a mutated oncogene or a mutated tumor suppressor gene. In some embodiments, a cancer-associated antigen may be an overexpressed cellular protein, a cancer antigen produced by an oncogenic virus, a carcinoembryonic antigen, or the product of a cell surface glycolipid or glycoprotein.

[0337] Cancer cell antigens are reviewed by Zarour HM, DeLeo A, Finn OJ, et al., Categories of Tumor Antigens. In: Kufe DW, Pollock RE, Weichselbaum RR, et al., editors. Holland-Frei Cancer Medicine. 6th edition. Hamilton (ON): BC Decker; 2003. Cancer cell antigens include carcinoembryonic antigen: CEA, immature laminin receptor, TAG-72; cancer virus antigens, e.g., HPV Overexpressed proteins include E6 and E7; BING-4, calcium-activated chloride channel 2, cyclin-B1, 9D7, Ep-CAM, EphA3, HER2 / neu, telomerase, mesothelin, SAP-1, and survivin; cancer-testis antigens include BAGE, CAGE, GAGE, MAGE, SAGE, XAGE, CT9, CT10, NY-ESO-1, PRAME, and SSX-2; lineage-restricted antigens include MART1, Gp100, tyrosinase, TRP-1 / 2, MC1R, and prostate-specific antigen; mutant antigens include β-catenin, BRCA1 / 2, CDK4, CML66, fibronectin, MART-2, p53, Ras, and TGF-βRII; and posttranslationally altered antigens include MUC1 and idiotypic antigens including Ig and TCR. Other cancer cell antigens include heat shock protein 70 (HSP70), heat shock protein 90 (HSP90), glucose-regulated protein 78 (GRP78), vimentin, nucleolin, fetoacinar pancreatic protein (FAPP), alkaline phosphatase placenta-like 2 (ALPPL-2), Siglec-5, stress-inducible phosphoprotein 1 (STIP1), protein tyrosine kinase 7 (PTK7), and cyclophilin B. In some embodiments, the cancer cell antigen is a cancer cell antigen described in Zhao and Cao, Front Immunol. (2019) 10:2250, which is incorporated herein by reference in its entirety.

[0338] In some embodiments, the target antigen is FAP (fibroblast activation protein), CEA (carcinoembryonic antigen), p95 (p95HER2), BCMA (B cell maturation antigen), EpCAM (epithelial cell adhesion molecule), MSLN (mesothelin), MCSP (melanoma chondroitin sulfate proteoglycan), HER-1 (human epidermal growth factor 1), HER-2 (human epidermal growth factor 2), HER-3 (human epidermal growth factor 3), CD19, CD20, CD22, CD33, CD38, CD52Flt3, folate receptor 1 (FOLR1), human The target antigen is selected from trophoblast cell surface antigen 2 (Trop-2), cancer antigen 12-5 (CA-12-5), human leukocyte antigen-antigen D-related (HLA-DR), MUC-1 (mucin-1), A33 antigen, PSMA (prostate-specific membrane antigen), FMS-like tyrosine kinase 3 (FLT-3), PSMA (prostate-specific membrane antigen), PSCA (prostate stem cell antigen), transferrin receptor, TNC (tenascin), carbonic anhydrase IX (CA-IX), and / or a peptide bound to a molecule of the human major histocompatibility complex (MHC). In some embodiments, the target antigen is CD19. In some embodiments, the target antigen is FOLR1.

[0339] It will be understood that the cells and compositions of the present disclosure may be used to treat / prevent any disease / condition that derives therapeutic or prophylactic benefit from reducing the level / activity of a given target antigen or reducing the number / proportion / activity of cells containing / expressing a given target antigen.

[0340] For example, the disease / condition may be one in which the target antigen or cells containing / expressing the target antigen are pathologically implicated, e.g., a disease / condition in which an increase in the level / activity of the target antigen or an increase in the number / proportion / activity of cells containing / expressing the target antigen is positively correlated with the onset, development, or progression of the disease / condition and / or the severity of one or more symptoms of the disease / condition. In some embodiments, an increase in the level / activity of the target antigen or an increase in the number / proportion / activity of cells containing / expressing the target antigen may be a risk factor for the onset, development, or progression of the disease / condition.

[0341] In some embodiments, the disease / condition treated / prevented in accordance with the present disclosure is one characterized by an increased level of expression or activity of a target antigen, e.g., compared to the level of expression / activity in the absence of the disease / condition. In some embodiments, the disease / condition treated / prevented is one characterized by an increased number / proportion / activity of cells expressing the target antigen, e.g., compared to the level / number / proportion / activity in the absence of the disease / condition (e.g., in a healthy subject or in an equivalent non-diseased tissue). When the disease / condition is cancer, the level of expression or activity of the target antigen may be greater than the level of expression or activity of the target antigen in an equivalent non-cancerous cell / non-tumor tissue. The cancer / its cells may contain one or more mutations (e.g., compared to an equivalent non-cancerous cell / non-tumor tissue) that cause upregulation of target antigen expression or activity.

[0342] Therapeutic / prophylactic intervention according to the present disclosure may achieve one or more of the following in a subject (compared to an equivalent untreated subject or a subject treated with a suitable control): a reduction in the level of the target antigen; a reduction in the activity of the target antigen; and / or a reduction in the number / proportion / activity of cells containing / expressing the target antigen.

[0343] In particular, the use of cells and compositions according to the present disclosure in methods of treating / preventing diseases / conditions by adoptive cell transfer (ACT) is contemplated.

[0344] Adoptive cell transfer generally refers to the process of obtaining cells (e.g., immune cells) from a subject, typically by collecting a blood sample from which the cells are isolated.The cells are then typically modified and / or expanded, and then administered to the same subject (in the case of adoptive transfer of autologous / autologous cells) or to a different subject (in the case of adoptive transfer of allogeneic cells).Treatment typically aims to provide a population of cells with specific desired properties to a subject, or to increase the frequency of such cells with such properties in the subject.Adoptive transfer can be carried out with the aim of introducing cells or cell populations into a subject and / or increasing the frequency of cells or cell populations in a subject.

[0345] Adoptive transfer of immune cells is described, for example, in Kalos and June (2013), Immunity 39(1):49-60, and Davis et al. (2015), Cancer J. 21(6):486-491, both of which are incorporated by reference in their entirety. Those skilled in the art can determine appropriate reagents and procedures for adoptive transfer of cells according to the present disclosure by referring, for example, to Dai et al., 2016 J Nat Cancer Inst 108(7):djv439, which is incorporated by reference in its entirety.

[0346] The cells and compositions according to the present disclosure may be used for the treatment / prevention of diseases / conditions by allogeneic or autologous transplantation.

[0347] As used herein, "allograft" refers to the transplantation of cells, tissues, or organs into a recipient subject that are not genetically identical to the recipient subject. The cells, tissues, or organs may be from or derived from the cells, tissues, or organs of a donor subject that is not genetically identical to the recipient subject. Allografting differs from autografting, which refers to the transplantation of cells, tissues, or organs from / derived from a donor subject that is genetically identical to the recipient subject (i.e., autologous material). It will be understood that adoptive transfer of allogeneic immune cells is a form of allografting, and adoptive transfer of autoimmune cells is a form of autografting.

[0348] The present disclosure provides methods that include administering cells and compositions according to the present disclosure to a subject.

[0349] In some embodiments, the method comprises modifying an immune cell to contain / express a polypeptide(s) according to the present disclosure (e.g., recombinant CD3-TCR complex polypeptide(s), complex polypeptide).

[0350] In some embodiments, the method comprises: Modifying immune cells to express or contain a CD3-TCR complex comprising one or more recombinant CD3-TCR complex polypeptides according to the present disclosure (e.g., as described herein); and administering the modified immune cells to a subject; Includes.

[0351] In some embodiments, the method comprises: The method further includes administering to a subject an antigen-binding molecule comprising a variant Fc domain according to the present disclosure, wherein the altered immune CD3-TCR complex comprises an antigen-binding portion that binds to the variant Fc domain of the antigen-binding molecule.

[0352] In some embodiments, the method comprises: The method further includes modifying the immune cells to reduce / prevent expression of a CD3-TCR complex polypeptide (e.g., as described herein), wherein the CD3-TCR complex polypeptide is a CD3-TCR complex polypeptide from which the CD3-TCR complex-associated domain of the recombinant CD3-TCR complex polypeptide of the CD3-TCR complex is derived.

[0353] It will be understood that the method steps recited in the previous three paragraphs may be performed in any suitable order.

[0354] In some embodiments, the method comprises: The method includes administering to a subject immune cells that have been modified to express or contain a CD3-TCR complex comprising one or more recombinant CD3-TCR complex polypeptides according to the present disclosure.

[0355] In some embodiments, the method comprises: administering to a subject immune cells that have been modified to (i) express or contain a CD3-TCR complex comprising one or more recombinant CD3-TCR complex polypeptides according to the present disclosure, and (ii) reduce / prevent expression of the CD3-TCR complex polypeptides. Including, The recombinant CD3-TCR complex polypeptide of the CD3-TCR complex of (i) comprises a CD3-TCR complex-associated domain derived from the CD3-TCR complex polypeptide of (ii).

[0356] In some embodiments, in accordance with the previous two paragraphs, the subject has been or will be administered an antigen binding molecule comprising a variant Fc domain according to the present disclosure, and the CD3-TCR complex comprises an antigen-binding portion that binds to the variant Fc domain of the antigen-binding molecule.

[0357] In some embodiments, the method comprises: (a) modifying an immune cell to express or contain a CD3-TCR complex comprising one or more recombinant CD3-TCR complex polypeptides according to the present disclosure (e.g., as described herein); and (b) administering to a subject an antigen-binding molecule comprising a variant Fc domain according to the present disclosure; and (c) administering the modified immune cells to a subject. Including, The CD3-TCR complex in (a) comprises an antigen-binding portion that binds to the variant Fc domain of the antigen-binding molecule in (b).

[0358] In some embodiments according to the method of the previous paragraph, step (c) may be performed before step (b).

[0359] In some embodiments, the method comprises: (a) modifying immune cells to reduce / prevent expression of CD3-TCR complex polypeptides; (b) modifying immune cells to express or contain a CD3-TCR complex comprising one or more recombinant CD3-TCR complex polypeptides according to the present disclosure (e.g., as described herein); and (c) administering to a subject an antigen-binding molecule comprising a variant Fc domain according to the present disclosure; and (d) administering the modified immune cells to a subject. Including, (b) a recombinant CD3-TCR complex polypeptide of the CD3-TCR complex, comprising a CD3-TCR complex-associated domain derived from the CD3-TCR complex polypeptide of (a); and The CD3-TCR complex in (b) comprises an antigen-binding portion that binds to the variant Fc domain of the antigen-binding molecule in (c).

[0360] In some embodiments according to the method of the previous paragraph, step (d) may be performed before step (c).

[0361] In some embodiments, the subject from which the immune cells are isolated / obtained is the same subject to which the cells are administered (i.e., the adoptive transfer can be of autologous / autologous cells). In some embodiments, the subject from which the immune cells are isolated / obtained is a different subject from the subject to which the cells are administered (i.e., the adoptive transfer can be of allogeneic cells).

[0362] In some embodiments, the method may further include one or more of the following: obtaining a blood sample from the subject; isolating immune cells (e.g., PBMCs) from a blood sample obtained from the subject; generating / expanding immune cell populations; culturing immune cells in in vitro or ex vivo cell culture; culturing immune cells expressing / containing a CD3-TCR complex comprising one or more recombinant CD3-TCR complex polypeptides according to the present disclosure in an in vitro or ex vivo cell culture; collecting / isolating immune cells that express / contain a CD3-TCR complex comprising one or more recombinant CD3-TCR complex polypeptides according to the present disclosure; Formulating immune cells expressing / containing a CD3-TCR complex, including one or more recombinant CD3-TCR complex polypeptides according to the present disclosure, into a pharmaceutical composition, for example, by mixing the cells with a pharmaceutically acceptable adjuvant, diluent or carrier.

[0363] The administration of the articles of the present disclosure is preferably a "therapeutically effective" or "prophylactically effective" amount, which is sufficient to provide a therapeutic or prophylactic benefit to the subject. The actual amount administered, as well as the rate and time course of administration, will depend on the nature and severity of the disease / symptoms and the specific article being administered. Prescribing treatment, such as determining dosage, is within the responsibility of general practitioners and other doctors, and typically takes into account the disease / disorder being treated, the individual subject's symptoms, the delivery site, the method of administration, and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's "The Science and Practice of Pharmacy" (ed. A. Adejare), 23rd Edition (2020), Academic Press.

[0364] The administration of the article of the present disclosure can be parenteral, systemic, intravenous, intraarterial, intramuscular, intracavitary, intrathecal, intraocular, intravitreal, intraconjunctival, subretinal, suprachoroidal, subcutaneous, intradermal, intrathecal, oral, nasal, topical, or transdermal. Administration can be by injection or infusion. Administration of the article of the present disclosure can be intratumoral. In some cases, the article of the present disclosure can be formulated for targeted delivery to specific cells, tissues, organs, and / or tumors.

[0365] Multiple doses of the articles of the present disclosure may be provided. The multiple doses may be separated by a predetermined time interval, which may be selected to be one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days, or 1, 2, 3, 4, 5, or 6 months.

[0366] Administration to a subject of cells or compositions according to the present disclosure using the antigen binding molecules described herein according to the therapeutic and prophylactic interventions described herein can be simultaneous or sequential.

[0367] Concomitant administration refers to the administration of (i) a cell or composition according to the present disclosure and (ii) an antigen-binding molecule described herein together, e.g., as a pharmaceutical composition containing both agents (i.e., a combined preparation), or shortly after each other, optionally via the same route of administration, e.g., into the same artery, vein, or other blood vessel.

[0368] Sequential administration refers to the administration of (i) a cell or composition according to the present disclosure and (ii) one of the antigen-binding molecules described herein, followed by the separate administration of another agent after a given time interval. Although in some embodiments, the two agents do not need to be administered by the same route. The time interval may be any time interval.

[0369] The present disclosure also provides a method of depleting or killing cells containing or expressing a target antigen, the method comprising: (i) an antigen-binding molecule comprising: (a) an antigen-binding domain that binds to a target antigen, and (b) a variant Fc domain according to the present disclosure; and (ii) an immune cell containing / expressing a CD3-TCR complex comprising one or more recombinant CD3-TCR complex polypeptides according to the present disclosure; The CD3-TCR complex of (ii) comprises an antigen-binding portion that binds to the variant Fc domain of the antigen-binding molecule of (i).

[0370] subject The subject according to various aspects of the present disclosure can be any animal or human. Therapeutic and prophylactic uses can be human or animal (veterinary use).

[0371] The subject to be administered with the article of the present disclosure (e.g., following a therapeutic or preventive intervention) may be a subject in need of such intervention. The subject is preferably a mammal, more preferably a human. The subject may be a non-human mammal, but is more preferably a human. The subject may be male or female. The subject may be a patient.

[0372] A subject may have (e.g., have been diagnosed with) a disease or condition described herein, may be suspected of having such a disease / condition, or may be at risk of developing / suffering such a disease / condition. In embodiments according to the present disclosure, a subject may be selected for treatment by a method based on characterization of one or more markers for such a disease / condition.

[0373] In some embodiments, a subject may be selected for a therapeutic or prophylactic intervention as described herein based on the detection of cells / tissues that express a target antigen (i.e., the target antigen of an antigen-binding molecule used in conjunction with a cell or composition according to the present disclosure) or that overexpress the target antigen, for example, in a sample obtained from the subject.

[0374] A subject may be an allogeneic or non-autologous subject with respect to an intervention according to the present disclosure. As used herein, when a subject is referred to herein as "allogeneic" or "non-autologous" with respect to an intervention, the subject is a subject other than the subject from which the cells of the intervention (i.e., the administered cells or the cells of the administered pharmaceutical composition / medication) are derived. A subject treated / prevented according to the present disclosure may not be genetically identical to the subject from which the cells administered to the subject (e.g., the cells of the pharmaceutical composition / medication) are derived. A subject treated / prevented according to the present disclosure may contain MHC / HLA genes encoding MHC / HLA molecules (e.g., MHC class Iα and / or MHC class II molecules) that are not identical to the MHC / HLA molecules (e.g., MHC class Iα and / or MHC class II molecules) encoded by the cells administered to the subject (e.g., the cells of the pharmaceutical composition / medication). A subject treated / prevented according to the present disclosure may be HLA-mismatched to the subject from which the cells administered to the subject (e.g., the cells of the pharmaceutical composition / medication) are derived.

[0375] The subject to whom cells are administered in accordance with the present disclosure may be allogeneic / non-autologous with respect to the source from which the cells administered to the subject (e.g., cells of a pharmaceutical composition / medication) are derived. The subject to whom cells are administered may be a different subject from the subject from which the cells were obtained for the production of the cells administered to the subject (e.g., cells of a pharmaceutical composition / medication). The subject to whom cells are administered may not be genetically identical to the subject from which the cells administered to the subject (e.g., cells of a pharmaceutical composition / medication) were obtained for the production of the cells.

[0376] A subject may be an autologous / self subject with respect to an intervention according to the present disclosure. As used herein, when a subject is referred to herein as "autologous" or "self" with respect to an intervention, the subject is the same subject from which the cells of the intervention (i.e., the administered cells or the cells of the administered pharmaceutical composition / medication) are derived. A subject treated / prevented according to the present disclosure may be genetically identical to the subject from which the cells administered to the subject (e.g., the cells of the pharmaceutical composition / medication) are derived. A subject treated / prevented according to the present disclosure may contain MHC / HLA genes encoding the same MHC / HLA molecules (e.g., MHC class Iα and / or MHC class II molecules) as the MHC / HLA molecules (e.g., MHC class Iα and / or MHC class II molecules) encoded by the cells administered to the subject (e.g., the cells of the pharmaceutical composition / medication). A subject treated / prevented according to the present disclosure may be HLA-matched to the subject from which the cells administered to the subject (e.g., the cells of the pharmaceutical composition / medication) are derived.

[0377] The subject to whom cells are administered according to the present disclosure may be autologous with respect to the source from which the cells (e.g., cells of a pharmaceutical composition / medication) administered to the subject are derived. The subject to whom cells are administered may be the same subject from which the cells were obtained for the production of the cells (e.g., cells of a pharmaceutical composition / medication) administered to the subject. The subject to whom cells are administered may be genetically identical to the subject from which the cells (e.g., cells of a pharmaceutical composition / medication) administered to the subject were obtained for the production of the cells.

[0378] kit The present disclosure also provides a kit of parts.

[0379] In some aspects and embodiments, the kit-of-parts according to the present disclosure includes (i) a cell according to the present disclosure and (ii) an antigen-binding molecule comprising (a) an antigen-binding domain that binds to a target antigen, and (b) a variant Fc domain according to the present disclosure. It will be understood that according to such aspects and embodiments, the cell of (i) comprises / expresses a CD3-TCR complex comprising an antigen-binding portion that binds to the variant Fc domain of the antigen-binding molecule of (ii).

[0380] In some aspects and embodiments, the kit-of-parts according to the present disclosure includes (i) a composition according to the present disclosure, and (ii) an antigen-binding molecule comprising (a) an antigen-binding domain that binds to a target antigen, and (b) a variant Fc domain according to the present disclosure. It will be understood that according to such aspects and embodiments, the composition of (i) includes a cell comprising / expressing a CD3-TCR complex comprising an antigen-binding portion that binds to the variant Fc domain of the antigen-binding molecule of (ii).

[0381] In some aspects and embodiments, the kit-of-parts according to the present disclosure includes (i) a nucleic acid(s) or expression vector(s) according to the present disclosure, and (ii) an antigen-binding molecule comprising (a) an antigen-binding domain that binds to a target antigen, and (b) a variant Fc domain according to the present disclosure. It will be understood that according to such aspects and embodiments, the nucleic acid(s) or expression vector(s) of (i) encode a polypeptide(s) for engineering a cell to contain / express a CD3-TCR complex comprising an antigen-binding portion that binds to the variant Fc domain of the antigen-binding molecule of (ii).

[0382] A kit-of-parts according to the present disclosure may include predetermined amounts of the items described in (i) and / or (ii), as described in the previous three paragraphs. In some embodiments, the items according to (i) and / or (ii) are provided in a container (e.g., in a vial or bottle). The kit may provide the items according to (i) and / or (ii) along with instructions (e.g., protocols) on how to use them in accordance with the therapeutic or prophylactic interventions described herein.

[0383] In some embodiments, the kit of parts includes materials for producing a polypeptide of the present disclosure, e.g., a recombinant CD3-TCR complex polypeptide of the present disclosure. In some embodiments, the kit of parts includes materials for producing a polypeptide complex according to the present disclosure, e.g., a CD3-TCR polypeptide complex comprising a recombinant CD3-TCR complex polypeptide according to the present disclosure. In some embodiments, the kit of parts includes materials for producing a cell according to the present disclosure, e.g., a cell comprising / expressing a CD3-TCR polypeptide complex comprising a recombinant CD3-TCR complex polypeptide according to the present disclosure. In some embodiments, the kit of parts includes materials for producing a composition according to the present disclosure, e.g., a pharmaceutical composition comprising a cell according to the present disclosure, e.g., a cell comprising / expressing a CD3-TCR polypeptide complex comprising a recombinant CD3-TCR complex polypeptide according to the present disclosure.

[0384] In some embodiments, the kit of parts may include a nucleic acid(s) or expression vector(s) according to the present disclosure, and, optionally, materials for introducing the nucleic acid(s) or expression vector(s) into cells. In some embodiments, the kit of parts may include a system for producing cells according to the present disclosure according to GMP conditions. In some embodiments, the kit of parts may include a (closed) bag cell incubation system in which the nucleic acid(s) or expression vector(s) according to the present disclosure can be introduced into cells, which can then be cultured under GMP conditions.

[0385] In some embodiments, the kit of parts may include materials for formulating cells according to the present disclosure into a pharmaceutical composition, such as a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant.

[0386] The manufacture of a kit-of-parts according to the present disclosure preferably follows standard procedures known to those skilled in the art.

[0387] Sequence identity As used herein, "sequence identity" refers to the percentage of nucleotides / amino acid residues in a subject sequence that are identical to the nucleotides / amino acid residues in a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity between the sequences. Pairwise and multiple sequence alignment for determining the percent sequence identity between two or more amino acid or nucleic acid sequences can be achieved by various methods known to those skilled in the art, for example, using publicly available computer software such as ClustalOmega (Soding, J. 2005, Bioinformatics 21, 951-960), T-coffee (Notredame et al. 2000, J. Mol. Biol. (2000) 302, 205-217), Kalign (Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6 (298)) and MAFFT (Katoh and Standley 2013, Molecular Biology and Evolution, 30 (4) 772-780) software.When using such software, it is preferable to use default parameters, for example, gap penalties and extension penalties.

[0388] Numbered paragraphs The following numbered paragraphs (para) provide further descriptions of features and combinations of features contemplated in connection with the present invention.

[0389] 1. A recombinant CD3-TCR complex polypeptide comprising: (i) an antigen-binding portion, or component thereof, that binds to a variant Fc domain having an amino acid sequence that includes at least one amino acid difference relative to a reference Fc domain to which the antigen-binding portion does not bind; and (ii) a CD3-TCR complex-associated domain having an amino acid sequence derived from a CD3-TCR complex polypeptide; A recombinant CD3-TCR complex polypeptide comprising:

[0390] 2. A recombinant CD3-TCR complex polypeptide according to paragraph 1, wherein the recombinant CD3-TCR complex polypeptide is capable of associating with one or more CD3-TCR complex polypeptides via its CD3-TCR complex association domain to form a CD3-TCR complex.

[0391] 3. A recombinant CD3-TCR complex polypeptide according to paragraph 1 or 2, wherein the amino acid sequence derived from the CD3-TCR complex polypeptide is derived from CD3ε, TCRα or TCRβ.

[0392] 4. A recombinant CD3-TCR complex polypeptide described in any one of paragraphs 1 to 3, wherein the CD3-TCR complex association domain comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity to one of SEQ ID NOs: 30, 52, 1, 53, 5, 54 or 9.

[0393] 5. A recombinant CD3-TCR complex polypeptide according to any one of paragraphs 1 to 4, wherein the amino acid sequence derived from the CD3-TCR complex polypeptide is derived from CD3ε.

[0394] 6. A recombinant CD3-TCR complex polypeptide according to any one of paragraphs 1 to 5, wherein the CD3-TCR complex association domain comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 30.

[0395] 7. A recombinant CD3-TCR complex polypeptide according to any one of paragraphs 1 to 4, wherein the amino acid sequence derived from the CD3-TCR complex polypeptide is derived from TCRα or TCRβ.

[0396] 8. The recombinant CD3-TCR complex polypeptide of any one of paragraphs 1 to 4 or paragraph 7, wherein the CD3-TCR complex association domain comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity to one of SEQ ID NOs: 52, 1, 53, 5, 54 or 9.

[0397] 9. The recombinant CD3-TCR complex polypeptide of paragraph 7 or 8, wherein the CD3-TCR complex association domain comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 52 or 1 and comprises a cysteine ​​residue at a position corresponding to position 47 numbered according to SEQ ID NO: 1.

[0398] 10. A recombinant CD3-TCR complex polypeptide according to any one of paragraphs 7 to 9, wherein the CD3-TCR complex association domain comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity to any one of SEQ ID NOs: 53, 5, 54 or 9, and comprises a cysteine ​​residue at a position corresponding to position 56 numbered according to SEQ ID NO: 5.

[0399] 11. A recombinant CD3-TCR complex polypeptide according to any one of paragraphs 1 to 10, wherein the antigen-binding portion that binds to the variant Fc domain comprises the heavy chain variable (VH) region and the light chain variable (VL) region of an antibody that binds to the variant Fc domain.

[0400] 12. A recombinant CD3-TCR complex polypeptide according to any one of paragraphs 1 to 11, wherein the antigen-binding portion is or comprises an Fv, scFv, Fab, Fab', Fab'-SH, F(ab')2, crossFab, scFab or dAb portion.

[0401] 13. A recombinant CD3-TCR complex polypeptide according to any one of paragraphs 1 to 12, wherein the antigen-binding portion is or comprises an scFv.

[0402] 14. A recombinant CD3-TCR complex polypeptide according to any one of paragraphs 1 to 13, wherein a component of the antigen-binding portion is or comprises a VH or VL region of an antibody that binds to the variant Fc domain.

[0403] 15. A recombinant CD3-TCR complex polypeptide according to any one of paragraphs 1 to 14, wherein the antigen-binding portion or component thereof is linked at its C-terminus to the N-terminus of the CD3-TCR complex association domain, optionally via a linker sequence.

[0404] 16. The recombinant CD3-TCR complex polypeptide of any one of paragraphs 1 to 15, wherein the variant Fc domain binds to an Fc receptor with an affinity that is lower than the affinity with which the reference Fc domain binds to the Fc receptor, and optionally, the Fc receptor is an Fcγ receptor or a neonatal Fc receptor (FcRn).

[0405] 17. The recombinant CD3-TCR complex polypeptide of any one of paragraphs 1 to 16, wherein the variant Fc domain comprises a CH2-CH3 region comprising an amino acid difference at one or more of the following positions relative to the amino acid sequence of the CH2-CH3 region of the reference Fc domain according to EU numbering: L234, L235, I253, N297, S298, H310, P329, E333, K334, or H435.

[0406] 18. The recombinant CD3-TCR complex polypeptide of any one of paragraphs 1 to 17, wherein the variant Fc domain comprises a CH2-CH3 region that includes G329 according to EU numbering.

[0407] 19. The antigen-binding portion comprises the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 69; LC-CDR2 having the amino acid sequence of SEQ ID NO: 70; and LC-CDR3 having the amino acid sequence of SEQ ID NO: 71; 19. A recombinant CD3-TCR complex polypeptide according to any one of paragraphs 1 to 18, comprising a VL region incorporating:

[0408] 20. The antigen-binding portion is (i) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 56; HC-CDR2 having the amino acid sequence of SEQ ID NO: 64; and HC-CDR3 having the amino acid sequence of SEQ ID NO: 58; or (ii) the following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 56; HC-CDR2 having the amino acid sequence of SEQ ID NO: 57; and HC-CDR3 having the amino acid sequence of SEQ ID NO: 58; 20. A recombinant CD3-TCR complex polypeptide according to any one of paragraphs 1 to 19, comprising a VH incorporating:

[0409] 21. The antigen-binding portion comprises: (a)(i) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 56; HC-CDR2 having the amino acid sequence of SEQ ID NO: 64; and HC-CDR3 having the amino acid sequence of SEQ ID NO: 68; and (ii) a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 69; LC-CDR2 having the amino acid sequence of SEQ ID NO: 70; and LC-CDR3 having the amino acid sequence of SEQ ID NO: 71; or (b)(i) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 56; HC-CDR2 having the amino acid sequence of SEQ ID NO: 57; and HC-CDR3 having the amino acid sequence of SEQ ID NO: 58; and (ii) a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 69; LC-CDR2 having the amino acid sequence of SEQ ID NO: 70; and LC-CDR3 having the amino acid sequence of SEQ ID NO: 71; 21. A recombinant CD3-TCR complex polypeptide according to any one of paragraphs 1 to 20, comprising:

[0410] 22. The recombinant CD3-TCR complex polypeptide of any one of paragraphs 1 to 21, wherein the antigen-binding portion comprises a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 68.

[0411] 23. The recombinant CD3-TCR complex polypeptide of any one of paragraphs 1 to 22, wherein the antigen-binding portion comprises a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 65, 63, or 55.

[0412] 24. The antigen-binding portion is (a)(i) a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 65; and (ii) a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 68; or (b)(i) a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 63; and (ii) a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 68; or (c)(i) a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 55; and (ii) a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 68; 24. A recombinant CD3-TCR complex polypeptide according to any one of paragraphs 1 to 23, comprising:

[0413] 25. The recombinant CD3-TCR complex polypeptide of any one of paragraphs 1 to 17, wherein the variant Fc domain comprises a CH2-CH3 region comprising A298, A333, and A334 according to EU numbering.

[0414] 26. The antigen-binding portion comprises the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 77; HC-CDR2 having the amino acid sequence of SEQ ID NO: 78; and HC-CDR3 having the amino acid sequence of SEQ ID NO: 79; 26. A recombinant CD3-TCR complex polypeptide according to any one of paragraphs 1 to 17 or paragraph 25, comprising a VH region incorporating:

[0415] 27. The antigen-binding portion comprises the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 85; LC-CDR2 having the amino acid sequence of SEQ ID NO: 86; and LC-CDR3 having the amino acid sequence of SEQ ID NO: 87; 27. A recombinant CD3-TCR complex polypeptide according to any one of paragraphs 1 to 17, paragraph 25 or paragraph 26, comprising a VL region incorporating:

[0416] 28. The antigen-binding portion is (i) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 77; HC-CDR2 having the amino acid sequence of SEQ ID NO: 78; and HC-CDR3 having the amino acid sequence of SEQ ID NO: 79; and (ii) a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 85; LC-CDR2 having the amino acid sequence of SEQ ID NO: 86; and LC-CDR3 having the amino acid sequence of SEQ ID NO: 87; 28. A recombinant CD3-TCR complex polypeptide according to any one of paragraphs 1 to 17 or 25 to 27, comprising:

[0417] 29. The recombinant CD3-TCR complex polypeptide of any one of paragraphs 1-17 or 25-28, wherein the antigen-binding portion comprises a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 76.

[0418] 30. The recombinant CD3-TCR complex polypeptide of any one of paragraphs 1-17 or 25-29, wherein the antigen-binding portion comprises a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 84.

[0419] 31. The antigen-binding portion is (i) a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 76; and (ii) a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 84; 31. A recombinant CD3-TCR complex polypeptide according to any one of paragraphs 1 to 17 or 25 to 30, comprising:

[0420] 32. A recombinant CD3-TCR complex polypeptide, comprising: (i) an antigen-binding moiety that is an scFv, wherein the antigen-binding moiety binds to a variant Fc domain having an amino acid sequence that includes at least one amino acid difference relative to a reference Fc domain to which the antigen-binding moiety does not bind, and the variant Fc domain includes a CH2-CH3 region that includes G329 according to EU numbering; and (ii) a CD3-TCR complex association domain having an amino acid sequence derived from a CD3-TCR complex polypeptide, wherein the amino acid sequence derived from a CD3-TCR complex polypeptide is derived from CD3ε; A recombinant CD3-TCR complex polypeptide comprising:

[0421] 33. A recombinant CD3-TCR complex polypeptide according to paragraph 32, wherein the recombinant CD3-TCR complex polypeptide is capable of associating with one or more CD3-TCR complex polypeptides via its CD3-TCR complex association domain to form a CD3-TCR complex.

[0422] 34. A recombinant CD3-TCR complex polypeptide according to paragraph 32 or paragraph 33, wherein the CD3-TCR complex association domain comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 30.

[0423] 35. A recombinant CD3-TCR complex polypeptide according to any one of paragraphs 32 to 34, wherein the antigen-binding portion is linked at its C-terminus to the N-terminus of the CD3-TCR complex association domain, optionally via a linker sequence.

[0424] 36. The antigen-binding portion comprises the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 69; LC-CDR2 having the amino acid sequence of SEQ ID NO: 70; and LC-CDR3 having the amino acid sequence of SEQ ID NO: 71; 36. A recombinant CD3-TCR complex polypeptide according to any one of paragraphs 32 to 35, comprising a VL region incorporating:

[0425] 37. The antigen-binding portion is (i) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 56; HC-CDR2 having the amino acid sequence of SEQ ID NO: 64; and HC-CDR3 having the amino acid sequence of SEQ ID NO: 58; or (ii) the following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 56; HC-CDR2 having the amino acid sequence of SEQ ID NO: 57; and HC-CDR3 having the amino acid sequence of SEQ ID NO: 58; 37. A recombinant CD3-TCR complex polypeptide according to any one of paragraphs 32 to 36, comprising a VH incorporating:

[0426] 38. The antigen-binding portion is (a)(i) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 56; HC-CDR2 having the amino acid sequence of SEQ ID NO: 64; and HC-CDR3 having the amino acid sequence of SEQ ID NO: 68; and (ii) a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 69; LC-CDR2 having the amino acid sequence of SEQ ID NO: 70; and LC-CDR3 having the amino acid sequence of SEQ ID NO: 71; or (b)(i) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 56; HC-CDR2 having the amino acid sequence of SEQ ID NO: 57; and HC-CDR3 having the amino acid sequence of SEQ ID NO: 58; and (ii) a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 69; LC-CDR2 having the amino acid sequence of SEQ ID NO: 70; and LC-CDR3 having the amino acid sequence of SEQ ID NO: 71; 38. A recombinant CD3-TCR complex polypeptide according to any one of paragraphs 32 to 37, comprising:

[0427] 39. The recombinant CD3-TCR complex polypeptide of any one of paragraphs 32 to 38, wherein the antigen-binding portion comprises a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 68.

[0428] 40. The recombinant CD3-TCR complex polypeptide of any one of paragraphs 32 to 39, wherein the antigen-binding portion comprises a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 65, 63, or 55.

[0429] 41. The antigen-binding portion is (a)(i) a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 65; and (ii) a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 68; or (b)(i) a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 63; and (ii) a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 68; or (c)(i) a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 55; and (ii) a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 68; 41. A recombinant CD3-TCR complex polypeptide according to any one of paragraphs 32 to 40, comprising:

[0430] 42. A polypeptide complex comprising an antigen-binding portion, wherein the antigen-binding portion binds to a variant Fc domain having an amino acid sequence that includes at least one amino acid difference relative to a reference Fc domain to which the antigen-binding portion does not bind, wherein the polypeptide complex comprises a first recombinant CD3-TCR complex polypeptide and a second recombinant CD3-TCR complex polypeptide, wherein the first and second recombinant CD3-TCR complex polypeptides each comprise (i) a CD3-TCR complex association domain having an amino acid sequence derived from the CD3-TCR complex polypeptide, and (ii) a component of the antigen-binding portion.

[0431] 43. A polypeptide complex comprising: (a) a first recombinant CD3-TCR complex polypeptide comprising: (i) a first component of the antigen-binding portion, wherein the antigen-binding portion binds to a variant Fc domain having an amino acid sequence that includes at least one amino acid difference relative to a reference Fc domain to which the antigen-binding portion does not bind; and (ii) a CD3-TCR complex-associated domain having an amino acid sequence derived from a CD3-TCR complex polypeptide; and (b) a second recombinant CD3-TCR complex polypeptide comprising: (i) a second component of the antigen-binding portion of(a)(i); and (ii) a CD3-TCR complex-associated domain having an amino acid sequence derived from a CD3-TCR complex polypeptide; Includes; A polypeptide complex, wherein the first and second recombinant CD3-TCR complex polypeptides are capable of associating via their CD3-TCR complex-associating domains to form an antigen-binding portion.

[0432] 44. The polypeptide complex of paragraph 42 or paragraph 43, wherein the first component of the antigen-binding portion is or comprises a heavy chain variable (VH) region of an antibody that binds to the variant Fc domain, and the second component of the antigen-binding portion is or comprises a light chain variable (VL) region of an antibody that binds to the variant Fc domain.

[0433] 45. (i) the CD3-TCR complex-associated domain of the first recombinant CD3-TCR complex polypeptide is derived from TCRα and the CD3-TCR complex-associated domain of the second recombinant CD3-TCR complex polypeptide is derived from TCRβ; or (ii) The polypeptide complex of any one of paragraphs 42 to 44, wherein the CD3-TCR complex association domain of the first recombinant CD3-TCR complex polypeptide is derived from TCRβ and the CD3-TCR complex association domain of the second recombinant CD3-TCR complex polypeptide is derived from TCRα.

[0434] 46. ​​A polypeptide complex according to paragraph 45, wherein the CD3-TCR complex association domain derived from TCRα comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 52 or 1.

[0435] 47. A polypeptide complex according to paragraph 46, wherein the CD3-TCR complex association domain derived from TCRα comprises a cysteine ​​residue at a position corresponding to position 47 numbered according to SEQ ID NO:1.

[0436] 48. A polypeptide complex according to any one of paragraphs 45 to 47, wherein the CD3-TCR complex association domain derived from TCRβ comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity to one of SEQ ID NOs: 53, 5, 54 or 9.

[0437] 49. A polypeptide complex according to paragraph 48, wherein the CD3-TCR complex association domain derived from TCRβ comprises a cysteine ​​residue at a position corresponding to position 56 numbered according to SEQ ID NO:5.

[0438] 50. (i) in the first recombinant CD3-TCR complex polypeptide, a first component of the antigen-binding portion is linked at its C-terminus to the N-terminus of the CD3-TCR complex-associated domain of the first recombinant CD3-TCR complex polypeptide, optionally via a linker sequence; and / or (ii) The polypeptide complex of any one of paragraphs 42 to 49, wherein in the second recombinant CD3-TCR complex polypeptide, the second component of the antigen-binding portion is linked at its C-terminus to the N-terminus of the CD3-TCR complex association domain of the second recombinant CD3-TCR complex polypeptide, optionally via a linker sequence.

[0439] 51. The polypeptide complex of any one of paragraphs 42 to 50, wherein the variant Fc domain binds to an Fc receptor with an affinity that is lower than the affinity with which the reference Fc domain binds to the Fc receptor, and optionally, the Fc receptor is an Fcγ receptor or a neonatal Fc receptor (FcRn).

[0440] 52. The polypeptide complex of any one of paragraphs 42 to 51, wherein the variant Fc domain comprises a CH2-CH3 region comprising an amino acid difference at one or more of the following positions relative to the amino acid sequence of the CH2-CH3 region of the reference Fc domain according to EU numbering: L234, L235, I253, N297, S298, H310, P329, E333, K334, or H435.

[0441] 53. The polypeptide complex of any one of paragraphs 42 to 52, wherein the variant Fc domain comprises a CH2-CH3 region that includes G329 according to EU numbering.

[0442] 54. The antigen-binding portion comprises the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 69; LC-CDR2 having the amino acid sequence of SEQ ID NO: 70; and LC-CDR3 having the amino acid sequence of SEQ ID NO: 71; 54. A polypeptide complex according to any one of paragraphs 42 to 53, comprising a VL region incorporating

[0443] 55. The antigen-binding portion is (i) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 56; HC-CDR2 having the amino acid sequence of SEQ ID NO: 64; and HC-CDR3 having the amino acid sequence of SEQ ID NO: 58; or (ii) the following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 56; HC-CDR2 having the amino acid sequence of SEQ ID NO: 57; and HC-CDR3 having the amino acid sequence of SEQ ID NO: 58; 55. A polypeptide complex according to any one of paragraphs 42 to 54, comprising a VH incorporating:

[0444] 56. The antigen-binding portion is (a)(i) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 56; HC-CDR2 having the amino acid sequence of SEQ ID NO: 64; and HC-CDR3 having the amino acid sequence of SEQ ID NO: 68; and (ii) a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 69; LC-CDR2 having the amino acid sequence of SEQ ID NO: 70; and LC-CDR3 having the amino acid sequence of SEQ ID NO: 71; or (b)(i) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 56; HC-CDR2 having the amino acid sequence of SEQ ID NO: 57; and HC-CDR3 having the amino acid sequence of SEQ ID NO: 58; and (ii) a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 69; LC-CDR2 having the amino acid sequence of SEQ ID NO: 70; and LC-CDR3 having the amino acid sequence of SEQ ID NO: 71; 56. The polypeptide complex according to any one of paragraphs 42 to 55, comprising:

[0445] 57. The polypeptide conjugate of any one of paragraphs 42 to 56, wherein the antigen-binding portion comprises a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 68.

[0446] 58. The polypeptide complex of any one of paragraphs 42 to 57, wherein the antigen-binding portion comprises a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 65, 63, or 55.

[0447] 59. The antigen-binding portion is (a)(i) a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 65; and (ii) a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 68; or (b)(i) a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 63; and (ii) a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 68; or (c)(i) a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 55; and (ii) a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 68; 59. The polypeptide complex according to any one of paragraphs 42 to 58, comprising:

[0448] 60. The polypeptide complex of any one of paragraphs 42 to 52, wherein the variant Fc domain comprises a CH2-CH3 region comprising A298, A333, and A334 according to EU numbering.

[0449] 61. The antigen-binding portion comprises the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 77; HC-CDR2 having the amino acid sequence of SEQ ID NO: 78; and HC-CDR3 having the amino acid sequence of SEQ ID NO: 79; 61. The polypeptide complex of any one of paragraphs 42 to 52 or paragraph 60, comprising a VH region incorporating

[0450] 62. The antigen-binding portion comprises the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 85; LC-CDR2 having the amino acid sequence of SEQ ID NO: 86; and LC-CDR3 having the amino acid sequence of SEQ ID NO: 87; 62. A polypeptide complex according to any one of paragraphs 42 to 52, paragraph 60 or paragraph 61, comprising a VL region incorporating

[0451] 63. The antigen-binding portion is (i) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 77; HC-CDR2 having the amino acid sequence of SEQ ID NO: 78; and HC-CDR3 having the amino acid sequence of SEQ ID NO: 79; and (ii) a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 85; LC-CDR2 having the amino acid sequence of SEQ ID NO: 86; and LC-CDR3 having the amino acid sequence of SEQ ID NO: 87; 63. A polypeptide complex according to any one of paragraphs 42 to 52 or 60 to 62, comprising:

[0452] 64. The polypeptide complex of any one of paragraphs 42-52 or 60-63, wherein the antigen-binding portion comprises a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 76.

[0453] 65. The polypeptide conjugate of any one of paragraphs 42-52 or 60-64, wherein the antigen-binding portion comprises a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:84.

[0454] 66. The antigen-binding portion is (i) a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 76; and (ii) a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 84; 66. A polypeptide complex according to any one of paragraphs 42 to 52 or 60 to 65, comprising:

[0455] 67. A polypeptide complex comprising an antigen-binding portion, wherein the antigen-binding portion binds to a variant Fc domain having an amino acid sequence comprising at least one amino acid difference relative to a reference Fc domain to which the antigen-binding portion does not bind, wherein the variant Fc domain comprises a CH2-CH3 region comprising G329 according to EU numbering, and wherein the polypeptide complex comprises a first recombinant CD3-TCR complex polypeptide and a second recombinant CD3-TCR complex polypeptide; (1)(a) a first recombinant CD3-TCR complex polypeptide comprising: (i) a first component of an antigen-binding portion, the first component of the antigen-binding portion comprising or consisting of a heavy chain variable (VH) region of an antibody that binds to a variant Fc domain; and (ii) a CD3-TCR complex-associated domain having an amino acid sequence derived from TCRα; (b) a second recombinant CD3-TCR complex polypeptide comprising: (i) a second component of an antigen-binding portion, the second component of the antigen-binding portion comprising or consisting of a light chain variable (VL) region of an antibody that binds to the variant Fc domain; and (ii) a CD3-TCR complex-associated domain having an amino acid sequence derived from TCRβ; Contains; or (2)(a) a first recombinant CD3-TCR complex polypeptide comprising: (i) a first component of an antigen-binding portion, the first component of the antigen-binding portion comprising or consisting of a heavy chain variable (VH) region of an antibody that binds to a variant Fc domain; and (ii) a CD3-TCR complex-associated domain having an amino acid sequence derived from TCRβ; (b) a second recombinant CD3-TCR complex polypeptide comprising: (i) a second component of an antigen-binding portion, the second component of the antigen-binding portion comprising or consisting of a light chain variable (VL) region of an antibody that binds to the variant Fc domain; and (ii) a CD3-TCR complex-associated domain having an amino acid sequence derived from TCRα; A polypeptide complex comprising:

[0456] 68. A polypeptide complex comprising: (a) a first recombinant CD3-TCR complex polypeptide comprising: (i) a first component of the antigen-binding portion, wherein the antigen-binding portion binds to a variant Fc domain having an amino acid sequence that includes at least one amino acid difference relative to a reference Fc domain to which the antigen-binding portion does not bind, the variant Fc domain including a CH2-CH3 region that includes G329 according to EU numbering; and (ii) a CD3-TCR complex-associated domain having an amino acid sequence derived from a CD3-TCR complex polypeptide; and (b) a second recombinant CD3-TCR complex polypeptide comprising: (i) a second component of the antigen-binding portion of(a)(i); and (ii) a CD3-TCR complex-associated domain having an amino acid sequence derived from a CD3-TCR complex polypeptide; Includes; the first and second recombinant CD3-TCR complex polypeptides are capable of associating via their CD3-TCR complex-associating domains to form an antigen-binding portion; a first component of the antigen-binding portion is or comprises a heavy chain variable (VH) region of an antibody that binds to the variant Fc domain, and a second component of the antigen-binding portion is or comprises a light chain variable (VL) region of an antibody that binds to the variant Fc domain; and (i) the CD3-TCR complex-associated domain of the first recombinant CD3-TCR complex polypeptide is derived from TCRα and the CD3-TCR complex-associated domain of the second recombinant CD3-TCR complex polypeptide is derived from TCRβ; or (ii) A polypeptide complex, wherein the CD3-TCR complex-associated domain of the first recombinant CD3-TCR complex polypeptide is derived from TCRβ and the CD3-TCR complex-associated domain of the second recombinant CD3-TCR complex polypeptide is derived from TCRα.

[0457] 69. A polypeptide complex according to paragraph 67 or paragraph 68, wherein the CD3-TCR complex association domain derived from TCRα comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 52 or 1.

[0458] 70. A polypeptide complex according to paragraph 69, wherein the CD3-TCR complex association domain derived from TCRα comprises a cysteine ​​residue at a position corresponding to position 47 numbered according to SEQ ID NO:1.

[0459] 71. A polypeptide complex according to any one of paragraphs 67 to 70, wherein the CD3-TCR complex association domain derived from TCRβ comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity to one of SEQ ID NOs: 53, 5, 54 or 9.

[0460] 72. A polypeptide complex according to paragraph 71, wherein the CD3-TCR complex association domain derived from TCRα comprises a cysteine ​​residue at a position corresponding to position 56 numbered according to SEQ ID NO:5.

[0461] 73. (i) in the first recombinant CD3-TCR complex polypeptide, a first component of the antigen-binding portion is linked at its C-terminus to the N-terminus of the CD3-TCR complex-associated domain of the first recombinant CD3-TCR complex polypeptide, optionally via a linker sequence; and / or (ii) The polypeptide complex of any one of paragraphs 67 to 72, wherein in the second recombinant CD3-TCR complex polypeptide, the second component of the antigen-binding portion is linked at its C-terminus to the N-terminus of the CD3-TCR complex association domain of the second recombinant CD3-TCR complex polypeptide, optionally via a linker sequence.

[0462] 74. The antigen-binding portion comprises the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 69; LC-CDR2 having the amino acid sequence of SEQ ID NO: 70; and LC-CDR3 having the amino acid sequence of SEQ ID NO: 71; 74. A polypeptide complex according to any one of paragraphs 67 to 73, comprising a VL region incorporating

[0463] 75. The antigen-binding portion is (i) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 56; HC-CDR2 having the amino acid sequence of SEQ ID NO: 64; and HC-CDR3 having the amino acid sequence of SEQ ID NO: 58; or (ii) the following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 56; HC-CDR2 having the amino acid sequence of SEQ ID NO: 57; and HC-CDR3 having the amino acid sequence of SEQ ID NO: 58; 75. A polypeptide complex according to any one of paragraphs 67 to 74, comprising a VH incorporating:

[0464] 76. The antigen-binding portion is (a)(i) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 56; HC-CDR2 having the amino acid sequence of SEQ ID NO: 64; and HC-CDR3 having the amino acid sequence of SEQ ID NO: 68; and (ii) a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 69; LC-CDR2 having the amino acid sequence of SEQ ID NO: 70; and LC-CDR3 having the amino acid sequence of SEQ ID NO: 71; or (b)(i) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 56; HC-CDR2 having the amino acid sequence of SEQ ID NO: 57; and HC-CDR3 having the amino acid sequence of SEQ ID NO: 58; and (ii) a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 69; LC-CDR2 having the amino acid sequence of SEQ ID NO: 70; and LC-CDR3 having the amino acid sequence of SEQ ID NO: 71; 76. A polypeptide complex according to any one of paragraphs 67 to 75, comprising:

[0465] 77. The polypeptide conjugate of any one of paragraphs 67 to 76, wherein the antigen-binding portion comprises a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:68.

[0466] 78. The polypeptide complex of any one of paragraphs 67 to 77, wherein the antigen-binding portion comprises a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 65, 63, or 55.

[0467] 79. The antigen-binding portion is (a)(i) a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 65; and (ii) a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 68; or (b)(i) a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 63; and (ii) a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 68; or (c)(i) a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 55; and (ii) a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 68; 79. A polypeptide complex according to any one of paragraphs 67 to 78, comprising:

[0468] 80. A CD3-TCR polypeptide complex comprising a recombinant CD3-TCR complex polypeptide according to any one of paragraphs 1 to 41, or a polypeptide complex according to any one of paragraphs 42 to 79.

[0469] 81. A composite polypeptide comprising: (a) an amino acid sequence encoding a first recombinant CD3-TCR complex polypeptide comprising: (i) a first component of the antigen-binding portion, wherein the antigen-binding portion binds to a variant Fc domain having an amino acid sequence that includes at least one amino acid difference relative to a reference Fc domain to which the antigen-binding portion does not bind; and (ii) a CD3-TCR complex-associated domain having an amino acid sequence derived from a CD3-TCR complex polypeptide; and (b) an amino acid sequence encoding a second recombinant CD3-TCR complex polypeptide comprising: (i) a second component of the antigen-binding portion of(a)(i); and (ii) a CD3-TCR complex-associated domain having an amino acid sequence derived from a CD3-TCR complex polypeptide; Includes; the first and second recombinant CD3-TCR complex polypeptides are capable of associating via their CD3-TCR complex association domains to form a CD3-TCR complex comprising an antigen-binding portion; The composite polypeptide further comprises a cleavage site between the amino acid sequences of (a) and (b).

[0470] 82. The composite polypeptide of para. 81, wherein a first component of the antigen-binding portion is or comprises a heavy chain variable (VH) region of an antibody that binds to the variant Fc domain, and a second component of the antigen-binding portion is or comprises a light chain variable (VL) region of an antibody that binds to the variant Fc domain.

[0471] 83. (i) the CD3-TCR complex-associated domain of the first recombinant CD3-TCR complex polypeptide is derived from TCRα and the CD3-TCR complex-associated domain of the second recombinant CD3-TCR complex polypeptide is derived from TCRβ; or (ii) The polypeptide complex of paragraph 81 or paragraph 82, wherein the CD3-TCR complex association domain of the first recombinant CD3-TCR complex polypeptide is derived from TCRβ and the CD3-TCR complex association domain of the second recombinant CD3-TCR complex polypeptide is derived from TCRα.

[0472] 84. A composite polypeptide according to para.83, wherein the CD3-TCR complex association domain derived from TCRα comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 52 or 1.

[0473] 85. A composite polypeptide according to paragraph 84, wherein the CD3-TCR complex association domain derived from TCRα comprises a cysteine ​​residue at a position corresponding to position 47 numbered according to SEQ ID NO:1.

[0474] 86. A composite polypeptide according to any one of paragraphs 83 to 85, wherein the CD3-TCR complex association domain derived from TCRβ comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity to one of SEQ ID NOs: 53, 5, 54 or 9.

[0475] 87. The composite polypeptide according to paragraph 86, wherein the CD3-TCR complex association domain derived from TCRβ comprises a cysteine ​​residue at a position corresponding to position 56 numbered according to SEQ ID NO:5.

[0476] 88. (i) in the first recombinant CD3-TCR complex polypeptide, a first component of the antigen-binding portion is linked at its C-terminus to the N-terminus of the CD3-TCR complex-associated domain of the first recombinant CD3-TCR complex polypeptide, optionally via a linker sequence; and / or (ii) The composite polypeptide of any one of paragraphs 81 to 87, wherein in the second recombinant CD3-TCR complex polypeptide, the second component of the antigen-binding portion is linked at its C-terminus to the N-terminus of the CD3-TCR complex-associated domain of the second recombinant CD3-TCR complex polypeptide, optionally via a linker sequence.

[0477] 89. The composite polypeptide of any one of paras. 81 to 88, wherein the variant Fc domain binds to an Fc receptor with an affinity that is lower than the affinity with which the reference Fc domain binds to the Fc receptor, and optionally, the Fc receptor is an Fcγ receptor or a neonatal Fc receptor (FcRn).

[0478] 90. The composite polypeptide of any one of paras. 81 to 89, wherein the variant Fc domain comprises a CH2-CH3 region comprising an amino acid difference at one or more of the following positions relative to the amino acid sequence of the CH2-CH3 region of the reference Fc domain according to EU numbering: L234, L235, I253, N297, S298, H310, P329, E333, K334, or H435.

[0479] 91. The composite polypeptide of any one of paragraphs 81 to 90, wherein the variant Fc domain comprises a CH2-CH3 region that includes G329 according to EU numbering.

[0480] 92. The antigen-binding portion comprises the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 69; LC-CDR2 having the amino acid sequence of SEQ ID NO: 70; and LC-CDR3 having the amino acid sequence of SEQ ID NO: 71; 92. The composite polypeptide of any one of paragraphs 81 to 91, comprising a VL region incorporating:

[0481] 93. The antigen-binding portion is (i) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 56; HC-CDR2 having the amino acid sequence of SEQ ID NO: 64; and HC-CDR3 having the amino acid sequence of SEQ ID NO: 58; or (ii) the following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 56; HC-CDR2 having the amino acid sequence of SEQ ID NO: 57; and HC-CDR3 having the amino acid sequence of SEQ ID NO: 58; 93. The composite polypeptide of any one of paragraphs 81 to 92, comprising a VH incorporating:

[0482] 94. The antigen-binding portion is (a)(i) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 56; HC-CDR2 having the amino acid sequence of SEQ ID NO: 64; and HC-CDR3 having the amino acid sequence of SEQ ID NO: 68; and (ii) a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 69; LC-CDR2 having the amino acid sequence of SEQ ID NO: 70; and LC-CDR3 having the amino acid sequence of SEQ ID NO: 71; or (b)(i) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 56; HC-CDR2 having the amino acid sequence of SEQ ID NO: 57; and HC-CDR3 having the amino acid sequence of SEQ ID NO: 58; and (ii) a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 69; LC-CDR2 having the amino acid sequence of SEQ ID NO: 70; and LC-CDR3 having the amino acid sequence of SEQ ID NO: 71; 94. The composite polypeptide according to any one of paragraphs 81 to 93, comprising:

[0483] 95. The composite polypeptide of any one of paragraphs 81 to 94, wherein the antigen-binding portion comprises a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:68.

[0484] 96. The composite polypeptide of any one of paragraphs 81 to 95, wherein the antigen-binding portion comprises a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 65, 63, or 55.

[0485] 97. The antigen-binding portion is (a)(i) a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 65; and (ii) a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 68; or (b)(i) a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 63; and (ii) a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 68; or (c)(i) a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 55; and (ii) a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 68; 97. The composite polypeptide according to any one of paragraphs 81 to 96, comprising:

[0486] 98. The composite polypeptide of any one of paragraphs 81 to 90, wherein the variant Fc domain comprises a CH2-CH3 region comprising A298, A333, and A334 according to EU numbering.

[0487] 99. The antigen-binding portion comprises the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 77; HC-CDR2 having the amino acid sequence of SEQ ID NO: 78; and HC-CDR3 having the amino acid sequence of SEQ ID NO: 79; 99. The composite polypeptide of any one of paragraphs 81 to 90 or paragraph 98, comprising a VH region incorporating:

[0488] 100. A composite polypeptide according to any one of paragraphs 81 to 90, 98 or 99, wherein the antigen-binding portion comprises the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 85; LC-CDR2 having the amino acid sequence of SEQ ID NO: 86; and LC-CDR3 having the amino acid sequence of SEQ ID NO: 87; A composite polypeptide comprising a VL region incorporating:

[0489] 101. The antigen-binding portion is (i) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 77; HC-CDR2 having the amino acid sequence of SEQ ID NO: 78; and HC-CDR3 having the amino acid sequence of SEQ ID NO: 79; and (ii) a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 85; LC-CDR2 having the amino acid sequence of SEQ ID NO: 86; and LC-CDR3 having the amino acid sequence of SEQ ID NO: 87; 8. The composite polypeptide of any one of paragraphs 81 to 90 or 98 to 100, comprising:

[0490] 102. The composite polypeptide of any one of paragraphs 81-90 or 98-101, wherein the antigen-binding portion comprises a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:76.

[0491] 103. The composite polypeptide of any one of paragraphs 81-90 or 98-102, wherein the antigen-binding portion comprises a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:84.

[0492] 104. The antigen-binding moiety is (i) a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 76; and (ii) a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 84; The composite polypeptide of any one of paragraphs 81 to 90 or 98 to 103, comprising:

[0493] 105. A nucleic acid or nucleic acids encoding a recombinant CD3-TCR complex polypeptide according to any one of paragraphs 1 to 41, a polypeptide complex according to any one of paragraphs 42 to 79, or a complex polypeptide according to any one of paragraphs 81 to 104.

[0494] 106. A nucleic acid or nucleic acids, (a) a first recombinant CD3-TCR complex polypeptide comprising: (i) a first component of the antigen-binding portion, wherein the antigen-binding portion binds to a variant Fc domain having an amino acid sequence that includes at least one amino acid difference relative to a reference Fc domain to which the antigen-binding portion does not bind; and (ii) a CD3-TCR complex-associated domain having an amino acid sequence derived from a CD3-TCR complex polypeptide; and (b) a second recombinant CD3-TCR complex polypeptide comprising: (i) a second component of the antigen-binding portion of(a)(i); and (ii) a CD3-TCR complex-associated domain having an amino acid sequence derived from a CD3-TCR complex polypeptide; Code; A nucleic acid or nucleic acids, wherein the first and second recombinant CD3-TCR complex polypeptides are capable of associating via their CD3-TCR complex association domains to form a CD3-TCR complex comprising an antigen-binding portion.

[0495] 107. The nucleic acid or nucleic acids according to para. 106, wherein the first component of the antigen-binding portion is or comprises a heavy chain variable (VH) region of an antibody that binds to the variant Fc domain, and the second component of the antigen-binding portion is or comprises a light chain variable (VL) region of an antibody that binds to the variant Fc domain.

[0496] 108. (i) the CD3-TCR complex-associated domain of the first recombinant CD3-TCR complex polypeptide is derived from TCRα and the CD3-TCR complex-associated domain of the second recombinant CD3-TCR complex polypeptide is derived from TCRβ; or (ii) the nucleic acid or nucleic acids of paragraph 106 or paragraph 107, wherein the CD3-TCR complex association domain of the first recombinant CD3-TCR complex polypeptide is derived from TCRβ and the CD3-TCR complex association domain of the second recombinant CD3-TCR complex polypeptide is derived from TCRα.

[0497] 109. The nucleic acid or nucleic acids according to paragraph 108, wherein the CD3-TCR complex association domain derived from TCRα comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 52 or 1.

[0498] 110. The nucleic acid or nucleic acids according to paragraph 109, wherein the CD3-TCR complex association domain derived from TCRα comprises a cysteine ​​residue at a position corresponding to position 47 numbered according to SEQ ID NO: 1.

[0499] 111. The nucleic acid or nucleic acids of any one of paragraphs 108 to 110, wherein the CD3-TCR complex association domain derived from TCRβ comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity to one of SEQ ID NOs: 53, 5, 54 or 9.

[0500] 112. The nucleic acid or nucleic acids according to paragraph 111, wherein the CD3-TCR complex association domain derived from TCRβ comprises a cysteine ​​residue at a position corresponding to position 56 numbered according to SEQ ID NO:5.

[0501] 113. (i) in the first recombinant CD3-TCR complex polypeptide, a first component of the antigen-binding portion is linked at its C-terminus to the N-terminus of the CD3-TCR complex-associated domain of the first recombinant CD3-TCR complex polypeptide, optionally via a linker sequence; and / or (ii) the nucleic acid or nucleic acids of any one of paragraphs 106 to 112, wherein in the second recombinant CD3-TCR complex polypeptide, the second component of the antigen-binding portion is linked at its C-terminus to the N-terminus of the CD3-TCR complex association domain of the second recombinant CD3-TCR complex polypeptide, optionally via a linker sequence.

[0502] 114. The nucleic acid or nucleic acids of any one of paras. 106 to 113, wherein the variant Fc domain binds to an Fc receptor with an affinity lower than the affinity with which the reference Fc domain binds to the Fc receptor, and optionally, the Fc receptor is an Fcγ receptor or a neonatal Fc receptor (FcRn).

[0503] 115. The nucleic acid or nucleic acids of any one of paragraphs 106 to 114, wherein the variant Fc domain comprises a CH2-CH3 region comprising an amino acid difference at one or more of the following positions relative to the amino acid sequence of the CH2-CH3 region of the reference Fc domain according to EU numbering: L234, L235, I253, N297, S298, H310, P329, E333, K334, or H435.

[0504] 116. The nucleic acid or nucleic acids of any one of paragraphs 106 to 115, wherein the variant Fc domain comprises a CH2-CH3 region that includes G3...

Claims

1. 1. A recombinant CD3-TCR complex polypeptide comprising: (i) an antigen-binding portion, or a component thereof, that binds to a variant Fc domain having an amino acid sequence that includes at least one amino acid difference relative to a reference Fc domain to which the antigen-binding portion does not bind; and (ii) a CD3-TCR complex-associated domain having an amino acid sequence derived from a CD3-TCR complex polypeptide; A recombinant CD3-TCR complex polypeptide comprising:

2. The recombinant CD3-TCR complex polypeptide of claim 1, wherein the recombinant CD3-TCR complex polypeptide is capable of associating with one or more CD3-TCR complex polypeptides via its CD3-TCR complex-associating domain to form a CD3-TCR complex.

3. The recombinant CD3-TCR complex polypeptide according to claim 1 or 2, wherein the amino acid sequence derived from a CD3-TCR complex polypeptide is derived from CD3ε, TCRα or TCRβ.

4. 4. The recombinant CD3-TCR complex polypeptide of any one of claims 1 to 3, wherein the CD3-TCR complex-associated domain comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity to one of SEQ ID NOs: 30, 52, 1, 53, 5, 54 or 9.

5. The recombinant CD3-TCR complex polypeptide according to any one of claims 1 to 4, wherein the amino acid sequence derived from a CD3-TCR complex polypeptide is derived from CD3ε.

6. 6. The recombinant CD3-TCR complex polypeptide of any one of claims 1 to 5, wherein the CD3-TCR complex-associated domain comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:

30.

7. The recombinant CD3-TCR complex polypeptide according to any one of claims 1 to 4, wherein the amino acid sequence derived from a CD3-TCR complex polypeptide is derived from TCRα or TCRβ.

8. 8. The recombinant CD3-TCR complex polypeptide of any one of claims 1 to 4 or claim 7, wherein the CD3-TCR complex-associated domain comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity to one of SEQ ID NOs: 52, 1, 53, 5, 54 or 9.

9. 9. The recombinant CD3-TCR complex polypeptide of any one of claims 1 to 8, wherein the antigen-binding portion that binds to the variant Fc domain comprises the heavy chain variable (VH) region and the light chain variable (VL) region of an antibody that binds to the variant Fc domain.

10. The antigen-binding portion is selected from the group consisting of Fv, scFv, Fab, Fab', Fab'-SH, F(ab') ２ 10. The recombinant CD3-TCR complex polypeptide of any one of claims 1 to 9, which is or comprises a crossFab, scFab or dAb portion.

11. The recombinant CD3-TCR complex polypeptide of any one of claims 1 to 10, wherein the antigen-binding portion is or comprises an scFv.

12. 12. The recombinant CD3-TCR complex polypeptide of any one of claims 1 to 11, wherein the component of the antigen-binding portion is or comprises the heavy chain variable (VH) region or the light chain variable (VL) region of an antibody that binds to the variant Fc domain.

13. 13. The recombinant CD3-TCR complex polypeptide of any one of claims 1 to 12, wherein the antigen-binding portion or component thereof is linked at its C-terminus to the N-terminus of the CD3-TCR complex-associated domain, optionally via a linker sequence.

14. 14. The recombinant CD3-TCR complex polypeptide of any one of claims 1 to 13, wherein the variant Fc domain binds to an Fc receptor with a lower affinity than the affinity with which the reference Fc domain binds to the Fc receptor, and optionally the Fc receptor is an Fcγ receptor or a neonatal Fc receptor (FcRn).

15. 15. The recombinant CD3-TCR complex polypeptide of any one of claims 1 to 14, wherein the variant Fc domain comprises a CH2-CH3 region that comprises an amino acid difference at one or more of the following positions relative to the amino acid sequence of the CH2-CH3 region of the reference Fc domain according to EU numbering: L234, L235, 1253, N297, S298, H310, P329, E333, K334, or H435.

16. 16. The recombinant CD3-TCR complex polypeptide of any one of claims 1 to 15, wherein the variant Fc domain comprises a CH2-CH3 region that comprises G329 according to EU numbering.

17. The antigen binding portion comprises the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 69; LC-CDR2 having the amino acid sequence of SEQ ID NO: 70; and LC-CDR3 having the amino acid sequence of SEQ ID NO: 71, 17. The recombinant CD3-TCR complex polypeptide of any one of claims 1 to 16, comprising a VL region incorporating:

18. the antigen-binding portion comprises: (i) the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO:56; HC-CDR2 having the amino acid sequence of SEQ ID NO: 64; and HC-CDR3 having the amino acid sequence of SEQ ID NO:58; or (ii) the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO:56; HC-CDR2 having the amino acid sequence of SEQ ID NO:57; and HC-CDR3 having the amino acid sequence of SEQ ID NO: 58; 18. The recombinant CD3-TCR complex polypeptide of any one of claims 1 to 17, comprising a VH incorporating:

19. the antigen-binding portion comprises: (a)(i) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO:56; HC-CDR2 having the amino acid sequence of SEQ ID NO: 64; and HC-CDR3 having the amino acid sequence of SEQ ID NO: 68; and (ii) a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 69; LC-CDR2 having the amino acid sequence of SEQ ID NO: 70; and LC-CDR3 having the amino acid sequence of SEQ ID NO: 71; or (b)(i) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO:56; HC-CDR2 having the amino acid sequence of SEQ ID NO:57; and HC-CDR3 having the amino acid sequence of SEQ ID NO:58; and (ii) a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 69; LC-CDR2 having the amino acid sequence of SEQ ID NO: 70; and LC-CDR3 having the amino acid sequence of SEQ ID NO: 71, 19. The recombinant CD3-TCR complex polypeptide according to any one of claims 1 to 18, comprising:

20. 20. The recombinant CD3-TCR complex polypeptide of any one of claims 1 to 19, wherein the antigen-binding portion comprises a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:

68.

21. 21. The recombinant CD3-TCR complex polypeptide of any one of claims 1 to 20, wherein the antigen-binding portion comprises a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 65, 63 or 55.

22. the antigen-binding portion comprises: (a) (i) a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 65; and (ii) a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 68; or (b)(i) a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 63; and (ii) a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 68; or (c) (i) a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 55; and (ii) a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 68; 22. The recombinant CD3-TCR complex polypeptide according to any one of claims 1 to 21, comprising:

23. 16. The recombinant CD3-TCR complex polypeptide of any one of claims 1 to 15, wherein the variant Fc domain comprises a CH2-CH3 region comprising A298, A333 and A334 according to EU numbering.

24. The antigen binding portion comprises the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 77; HC-CDR2 having the amino acid sequence of SEQ ID NO: 78; and HC-CDR3 having the amino acid sequence of SEQ ID NO: 79; 24. The recombinant CD3-TCR complex polypeptide of any one of claims 1 to 15 or claim 23, comprising a VH region incorporating:

25. The antigen binding portion comprises the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 85; LC-CDR2 having the amino acid sequence of SEQ ID NO: 86; and LC-CDR3 having the amino acid sequence of SEQ ID NO: 87, 25. The recombinant CD3-TCR complex polypeptide of any one of claims 1 to 15, claim 23 or claim 24, comprising a VL region incorporating:

26. the antigen-binding portion comprises: (i) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 77; HC-CDR2 having the amino acid sequence of SEQ ID NO: 78; and HC-CDR3 having the amino acid sequence of SEQ ID NO: 79; and (ii) a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 85; LC-CDR2 having the amino acid sequence of SEQ ID NO: 86; and LC-CDR3 having the amino acid sequence of SEQ ID NO: 87, 26. The recombinant CD3-TCR complex polypeptide of any one of claims 1 to 15 or 23 to 25, comprising:

27. 27. The recombinant CD3-TCR complex polypeptide of any one of claims 1 to 15 or 23 to 26, wherein the antigen-binding portion comprises a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:

76.

28. 28. The recombinant CD3-TCR complex polypeptide of any one of claims 1 to 15 or 23 to 27, wherein the antigen-binding portion comprises a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:

84.

29. the antigen-binding portion comprises: (i) a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 76; and (ii) a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 84; 29. The recombinant CD3-TCR complex polypeptide of any one of claims 1 to 15 or 23 to 28, comprising:

30. 1. A recombinant CD3-TCR complex polypeptide comprising: (i) an antigen-binding moiety that is an scFv, wherein the antigen-binding moiety binds to a variant Fc domain having an amino acid sequence that includes at least one amino acid difference relative to a reference Fc domain to which the antigen-binding moiety does not bind, and the variant Fc domain comprises a CH2-CH3 region that includes G329 according to EU numbering; and (ii) a CD3-TCR complex-associated domain having an amino acid sequence derived from a CD3-TCR complex polypeptide, wherein the amino acid sequence derived from a CD3-TCR complex polypeptide is derived from CD3ε; A recombinant CD3-TCR complex polypeptide comprising:

31. 1. A polypeptide complex comprising: (a) a first recombinant CD3-TCR complex polypeptide comprising: (i) a first component of an antigen-binding portion, wherein the antigen-binding portion binds to a variant Fc domain having an amino acid sequence that includes at least one amino acid difference relative to a reference Fc domain to which the antigen-binding portion does not bind; and (ii) a CD3-TCR complex-associated domain having an amino acid sequence derived from a CD3-TCR complex polypeptide; and (b) a second recombinant CD3-TCR complex polypeptide comprising: (i) a second component of the antigen-binding portion of (a)(i); and (ii) a CD3-TCR complex-associated domain having an amino acid sequence derived from a CD3-TCR complex polypeptide; Including; A polypeptide complex wherein said first and second recombinant CD3-TCR complex polypeptides are capable of associating via their CD3-TCR complex-associating domains to form said antigen-binding portion.

32. 32. The polypeptide complex of claim 31 , wherein the first component of the antigen-binding portion is or comprises a heavy chain variable (VH) region of an antibody that binds to the variant Fc domain, and the second component of the antigen-binding portion is or comprises a light chain variable (VL) region of the antibody that binds to the variant Fc domain.

33. (i) the CD3-TCR complex-associated domain of the first recombinant CD3-TCR complex polypeptide is derived from TCRα and the CD3-TCR complex-associated domain of the second recombinant CD3-TCR complex polypeptide is derived from TCRβ; or (ii) the CD3-TCR complex-associated domain of the first recombinant CD3-TCR complex polypeptide is derived from TCRβ, and the CD3-TCR complex-associated domain of the second recombinant CD3-TCR complex polypeptide is derived from TCRα.

34. 34. The polypeptide complex of claim 33, wherein the CD3-TCR complex-associated domain derived from TCRα comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 52 or 1.

35. 35. The polypeptide complex of claim 33 or claim 34, wherein the CD3-TCR complex-associated domain derived from TCRβ comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity to one of SEQ ID NOs: 53, 5, 54 or 9.

36. 1. A polypeptide complex comprising: (a) a first recombinant CD3-TCR complex polypeptide comprising: (i) a first component of an antigen-binding portion, wherein the antigen-binding portion binds to a variant Fc domain having an amino acid sequence that includes at least one amino acid difference relative to a reference Fc domain to which the antigen-binding portion does not bind, the variant Fc domain comprising a CH2-CH3 region that includes G329 according to EU numbering; and (ii) a CD3-TCR complex-associated domain having an amino acid sequence derived from a CD3-TCR complex polypeptide; and (b) a second recombinant CD3-TCR complex polypeptide comprising: (i) a second component of the antigen-binding portion of (a)(i); and (ii) a CD3-TCR complex-associated domain having an amino acid sequence derived from a CD3-TCR complex polypeptide; Including; said first and second recombinant CD3-TCR complex polypeptides are capable of associating via their CD3-TCR complex-associating domains to form said antigen-binding portion; the first component of the antigen-binding portion is or comprises a heavy chain variable (VH) region of an antibody that binds to the variant Fc domain, and the second component of the antigen-binding portion is or comprises a light chain variable (VL) region of the antibody that binds to the variant Fc domain; and (i) the CD3-TCR complex-associated domain of the first recombinant CD3-TCR complex polypeptide is derived from TCRα and the CD3-TCR complex-associated domain of the second recombinant CD3-TCR complex polypeptide is derived from TCRβ; or (ii) a polypeptide complex, wherein the CD3-TCR complex-associated domain of the first recombinant CD3-TCR complex polypeptide is derived from TCRβ, and the CD3-TCR complex-associated domain of the second recombinant CD3-TCR complex polypeptide is derived from TCRα.

37. A CD3-TCR polypeptide complex comprising a recombinant CD3-TCR complex polypeptide according to any one of claims 1 to 30, or a polypeptide complex according to any one of claims 31 to 36.

38. A composite polypeptide comprising: (a) an amino acid sequence encoding a first recombinant CD3-TCR complex polypeptide comprising: (i) a first component of an antigen-binding portion, wherein the antigen-binding portion binds to a variant Fc domain having an amino acid sequence that includes at least one amino acid difference relative to a reference Fc domain to which the antigen-binding portion does not bind; and (ii) a CD3-TCR complex-associated domain having an amino acid sequence derived from a CD3-TCR complex polypeptide; and (b) an amino acid sequence encoding a second recombinant CD3-TCR complex polypeptide comprising: (i) a second component of the antigen-binding portion of (a)(i); and (ii) a CD3-TCR complex-associated domain having an amino acid sequence derived from a CD3-TCR complex polypeptide; Including; said first and second recombinant CD3-TCR complex polypeptides are capable of associating via their CD3-TCR complex-associating domains to form a CD3-TCR complex comprising said antigen-binding portion; The composite polypeptide further comprises a cleavage site between the amino acid sequences of (a) and (b).

39. 39. The composite polypeptide of claim 38, wherein the first component of the antigen-binding portion is or comprises a heavy chain variable (VH) region of an antibody that binds to the variant Fc domain, and the second component of the antigen-binding portion is or comprises a light chain variable (VL) region of the antibody that binds to the variant Fc domain.

40. (i) the CD3-TCR complex-associated domain of the first recombinant CD3-TCR complex polypeptide is derived from TCRα and the CD3-TCR complex-associated domain of the second recombinant CD3-TCR complex polypeptide is derived from TCRβ; or (ii) the CD3-TCR complex-associated domain of the first recombinant CD3-TCR complex polypeptide is derived from TCRβ, and the CD3-TCR complex-associated domain of the second recombinant CD3-TCR complex polypeptide is derived from TCRα.

41. 41. The composite polypeptide of claim 40, wherein the CD3-TCR complex-associated domain derived from TCRα comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 52 or 1.

42. 42. The composite polypeptide of claim 40 or claim 41, wherein the CD3-TCR complex-associated domain derived from TCRβ comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity to one of SEQ ID NOs: 53, 5, 54 or 9.

43. A nucleic acid or a plurality of nucleic acids encoding a recombinant CD3-TCR complex polypeptide according to any one of claims 1 to 30, a polypeptide complex according to any one of claims 31 to 36, or a complex polypeptide according to any one of claims 38 to 42.

44. a nucleic acid or nucleic acids, (a) a first recombinant CD3-TCR complex polypeptide comprising: (i) a first component of an antigen-binding portion, wherein the antigen-binding portion binds to a variant Fc domain having an amino acid sequence that includes at least one amino acid difference relative to a reference Fc domain to which the antigen-binding portion does not bind; and (ii) a CD3-TCR complex-associated domain having an amino acid sequence derived from a CD3-TCR complex polypeptide; and (b) a second recombinant CD3-TCR complex polypeptide comprising: (i) a second component of the antigen-binding portion of (a)(i); and (ii) a CD3-TCR complex-associated domain having an amino acid sequence derived from a CD3-TCR complex polypeptide; Code; A nucleic acid or nucleic acids, wherein said first and second recombinant CD3-TCR complex polypeptides are capable of associating via their CD3-TCR complex-associating domains to form a CD3-TCR complex comprising said antigen-binding portion.

45. 45. An expression vector or vectors comprising the nucleic acid or nucleic acids of claim 43 or claim 44.

46. A cell comprising a recombinant CD3-TCR complex polypeptide according to any one of claims 1 to 30, a polypeptide complex according to any one of claims 31 to 36, a CD3-TCR polypeptide complex according to claim 15, a composite polypeptide according to any one of claims 38 to 42, a nucleic acid or nucleic acids according to claim 43 or 44, or an expression vector or expression vectors according to claim 45.

47. A pharmaceutical composition comprising the cells of claim 46.

48. 48. A cell according to claim 46 or a pharmaceutical composition according to claim 47 for use in a method of medical treatment or prevention.

49. 48. The cell of claim 46, or the pharmaceutical composition of claim 47, for use in a method for treating or preventing a disease in which cells containing or expressing a target antigen are pathologically involved, the method comprising administering the cells or pharmaceutical composition to a subject who has been or will be administered an antigen-binding molecule; The antigen-binding molecule comprises: (a) an antigen-binding domain that binds to the target antigen; and (b) a variant Fc domain having an amino acid sequence that includes at least one amino acid difference relative to a reference Fc domain; 48. A cell or pharmaceutical composition wherein the antigen-binding portion of the recombinant CD3-TCR complex polypeptide, polypeptide complex, CD3-TCR polypeptide complex or composite polypeptide contained in the cell of claim 46 or contained in the pharmaceutical composition of claim 47, or the antigen-binding portion of the CD3-TCR complex polypeptide, polypeptide complex, CD3-TCR polypeptide complex or composite polypeptide encoded by the nucleic acid(s) or nucleic acids or expression vector(s) contained in the cell of claim 46 or contained in the pharmaceutical composition of claim 47, binds to the variant Fc domain.

50. A method for depleting or killing cells containing or expressing a target antigen, comprising: (i) a cell according to claim 46 or a pharmaceutical composition according to claim 47; and (ii) an antigen-binding molecule comprising: (a) an antigen-binding domain that binds to said target antigen; and (b) a variant Fc domain having an amino acid sequence that includes at least one amino acid difference relative to a reference Fc domain. contacting the 48. A method wherein the antigen-binding portion of the recombinant CD3-TCR complex polypeptide, polypeptide complex, CD3-TCR polypeptide complex or composite polypeptide contained in the cell of claim 46 or in a cell comprised in the pharmaceutical composition of claim 47, or the antigen-binding portion of the CD3-TCR complex polypeptide, polypeptide complex, CD3-TCR polypeptide complex or composite polypeptide encoded by the nucleic acid(s) or nucleic acids or expression vector(s) contained in the cell of claim 46 or in a cell comprised in the pharmaceutical composition of claim 47, binds to the variant Fc domain.

51. A kit comprising: (i) a cell according to claim 46 or a pharmaceutical composition according to claim 47; and (ii) an antigen-binding molecule comprising: (a) an antigen-binding domain that binds to a target antigen; and (b) a variant Fc domain having an amino acid sequence that includes at least one amino acid difference relative to a reference Fc domain. Including; 48. A kit wherein the antigen-binding portion of the recombinant CD3-TCR complex polypeptide, polypeptide complex, CD3-TCR polypeptide complex or composite polypeptide contained in the cell of claim 46 or in the cell comprised in the pharmaceutical composition of claim 47, or the antigen-binding portion of the CD3-TCR complex polypeptide, polypeptide complex, CD3-TCR polypeptide complex or composite polypeptide encoded by the nucleic acid(s) or nucleic acids or expression vector(s) contained in the cell of claim 46 or in the cell comprised in the pharmaceutical composition of claim 47, binds to the variant Fc domain.

52. A kit comprising: (i) a nucleic acid or nucleic acids according to claim 43 or claim 44, or an expression vector or vectors according to claim 45; and (ii) an antigen-binding molecule comprising: (a) an antigen-binding domain that binds to a target antigen; and (b) a variant Fc domain having an amino acid sequence that includes at least one amino acid difference relative to a reference Fc domain. Including; 46. ​​A kit wherein the antigen binding portion of the CD3-TCR complex polypeptide, polypeptide complex, CD3-TCR polypeptide complex or composite polypeptide encoded by a nucleic acid or nucleic acids of claim 43 or claim 44 or contained in an expression vector or vectors of claim 45 binds to the variant Fc domain.

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