Recombinant T cell receptors

Abstract

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

Description

Technical Field

[0001] The present disclosure relates to the field of molecular biology, more specifically to antigen binding molecule technology. The present disclosure also relates to methods of medical treatment and prevention, in particular cellular immunotherapy. Background Art

[0002] T cells expressing chimeric antigen receptors (CARs) have shown clinical efficacy in different hematological malignancies. For solid tumor indications, CAR-T cell therapy has not yet provided meaningful clinical benefits over traditional chemotherapy and immunotherapy. Various strategies have been adopted to overcome the limitations of CAR-T cell therapy, which can be summarized as systemic toxicity and insufficient anti-tumor immune responses mediated by CAR-T cells. The most widespread CAR format to date is the second-generation CAR, which is characterized by a co-stimulatory signaling domain (usually from CD28 or 4-1BB co-stimulatory receptors) and a CD3ζT cell receptor (TCR) signaling domain. Although this format is widely used, it exhibits inherent limitations that are well documented. The expression levels of second-generation CARs are higher than those of natural T cell receptors, and some commonly used antibody-derived single-chain fragment variable (scFv) domains can exhibit a tendency to mispair and aggregate on the cell surface under these conditions. This may lead to constitutive CAR signaling in the absence of tumor antigens, causing toxicity and T cell exhaustion. Therefore, research efforts have been devoted to generating CAR formats that exhibit more physiological and strictly antigen-dependent CAR signaling.

[0003] An early described strategy aims to add or replace the antigen specificity of the natural TCR complex. Different methods for achieving this have been described. By adding antibody variable domains to the CD3ε domain of 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 T cell activation independent of peptide-human leukocyte antigen (pHLA) via the TCR complex. Another approach aims to replace the variable α and β domains of TCR with variable light chain domains and variable heavy chain domains derived from antibodies (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 is combined with enzyme-mediated gene knockout of endogenous TCR α and β chain encoding genes, causing the loss of natural T cell specificity and obtaining new antigen specificity of interest. In view of the above two forms and the second-generation CAR forms described previously, unless several different CAR genes are introduced, the final recombinant T cell product will be characterized by a single new antigen specificity.

[0004] We and others have previously described modular CARs that can bind to defined adapter molecules and have specificity for different tumor antigens (Darowski et al. MAbs (2019) 11(4): 621-631). T cells expressing chimeric antigen receptor (CAR) constructs (which include antigen binding portions specific for variant Fc domains) are disclosed in WO 2018 / 177966A1. The CAR construct is a second-generation CAR that includes a costimulatory sequence associated with the intracellular domain of CD3ζ. Summary of the invention

[0005] In a first aspect, the present disclosure provides a recombinant CD3-TCR complex polypeptide comprising:

[0006] (i) an antigen binding portion or a component thereof, 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; and

[0007] (ii) a CD3-TCR complex association domain having an amino acid sequence derived from a CD3-TCR complex polypeptide.

[0008] In some embodiments, the recombinant CD3-TCR complex polypeptide is capable of associating with one or more CD3-TCR complex polypeptides through its CD3-TCR complex association domain to form a CD3-TCR complex.

[0009] In some embodiments, the amino acid sequence derived from a CD3-TCR complex polypeptide is derived from CD3ε, TCRα, or TCRβ.

[0010] 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 NO: 30, 52, 1, 53, 5, 54, or 9.

[0011] In some embodiments, the amino acid sequence derived from a CD3-TCR complex polypeptide is derived from CD3 epsilon.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.

[0012] 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 NO: 52, 1, 53, 5, 54 or 9.

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

[0014] In some embodiments, the antigen binding portion is or comprises Fv, scFv, Fab, Fab', Fab'-SH, F(ab') 2 , crossFab, scFab or dAb portion. In some embodiments, the antigen binding portion is or comprises scFv.

[0015] In some embodiments, the component of the antigen binding moiety is or comprises a heavy chain variable (VH) region or a light chain variable (VL) region of an antibody bound to a variant Fc domain.

[0016] 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 associating domain, optionally via a linker sequence.

[0017] In some embodiments, the variant Fc domain binds to an Fc receptor with a lower affinity than the reference Fc domain binds to the Fc receptor, optionally wherein the Fc receptor is an Fc gamma receptor or a neonatal Fc receptor (FcRn).

[0018] In some embodiments, the variant Fc domain comprises a CH2-CH3 region comprising an amino acid difference relative to the amino acid sequence of the CH2-CH3 region of a reference Fc domain at one or more of the following positions according to EU numbering: L234, L235, 1253, N297, S298, H310, P329, E333, K334, or H435.

[0019] In some embodiments, the variant Fc domain comprises a CH2-CH3 region comprising G329 according to EU numbering.

[0020] In some embodiments, the antigen binding portion comprises a VL region incorporating the following CDRs:

[0021] LC-CDR1 having the amino acid sequence of SEQ ID NO:69;

[0022] LC-CDR2 having the amino acid sequence of SEQ ID NO:70; and

[0023] LC-CDR3 having the amino acid sequence of SEQ ID NO:71.

[0024] In one embodiment, the antigen binding portion comprises a VH region incorporating:

[0025] (i) the following CDRs:

[0026] HC-CDR1 having the amino acid sequence of SEQ ID NO:56;

[0027] an HC-CDR2 having the amino acid sequence of SEQ ID NO: 64; and

[0028] an HC-CDR3 having the amino acid sequence of SEQ ID NO: 58;

[0029] or

[0030] (ii) the following CDRs:

[0031] HC-CDR1 having the amino acid sequence of SEQ ID NO:56;

[0032] an HC-CDR2 having the amino acid sequence of SEQ ID NO: 57; and

[0033] HC-CDR3 having the amino acid sequence of SEQ ID NO:58.

[0034] In one embodiment, the antigen binding portion comprises:

[0035] (a)(i) A VH region incorporating the following CDRs:

[0036] HC-CDR1 having the amino acid sequence of SEQ ID NO:56;

[0037] an HC-CDR2 having the amino acid sequence of SEQ ID NO: 64; and

[0038] an HC-CDR3 having the amino acid sequence of SEQ ID NO: 68;

[0039] and

[0040] (ii) a VL region incorporating the following CDRs:

[0041] LC-CDR1 having the amino acid sequence of SEQ ID NO:69;

[0042] LC-CDR2 having the amino acid sequence of SEQ ID NO:70; and

[0043] a LC-CDR3 having the amino acid sequence of SEQ ID NO: 71;

[0044] or

[0045] (b)(i) a VH region incorporating the following CDRs:

[0046] HC-CDR1 having the amino acid sequence of SEQ ID NO:56;

[0047] an HC-CDR2 having the amino acid sequence of SEQ ID NO: 57; and

[0048] an HC-CDR3 having the amino acid sequence of SEQ ID NO: 58;

[0049] and

[0050] (ii) a VL region incorporating the following CDRs:

[0051] LC-CDR1 having the amino acid sequence of SEQ ID NO:69;

[0052] LC-CDR2 having the amino acid sequence of SEQ ID NO:70; and

[0053] LC-CDR3 having the amino acid sequence of SEQ ID NO:71.

[0054] 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.

[0055] 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.

[0056] In one embodiment, the antigen binding portion comprises:

[0057] (a)(i) a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:65; and

[0058] (ii) a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 68;

[0059] or

[0060] (b)(i) a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:63; and

[0061] (ii) a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 68;

[0062] or

[0063] (c)(i) a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:55; and

[0064] (ii) a VL having an amino acid sequence that has at least 70% amino acid sequence identity to SEQ ID NO:68.

[0065] In some embodiments, the variant Fc domain comprises a CH2-CH3 region containing A298, A333, and A334 according to EU numbering.

[0066] In some embodiments, the antigen binding portion comprises a VH region incorporating the following CDRs:

[0067] HC-CDR1 having the amino acid sequence of SEQ ID NO: 77;

[0068] an HC-CDR2 having the amino acid sequence of SEQ ID NO: 78; and

[0069] HC-CDR3 having the amino acid sequence of SEQ ID NO:79.

[0070] In some embodiments, the antigen binding portion comprises a VL region incorporating the following CDRs:

[0071] LC-CDR1 having the amino acid sequence of SEQ ID NO:85;

[0072] LC-CDR2 having the amino acid sequence of SEQ ID NO:86; and

[0073] LC-CDR3 having the amino acid sequence of SEQ ID NO:87.

[0074] In one embodiment, the antigen binding portion comprises:

[0075] (i) a VH region incorporating the following CDRs:

[0076] HC-CDR1 having the amino acid sequence of SEQ ID NO: 77;

[0077] an HC-CDR2 having the amino acid sequence of SEQ ID NO: 78; and

[0078] an HC-CDR3 having the amino acid sequence of SEQ ID NO: 79;

[0079] and

[0080] (ii) a VL region incorporating the following CDRs:

[0081] LC-CDR1 having the amino acid sequence of SEQ ID NO:85;

[0082] LC-CDR2 having the amino acid sequence of SEQ ID NO:86; and

[0083] LC-CDR3 having the amino acid sequence of SEQ ID NO:87.

[0084] In some embodiments, the antigen binding portion comprises a VH having an amino acid sequence that has at least 70% amino acid sequence identity to SEQ ID NO:76.

[0085] 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.

[0086] In one embodiment, the antigen binding portion comprises:

[0087] (i) a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 76; and

[0088] (ii) a VL having an amino acid sequence that has at least 70% amino acid sequence identity to SEQ ID NO:84.

[0089] The present disclosure also provides a recombinant CD3-TCR complex polypeptide, comprising:

[0090] (i) an antigen binding portion which is an scFv or a component thereof, 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

[0091] (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.

[0092] The present disclosure also provides a polypeptide complex comprising:

[0093] (a) a first recombinant CD3-TCR complex polypeptide comprising:

[0094] (i) a first component of an antigen binding moiety, wherein the antigen binding moiety 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 moiety does not bind; and

[0095] (ii) a CD3-TCR complex association domain having an amino acid sequence derived from a CD3-TCR complex polypeptide; and

[0096] (b) a second recombinant CD3-TCR complex polypeptide comprising:

[0097] (i) a second component of the antigen binding portion of (a) (i); and

[0098] (ii) a CD3-TCR complex association domain having an amino acid sequence derived from a CD3-TCR complex polypeptide;

[0099] The first recombinant CD3-TCR complex polypeptide and the second recombinant CD3-TCR complex polypeptide are capable of associating via their CD3-TCR complex association domains to form the antigen binding portion.

[0100] In some embodiments, the first component of the antigen binding moiety is or comprises a heavy chain variable (VH) region of an antibody bound to a variant Fc domain, and wherein the second component of the antigen binding moiety is or comprises a light chain variable (VL) region of an antibody bound to the variant Fc domain.

[0101] In some embodiments:

[0102] (i) the CD3-TCR complex association domain of the first recombinant CD3-TCR complex polypeptide is derived from TCRα, and the CD3-TCR complex association domain of the second recombinant CD3-TCR complex polypeptide is derived from TCRβ, or

[0103] (ii) 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.

[0104] In some embodiments, 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.

[0105] In some embodiments, 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 NO: 53, 5, 54, or 9.

[0106] The present disclosure also provides a polypeptide complex comprising:

[0107] (a) a first recombinant CD3-TCR complex polypeptide comprising:

[0108] (i) a first component of an antigen binding moiety, wherein the antigen binding moiety 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 moiety does not bind, wherein the variant Fc domain comprises a CH2-CH3 region comprising G329 according to EU numbering; and

[0109] (ii) a CD3-TCR complex association domain having an amino acid sequence derived from a CD3-TCR complex polypeptide; and

[0110] (b) a second recombinant CD3-TCR complex polypeptide comprising:

[0111] (i) a second component of the antigen binding portion of (a) (i); and

[0112] (ii) a CD3-TCR complex association domain having an amino acid sequence derived from a CD3-TCR complex polypeptide;

[0113] wherein the first recombinant CD3-TCR complex polypeptide and the second recombinant CD3-TCR complex polypeptide are capable of associating via their CD3-TCR complex association domains to form the antigen binding portion;

[0114] wherein the first component of the antigen binding moiety is or comprises a heavy chain variable (VH) region of an antibody that binds to the variant Fc domain, and wherein the second component of the antigen binding moiety is or comprises a light chain variable (VL) region of an antibody that binds to the variant Fc domain;

[0115] And among them:

[0116] (i) the CD3-TCR complex association domain of the first recombinant CD3-TCR complex polypeptide is derived from TCRα, and the CD3-TCR complex association domain of the second recombinant CD3-TCR complex polypeptide is derived from TCRβ, or

[0117] (ii) 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.

[0118] The present disclosure also provides a CD3-TCR polypeptide complex, wherein the CD3-TCR polypeptide complex comprises a recombinant CD3-TCR complex polypeptide or polypeptide complex according to the present disclosure.

[0119] The present disclosure also provides a composite polypeptide comprising:

[0120] (a) an amino acid sequence encoding a first recombinant CD3-TCR complex polypeptide, the first recombinant CD3-TCR complex polypeptide comprising:

[0121] (i) a first component of an antigen binding moiety, wherein the antigen binding moiety 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 moiety does not bind; and

[0122] (ii) a CD3-TCR complex association domain having an amino acid sequence derived from a CD3-TCR complex polypeptide; and

[0123] (b) an amino acid sequence encoding a second recombinant CD3-TCR complex polypeptide, the second recombinant CD3-TCR complex polypeptide comprising:

[0124] (i) a second component of the antigen binding portion of (a) (i); and

[0125] (ii) a CD3-TCR complex association domain having an amino acid sequence derived from a CD3-TCR complex polypeptide;

[0126] wherein the first recombinant CD3-TCR complex polypeptide and the second recombinant CD3-TCR complex polypeptide are capable of associating via their CD3-TCR complex association domains to form a CD3-TCR complex comprising the antigen binding portion;

[0127] And wherein the composite polypeptide further comprises a cleavage site between the amino acid sequences of (a) and (b).

[0128] In some embodiments, the first component of the antigen binding moiety is or comprises a heavy chain variable (VH) region of an antibody bound to a variant Fc domain, and wherein the second component of the antigen binding moiety is or comprises a light chain variable (VL) region of an antibody bound to the variant Fc domain.

[0129] In some embodiments:

[0130] (i) the CD3-TCR complex association domain of the first recombinant CD3-TCR complex polypeptide is derived from TCRα, and the CD3-TCR complex association domain of the second recombinant CD3-TCR complex polypeptide is derived from TCRβ, or

[0131] (ii) 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.

[0132] In some embodiments, 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.

[0133] In some embodiments, 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 NO: 53, 5, 54, or 9.

[0134] The present disclosure also provides a nucleic acid or multiple nucleic acids encoding a recombinant CD3-TCR complex polypeptide, a polypeptide complex or a composite polypeptide according to the present disclosure.

[0135] The present disclosure also provides a nucleic acid or multiple nucleic acids encoding:

[0136] (a) a first recombinant CD3-TCR complex polypeptide comprising:

[0137] (i) a first component of an antigen binding moiety, wherein the antigen binding moiety 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 moiety does not bind; and

[0138] (ii) a CD3-TCR complex association domain having an amino acid sequence derived from a CD3-TCR complex polypeptide; and

[0139] (b) a second recombinant CD3-TCR complex polypeptide comprising:

[0140] (i) a second component of the antigen binding portion of (a) (i); and

[0141] (ii) a CD3-TCR complex association domain having an amino acid sequence derived from a CD3-TCR complex polypeptide;

[0142] The first recombinant CD3-TCR complex polypeptide and the second recombinant CD3-TCR complex polypeptide are capable of associating through their CD3-TCR complex association domains to form a CD3-TCR complex comprising the antigen binding portion.

[0143] The present disclosure also provides an expression vector or multiple expression vectors comprising a nucleic acid or multiple nucleic acids according to the present disclosure.

[0144] The present disclosure also provides a cell comprising a recombinant CD3-TCR complex polypeptide, a polypeptide complex, a CD3-TCR polypeptide complex, a composite polypeptide, a nucleic acid or multiple nucleic acids, or an expression vector or multiple expression vectors according to the present disclosure.

[0145] The present disclosure also provides a pharmaceutical composition comprising the cell according to the present disclosure.

[0146] The present disclosure also provides a cell or a pharmaceutical composition according to the present disclosure for use in a method of medical treatment or prevention.

[0147] 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 a cell comprising or expressing a target antigen is pathologically affected, wherein the method comprises administering the cell or pharmaceutical composition to a subject to whom an antigen binding molecule has been or is to be administered;

[0148] wherein 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 comprising at least one amino acid difference relative to a reference Fc domain;

[0149] and wherein a recombinant CD3-TCR complex polypeptide, polypeptide complex, CD3-TCR polypeptide complex or an antigen-binding portion of a composite polypeptide comprised in a cell according to the present disclosure or in a cell comprised in a pharmaceutical composition according to the present disclosure, or a CD3-TCR complex polypeptide, polypeptide complex, CD3-TCR polypeptide complex or an antigen-binding portion of a composite polypeptide encoded by a nucleic acid or multiple nucleic acids or an expression vector or multiple expression vectors comprised in a cell according to the present disclosure or in a cell comprised in a pharmaceutical composition according to the present disclosure, binds to the variant Fc domain.

[0150] The present disclosure also provides a method for depleting or killing cells containing or expressing a target antigen, the method comprising contacting the cells containing / expressing the target antigen with:

[0151] (i) a cell or a pharmaceutical composition according to the present disclosure; and

[0152] (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 comprising at least one amino acid difference relative to a reference Fc domain;

[0153] wherein a recombinant CD3-TCR complex polypeptide, polypeptide complex, CD3-TCR polypeptide complex or composite polypeptide antigen-binding portion contained in a cell according to the present disclosure or a cell contained in a pharmaceutical composition according to the present disclosure, or a CD3-TCR complex polypeptide, polypeptide complex, CD3-TCR polypeptide complex or composite polypeptide antigen-binding portion encoded by a nucleic acid or multiple nucleic acids or an expression vector or multiple expression vectors contained in a cell according to the present disclosure or a cell contained in a pharmaceutical composition according to the present disclosure, binds to the variant Fc domain.

[0154] The present disclosure also provides a kit, which comprises:

[0155] (i) a cell or a pharmaceutical composition according to the present disclosure; and

[0156] (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 comprising at least one amino acid difference relative to a reference Fc domain;

[0157] wherein a recombinant CD3-TCR complex polypeptide, polypeptide complex, CD3-TCR polypeptide complex or composite polypeptide antigen-binding portion contained in a cell according to the present disclosure or a cell contained in a pharmaceutical composition according to the present disclosure, or a CD3-TCR complex polypeptide, polypeptide complex, CD3-TCR polypeptide complex or composite polypeptide antigen-binding portion encoded by a nucleic acid or multiple nucleic acids or an expression vector or multiple expression vectors contained in a cell according to the present disclosure or a cell contained in a pharmaceutical composition according to the present disclosure, binds to the variant Fc domain.

[0158] The present disclosure also provides a kit, which comprises:

[0159] (i) one or more nucleic acids according to the disclosure, or one or more expression vectors according to the disclosure; and

[0160] (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 comprising at least one amino acid difference relative to a reference Fc domain;

[0161] wherein an antigen-binding portion of a CD3-TCR complex polypeptide, a polypeptide complex, a CD3-TCR polypeptide complex or a composite polypeptide encoded by a nucleic acid or multiple nucleic acids according to the present disclosure or encoded by a nucleic acid or multiple nucleic acids contained in an expression vector or multiple expression vectors according to the present disclosure binds to the variant Fc domain. DETAILED DESCRIPTION

[0162] The present disclosure provides recombinant CD3-TCR complex polypeptides, polypeptide complexes comprising such recombinant CD3-TCR complex polypeptides (including CD3-TCR polypeptide complexes), 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.

[0163] More specifically, the present disclosure relates to novel recombinant CD3-TCR complex polypeptides and polypeptide complexes comprising the recombinant CD3-TCR complex polypeptides, the recombinant CD3-TCR complex polypeptides having an antigen binding portion specific for a variant Fc domain and comprising a CD3-TCR complex association domain, such that when they are expressed by cells (particularly T cells), the recombinant CD3-TCR complex polypeptides are able to associate with the CD3-TCR complex polypeptides to form a CD3-TCR complex that recognizes the variant Fc domain. When the antigen binding portion binds to its cognate variant Fc domain, CD3-TCR complex-mediated signaling is triggered, thereby activating cells expressing the CD3-TCR polypeptide complex.

[0164] Unexpectedly, the present disclosure demonstrates that novel recombinant CD3-TCR complex polypeptides that do not contain a domain capable of intrinsically triggering CD3-TCR complex-mediated signaling (e.g., a CD3ζ intracellular domain) are capable of associating with endogenous CD3-TCR complex polypeptides expressed by cells to form functional CD3-TCR complexes that still cause T cell activation in response to the variant Fc domain to which the antigen binding moiety is bound. In addition, the present disclosure demonstrates that this technical effect is achieved through different embodiments, particularly (i) embodiments in which the recombinant CD3-TCR complex polypeptide comprises an scFv bound to a variant Fc domain and a CD3-TCR complex associating 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 comprising CD3-TCR complex associating domains derived from TRAC and TRBC1, respectively.

[0165] More unexpectedly, in experiments providing a direct comparison of T cell activation levels in response to cells presenting variant Fc domains, T cells expressing the novel CD3-TCR complexes according to (i) and (ii) in the preceding paragraph were shown to be activated to a degree similar to or greater than that of T cells expressing a CAR construct having the same antigen binding portion (in scFv format) comprising co-stimulatory and CD3ζ intracellular signaling domains (disclosed in WO 2018 / 177966 A1) - see Figures 6, 7, 10 and 11.

[0166] Recombinant CD3-TCR complex polypeptide

[0167] Aspects and embodiments of the present disclosure relate to recombinant CD3-TCR complex polypeptides.

[0168] As mentioned herein, a "recombinant" polypeptide refers to a non-naturally occurring polypeptide. A recombinant polypeptide may also be referred to as a "synthetic" polypeptide. A recombinant polypeptide may 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 may comprise or consist of an amino acid sequence that is not contained in the amino acid sequence of a polypeptide produced by a naturally occurring organism. A recombinant polypeptide may be encoded by a nucleic acid produced using recombinant nucleic acid technology. A recombinant polypeptide may be produced by expressing (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 nucleotide sequences of nucleic acids, and includes molecular cloning.

[0169] The CD3-TCR complex (sometimes also referred to as the TCR-CD3 complex, see, e.g., Dong et al., Nature (2019) 573(7775):546-552) is a polypeptide complex expressed at the cell surface of T cells that is involved in antigen-specific T cell activation. The structure and function of the CD3-TCR complex have been reviewed, e.g., in Mariuzza et al., J Biol Chem. 2020 Jan 24; 295(4):914–925, the entire contents of which are incorporated herein by reference.

[0170] In mammals, the CD3-TCR complex contains a variety of TCR polypeptides that together form a heterodimeric TCR (TCRα and TCRβ, or TCRy and TCRδ) for antigen recognition, provided in a non-covalent association with the invariant CD3ε, CD3δ, CD3γ and CD3ζ polypeptides. The classic octameric CD3-TCR complex contains TCRα and TCRβ heterodimers (ie, TCRαβ) or TCRy and TCRδ heterodimers (ie, TCRγδ), heterodimers containing CD3ε and CD3δ (ie, CD3δε), heterodimers containing CD3ε and CD3γ (ie, CD3γε), and CD3ζ homodimers (ie, CD3ζζ). Such TCR-CD3 complexes can be expressed as CD3γε / CD3δε / CD3ζζ / TCRαβ and CD3γε / CD3δε / CD3ζζ / TCRγδ, respectively (see, e.g., Zheng et al., Nature (2019) 573(7775):546–552).

[0171] In some embodiments, the CD3-TCR complex is a CD3-TCRα / β complex. In some embodiments, the CD3-TCR complex is a CD3-TCRy / δ complex. The CD3-TCRα / β complex may include TCRα and TCRβ polypeptides, and may also include CD3γ, CD3ε, CD3δ and / or CD3ζ polypeptides. The CD3-TCRα / β complex may include CD3γε / CD3δε / CD3ζζ / TCRαβ or may be composed thereof. The CD3-TCRγ / δ complex may include TCRγ and TCRδ polypeptides, and may also include CD3γ, CD3ε, CD3δ and / or CD3ζ polypeptides. The CD3-TCRγ / δ complex may include CD3γε / CD3δε / CD3ζζ / TCRγ / δ or may be composed thereof.

[0172] Herein, "CD3-TCR complex polypeptide" refers to a constituent polypeptide of the 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 as described herein.

[0173] In the present specification, "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 from any species, respectively, and include isoforms, fragments, variants or homologs from any species. In some embodiments, TCRα, TCRβ, TCRγ, TCRδ, TRAC, TRBC1, TRBC2, TRGC1, TRGC2, TRDC, CD3ε, CD3δ, CD3γ, CD3ζ and CD3η from any species, respectively. AC, TRBC1, TRBC2, TRGC1, TRGC2, TRDC, CD3ε, CD3δ, CD3γ, CD3ζ or CD3η are from mammals (e.g., therian, placental, epitherian, preptotheria, archontan, primate (rhesus monkey, cynomolgus monkey, non-human primate or human). In some embodiments, TCRα, TCRβ, TCRγ, TCRδ, TRAC, TRBC1, TRBC2, TRGC1, TRGC2, TRDC, CD3ε, CD3δ, CD3γ, CD3ζ or CD3η are human.

[0174] 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) can be characterized as having at least 70% sequence identity, preferably one of ≥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.

[0175] A "fragment" generally refers to a portion 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 relative to the amino acid sequence of a reference protein, but retains a substantial 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 expressed by the same species as the species of the reference protein. A "homolog" generally refers to a variant of a reference protein produced by a different species than the species of the reference protein. Homologs include orthologs.

[0176] Isoforms, fragments, variants or homologs of a given reference protein may optionally be characterized as having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to the amino acid sequence of a specified isoform of the related protein from a given species (e.g., human) in its immature or mature (after processing to remove the signal peptide) form.

[0177] In some embodiments, 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, 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.

[0178] In some embodiments, the TCRβ comprises an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to SEQ ID NO: 5 or 9. In some embodiments, TRBC1 comprises or consists of an amino acid sequence having at least 70% (preferably one of ≥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, TRBC2 comprises or consists of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO:9.

[0179] 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 one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO:17.

[0180] In some embodiments, the TCRδ comprises an amino acid sequence having at least 70% (preferably one of ≥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, the TRDC comprises or consists of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to SEQ ID NO: 21.

[0181] In some embodiments, CD3ε comprises or consists of an amino acid sequence having at least 70% (preferably one of ≥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.

[0182] In some embodiments, CD3δ comprises or consists of an amino acid sequence having at least 70% (preferably one of ≥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.

[0183] In some embodiments, CD3γ comprises or consists of an amino acid sequence having at least 70% (preferably one of ≥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.

[0184] In some embodiments, CD3ζ comprises or consists of an amino acid sequence having at least 70% (preferably one of ≥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.

[0185] In some embodiments, CD3η comprises or consists of an amino acid sequence having at least 70% (preferably one of ≥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.

[0186] In some embodiments, a recombinant CD3-TCR complex polypeptide according to the present disclosure comprises:

[0187] (i) an antigen binding portion or component thereof as described herein; and

[0188] (ii) a CD3-TCR complex association domain as described herein.

[0189] In some embodiments, in the amino acid sequence of the recombinant CD3-TCR complex polypeptide, the amino acid sequence of the antigen binding portion / component thereof is located at the N-terminus of the amino acid sequence of the CD3-TCR complex associating domain. That is, in some embodiments, the recombinant CD3-TCR complex polypeptide comprises the following structure: N-terminus-[...]-[antigen binding portion / component thereof]-[CD3-TCR complex associating domain]-[...]-C-terminus.

[0190] In some embodiments, the recombinant CD3-TCR complex polypeptide according to the present disclosure does not comprise a domain or amino acid sequence containing an immunoreceptor tyrosine-based activation motif (ITAM). ITAM comprises an amino acid sequence according to YXXL / I (SEQ ID NO: 222), wherein "X" represents any amino acid. In proteins containing ITAM, sequences according to YXXL / I are typically separated by 6 to 8 amino acids (i.e., they conform to the formula: YXXL / I(X) 6-8 YXXL / I; SEQ ID NO: 223). When a phosphate group is added to the tyrosine residue of ITAM by tyrosine kinase, a signaling cascade is initiated in the cell. ITAM-containing sequences include the intracellular domains of CD3ζ and FcγRI. In some embodiments, the recombinant CD3-TCR complex polypeptide according to the present disclosure does not comprise the amino acid sequence shown in SEQ ID NO: 47. In some embodiments, the recombinant CD3-TCR complex polypeptide does not comprise the amino acid sequence according to SEQ ID NO: 223. In some embodiments, the recombinant CD3-TCR complex polypeptide does not comprise the amino acid sequence according to SEQ ID NO: 222.

[0191] In some embodiments, the recombinant CD3-TCR complex polypeptide according to the present disclosure does not include a costimulatory sequence. As mentioned herein, "costimulatory sequence" refers to an amino acid sequence that provides costimulation of immune cells expressing recombinant CD3-TCR complex polypeptides. Costimulation promotes proliferation and survival, and can also promote cytokine production, differentiation, cytotoxic function and memory formation. The molecular mechanism of T cell costimulation is reviewed, for example, in Chen and Flies, (2013) Nat Rev Immunol 13 (4): 227-242. The costimulatory sequence can be or can be derived from the intracellular domain of costimulatory proteins. Costimulatory proteins include CD28, 4-1BB, ICOS, CD27, OX40, HVEM, CD2, SLAM, TIM-1, CD30, GITR, DR3, CD226 and LIGHT. In some embodiments, the recombinant CD3-TCR complex polypeptide according to the present disclosure does not include the amino acid sequence shown in SEQ ID NO:101 (the intracellular domain of human 4-1BB).

[0192] CD3-TCR complex association domain

[0193] The recombinant CD3-TCR complex polypeptides of the present disclosure comprise a CD3-TCR complex association domain. The basic function of the CD3-TCR complex association domain 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.

[0194] "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 above). Therefore, the CD3-TCR complex association domain according to the present disclosure comprises or consists of an amino acid sequence that confers the ability of a polypeptide comprising the domain to associate with a CD3-TCR complex polypeptide to form a polypeptide complex comprising the CD3-TCR complex polypeptide and a polypeptide carrying the CD3-TCR complex association domain.

[0195] The association between a CD3-TCR complex association domain / a polypeptide comprising a CD3-TCR complex association domain and a CD3-TCR complex polypeptide can be characterized by non-covalent protein:protein interactions. In some embodiments, the association comprises electrostatic interactions (e.g., ionic bonds, hydrogen bonds) and / or van der Waals forces.

[0196] In some embodiments, the CD3-TCR complex association domain is or is derived from the amino acid sequence of a CD3-TCR complex polypeptide. It should be understood that the CD3-TCR complex association domain can be or can be derived from a region of a CD3-TCR complex polypeptide through which the CD3-TCR complex polypeptide interacts with other CD3-TCR complex polypeptides to form a polypeptide complex. In some embodiments, the 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 required for the association between the CD3-TCR complex polypeptide and other CD3-TCR complex polypeptides to form a polypeptide complex comprising such polypeptides.

[0197] The regions of the CD3-TCR complex polypeptides required for such interactions can be determined by site-directed mutagenesis and / or truncation studies, in which the amino acid sequence of the CD3-TCR complex polypeptide is altered or truncated, and the effect of such alterations / truncations on the ability of the CD3-TCR complex polypeptide to bind to other CD3-TCR complex polypeptides is assessed. Suitable techniques for studying such protein:protein interactions include, for example, resonance energy transfer techniques, such as fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET), using appropriately labeled interaction partners, for example as described in Ciruela, Curr. Opin. Biotechnol. (2008) 19(4): 338-43.

[0198] 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 the functional and / or structural properties of the reference polypeptide / domain / amino acid sequence.

[0199] In some embodiments, the CD3-TCR complex association domain comprises modifications that promote association with CD3-TCR complex polypeptides. In some embodiments, the CD3-TCR complex association domain comprises modifications that promote heteromerization (ie, association with non-identical CD3-TCR complex polypeptides).

[0200] As used herein, "modification" refers to the difference relative to a reference amino acid sequence. The reference amino acid sequence can be an amino acid sequence encoded by the most common nucleotide sequence of a gene encoding a related protein. In the embodiments herein (and more generally in the art), "modification" may also be referred to as "substitution" or "mutation". Modifications generally include substitutions of amino acid residues. Substitution of amino acid residues includes substitution of amino acid residues with non-identical "replacement" amino acid residues. The replacement amino acid residue modified according to the present disclosure can be a naturally occurring amino acid residue (i.e., encoded by the genetic code) that is different from the amino acid residue at the relevant position of the amino acid sequence before 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 modified can be a non-naturally occurring amino acid residue, i.e., an amino acid residue other than the amino acid residues listed in the previous 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.

[0201] For example, in the embodiments herein, the CD3-TCR complex association domain derived from TRAC comprises a modification of replacing 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 comprises a modification of replacing the serine residue at position 56 (numbered relative to SEQ ID NO: 53) with a cysteine ​​residue (SEQ ID NO: 5). The introduction of these cysteine ​​residues promotes heteromerization between the modified domains through the formation of interchain disulfide bridges.

[0202] Embodiments in which the CD3-TCR complex association domain further comprises modifications to promote association with a CD3-TCR complex polypeptide are contemplated to be particularly relevant 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 contemplated to be particularly useful 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.

[0203] 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 the region of CD3ε required 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 with SEQ ID NO: 30.

[0204] 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 the region of TRAC required 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 a modification that promotes 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 the position corresponding to position 47 according to SEQ ID NO: 1 numbering. 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.

[0205] 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 the region of TRBC1 required for association with TCRα and / or TRAC (i.e., to form 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 with SEQ ID NO:5. In some embodiments, the CD3-TCR complex association domain is derived from TRBC1 and further comprises a modification that promotes 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 the position corresponding to position 56 according to the numbering of 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.

[0206] 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 the region of TRBC2 required for association with TCRα and / or TRAC (i.e., to form 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 with SEQ ID NO: 9. In some embodiments, the CD3-TCR complex association domain is derived from TRBC2 and further comprises a modification that promotes 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 the position corresponding to position 56 according to the numbering of 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.

[0207] Antigen-binding portion

[0208] The recombinant CD3-TCR complex polypeptides of the present disclosure comprise an antigen binding portion or a component thereof. The basic function of the antigen binding portion is to provide binding to a variant Fc domain, as described below.

[0209] As used herein, "antigen binding portion" refers to a portion that binds to a given target antigen.

[0210] Antigen binding moieties include antibodies (i.e., immunoglobulins (Ig)) and antigen binding fragments and derivatives thereof. In some embodiments, antigen binding moieties according to the present disclosure include monoclonal antibodies, monospecific antibodies, multispecific (e.g., bispecific, trispecific, etc.) antibodies, variable fragment (Fv) moieties, single-chain Fv (scFv) moieties, fragment antigen binding (Fab) moieties, single-chain Fab moieties (scFab), crossFab moieties, Fab' moieties, Fab'-SH moieties, F(ab') 2 The invention relates to a method for preparing an antibody comprising: comprising or consisting of a 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.

[0211] 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, fynomers, atrimers, DARPins, affibodies, affilins, armadillo repeat proteins (ArmRPs), OBodys, and adnectins (reviewed, for example, in Reverdatto et al., Curr Top Med Chem. 2015; 15(12): 1082–1101, the entire contents of which are incorporated herein by reference (see also, for example, 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 (e.g., reviewed in the following literature: 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 between about 300 and 700 Daltons) organic compounds).

[0212] The antigen binding portion of the recombinant CD3-TCR complex polypeptide of the present disclosure is capable of binding to the variant Fc domain according to the present disclosure. The antigen binding portion capable of binding to the variant Fc domain according to the present disclosure can also be described as an antigen binding portion that binds to the variant Fc domain according to the present disclosure.

[0213] The antigen binding moieties described herein preferably exhibit specific binding to the variant Fc domains 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 greater affinity and / or longer duration than when it binds to other non-target antigens.

[0214] The ability of a given moiety to specifically bind to a given target antigen can be determined by analysis according to methods known in the art, such as by 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 by radiolabeled antigen binding assay (RIA) enzyme-linked immunosorbent assay. By such analysis, binding to a given target antigen can be measured and quantified. In some embodiments, the level of binding can be the response detected in a given assay.

[0215] In some embodiments, the antigen binding portions described herein bind with affinities (i.e., K in the micromolar range). D =9.9x10 -4 Up to 1x10 -6 M) (e.g., as determined by SPR or BLI) binds to a variant Fc domain according to the present disclosure. In some embodiments, the antigen binding moieties described herein bind with submicromolar affinity (i.e., K D <1x10 -6 M) is combined with a variant Fc domain according to the present disclosure. In some embodiments, the antigen binding portions described herein bind with an affinity in the nanomolar range (i.e., K D =9.9x10 -7 Up to 1x10 -9 M) binds to a variant Fc domain according to the present disclosure. In some embodiments, the antigen binding moieties described herein bind with subnanomolar affinity (i.e., K D <1x10 -9 M) is combined with a variant Fc domain according to the present disclosure. In some embodiments, the antigen binding portions described herein bind with an affinity in the picomolar range (i.e., K D =9.9x10 -10 Up to 1x10 -12 M) binds to a variant Fc domain according to the present disclosure. In some embodiments, the antigen binding moieties described herein bind with sub-picomolar affinity (i.e., K D <1x10-12 M) in combination with a variant Fc domain according to the disclosure.

[0216] The antigen binding portion of the 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 a reference Fc domain according to the present disclosure, or exhibits substantially no binding thereto.

[0217] An antigen-binding portion 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 to which the antigen-binding portion is known not to bind or is known not to bind specifically (e.g., a non-target antigen). In some embodiments, the level of binding to a given antigen by an antigen-binding portion that does not bind or exhibits substantially no binding is ≥0.5-fold and ≤2-fold (e.g., ≥0.75-fold and ≤1.5-fold, ≥0.8-fold and ≤1.4-fold, ≥0.85-fold and ≤1.3-fold, ≥0.9-fold and ≤1.2-fold, ≥0.95-fold and ≤1.1-fold) relative to the level of binding exhibited by the antigen-binding portion to an antigen to which the antigen-binding portion is known not to bind or is known not to bind specifically (e.g., a non-target antigen).

[0218] In some embodiments, the level of binding of the antigen binding moiety to a reference Fc domain according to the present disclosure is ≤ 10% 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 is ≤ 10% 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. D ; for example, as determined by SPR or BLI) at least 0.1 order of magnitude greater D In combination with a reference Fc domain according to the present disclosure.

[0219] The antigen binding moiety according to the present disclosure may be or may comprise an antigen binding peptide / polypeptide or an antigen binding peptide / polypeptide complex. The antigen binding moiety may comprise more than one peptide / polypeptide, which together form an antigen binding domain. The peptide / polypeptide may be covalently or non-covalently associated. In some embodiments, the peptide / polypeptide forms part of a larger polypeptide comprising the peptide / polypeptide (e.g., in the case of a scFv portion comprising a VH region and a VL region, or in the case of a scFab comprising a VH-CH1 and a VL-CL).

[0220] 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 of an antibody capable of binding to a given target antigen. In some embodiments, the antigen binding portion comprises or consists of an Fv portion formed by a VH region and a VL region of an antibody capable of binding to a given target antigen. In some embodiments, the VH region and the VL region may be provided in the same polypeptide and joined by a linker sequence. In some embodiments, the antigen binding portion comprises or consists of a scFv portion that binds to a given target antigen.

[0221] The antigen binding moieties of the present disclosure typically comprise six complementary determining regions (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 portion that binds to the target antigen.

[0222] The VH region and the VL region include framework regions (FRs) on both sides of each CDR, which provide a scaffold for the CDR. From N-terminus to C-terminus, the VH region includes the following structure: N-terminus-[HC-FR1]-[HC-CDR1]-[HC-FR2]-[HC-CDR2]-[HC-FR3]-[HC-CDR3]-[HC-FR4]-C-terminus; and the VL region includes the following structure: N-terminus-[LC-FR1]-[LC-CDR1]-[LC-FR2]-[LC-CDR2]-[LC-FR3]-[LC-CDR3]-[LC-FR4]-C-terminus.

[0223] There are several different conventions for defining antibody CDRs and FRs, such as (i) the Kabat 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 as described in Lefranc et al., Dev. Comp. Immunol. (2003) 27:55-77.

[0224] The CDRs and FRs of the VH and VL regions of the antigen binding portions described herein are defined according to the Kabat system.

[0225] In some embodiments, the antigen binding portion includes the CDRs of the antigen binding portion that binds to the variant Fc domain of the present disclosure. In some embodiments, the antigen binding portion includes the FRs of the antigen binding portion that binds to the variant Fc domain of the present disclosure. In some embodiments, the antigen binding portion includes the CDRs and FRs of the antigen binding portion that binds to the variant Fc domain of the present disclosure. That is, in some embodiments, the antigen binding portion includes the VH region and VL region of the antigen binding portion that binds to the variant Fc domain of the present disclosure.

[0226] Wessels et al. Bioanal. (2017) 9(11): 849–59 describe the identification of an antibody that binds to an antibody comprising an Fc domain derived from human IgG1 comprising P329G, but does not bind to an antibody comprising an equivalent Fc domain lacking the P329G substitution. The antibody also binds to an antibody having an hIgG1-derived Fc region comprising P329G and further comprising 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 structures of anti-P329G Fabs having Fc comprising P329G, L234A, and L235A. Anti-P329G Fab interacts with Fc comprising P329G, L234A and L235A in a 1: 1 stoichiometry.The epitope is disclosed as including positions N325 to P331 (including G329), and S267 to E272.

[0227] In some embodiments, the antigen binding portion comprises a CDR, FR and / or VH and / or VL region of an antigen binding molecule described herein that is bound to a variant Fc domain according to the present disclosure, or comprises a CDR, FR and / or VH and / or VL region that is derived from those regions of an antigen binding molecule described herein that is bound to a variant Fc domain according to the present disclosure. In some embodiments, the antigen binding molecule bound to a variant Fc domain according to the present disclosure is selected from: αP329G_VH1 / VL1, αP329G_VH2 / VL1, αP329G_VH3 / VL1, and αAAA.

[0228] In some embodiments, the antigen binding portion comprises a VH region according to (1) or (2) below:

[0229] (1) A VH region incorporating the following CDRs:

[0230] HC-CDR1 having the amino acid sequence of SEQ ID NO: 56

[0231] HC-CDR2 having the amino acid sequence of SEQ ID NO:57

[0232] A HC-CDR3 having the amino acid sequence of SEQ ID NO: 58, or a variant thereof, wherein 1 or 2 or 3 amino acids in HC-CDR1, and / or wherein 1 or 2 or 3 amino acids in HC-CDR2, and / or wherein 1 or 2 or 3 amino acids in HC-CDR3 are substituted with another amino acid.

[0233] (2) VH region incorporating the following CDRs:

[0234] HC-CDR1 having the amino acid sequence of SEQ ID NO: 56

[0235] HC-CDR2 having the amino acid sequence of SEQ ID NO: 64

[0236] A HC-CDR3 having the amino acid sequence of SEQ ID NO: 58, or a variant thereof, wherein 1 or 2 or 3 amino acids in HC-CDR1, and / or wherein 1 or 2 or 3 amino acids in HC-CDR2, and / or wherein 1 or 2 or 3 amino acids in HC-CDR3 are substituted with another amino acid.

[0237] In some embodiments, the antigen binding portion comprises a VH region according to (3) or (4) below:

[0238] (3) VH region incorporating the following FRs:

[0239] HC-FR1 having the amino acid sequence of SEQ ID NO:59

[0240] HC-FR2 having the amino acid sequence of SEQ ID NO: 60

[0241] HC-FR3 having the amino acid sequence of SEQ ID NO: 61

[0242] HC-FR4 having the amino acid sequence of SEQ ID NO:62,

[0243] or a variant thereof, wherein 1 or 2 or 3 amino acids in HC-FR1, and / or wherein 1 or 2 or 3 amino acids in HC-FR2, and / or wherein 1 or 2 or 3 amino acids in HC-FR3, and / or wherein 1 or 2 or 3 amino acids in HC-FR4 are substituted with another amino acid.

[0244] (4) VH region incorporating the following FRs:

[0245] HC-FR1 having the amino acid sequence of SEQ ID NO: 66

[0246] HC-FR2 having the amino acid sequence of SEQ ID NO: 60

[0247] HC-FR3 having the amino acid sequence of SEQ ID NO: 67

[0248] HC-FR4 having the amino acid sequence of SEQ ID NO:62,

[0249] or a variant thereof, wherein 1 or 2 or 3 amino acids in HC-FR1, and / or wherein 1 or 2 or 3 amino acids in HC-FR2, and / or wherein 1 or 2 or 3 amino acids in HC-FR3, and / or wherein 1 or 2 or 3 amino acids in HC-FR4 are substituted with another amino acid.

[0250] In some embodiments, the antigen binding portion comprises a VH region comprising CDRs according to (1) or (2) above and FRs according to (3) or (4) above.

[0251] In some embodiments, the antigen binding portion comprises a VH region according to (5) or (6) below:

[0252] (5) A VH region comprising the CDR according to (1) and the FR according to (3).

[0253] (6) A VH region comprising the CDR according to (2) and the FR according to (4).

[0254] (7) A VH region comprising the CDR according to (2) and the FR according to (3).

[0255] In some embodiments, the antigen binding portion comprises a VH region according to one of the following (8) to (10):

[0256] (8) a VH region comprising an amino acid sequence having at least 70% sequence identity (more preferably at least one of ≥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:55.

[0257] (9) a VH region comprising an amino acid sequence having at least 70% sequence identity (more preferably at least one of ≥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:63.

[0258] (10) a VH region comprising an amino acid sequence having at least 70% sequence identity (more preferably at least one of ≥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:65.

[0259] In some embodiments, the antigen binding portion comprises a VL region according to (11):

[0260] (11) A VL region incorporating the following CDRs:

[0261] LC-CDR1 having the amino acid sequence of SEQ ID NO: 69

[0262] LC-CDR2 having the amino acid sequence of SEQ ID NO: 70

[0263] An LC-CDR3 having the amino acid sequence of SEQ ID NO:71, or a variant thereof, wherein 1 or 2 or 3 amino acids in LC-CDR1, and / or wherein 1 or 2 or 3 amino acids in LC-CDR2, and / or wherein 1 or 2 or 3 amino acids in LC-CDR3 are substituted by another amino acid.

[0264] In some embodiments, the antigen binding portion comprises a VL region according to (12) below:

[0265] (12) VL region incorporating the following FR:

[0266] LC-FR1 having the amino acid sequence of SEQ ID NO: 72

[0267] LC-FR2 having the amino acid sequence of SEQ ID NO: 73

[0268] LC-FR3 having the amino acid sequence of SEQ ID NO: 74

[0269] LC-FR4 having the amino acid sequence of SEQ ID NO:75,

[0270] or a variant thereof, wherein 1 or 2 or 3 amino acids in LC-FR1, and / or wherein 1 or 2 or 3 amino acids in LC-FR2, and / or wherein 1 or 2 or 3 amino acids in LC-FR3, and / or wherein 1 or 2 or 3 amino acids in LC-FR4 are substituted with another amino acid.

[0271] In some embodiments, the antigen binding portion comprises a VL region according to (13) below:

[0272] (13) A VL region comprising the CDR according to (11) and the FR according to (12).

[0273] In some embodiments, the antigen binding portion comprises a VL region according to (14) below:

[0274] (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) to the amino acid sequence of SEQ ID NO:68.

[0275] In some embodiments, the antigen binding portion comprises a VH region according to any one of (1) to (10) above and a VL region according to any one of (11) to (14) above.

[0276] In some embodiments, the antigen binding portion comprises a VH region according to (15):

[0277] (15) A VH region incorporating the following CDRs:

[0278] HC-CDR1 having the amino acid sequence of SEQ ID NO: 77

[0279] HC-CDR2 having the amino acid sequence of SEQ ID NO: 78

[0280] A HC-CDR3 having the amino acid sequence of SEQ ID NO: 79, or a variant thereof, wherein 1 or 2 or 3 amino acids in HC-CDR1, and / or wherein 1 or 2 or 3 amino acids in HC-CDR2, and / or wherein 1 or 2 or 3 amino acids in HC-CDR3 are substituted with another amino acid.

[0281] In some embodiments, the antigen binding portion comprises a VH region according to (16):

[0282] (16) VH region incorporating the following FRs:

[0283] HC-FR1 having the amino acid sequence of SEQ ID NO: 80

[0284] HC-FR2 having the amino acid sequence of SEQ ID NO: 81

[0285] HC-FR3 having the amino acid sequence of SEQ ID NO: 82

[0286] HC-FR4 having the amino acid sequence of SEQ ID NO:83,

[0287] or a variant thereof, wherein 1 or 2 or 3 amino acids in HC-FR1, and / or wherein 1 or 2 or 3 amino acids in HC-FR2, and / or wherein 1 or 2 or 3 amino acids in HC-FR3, and / or wherein 1 or 2 or 3 amino acids in HC-FR4 are substituted with another amino acid.

[0288] In some embodiments, the antigen binding portion comprises a VH region according to (17):

[0289] (17) A VH region comprising the CDR according to (15) and the FR according to (16).

[0290] In some embodiments, the antigen binding portion comprises a VH region according to (18):

[0291] (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) to the amino acid sequence of SEQ ID NO:76.

[0292] In some embodiments, the antigen binding portion comprises a VL region according to (19):

[0293] (19) A VL region incorporating the following CDRs:

[0294] LC-CDR1 having the amino acid sequence of SEQ ID NO: 85

[0295] LC-CDR2 having the amino acid sequence of SEQ ID NO: 86

[0296] An LC-CDR3 having the amino acid sequence of SEQ ID NO: 87, or a variant thereof, wherein 1 or 2 or 3 amino acids in LC-CDR1, and / or wherein 1 or 2 or 3 amino acids in LC-CDR2, and / or wherein 1 or 2 or 3 amino acids in LC-CDR3 are substituted by another amino acid.

[0297] In some embodiments, the antigen binding portion comprises a VL region according to (20):

[0298] (20) VL region incorporating the following FR:

[0299] LC-FR1 having the amino acid sequence of SEQ ID NO: 88

[0300] LC-FR2 having the amino acid sequence of SEQ ID NO: 89

[0301] LC-FR3 having the amino acid sequence of SEQ ID NO:90

[0302] LC-FR4 having the amino acid sequence of SEQ ID NO:91,

[0303] or a variant thereof, wherein 1 or 2 or 3 amino acids in LC-FR1, and / or wherein 1 or 2 or 3 amino acids in LC-FR2, and / or wherein 1 or 2 or 3 amino acids in LC-FR3, and / or wherein 1 or 2 or 3 amino acids in LC-FR4 are substituted with another amino acid.

[0304] In some embodiments, the antigen binding portion comprises a VL region according to (21):

[0305] (21) A VL region comprising the CDR according to (19) and the FR according to (20).

[0306] In some embodiments, the antigen binding portion comprises a VL region according to (22):

[0307] (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) to the amino acid sequence of SEQ ID NO:84.

[0308] In some embodiments, the antigen binding portion comprises a VH region according to any one of (15) to (18) above and a VL region according to any one of (19) to (22) above.

[0309] Amino acid substitutions according to the present disclosure may be biochemically conservative. In some embodiments, if the amino acid to be substituted is provided in a row in rows 1 to 5 of the following table, the substituted amino acid is another non-identical amino acid provided in the same row:

[0310]

[0311] For example, in some embodiments where the substitution is to a Met residue, the replacement amino acid can be selected from Ala, Val, Leu, He, Trp, Tyr, Phe, and norleucine.

[0312] 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 charge (at pH 7.4) as the amino acid residue it replaces:

[0313]

[0314]

[0315] That is, in some embodiments, a non-polar amino acid is replaced by another non-polar amino acid that is not identical. In some embodiments, a polar amino acid is replaced by another non-polar amino acid that is not identical. In some embodiments, an acidic polar amino acid is replaced by another non-identical acidic polar amino acid. In some embodiments, a basic polar amino acid is replaced by another non-identical basic polar amino acid. In some embodiments, a neutral amino acid is replaced by another non-identical neutral amino acid. In some embodiments, a positively charged amino acid is replaced by another non-identical positively charged amino acid. In some embodiments, a negatively charged amino acid is replaced by another non-identical negatively charged amino acid.

[0316] In some embodiments, the substitution may be functionally conservative. That is, in some embodiments, the substitution may not affect (or substantially not affect) one or more functional properties (e.g., target antigen binding) of the antigen-binding portion comprising the substitution compared to an equivalent unsubstituted molecule.

[0317] In some embodiments, the antigen binding portion of the present disclosure comprises a VH as described herein. In some embodiments, the antigen binding portion comprises 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 a CH1, CH2 region and / or CH3 region of an immunoglobulin (Ig). In some embodiments, the antigen binding portion comprises a linker sequence as described herein.

[0318] In some embodiments, the antigen binding portion of the present disclosure comprises one or more polypeptides comprising: (i) a VH region comprising HC-CDR1, HC-CDR2, and HC-CDR3 as indicated in column A of Table A, and (ii) a VL region comprising LC-CDR1, LC-CDR2, and LC-CDR3 as indicated in column B of Table A, wherein the sequences of columns A and B are selected from the same row of Table A.

[0319] In some embodiments, the antigen binding portion of the present disclosure comprises one or more polypeptides comprising: (i) a VH region comprising an HC-CDR1 according to SEQ ID NO:56, an HC-CDR2 according to SEQ ID NO:64, and an HC-CDR3 according to SEQ ID NO:58, and (ii) a VL region comprising an LC-CDR1 according to SEQ ID NO:69, an LC-CDR2 according to SEQ ID NO:70, and an LC-CDR3 according to SEQ ID NO:71.

[0320] In some embodiments, the antigen binding portion of the present disclosure comprises one or more polypeptides comprising: (i) a VH region comprising HC-FR1, HC-FR2, HC-FR3, and HC-FR4 as indicated in column A of Table B, and (ii) a VL region comprising LC-FR1, LC-FR2, LC-FR3, and LC-FR4 as indicated in column B of Table B, wherein the sequences of columns A and B are selected from the same row of Table B.

[0321] In some embodiments, the antigen binding portion of the present disclosure comprises one or more polypeptides comprising: (i) a VH region comprising HC-FR1 according to SEQ ID NO:66, HC-FR2 according to SEQ ID NO:60, HC-FR3 according to SEQ ID NO:67, and HC-FR4 according to SEQ ID NO:62, and (ii) a VL region comprising LC-FR1 according to SEQ ID NO:72, LC-FR2 according to SEQ ID NO:73, LC-FR3 according to SEQ ID NO:74, and LC-FR4 according to SEQ ID NO:75.

[0322] In some embodiments, the antigen binding portion of the present disclosure comprises one or more polypeptides comprising: (i) an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to the amino acid sequence indicated in column A of Table C, and (ii) an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to the amino acid sequence indicated in column B of Table C, wherein the sequences in column A and column B are selected from the same row of Table C.

[0323] In some embodiments, the antigen binding portions of the present disclosure comprise amino acids having at least 70% (preferably one of ≥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, the antigen binding portions of the present disclosure comprise amino acids having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to SEQ ID NO: 68.

[0324] In some embodiments, the antigen binding portion of the present disclosure comprises an amino acid having at least 70% (preferably one of ≥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, the antigen binding portion of the present disclosure comprises an amino acid having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 68.

[0325] In some embodiments, the antigen binding portion of the present disclosure comprises an amino acid having at least 70% (preferably one of ≥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, the antigen binding portion of the present disclosure comprises an amino acid having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 84.

[0326] In some embodiments, the antigen binding portion of the present disclosure comprises or consists of an amino acid 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, the antigen binding portion comprises or consists of an amino acid 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 one of ≥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 one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to SEQ ID NO: 97.

[0327] 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 the recombinant CD3-TCR complex polypeptide is intended to be used together with another non-identical and complementary recombinant CD3-TCR complex polypeptide. In such aspects and embodiments, the two non-identical and complementary polypeptides preferably associate with each other to form a polypeptide complex comprising an antigen binding portion. That is, the association between the recombinant CD3-TCR complex polypeptides reconstructs a functional antigen binding portion.

[0328] For example, in the embodiments described herein, the recombinant CD3-TCR complex polypeptide comprises a VH region of an antigen binding portion having specificity for a variant Fc domain and the ECD, TMD and ICD of TRAC (T47C), and it is contemplated that the recombinant CD3-TCR complex polypeptide is used in conjunction with a recombinant CD3-TCR complex polypeptide comprising a VL region of an antigen binding portion having specificity 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 having specificity for a variant Fc domain, the Fv being formed by the VH region from the first polypeptide and the VL region from the second polypeptide.

[0329] In some aspects and embodiments of the present disclosure, a first component and a second component of an antigen binding moiety are provided. According to such aspects and embodiments, it will be understood that the first component and the second component of the antigen binding moiety are complementary and can associate to form a (complete, functional) antigen binding moiety.

[0330] In some embodiments according to the present disclosure, the component of the antigen binding moiety may be or comprise a VH region of an antigen binding moiety that is specific for a variant Fc domain (e.g., as described herein). In some embodiments, the component of the antigen binding moiety may be or comprise a VL region of an antigen binding moiety that is specific for a variant Fc domain (e.g., as described herein). In preferred embodiments, the VH region and the VL region may be from the same antigen binding moiety.

[0331] In some embodiments, the components of the antigen binding portion comprise VH as described herein or consist thereof. In some embodiments, the components of the antigen binding portion comprise VL as described herein or consist thereof. In some embodiments, the components of the antigen binding portion comprise one or more antibody heavy chain constant regions (CH). In some embodiments, the components of the antigen binding portion comprise one or more antibody light chain constant regions (CL). In some embodiments, the components of the antigen binding portion comprise CH1, CH2 and / or CH3 regions of immunoglobulins (Ig).

[0332] In some embodiments, the components of the antigen binding portion comprise or consist of one or more polypeptides comprising a VH region comprising HC-CDR1, HC-CDR2, and HC-CDR3 as indicated in column A of Table A. In some embodiments, the components of the antigen binding portion comprise or consist of one or more polypeptides comprising a VL region comprising LC-CDR1, LC-CDR2, and LC-CDR3 as indicated in column B of Table A.

[0333] In some embodiments, the components of the antigen binding portion comprise or consist of one or more polypeptides comprising a VH region comprising HC-FR1, HC-FR2, HC-FR3, and HC-FR4 as indicated in column A of Table B. In some embodiments, the components of the antigen binding portion comprise or consist of one or more polypeptides comprising a VL region comprising LC-FR1, LC-FR2, LC-FR3, and LC-FR4 as indicated in column B of Table B.

[0334] In some embodiments, the components of the antigen binding portion comprise or consist of one or more polypeptides 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 the amino acid sequence indicated in column A of Table C. In some embodiments, the components of the antigen binding portion comprise one or more polypeptides 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 the amino acid sequence indicated in column B of Table C.

[0335] In some embodiments, the component of the antigen binding portion comprises or consists of an amino acid having at least 70% (preferably one of ≥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, the component of the antigen binding portion comprises or consists of an amino acid having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to SEQ ID NO: 68.

[0336] In some embodiments, the component of the antigen binding portion comprises or consists of an amino acid 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, the component of the antigen binding portion comprises or consists of an amino acid 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.

[0337] It will be appreciated that where components of an antigen binding moiety are provided in aspects and embodiments of the present disclosure, it is intended that the components provided are complementary and capable of associating to form a (complete, functional) antigen binding moiety.

[0338] Variant Fc domain

[0339] The antigen binding portion of the recombinant CD3-TCR complex polypeptides of the present disclosure provides binding to a variant Fc domain.

[0340] As used herein, "Fc domain" refers to a polypeptide complex formed by the interaction between two polypeptides, each of which comprises the CH2-CH3 region of an immunoglobulin (Ig) heavy chain constant sequence.

[0341] G-type immunoglobulins (i.e., IgG) are glycoproteins of approximately 150 kDa that include two heavy chains and two light chains. From N-terminus to C-terminus, the heavy chain includes VH, followed by a heavy chain constant region including three constant domains (CH1, CH2, and CH3), and similarly, the light chain includes VL, followed by 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).

[0342] Herein, "CH2 domain" refers to the amino acid sequence corresponding to the CH2 domain of an immunoglobulin (Ig). According to the EU numbering system described in Edelman et al., Proc Natl Acad Sci USA (1969) 63 (1): 78-85, the CH2 domain is the region of Ig formed by positions 231 to 340 of an immunoglobulin constant domain. "CH3 domain" refers to the amino acid sequence corresponding to the CH3 domain of an immunoglobulin (Ig). According to the EU numbering system described in Edelman et al., Proc Natl Acad Sci USA (1969) 63 (1): 78-85, the CH3 domain is the region of Ig formed by positions 341 to 447 of an immunoglobulin constant domain. "CH2-CH3 region" refers to the amino acid sequence corresponding to the CH2 and CH3 domains of an immunoglobulin (Ig). According to the EU numbering system described in Edelman et al., Proc Natl Acad Sci USA (1969) 63(1):78-85, the CH2-CH3 region is the region of an Ig formed by positions 231 to 447 of an immunoglobulin constant domain.

[0343] In some embodiments, the CH2 domain, CH3 domain and / or CH2-CH3 region according to the present disclosure corresponds to the CH2 domain / CH3 domain / CH2-CH3 region of IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE or IgM. In some embodiments, the CH2 domain, CH3 domain and / or CH2-CH3 region corresponds to the CH2 domain / CH3 domain / CH2-CH3 region of human IgG (e.g., hIgG1, hIgG2, hIgG3, hIgG4), hIgA (e.g., hIgA1, hIgA2), hIgD, hIgE or hIgM. In some embodiments, the CH2 domain, CH3 domain and / or CH2-CH3 region corresponds to the CH2 domain / CH3 domain / CH2-CH3 region of human IgG1 allotype (e.g., G1m1, G1m2, G1m3 or G1m17).

[0344] It should be understood that the Fc domain according to the present disclosure may form part of a larger molecule comprising the 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) comprising an antigen binding portion specific for a target antigen and a variant Fc domain according to the present disclosure.

[0345] The Fc domain provides interactions with Fc receptors and other molecules of the immune system to bring about functional effects. The effector functions mediated by Fc have been reviewed, for example, in Jefferis et al., Immunol Rev 1998 163: 59-76 (the entire contents of which are hereby incorporated by reference), and are brought about by: the recruitment and activation of Fc-mediated immune cells (e.g., macrophages, dendritic cells, neutrophils, basophils, eosinophils, platelets, mast cells, NK cells, and T cells) performed by the interaction between the Fc region and the Fc receptors expressed by the immune cells, the recruitment of complement pathway components performed by the combination of the Fc region and the complement protein C1q, and the subsequent 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.

[0346] The CH2-CH3 region sequence of human IgG1 G1m1 is shown in SEQ ID NO: 206. The CH2-CH3 region sequence of human IgG1 G1m3 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.

[0347] A variant Fc domain according to the present disclosure comprises an amino acid sequence comprising at least one amino acid difference relative to a reference Fc domain.

[0348] In some embodiments, the reference Fc domain according to the present disclosure comprises two polypeptides, wherein each polypeptide comprises a CH2-CH3 region comprising or consisting of an amino acid sequence having at least 70% sequence identity (more preferably at least one of ≥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, the reference Fc domain comprises two polypeptides, wherein each polypeptide comprises a CH2-CH3 region comprising SEQ ID NO: 206 or consisting thereof.

[0349] In some embodiments, the reference Fc domain comprises two polypeptides, wherein each polypeptide comprises a CH2-CH3 region comprising or consisting of an amino acid sequence having at least 70% sequence identity (more preferably at least one of ≥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, wherein each polypeptide comprises a CH2-CH3 region comprising SEQ ID NO: 207 or consisting thereof.

[0350] In some embodiments, the reference Fc domain comprises two polypeptides, wherein each polypeptide comprises a CH2-CH3 region comprising or consisting of an amino acid sequence having at least 70% sequence identity (more preferably at least one of ≥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, wherein each polypeptide comprises a CH2-CH3 region comprising SEQ ID NO: 208 or consisting thereof.

[0351] In some embodiments, the reference Fc domain comprises two polypeptides, wherein each polypeptide comprises a CH2-CH3 region comprising or consisting of an amino acid sequence having at least 70% sequence identity (more preferably at least one of ≥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, wherein each polypeptide comprises a CH2-CH3 region comprising SEQ ID NO: 209 or consisting thereof.

[0352] In some embodiments, the reference Fc domain comprises two polypeptides, wherein each polypeptide comprises a CH2-CH3 region comprising or consisting of an amino acid sequence having at least 70% sequence identity (more preferably at least one of ≥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, wherein each polypeptide comprises a CH2-CH3 region comprising SEQ ID NO: 210 or consisting thereof.

[0353] A variant Fc domain according to the present disclosure may comprise amino acid differences relative to the amino acid sequence of one or both polypeptides of a reference Fc domain according to the present disclosure.

[0354] In some embodiments, the variant Fc domain according to the present disclosure comprises two polypeptides, each polypeptide comprising a CH2-CH3 region, and wherein one or both of the CH2-CH3 regions comprise an amino acid sequence that is not identical to SEQ ID NO: 206. In some embodiments, the variant Fc domain according to the present disclosure comprises two polypeptides, each polypeptide comprising a CH2-CH3 region, and wherein one or both of the CH2-CH3 regions comprise an amino acid sequence having 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 (more preferably at least one of ≥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, wherein one or both of the CH2-CH3 regions comprise 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 one of ≥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, wherein one or both of the CH2-CH3 regions comprise an amino acid sequence having one or more (e.g., 1, 2, 3, 4, 5 or more) amino acid differences relative to SEQ ID NO: 206.

[0355] In some embodiments, the variant Fc domain according to the present disclosure comprises two polypeptides, each polypeptide comprising a CH2-CH3 region, and wherein one or both of the CH2-CH3 regions comprise an amino acid sequence that is not identical to SEQ ID NO: 207. In some embodiments, the variant Fc domain according to the present disclosure comprises two polypeptides, each polypeptide comprising a CH2-CH3 region, and wherein one or both of the CH2-CH3 regions comprise an amino acid sequence having 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 (more preferably at least one of ≥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, wherein one or both of the CH2-CH3 regions comprise 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 one of ≥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, wherein one or both of the CH2-CH3 regions comprise an amino acid sequence having one or more (e.g., 1, 2, 3, 4, 5 or more) amino acid differences relative to SEQ ID NO: 207.

[0356] In some embodiments, the variant Fc domain according to the present disclosure comprises two polypeptides, each polypeptide comprising a CH2-CH3 region, and wherein one or both of the CH2-CH3 regions comprise an amino acid sequence that is not identical to SEQ ID NO: 208. In some embodiments, the variant Fc domain according to the present disclosure comprises two polypeptides, each polypeptide comprising a CH2-CH3 region, and wherein one or both of the CH2-CH3 regions comprise an amino acid sequence having 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 (more preferably at least one of ≥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, wherein one or both of the CH2-CH3 regions comprise 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 one of ≥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, wherein one or both of the CH2-CH3 regions comprise an amino acid sequence having one or more (e.g., 1, 2, 3, 4, 5 or more) amino acid differences relative to SEQ ID NO: 208.

[0357] In some embodiments, the variant Fc domain according to the present disclosure comprises two polypeptides, each polypeptide comprising a CH2-CH3 region, and wherein one or both of the CH2-CH3 regions comprise an amino acid sequence that is not identical to SEQ ID NO: 209. In some embodiments, the variant Fc domain according to the present disclosure comprises two polypeptides, each polypeptide comprising a CH2-CH3 region, and wherein one or both of the CH2-CH3 regions comprise an amino acid sequence having 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 (more preferably at least one of ≥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, wherein one or both of the CH2-CH3 regions comprise 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 one of ≥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, wherein one or both of the CH2-CH3 regions comprise an amino acid sequence having one or more (e.g., 1, 2, 3, 4, 5 or more) amino acid differences relative to SEQ ID NO: 209.

[0358] In some embodiments, the variant Fc domain according to the present disclosure comprises two polypeptides, each polypeptide comprising a CH2-CH3 region, and wherein one or both of the CH2-CH3 regions comprise an amino acid sequence that is not identical to SEQ ID NO: 210. In some embodiments, the variant Fc domain according to the present disclosure comprises two polypeptides, each polypeptide comprising a CH2-CH3 region, and wherein one or both of the CH2-CH3 regions comprise an amino acid sequence having 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 (more preferably at least one of ≥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, wherein one or both of the CH2-CH3 regions comprise 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 one of ≥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, wherein one or both of the CH2-CH3 regions comprise an amino acid sequence having one or more (e.g., 1, 2, 3, 4, 5 or more) amino acid differences relative to SEQ ID NO: 210.

[0359] In some embodiments, each CH2-CH3 region of a variant Fc domain according to the present disclosure comprises amino acid differences relative to a reference Fc domain according to the present disclosure. In some embodiments, the amino acid sequences of the CH2-CH3 regions of the constituent polypeptides of a variant Fc domain according to the present disclosure are identical (i.e., they have the same amino acid sequence).

[0360] Amino acid differences in a variant Fc domain according to the present disclosure (relative to a reference Fc domain) may affect Fc-mediated functions.

[0361] Modifications of Fc domains that affect Fc-mediated functions are known in the art, such as, for example, 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 entirety. Exemplary Fc domain modifications known to affect Fc-mediated functions 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 comprising amino acid differences relative to a reference Fc domain (e.g., a reference Fc according to the present disclosure) that increase or decrease Fc-mediated functions.

[0362] In some embodiments, the variant Fc domain comprises amino acid differences relative to the reference Fc domain, and the amino acid differences increase Fc-mediated functions. In some embodiments, the variant Fc domain comprises amino acid differences relative to the reference Fc domain, and the amino acid differences increase ADCC, ADCP and / or CDC. Therefore, in some embodiments, the variant Fc domain exhibits an increased level of Fc-mediated functions 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.

[0363] In some embodiments, the variant Fc domain comprises amino acid differences relative to the reference Fc domain, and the amino acid differences increase the binding to Fc receptors (e.g., Fcγ receptors, such as FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa and / or FcγRIIIb). In some embodiments, the variant Fc domain comprises amino acid differences relative to the reference Fc domain, and the amino acid differences increase the binding to FcRn. In some embodiments, the variant Fc domain comprises amino acid differences relative to the reference Fc domain, and the amino acid differences increase the binding to complement proteins (e.g., C1q). In some embodiments, the variant Fc domain comprises amino acid differences relative to the reference Fc domain to increase the hexamerization of the antigen binding molecules comprising the variant Fc domain. In some embodiments, the Fc domain comprises amino acid differences relative to the reference Fc domain, and the amino acid differences increase the half-life of the antigen binding molecules comprising the variant Fc domain. Thus, in some embodiments, the variant Fc domain exhibits increased binding to Fc receptors (e.g., Fcγ receptors, such as 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 complement proteins (e.g., C1q) compared to a reference Fc domain. In some embodiments, the antigen binding molecules comprising the variant Fc domain exhibit increased hexamerization compared to an antigen binding molecule comprising a reference Fc domain. In some embodiments, the antigen binding molecules comprising the variant Fc domain exhibit increased half-life compared to an antigen binding molecule comprising a reference Fc domain.

[0364] In some embodiments, the variant Fc domain comprises amino acid differences relative to the reference Fc domain, and the amino acid differences reduce Fc-mediated functions. In some embodiments, the variant Fc domain comprises amino acid differences relative to the reference Fc domain, and the amino acid differences reduce ADCC, ADCP and / or CDC. Therefore, 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.

[0365] In some embodiments, the variant Fc domain comprises amino acid differences relative to the reference Fc domain, and the amino acid differences reduce binding to Fc receptors (e.g., Fcγ receptors, such as FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa and / or FcγRIIIb). In some embodiments, the variant Fc domain comprises amino acid differences relative to the reference Fc domain, and the amino acid differences reduce binding to FcRn. In some embodiments, the variant Fc domain comprises amino acid differences relative to the reference Fc domain, and the amino acid differences reduce binding to complement proteins (e.g., C1q). In some embodiments, the variant Fc domain comprises amino acid differences relative to the reference Fc domain to increase the hexamerization of the antigen binding molecules comprising the variant Fc domain. In some embodiments, the Fc domain comprises amino acid differences relative to the reference Fc domain, and the amino acid differences reduce the half-life of the antigen binding molecules comprising the variant Fc domain. Thus, in some embodiments, the variant Fc domain exhibits reduced binding to Fc receptors (e.g., Fcγ receptors, such as 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, the antigen binding molecules comprising the variant Fc domain exhibit reduced hexamerization compared to an antigen binding molecule comprising a reference Fc domain. In some embodiments, the antigen binding molecules comprising the variant Fc domain exhibit reduced half-life compared to an antigen binding molecule comprising a reference Fc domain.

[0366] In some embodiments, the variant Fc domain comprises a CH2-CH3 region comprising an amino acid difference relative to the amino acid sequence of a CH2-CH3 region of a reference Fc domain at one or more of the following positions: 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 comprising an amino acid difference relative to the amino acid sequence of a CH2-CH3 region of a reference Fc domain at one or more of the following positions: 234, 235, 253, 297, 298, 310, 329, 333, 334, or 435. In some embodiments, the variant Fc domain comprises a CH2-CH3 region comprising an amino acid difference relative to the amino acid sequence of a CH2-CH3 region of a reference Fc domain at position 329. In some embodiments, the variant Fc domain comprises a CH2-CH3 region comprising amino acid differences at positions 234, 235, and 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 comprising amino acid differences at positions 298, 333, and 334 relative to the amino acid sequence of the CH2-CH3 region of a reference Fc domain.

[0367] 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 an amino acid difference at positions L234, L235, and 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 an amino acid difference at positions S298, E333, and K334 relative to the amino acid sequence of the CH2-CH3 region of a reference Fc domain.

[0368] In some embodiments, the variant Fc domain comprises a CH2-CH3 region comprising one or more of the following specified 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 specified 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.

[0369] In some embodiments, the variant Fc domain comprises a CH2-CH3 region comprising 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 comprising 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 comprising an amino acid substitution P329G 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 an amino acid substitution L234A, L235A and P329G 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 an amino acid substitution S298A, E333A and K334A relative to the amino acid sequence of the CH2-CH3 region of a reference Fc domain.

[0370] In some embodiments, the 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 one of ≥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, the variant Fc domain according to the present 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, the variant Fc domain according to the present disclosure comprises one or more (e.g., two) polypeptides comprising the amino acid sequence of SEQ ID NO: 211 or 214.

[0371] In some embodiments, the 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 one of ≥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, the variant Fc domain according to the present 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, the variant Fc domain according to the present disclosure comprises one or more (e.g., two) polypeptides comprising the amino acid sequence of SEQ ID NO: 213 or 216.

[0372] In some embodiments, the 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 one of ≥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, the variant Fc domain according to the present 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, the variant Fc domain according to the present disclosure comprises one or more (e.g., two) polypeptides comprising the amino acid sequence of SEQ ID NO: 212 or 215.

[0373] Composite polypeptide

[0374] Aspects and embodiments of the present disclosure pertain to composite polypeptides.

[0375] The 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 series. As described below, a cleavage site may be provided in the composite polypeptide between the amino acid sequences of the recombinant CD3-TCR complex polypeptides.

[0376] In some embodiments, the individual recombinant CD3-TCR complex polypeptides contained in the composite polypeptides of the present disclosure are not identical. In some embodiments, the recombinant CD3-TCR complex polypeptides contained in the composite polypeptides of the present disclosure are complementary. That is, in some embodiments, the recombinant CD3-TCR complex polypeptides contained in the composite polypeptides of the present disclosure are able to associate with each other (e.g., via non-covalent, protein: protein interactions) to form a polypeptide complex (e.g., a polypeptide complex as described herein).

[0377] In a further preferred embodiment, the single recombinant CD3-TCR complex polypeptides contained in the composite polypeptide of the present disclosure are able to associate with each other to form an antigen binding portion according to the present disclosure. In some embodiments, the recombinant CD3-TCR complex polypeptides contained in the composite polypeptide of the present disclosure include complementary components of the antigen binding portion according to the present disclosure. The complementary components of the antigen binding portion can associate (e.g., via non-covalent, protein: protein interactions) to form an antigen binding portion. In some embodiments, the association between the recombinant CD3-TCR complex polypeptides contained in the composite polypeptide of the present disclosure leads to the formation of an antigen binding portion according to the present disclosure. In some embodiments, the recombinant CD3-TCR complex polypeptide of the composite polypeptide of the present disclosure includes a VH region of an antigen binding portion bound to a variant Fc domain (e.g., as described herein), and another CD3-TCR composite polypeptide of the composite polypeptide includes a VL region of an antigen binding portion.

[0378] For example, in the embodiments described herein, the composite polypeptide comprises (i) a recombinant CD3-TCR complex polypeptide comprising a VH having 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 having an antigen binding portion specific for a variant Fc domain, and the ECD, TMD and ICD of TRBC1 (S56C), and (iii) further comprising 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 having specificity for a variant Fc domain, the Fv being formed by the VH from (i) and the VL from (ii).

[0379] Linkers, tags, and conjugates

[0380] The polypeptides of the present disclosure (eg, recombinant CD3-TCR complex polypeptides and composite polypeptides) may additionally comprise additional amino acids or sequences of amino acids.

[0381] The polypeptide may comprise one or more linker sequences between the sequences of amino acids. For 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 associating domain). By further example, a linker sequence may be provided between subsequences of domains of a recombinant CD3-TCR complex polypeptide (e.g., between a VH region and a VL region of an antigen binding portion).

[0382] The linker sequence is known to those skilled in the art, and is 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 may be a flexible linker sequence. The flexible linker sequence allows relative movement of the amino acid sequence connected by the linker sequence. Flexible linkers are known to those skilled in the art, and several flexible linkers have been identified in Chen et al., Adv Drug Deliv Rev (2013) 65 (10): 1357-1369. Flexible linker sequences typically contain a high proportion of glycine and / or serine residues.

[0383] In some embodiments, the linker sequence comprises at least one glycine residue and / or at least one serine residue. In some embodiments, the linker comprises or consists of glycine and serine residues. In some embodiments, the linker sequence has the following structure: (GxS)n or (GxS)nGm; wherein 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 the sequence motif G 4 One or more (e.g., 1, 2, 3, 4, 5, or 6) copies (e.g., in tandem) of S. In some embodiments, the linker sequence comprises (G 4 S) 3 or (G 4 S) 4 Or consisting of. In one embodiment, the length of the linker sequence is 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.

[0384] In some embodiments, the linker sequence comprises one or more copies of an 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 an amino acid sequence according to SEQ ID NO: 92.

[0385] In some embodiments, the linker sequence comprises or consists of an amino acid sequence having 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 to SEQ ID NO:93.

[0386] In some embodiments, the linker sequence comprises a cleavage site, eg, a cleavage site as described below.

[0387] The polypeptides of the present disclosure may comprise amino acid sequences that facilitate expression, folding, transport, processing, purification or detection of antigen binding molecules / polypeptides. For example, the recombinant CD3-TCR complex polypeptides of the present disclosure may additionally comprise amino acid sequences that form a detectable portion, such as described below.

[0388] Polypeptide can additionally comprise signal peptide (also referred to as leader sequence or signal sequence).Signal peptide is usually composed of 5 to 30 hydrophobic amino acid sequences, and these amino acids form single alpha helix.Proteins secreted and expressed at cell surface usually comprise signal peptide.Signal peptide is known for many proteins, and is recorded in databases such as GenBank, UniProt and Ensembl, and / or can be identified / predicted, for example, 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).

[0389] The signal peptide may be present at the N-terminus of the recombinant CD3-TCR complex polypeptide and may be present in a newly synthesized polypeptide. The signal peptide provides for efficient transport of the recombinant CD3-TCR complex polypeptide. The signal peptide is typically removed by cleavage and is therefore not included in a mature recombinant CD3-TCR complex polypeptide.

[0390] 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, for example, 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).

[0391] In some embodiments, the signal peptide comprises or consists of an amino acid sequence having 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 to SEQ ID NO:98.

[0392] In some embodiments, the polypeptides of the present disclosure (e.g., recombinant CD3-TCR complex polypeptides and composite polypeptides) comprise a detectable moiety. In some embodiments, the detectable moiety is provided at the N-terminus and / or C-terminus of the polypeptide.

[0393] 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 enzyme label. The recombinant CD3-TCR complex polypeptide can be covalently or non-covalently labeled with a detectable moiety.

[0394] 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 radioactive isotopes such as 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 ,bismuth 217 and antimony 211Luminescent labels include radioluminescent, chemiluminescent (e.g., acridinium esters, luminol, isoluminol), and bioluminescent labels. Immunodetectable labels include haptens, peptides / polypeptides, antibodies, receptors, and ligands such as biotin, avidin, streptavidin, or digoxigenin. Nucleic acid labels include aptamers.

[0395] In some embodiments, the recombinant CD3-TCR complex polypeptide comprises an epitope tag, such as aHis, (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 hapten (e.g., biotin, digoxigenin, dinitrophenol), optionally located at the N-terminus or C-terminus of the recombinant CD3-TCR complex polypeptide.

[0396] In some embodiments, the recombinant CD3-TCR complex polypeptide comprises a portion having detectable activity, such as an enzyme portion. The enzyme portion includes, for example, luciferase, glucose oxidase, β-galactosidase (e.g., β-galactosidase), glucosidase, phosphatase (e.g., alkaline phosphatase), peroxidase (e.g., horseradish peroxidase), and cholinesterase.

[0397] In some embodiments, the polypeptides of the present disclosure comprise a fluorescent marker. In some embodiments, the polypeptides comprise an eGFP portion. In some embodiments, the polypeptides comprise 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.

[0398] The polypeptides of the present disclosure may also comprise one or more cleavage sites. A cleavage site refers to an amino acid sequence that serves as a substrate for an enzyme capable of cleaving a peptide bond.

[0399] The present invention relates to the invention of the present invention.Many such cleavage sites are known to and can be used by the technicians in the field of molecular biology.In certain embodiments, the cleavage sequence comprises an automatic cleavage site.The automatic cleavage site comprises the 2A cleavage sequence from the picornavirus " NPGP ", which cuts at the " G / P " place.More automatic cleavage sites are for example described in Kim et al., PLoS ONE (2011) 6: e18556 (its entire content is incorporated herein by reference), and include for example T2A, P2A, E2A and F2A cleavage sites.The amino acid sequences of T2A, P2A, E2A and F2A cleavage sites are shown in SEQ ID NO:104, 220, 109 and 221 respectively.

[0400] A cleavage site may be included in a polypeptide according to the present disclosure to provide for removal of a portion or domain. It may be necessary to remove a given portion or domain so that it is not included in a polypeptide complex formed by the polypeptide. For example, in an embodiment of a recombinant CD3-TCR complex polypeptide disclosed herein, a cleavage site (specifically, a T2A cleavage site) is provided upstream of the eGFP portion to provide for its removal so that the eGFP portion is not included in the CD3-TCR complex comprising the CD3-TCR complex polypeptide. Therefore, in some embodiments, a polypeptide according to the present disclosure includes a cleavage site adjacent to (i.e., in the amino acid sequence of the polypeptide, e.g., immediately upstream or downstream thereof) a detectable portion according to the present disclosure.

[0401] A cleavage site may be included in a composite polypeptide according to the present disclosure to provide separation of the recombinant CD3-TCR complex polypeptides contained in the composite polypeptide. For example, in an embodiment of a composite polypeptide of the present disclosure, a cleavage site (specifically, a T2A cleavage site) is provided between the amino acid sequences encoding complementary recombinant CD3-TCR complex polypeptides, and physical separation of the recombinant CD3-TCR complex polypeptides is provided. Therefore, in some embodiments, a polypeptide according to the present disclosure comprises a cleavage site adjacent to (i.e., in the amino acid sequence of the polypeptide, e.g., immediately upstream or downstream thereof) an amino acid sequence encoding a recombinant CD3-TCR complex polypeptide according to the present disclosure.

[0402] 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.

[0403] In some embodiments, the polypeptides according to the present disclosure comprise an amino acid sequence having 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 to SEQ ID NO: 104, 220, 109 or 221. In some embodiments, the polypeptides according to the present disclosure comprise an amino acid sequence having 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 to SEQ ID NO: 104.

[0404] Polypeptide complex

[0405] The present disclosure also provides polypeptide complexes. "Polypeptide complexes" refer to molecules formed by non-covalent protein:protein interactions between two or more polypeptides. In some embodiments, the association includes electrostatic interactions (e.g., ionic bonds, hydrogen bonds) and / or van der Waals forces.

[0406] 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.

[0407] In some embodiments, the constituent polypeptides of such polypeptide complexes include complementary CD3-TCR complex association domains. In some embodiments, the association between the constituent polypeptides of such polypeptide complexes includes interactions 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 include a CD3-TCR complex association domain derived from TRAC, and the other may include 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 include complementary modifications to promote their association. In some embodiments, the CD3-TCR complex association domains of the recombinant CD3-TCR complex polypeptides include modifications that introduce cysteine ​​residues for forming interchain disulfide bonds between CD3-TCR complex association domains.

[0408] For example, the polypeptide complex according to the present disclosure may comprise (i) a recombinant CD3-TCR complex polypeptide comprising a CD3-TCR complex association domain derived from TRAC, further comprising a modification of T47C, and (ii) a recombinant CD3-TCR complex polypeptide comprising a CD3-TCR complex association domain derived from TRBC1 or TRBC2, further comprising a modification of S56C.

[0409] In some embodiments, the constituent polypeptides of such polypeptide complexes comprise complementary components of an antigen binding portion that is specific for a variant Fc domain (e.g., as described herein). In some embodiments, the association between the constituent polypeptides comprises interactions between individual components of the antigen binding portion 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 that is specific for a variant Fc domain, and the other may comprise a VL region of an antigen binding portion.

[0410] Aspects and embodiments of the present disclosure also relate to CD3-TCR polypeptide complexes.

[0411] The CD3-TCR polypeptide complex according to the present disclosure comprises a recombinant CD3-TCR complex polypeptide according to the present disclosure. In some embodiments, the CD3-TCR polypeptide complex may comprise one or more (e.g., two, three, four or more) recombinant CD3-TCR complex polypeptides according to the present disclosure.

[0412] In some aspects and embodiments in which the CD3-TCR polypeptide complex comprises a plurality of recombinant CD3-TCR complex polypeptides according to the present disclosure, the recombinant CD3-TCR complex polypeptides may be identical.

[0413] In some aspects and embodiments, the CD3-TCR polypeptide complex comprises non-identical recombinant CD3-TCR complex polypeptides 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 association of complementary recombinant CD3-TCR complex polypeptides according to the present disclosure). That is, the polypeptide complex can be a CD3-TCR polypeptide complex, or can form part of a CD3-TCR polypeptide complex.

[0414] It should be understood that in addition to (a) a recombinant CD3-TCR complex polypeptide, the CD3-TCR polypeptide complex according to the present disclosure may contain (b) one or more additional CD3-TCR complex polypeptides, for example selected from TCRα, TCRβ, TCRγ, TCRδ, TRAC, TRBC1, TRBC2, TRGC1, TRGC2, TRDC, CD3ε, CD3δ, CD3γ, CD3ζ and CD3η.

[0415] The CD3-TCR polypeptide complex according to the present disclosure may optionally be characterized by the ability to elicit one or more of the following by an immune cell (e.g., 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 comprising or expressing the antigen): CD3-TCR complex-mediated signaling, proliferation / population expansion, expression of growth factors (e.g., IL-2), expression of IFNγ, expression of CD107a, expression of TNFα, expression of GM-CSF, expression of perforin, expression of granzymes, expression of granulysin, and / or expression of FAS ligand (FASL).

[0416] 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 / an antigen binding portion thereof comprised in a CD3-TCR polypeptide complex) or in response to a cell comprising / expressing such a variant Fc domain.

[0417] Specific exemplary CD3-TCR complex polypeptides, composite polypeptides, and polypeptide complexes

[0418] In some embodiments, the CD3-TCR complex polypeptide according to the present disclosure comprises or consists of one of the following structures:

[0419] N-terminus - [Signal peptide] - [Antigen binding portion or component thereof] - [CD3-TCR complex association domain] - C-terminus

[0420] N-terminus - [Antigen binding portion or component thereof] - [CD3-TCR complex associating domain] - C-terminus

[0421] N-terminus - [Signal peptide] - [Antigen binding moiety or component thereof] - [CD3-TCR complex association domain] - [Cleavage site] - [Detectable moiety] - C-terminus

[0422] N-terminus - [Antigen binding portion or component thereof] - [CD3-TCR complex association domain] - [Cleavage site] - [Detectable portion] - C-terminus

[0423] In some embodiments, a composite polypeptide according to the present disclosure comprises or consists of one of the following structures:

[0424] N-terminus-[Signal peptide]-[Antigen-binding moiety]-[CD3-TCR complex associating domain]-[Cleavage site]-[Signal peptide]-[Antigen-binding moiety]-[CD3-TCR complex associating domain]-C-terminusN-terminus-[Signal peptide]-[Antigen-binding moiety]-[CD3-TCR complex associating domain]-[Cleavage site]-[Signal peptide]-[Antigen-binding moiety]-[CD3-TCR complex associating domain]-[Cleavage site]-[Detectable moiety]-C-terminus

[0425] 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):

[0426] (1) (i) an amino acid sequence encoding a signal peptide, for example, 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;

[0427] (ii) an amino acid sequence having at least 70% (preferably one of ≥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; and

[0428] (iii) an amino acid sequence having at least 70% (preferably one of ≥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;

[0429] Wherein 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.

[0430] (2)(i) an amino acid sequence having at least 70% (preferably one of ≥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; and

[0431] (ii) an amino acid sequence having at least 70% (preferably one of ≥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;

[0432] Wherein 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.

[0433] (3) (i) an amino acid sequence encoding a signal peptide, for example, 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;

[0434] (ii) an amino acid sequence having at least 70% (preferably one of ≥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;

[0435] (iii) an amino acid sequence having at least 70% (preferably one of ≥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;

[0436] (iv) an amino acid sequence encoding a cleavage site, e.g., an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 104; and

[0437] (v) an amino acid sequence encoding a detectable portion, such as an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to SEQ ID NO: 103;

[0438] Wherein 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.

[0439] (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 with a sequence selected from column A of Table 1;

[0440] (ii) an amino acid sequence having at least 70% (preferably one of ≥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;

[0441] (iii) an amino acid sequence encoding a cleavage site, e.g., an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 104; and

[0442] (iv) an amino acid sequence encoding a detectable portion, such as an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to SEQ ID NO: 103;

[0443] Wherein 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.

[0444] Table 1

[0445]

[0446]

[0447] In some embodiments, the CD3-TCR complex 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: 110 to 173. In some embodiments, the CD3-TCR complex 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: 121.

[0448] In some embodiments, a composite polypeptide according to the present disclosure comprises or consists of (e.g., from N-terminus to C-terminus):

[0449] (1) (i) an amino acid sequence encoding a signal peptide, for example, 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;

[0450] (ii) an amino acid sequence having at least 70% (preferably one of ≥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 2; and

[0451] (iii) an amino acid sequence having at least 70% (preferably one of ≥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;

[0452] (iv) an amino acid sequence encoding a cleavage site, such as an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 104;

[0453] (v) an amino acid sequence encoding a signal peptide, for example, an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO:98;

[0454] (vi) an amino acid sequence having at least 70% (preferably one of ≥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

[0455] (vii) an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to a sequence selected from column D of Table 2;

[0456] Wherein the sequence selected from column A of Table 2, the sequence selected from column B of Table 2, 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.

[0457] (2) (i) an amino acid sequence encoding a signal peptide, for example, 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;

[0458] (ii) an amino acid sequence having at least 70% (preferably one of ≥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 2; and

[0459] (iii) an amino acid sequence having at least 70% (preferably one of ≥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;

[0460] (iv) an amino acid sequence encoding a cleavage site, such as an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 104;

[0461] (v) an amino acid sequence encoding a signal peptide, for example, an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO:98;

[0462] (vi) an amino acid sequence having at least 70% (preferably one of ≥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;

[0463] (vii) an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to a sequence selected from column D of Table 2;

[0464] (viii) an amino acid sequence encoding a cleavage site, e.g., an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 104; and

[0465] (ix) an amino acid sequence encoding a detectable portion, such as an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 103;

[0466] Wherein the sequence selected from column A of Table 2, the sequence selected from column B of Table 2, 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.

[0467] Table 2

[0468]

[0469]

[0470]

[0471] In some embodiments, the composite polypeptide according to the present disclosure comprises or consists of an amino acid sequence having at least 70% (preferably one of ≥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 to 205. In some embodiments, the composite polypeptide according to the present disclosure comprises or consists of an amino acid sequence having at least 70% (preferably one of ≥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 composite polypeptide according to the present disclosure comprises or consists of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO:186.

[0472] In some embodiments, a polypeptide complex according to the present disclosure comprises a CD3-TCR complex polypeptide according to the embodiments described herein.

[0473] In some embodiments, a polypeptide complex according to the present disclosure comprises:

[0474] (a) a polypeptide comprising (e.g., from the N-terminus to the C-terminus):

[0475] (i) have at least 70% (preferably ≥ 80%),

[0476] ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%,

[0477] ≥98%, ≥99% or 100% amino acid sequence identity;

[0478] and

[0479] (ii) have at least 70% (preferably ≥ 80%),

[0480] ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%,

[0481] ≥98%, ≥99%, or 100% amino acid sequence identity; and

[0482] (b) a polypeptide comprising (e.g., from the N-terminus to the C-terminus):

[0483] (i) have at least 70% (preferably ≥ 80%),

[0484] ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%,

[0485] ≥98%, ≥99% or 100% amino acid sequence identity;

[0486] and

[0487] (ii) have at least 70% (preferably ≥ 80%),

[0488] ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%,

[0489] ≥98%, ≥99% or 100%) amino acid sequence identity; wherein 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.

[0490] In some embodiments, a polypeptide complex according to the present disclosure comprises:

[0491] (1)(i) a polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 129; and

[0492] (ii) a polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 165;

[0493] or

[0494] (2)(i) a polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 133; and

[0495] (ii) a polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 165;

[0496] or

[0497] (3)(i) a polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 137; and

[0498] (ii) a polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 165;

[0499] or

[0500] (4)(i) a polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 145; and

[0501] (ii) a polypeptide comprising, or consisting of, an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 173;

[0502] or

[0503] (5)(i) a polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 141; and

[0504] (ii) a polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 153;

[0505] or

[0506] (6)(i) a polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 141; and

[0507] (ii) a polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 157;

[0508] or

[0509] (7)(i) a polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 141; and

[0510] (ii) a polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 161;

[0511] or

[0512] (8)(i) A polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 149; and

[0513] (ii) A polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 169.

[0514] In a preferred embodiment, the polypeptide complex according to the present disclosure comprises:

[0515] (i) A polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 137; and

[0516] (ii) A polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 165.

[0517] In a preferred embodiment, the polypeptide complex according to the present disclosure comprises:

[0518] (i) A polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 141; and

[0519] (ii) A polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 161.

[0520] Nucleic acids and vectors

[0521] The present disclosure provides a nucleic acid or multiple nucleic acids encoding a recombinant CD3-TCR complex polypeptide, a composite polypeptide or a polypeptide complex according to the present disclosure. In some embodiments, the nucleic acid comprises or consists of DNA and / or RNA.

[0522] The recombinant CD3-TCR complex polypeptide, composite polypeptide or polypeptide complex according to the present disclosure can be produced in a cell by translation of RNA encoding the recombinant CD3-TCR complex polypeptide, composite polypeptide or polypeptide complex. The recombinant CD3-TCR complex polypeptide, composite polypeptide or polypeptide complex according to the present disclosure can be produced in a cell 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.

[0523] In some embodiments, the nucleic acid can be or can be contained in a vector or vectors.As used herein, a "vector" is a nucleic acid molecule used as a vehicle for transferring exogenous nucleic acid into a cell.

[0524] Therefore, the present disclosure also provides a vector or multiple vectors, which contain a nucleic acid or multiple nucleic acids according to the present disclosure. The vector can facilitate the delivery of a nucleic acid encoding a recombinant CD3-TCR complex polypeptide, a composite polypeptide or a polypeptide complex according to the present disclosure to a cell. The vector can be an expression vector, which contains the elements required for expressing a recombinant CD3-TCR complex polypeptide, a composite polypeptide or a polypeptide complex according to the present disclosure. The vector may contain elements that facilitate integration of the nucleic acid into the genomic DNA of the cell into which the vector is introduced.

[0525] Nucleic acids and vectors according to the present disclosure may be provided in purified or isolated form, ie, from other nucleic acids or naturally occurring biological material.

[0526] The vector may be a vector for expressing nucleic acid in a cell (i.e., an expression vector). Such a vector may include a promoter sequence operably connected to a nucleotide sequence encoding a recombinant CD3-TCR complex polypeptide, a composite polypeptide, or a polypeptide complex according to the present disclosure. The vector may also include a stop codon (i.e., located 3' of the nucleotide sequence encoding the recombinant CD3-TCR complex polypeptide / complex polypeptide / polypeptide complex in the nucleotide sequence of the vector) and an expression enhancer. Any suitable vector, promoter, enhancer, and stop codon known in the art may be used to express a peptide or polypeptide from a vector according to the present disclosure.

[0527] The term "operably linked" may include situations in which a nucleic acid encoding a recombinant CD3-TCR complex polypeptide, a composite polypeptide or a polypeptide complex according to the present disclosure and a regulatory nucleic acid sequence (promoter and / or enhancer) are covalently linked in such a way as to place the expression of the nucleic acid encoding the recombinant CD3-TCR complex polypeptide, a composite polypeptide or a polypeptide complex under the influence or control of the regulatory nucleic acid sequence (thereby forming an expression cassette). Thus, if a regulatory sequence is capable of influencing the transcription of a selected nucleic acid sequence, the regulatory sequence is operably linked to the nucleic acid sequence. The resulting transcript may then be translated into the desired polypeptide.

[0528] Vectors contemplated for use 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., gammaretroviral 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, e.g., in Maus et al., Annu Rev Immunol (2014) 32: 189-225 and Morgan and Boyerinas, Biomedicines (2016) 4: 9, the entire contents of which are incorporated herein by reference. In some embodiments, the vector according to the present disclosure is a lentiviral vector.

[0529] In some embodiments, the vector can be a eukaryotic vector, i.e., a vector comprising the elements necessary for expressing proteins from the vector in eukaryotic cells. In some embodiments, the vector can be a mammalian vector, which, for example, comprises a cytomegalovirus (CMV) or SV40 promoter to drive protein expression.

[0530] In some embodiments, the nucleic acid / nucleic acids or vector / vectors according to the present disclosure comprise an EF1α promoter.

[0531] In some embodiments, the nucleic acid / multiple nucleic acids or vector / multiple vectors according to the present disclosure encode a CD3-TCR complex polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥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 to 173. In some embodiments, the nucleic acid / multiple nucleic acids or vector / multiple vectors according to the present disclosure encode a CD3-TCR complex polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 121.

[0532] In a preferred embodiment, the nucleic acid / multiple nucleic acids or vector / multiple vectors according to the present disclosure comprise the nucleotide sequence of SEQ ID NO: 224, or a codon-degenerate nucleotide sequence thereof encoding the amino acid sequence encoded by SEQ ID NO: 224.

[0533] As used herein, a "codon-degenerate nucleotide sequence" of a reference nucleotide sequence refers to a nucleotide sequence that has nucleotides that are different from those of the reference nucleotide sequence but that, as a result of the degeneracy of the genetic code, encodes the same amino acid sequence as that encoded by the reference nucleotide sequence.

[0534] The constituent polypeptides of the polypeptide complex according to the present disclosure may be encoded by different nucleic acids among the plurality of nucleic acids according to the present disclosure, or encoded by different vectors of the plurality of nucleic acids according to the present disclosure.

[0535] In aspects and embodiments of the present disclosure, one or more nucleic acids according to the present disclosure encode two or more (e.g., 2, 3, 4 or more) recombinant CD3-TCR complex polypeptides according to the present disclosure. In aspects and embodiments of the present disclosure, one or more vectors according to the present disclosure encode two or more (e.g., 2, 3, 4 or more) recombinant CD3-TCR complex polypeptides according to the present disclosure.

[0536] In some embodiments in which the nucleic acid / multiple nucleic acids or vector / multiple vectors 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 / multiple nucleic acids or vector / multiple vectors encode complementary recombinant CD3-TCR complex polypeptides. That is, in some embodiments, the nucleic acid / multiple nucleic acids or vector / multiple vectors encode CD3-TCR complex polypeptides that are capable of associating with each other (e.g., by non-covalent, protein:protein interactions) to form a polypeptide complex (e.g., a polypeptide complex as described herein).

[0537] In some embodiments, the nucleic acid / nucleic acids or vector / vectors encode CD3-TCR complex polypeptides that are capable of associating with each other to form an antigen binding portion according to the present disclosure. In some embodiments, the nucleic acid / nucleic acids or vector / vectors encode CD3-TCR complex polypeptides comprising complementary components of an antigen binding portion according to the present disclosure (i.e., components of an antigen binding portion that are capable of associating (e.g., by non-covalent, protein:protein interactions) to form an antigen binding portion).

[0538] For example, in one embodiment, the nucleic acid / nucleic acids or vector / vectors encode (i) a recombinant CD3-TCR complex polypeptide comprising a VH having an antigen binding portion specific for a variant Fc domain, and an ECD, TMD, and ICD of TRAC (T47C), and (ii) a recombinant CD3-TCR complex polypeptide comprising a VL region having an antigen binding portion specific for a variant Fc domain, and an ECD, TMD, and ICD of TRBC1 (S56C). After expression from the nucleic acid / nucleic acids or vector / vectors, polypeptides (i) and (ii) associate to form a polypeptide complex comprising an Fv having specificity for a variant Fc domain, the Fv being formed by the VH from (i) and the VL from (ii).

[0539] In some embodiments, a nucleic acid / nucleic acids or vector / vectors according to the present disclosure encode:

[0540] (1)(i) a polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 129; and

[0541] (ii) a polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 165;

[0542] or

[0543] (2)(i) a polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 133; and

[0544] (ii) a polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 165;

[0545] or

[0546] (3)(i) a polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 137; and

[0547] (ii) a polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 165;

[0548] or

[0549] (4)(i) a polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 145; and

[0550] (ii) a polypeptide comprising, or consisting of, an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 173;

[0551] or

[0552] (5)(i) a polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 141; and

[0553] (ii) a polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 153;

[0554] or

[0555] (6)(i) a polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 141; and

[0556] (ii) a polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 157;

[0557] or

[0558] (7)(i) a polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 141; and

[0559] (ii) a polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 161;

[0560] or

[0561] (8)(i) a polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 149; and

[0562] (ii) a polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 169.

[0563] In a preferred embodiment, the nucleic acid / nucleic acids or vector / vectors according to the present disclosure encode:

[0564] (i) a polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 137; and

[0565] (ii) a polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 165.

[0566] In a preferred embodiment, the nucleic acid / nucleic acids or vector / vectors according to the present disclosure encode:

[0567] (i) a polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 141; and

[0568] (ii) a polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 161.

[0569] In a preferred embodiment, the nucleic acid / nucleic acids or vector / vectors according to the present disclosure comprise:

[0570] (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

[0571] (i) the nucleotide sequence of SEQ ID NO: 226, or a codon-degenerate nucleotide sequence thereof encoding the amino acid sequence encoded by SEQ ID NO: 226.

[0572] In a preferred embodiment, the nucleic acid / nucleic acids or vector / vectors according to the present disclosure comprise:

[0573] (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

[0574] (i) the nucleotide sequence of SEQ ID NO: 228, or a codon-degenerate nucleotide sequence thereof encoding the amino acid sequence encoded by SEQ ID NO: 228.

[0575] In some embodiments, the nucleic acid / nucleic acids or vector / vectors 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 to 205. In some embodiments, the nucleic acid / nucleic acids or vector / vectors 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 to 205. In some embodiments, the nucleic acid / multiple nucleic acids or vector / multiple vectors according to the present disclosure encode a composite polypeptide, which 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.

[0576] In some embodiments, wherein the nucleic acid / multiple or vector / multiple vectors encode two or more (e.g., 2, 3, 4 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.

[0577] In some embodiments, the nucleic acid / nucleic acids or vector / vectors comprise a nucleic acid encoding an internal ribosome entry site (IRES). In some embodiments, an IRES is provided between nucleotide sequences encoding recombinant CD3-TCR complex polypeptides. In some embodiments, the nucleic acid / nucleic acids or vector / vectors comprise a nucleic acid that allows two or more recombinant CD3-TCR complex polypeptides to be translated separately from the same RNA transcript.

[0578] In some embodiments, two or more recombinant CD3-TCR complex polypeptides are encoded by nucleotide sequences provided in the same reading frame. In some embodiments, nucleic acid / multiple nucleic acids or vector / multiple vectors encode fusion proteins comprising two or more recombinant CD3-TCR complex polypeptides. In some embodiments, the fusion protein encoded by nucleic acid / multiple nucleic acids or vector / multiple vectors comprises a cleavage site (e.g., a cleavage site as described herein) between the amino acid sequences of the recombinant CD3-TCR complex polypeptides. In some embodiments, nucleic acid / multiple nucleic acids or vector / multiple vectors encode composite polypeptides according to the present disclosure.

[0579] In some embodiments, transcription of nucleic acids encoding two or more recombinant CD3-TCR complex polypeptides is under the control of different promoters.

[0580] In some embodiments, the nucleic acid / nucleic acids or vector / vectors are polycistronic (e.g., bicistronic, tricistronic, etc.). That is, in some embodiments, the nucleic acid / nucleic acids or vector / vectors comprise a plurality of nucleotide sequences encoding polypeptides. In some embodiments, nucleic acids encoding two or more recombinant CD3-TCR complex polypeptides are provided in different cistrons.

[0581] Cells comprising / expressing the polypeptides, polypeptide complexes, and nucleic acids / vectors of the present disclosure

[0582] The present disclosure also provides a cell comprising a recombinant CD3-TCR complex polypeptide, a composite polypeptide or a polypeptide complex according to the present disclosure or a nucleic acid / multiple nucleic acids or a vector / multiple vectors according to the present disclosure.

[0583] It will be understood that when a cell is referred to herein in the singular (ie, "a / the cell"), a plurality of cells / populations of such cells are also contemplated.

[0584] The cell can be a eukaryotic cell, such as a mammalian cell. The mammal can 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 in the order Rodentia), cat, dog, pig, sheep, goat, cattle (including cows, such as dairy cows) or any animal in the order Bos), horse (including any animal in the family Equine), donkey, and non-human primate). In a preferred embodiment, the cell is a human cell.

[0585] In some embodiments, the cell is an immune cell. The immune cell can be a cell of hematopoietic origin, such as a neutrophil, an eosinophil, a basophil, a dendritic cell, a lymphocyte or a monocyte. Lymphocytes can be, for example, T cells, B cells, NK cells, NKT cells or innate lymphoid cells (ILC) or their precursors. 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. Immune cells can express CD27, CD28, CD4 and / or CD8. In some embodiments, immune cells are T cells, such as CD3+T cells. In some embodiments, T cells are CD3+, CD4+T cells. In some embodiments, T cells are CD3+, CD8+T cells. In some embodiments, T cells are T helper cells (T H In some embodiments, the T cell is a cytotoxic T cell (eg, a cytotoxic T lymphocyte (CTL)).

[0586] 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.

[0587] In some aspects and embodiments, the cells according to the present disclosure express / present the recombinant CD3-TCR complex polypeptide or polypeptide complex according to the present disclosure at the cell surface. That is, the recombinant CD3-TCR complex polypeptide or polypeptide complex may be present in or at the cell membrane. Cells may be evaluated for surface expression of CD3-TCR complex polypeptides and polypeptide complexes (after introducing nucleic acids encoding them into cells), for example, using antibody-based methods such as flow cytometry (e.g., as described in Example 1 of the present disclosure).

[0588] In aspects and embodiments of the present disclosure, the 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, the 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, the cell according to the present disclosure comprises a nucleic acid / multiple nucleic acids or a vector / multiple vectors according to the present disclosure.

[0589] In aspects and embodiments of the present disclosure, a cell according to the present disclosure comprises or expresses a polypeptide complex according to the present disclosure, which is bound to a variant Fc domain as described herein. It should be understood that the binding to the variant Fc domain is achieved through the binding portion of the recombinant CD3-TCR complex polypeptide / polypeptide complex. As a result of the expression of a nucleic acid encoding such a polypeptide complex, a cell may express / comprising a polypeptide complex according to the present disclosure. A cell may be engineered to comprise a nucleic acid encoding such a polypeptide complex.

[0590] In some embodiments, cells according to the present disclosure may comprise modifications to reduce expression of CD3-TCR complex polypeptides (i.e., compared to the expression level of CD3-TCR complex polypeptides by equivalent unmodified cells). In some embodiments, cells comprise modifications to reduce expression of endogenous CD3-TCR complex polypeptides (i.e., CD3-TCR complex polypeptides encoded by the genome of equivalent unmodified cells).

[0591] In some embodiments, the cell comprises a modification to reduce the 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. For example, in embodiments in which the cell comprises or expresses a recombinant CD3-TCR complex polypeptide comprising a CD3-TCR complex association domain derived from CD3ε (or a composite polypeptide or polypeptide complex comprising such a recombinant CD3-TCR complex polypeptide), the cell may comprise a modification to reduce the expression of CD3ε (i.e., endogenous CD3ε) by the cell. By further example, in embodiments in which the cell comprises or expresses a recombinant CD3-TCR complex polypeptide comprising a CD3-TCR complex association domain derived from TRAC, TRBC1 and / or TRBC2 (or a composite polypeptide or polypeptide complex comprising such a recombinant CD3-TCR complex polypeptide), the cell may comprise a modification to reduce the expression of TRAC / TRBC1 / TRBC2 (i.e., endogenous TRAC / TRBC1 / TRBC2) by the cell.

[0592] In some embodiments, the cell comprises a modification of 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 a cell are modified. In some embodiments, the modification includes insertion, substitution, or deletion in the nucleotide sequence of a nucleic acid encoding a CD3-TCR complex polypeptide. In some embodiments, the modification reduces or prevents the 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 an early termination codon in the nucleotide sequence of an RNA transcribed from an endogenous nucleic acid encoding a CD3-TCR complex polypeptide. In some embodiments, the nucleotide sequence of the modified nucleic acid encodes a truncated and / or non-functional form of a CD3-TCR complex polypeptide. In some embodiments, the nucleotide sequence of the modified nucleic acid encodes a form of a CD3-TCR complex polypeptide that is misfolded and / or degraded. In some embodiments, the nucleotide sequence of the modified nucleic acid encodes a form of a CD3-TCR complex polypeptide that cannot participate in a functional CD3-TCR polypeptide complex.

[0593] In some embodiments, the cell comprises a modification of a nucleic acid (e.g., an endogenous nucleic acid) encoding CD3ε (e.g., a polypeptide having a 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 the 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 an early stop codon in the nucleotide sequence of RNA transcribed from an 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 form of CD3ε that is misfolded and / or degraded. In some embodiments, the nucleotide sequence of the modified nucleic acid encodes a form of CD3ε that cannot participate in a functional CD3-TCR polypeptide complex.

[0594] In some embodiments, the cell comprises a modification of a nucleic acid (e.g., an endogenous nucleic acid) encoding TRAC (e.g., a polypeptide having a sequence of SEQ ID NO: 1), TRBC1 (e.g., a polypeptide having a sequence of SEQ ID NO: 5), and / or TRBC2 (e.g., a polypeptide having a sequence of SEQ ID NO: 9). In some embodiments, the cell comprises a modification of 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 cell. In some embodiments, the modified cell lacks endogenous nucleic acids encoding TRAC, TRBC1, and / or TRBC2 (e.g., TRAC and TRBC1). In some embodiments, the modification introduces a premature stop codon in 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 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 misfolded and / or degraded 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 form of TRAC, TRBC1 and / or TRBC2 (e.g., TRAC and TRBC1) that cannot participate in a functional CD3-TCR polypeptide complex.

[0595] Immune cells (e.g., T cells) according to the present disclosure can be characterized by certain functional properties in response to an antigen for which the CD3-TCR polypeptide complex comprises an antigen binding portion (or in response to a cell comprising or expressing the antigen): CD3-TCR complex-mediated signaling; cell proliferation / population expansion, expression of growth factors (e.g., IL-2), expression of IFNγ, expression of CD107a, expression of TNFα, expression of GM-CSF, expression of perforin, expression of granzymes, expression of granulysin, and / or expression of FAS ligand (FASL).

[0596] 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 section in response to a variant Fc domain according to the present disclosure (i.e., a variant Fc domain bound by an antigen binding portion comprised in a recombinant CD3-TCR polypeptide complex expressed by the cell) or in response to a cell comprising / expressing such a variant Fc domain.

[0597] The signal transduction of CD3-TCR complex mediation can be studied by analyzing one or more related objects of CD3-TCR complex mediated signal transduction.For example, the signal transduction of CD3-TCR complex mediation can be studied by the phosphorylation of one or more signal transduction molecules of CD3-TCR complex signal transduction pathway assessment.The level of signal transduction of CD3-TCR complex mediation can be analyzed by detecting and quantifying the phosphorylation level of CD3ζ, ZAP-70, Lck, LAT and / or SLP-76.The level of signal transduction of CD3-TCR complex mediation can also be analyzed using a reporter gene-based method, such as a method for quantitatively expressing the activity of transcription factors (such as NFAT, NF-κB and / or AP-1) that are upregulated by the signal transduction of CD3-TCR complex, or by expressing the gene (such as IL2) that is upregulated by the signal transduction of CD3-TCR complex. For example, CD3-TCR complex-mediated signaling can be studied using a reporter cell line that stably expresses a luciferase reporter gene driven by CD3-TCR complex-mediated signaling (e.g., GloResponse Jurkat NFAT-RE-luc2P (Promega #CS176501) or T cell activation bioassay TCRαβ-KO CD4+ (Promega #GA1172), as described in Example 1 of the present disclosure.

[0598] Cell proliferation / population expansion can be studied by analyzing cell division or the number of cells over a period of time. 3 Cell division can be analyzed by incorporation of H-thymidine or by 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 analyzing the incorporation of 5-ethynyl-2'-deoxyuridine (EdU), as described in, for example, 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 both of which are incorporated herein by reference.

[0599] As used herein, "expression" can be gene or protein expression. Gene expression encompasses transcription of DNA to RNA, and can be measured by various methods known to those skilled in the art, such as by quantitative real-time PCR (qRT-PCR) to measure the level of mRNA, or using a reporter gene-based method. Similarly, protein expression can be measured by various methods well known in the art, such as antibody-based methods, such as by Western blotting, immunohistochemistry, immunocytochemistry, flow cytometry, ELISA, ELISPOT or reporter gene-based methods.

[0600] Immune cells (e.g., T cells) according to the present disclosure may exhibit cytotoxicity to cells comprising / expressing variant Fc domains according to the present disclosure. That is, immune cells (e.g., T cells) according to the present disclosure may have the ability to kill cells comprising / expressing variant Fc domains according to the present disclosure.

[0601] As a result of the antigen binding molecule comprising the variant Fc domain binding to an antigen expressed by the cell (e.g., at the cell surface, i.e., within or at the cell membrane), the cell comprising the variant Fc domain according to the present disclosure may do this. In some embodiments, the cell comprising the variant Fc domain according to the present disclosure comprises (e.g., at the cell surface) a polypeptide complex comprising (i) an antigen binding molecule comprising a variant Fc domain, and (ii) a target antigen of the antigen binding molecule.

[0602] For example, cytotoxicity and cell killing can be studied using any of the methods reviewed in Zaritskaya et al., Expert Rev Vaccines (2011), 9(6):601-616, the entire contents of which are incorporated herein by reference. Examples of in vitro cytotoxicity / cell killing assays include release assays such as 51Cr 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 are based on detecting factors released from lysed cells to measure cell killing. Cell killing of a given test cell type (e.g., immune cells (e.g., T cells) of the present disclosure) can be analyzed, for example, by co-culturing the test cells with a given target cell type (e.g., cells comprising a variant Fc domain according to the present disclosure) and measuring the number / ratio of surviving (i.e., unlysed) / dead (e.g., lysed) target cells after an appropriate period of time. Other suitable assays include the xCELLigence real-time cell lysis in vitro potency assay described in Cerignoli et al., PLoS One. (2018) 13(3): e0193498 (the entire contents of which are incorporated herein by reference), and the Incucyte immune cell killing assay employed in the experimental examples of the present disclosure.

[0603] Compared to a CAR comprising the same antigen binding portion (i.e., an antigen binding portion of a recombinant CD3-TCR complex polypeptide / polypeptide complex expressed by a cell), an immune cell (e.g., a T cell) according to the present disclosure may have one or more novel, similar, or improved functional properties. In some embodiments, compared to a CAR-expressing cell described in WO 2018 / 177966 A1 (the entire contents of which are incorporated herein by reference), an immune cell according to the present disclosure may have one or more novel, similar, or improved functional properties.

[0604] In some embodiments, an immune cell (e.g., T cell) according to the present disclosure may exhibit a level of CD3-TCR complex-mediated signaling in response to an antigen for which the CD3-TCR polypeptide complex comprises an antigen binding portion, or in response to a cell comprising or expressing the 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 in a CD3-TCR polypeptide complex expressed by the cell), or in response to a cell comprising / expressing such a variant Fc domain) 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.

[0605] The level of CD3-TCR complex-mediated signaling that is "similar to" a reference level of CD3-TCR complex-mediated signaling can be ≥0.5 times and ≤2 times, such as ≥0.55 times and ≤1.9 times, ≥0.6 times and ≤1.8 times, ≥0.65 times and ≤1.7 times, ≥0.7 times and ≤1.6 times, ≥0.75 times and ≤1.5 times, ≥0.8 times and ≤1.4 times, ≥0.85 times and ≤1.3 times, ≥0.9 times and ≤1.2 times, ≥0.95 times and ≤1.1 times, of 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 greater than 1 times, e.g., ≥1.01 times, ≥1.02 times, ≥1.03 times, ≥1.04 times, ≥1.05 times, ≥1.1 times, ≥1.2 times, ≥1.3 times, ≥1.4 times, ≥1.5 times, ≥1.6 times, ≥1.7 times, ≥1.8 times, ≥1.9 times, ≥2 times, ≥3 times, ≥4 times, or ≥5 times, of the reference level of CD3-TCR complex-mediated signaling.

[0606] 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 can be evaluated in assays comprising:

[0607] (i) contacting a T cell expressing a recombinant CD3-TCR complex polypeptide / polypeptide complex according to the present disclosure comprising a VH region according to SEQ ID NO: 65 and a VL region according to SEQ ID NO: 68 with an antigen presenting cell, which is a cell expressing a given target antigen that has been contacted with an antigen binding molecule that binds to the given target antigen and comprises an Fc domain having a CH2-CH3 region according to SEQ ID NO: 213; and subsequently analyzing the level of signaling mediated by the CD3-TCR complex of the T cell;

[0608] (ii) contacting a T cell expressing a CAR according to SEQ ID NO: 108 (e.g., an equivalent T cell, i.e., derived from the same source as the T cell of (i)) with an antigen presenting cell as defined in (i), and subsequently analyzing the level of signaling mediated by the CD3-TCR complex of the T cell; and

[0609] (iii) comparing the level of CD3-TCR complex-mediated signaling by the T cells of (i) with the T cells of (ii).

[0610] In some embodiments, in an assay performed as described in the previous paragraph, T cells according to (i) exhibit a level of CD3-TCR complex-mediated signaling that is ≥0.5 times and ≤2 times, for example, ≥0.55 times and ≤1.9 times, ≥0.6 times and ≤1.8 times, ≥0.65 times and ≤1.7 times, ≥0.7 times and ≤1.6 times, ≥0.75 times and ≤1.5 times, ≥0.8 times and ≤1.4 times, ≥0.85 times and ≤1.3 times, ≥0.9 times and ≤1.2 times, ≥0.95 times and ≤1.1 times, of the level of CD3-TCR complex-mediated signaling exhibited by T cells according to (ii). In some embodiments, in an assay performed as described in the preceding paragraph, T cells according to (i) exhibit a level of CD3-TCR complex-mediated signaling that is greater than 1-fold, e.g., ≥1.01-fold, ≥1.02-fold, ≥1.03-fold, ≥1.04-fold, ≥1.05-fold, ≥1.1-fold, ≥1.2-fold, ≥1.3-fold, ≥1.4-fold, ≥1.5-fold, ≥1.6-fold, ≥1.7-fold, ≥1.8-fold, ≥1.9-fold, ≥2-fold, ≥3-fold, ≥4-fold, or ≥5-fold, of the level of CD3-TCR complex-mediated signaling exhibited by T cells according to (ii).

[0611] The present disclosure also provides methods for producing cells according to the present disclosure, and cells obtained or obtainable by such methods.

[0612] Methods for producing cells comprising / expressing a polypeptide / polypeptide complex of interest are well known to the skilled person and generally comprise introducing into the cells a nucleic acid / vector encoding the polypeptide of interest.

[0613] 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 encoding a polypeptide of interest may be integrated into the genomic DNA of the cell or form a part of the genomic DNA of the cell. In some embodiments, after introduction into a cell, the nucleic acid encoding a polypeptide of interest may be maintained outside the chromosome.

[0614] Any suitable genetic engineering platform can be used, and includes gamma retroviral vectors, lentiviral vectors, adenoviral vectors, DNA transfection, transposon-based gene delivery and RNA transfection, for example, as described in Maus et al., Annu Rev Immunol (2014) 32: 189-225, the entire contents of which are incorporated herein by reference. Methods also include those described in, for example, Wang and Rivière Mol Ther Oncolytics. (2016) 3: 16015, the entire contents of which are incorporated herein by reference. Suitable methods for introducing nucleic acids / vectors into cells include transduction, transfection and electroporation.

[0615] Methods for in vitro / ex vivo production / amplification of cell populations containing / expressing polypeptides of interest are well known to technicians. Suitable culture conditions (i.e., cell culture medium, additives, stimulation, temperature, gas atmosphere), cell number, culture cycle, and methods for introducing nucleic acids / vectors encoding polypeptides of interest into cells, etc. can be determined by reference to, for example, WO 2018 / 177966 A1. In some embodiments, cells / cell populations according to the present disclosure are prepared under GMP (good manufacturing practice; for example, as in the Good Manufacturing Practice Guidelines issued by the European Commission (Volume 4 of the rules governing medicinal products in the European Union contains guidance on the interpretation of the principles and guidelines for good manufacturing practices for human and veterinary medicines in Commission Directive 91 / 356 / EEC (as amended by Directives 2003 / 94 / EC and 91 / 412 / EEC, respectively)) conditions.

[0616] Conveniently, cell cultures according to the present disclosure may be grown in a medium containing 5% CO 2 The cells of the cell culture can be established and / or maintained at 37° C. in a humidified atmosphere of . The cells of the cell culture can be established and / or maintained at any suitable density, as can be easily determined by the technician. Cultivation can be carried out in any container suitable for the culture volume (e.g., in a cell culture plate, a cell culture bottle, a well of a bioreactor, etc.). In some embodiments, the cells are cultured in a bioreactor, such as 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 before the introduction of a nucleic acid encoding a polypeptide of interest. For example, in the presence of IL-2, T cells within a PBMC population can be non-specifically activated by in vitro stimulation with agonist anti-CD3 and agonist anti-CD28 antibodies.

[0617] Introducing nucleic acid into cells may include transduction, such as lentiviral transduction. Transduction of immune cells with 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.

[0618] Reagents can be used to improve transduction efficiency. Hexadimethrine bromide (polybrene) is a cationic polymer that is commonly used to improve transduction by neutralizing the charge repulsion between sialic acid residues expressed on the cell surface of viral particles. Other reagents commonly used to enhance transduction include, for example, poloxamer-based reagents such as LentiBOOST (Sirion Biotech), Retronectin (Takara), Vectofusin (Miltenyi Biotech) and SureENTRY (Qiagen) and ViraDuctin (Cell Biolabs). In certain embodiments, the method includes centrifuging cells (referred to as "spinfection" in the art) into which nucleic acids encoding polypeptides of interest are to be introduced in the presence of a cell culture medium containing a viral vector containing nucleic acid.

[0619] The method generally includes introducing a nucleic acid encoding a polypeptide of interest into a cell and culturing the cell under conditions suitable for the cell to express the polypeptide of interest. In some embodiments, the method includes culturing immune cells into which a nucleic acid encoding a polypeptide of interest has been introduced to expand their number.

[0620] In some embodiments, the method comprises analyzing the cell to confirm that the nucleic acid is successfully introduced into the cell. In some embodiments, the method comprises analyzing the cell to confirm the expression of the polypeptide of interest by the cell (e.g., via assessment of a detectable entity).

[0621] In some embodiments, the method further comprises cells expressing the polypeptide of interest, for example, from other cells (e.g., cells that do not express the polypeptide of interest). Methods for purifying / separating immune cells from heterogeneous cell populations are well known in the art, and methods such as FACS or MACS-based methods can be used to sort the cell population according to the expression of immune cell markers. In some embodiments, the method purifies / separates specific types of cells, such as CD8+T cells or CTLs expressing the polypeptide of interest.

[0622] Methods for producing cells according to the present disclosure may include modifying the cells to reduce expression of CD3-TCR complex polypeptides. In some embodiments, the method includes modifying a nucleic acid (eg, an endogenous nucleic acid) encoding a CD3-TCR complex polypeptide.

[0623] Modification of a given target nucleic acid can be achieved in a variety of ways known to the skilled artisan, including modification of the target nucleic acid by homologous recombination, and editing of the target nucleic acid using site-specific nucleases (SSNs).

[0624] Suitable methods may employ targeting by homologous recombination, which is reviewed, for example, in 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 exchanging nucleic acid sequences by crossover events guided by homologous sequences. Other suitable techniques include nucleic acid editing using SSNs. Gene editing using SSNs is reviewed, for example, in Eid and Mahfouz, Exp Mol Med. 2016 Oct; 48 (10): e265, the entirety of which is incorporated herein by reference. Enzymes capable of producing site-specific double-strand breaks (DSBs) can be engineered to introduce DSBs into a target nucleic acid sequence of interest. DSBs can be repaired by error-prone non-homologous end joining (NHEJ), in which the two ends of the break are rejoined, usually by 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 site is provided and introduced at the DSB site.

[0625] SSNs that can be engineered to produce 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. The ZFN system is reviewed, 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 include programmable zinc finger DNA binding domains and DNA cleavage domains (e.g., FokI endonuclease domains). DNA binding domains can be identified by screening zinc finger arrays that can bind to target nucleic acid sequences. The TALEN system is reviewed, for example, in Mahfouz et al., Plant Biotechnol J. (2014) 12 (8): 1006-14, the entire contents of which are incorporated herein by reference. TALEN includes programmable DNA binding TALE domains and DNA cleavage domains (e.g., FokI endonuclease domains). TALEs contain repeat domains consisting of 33 to 39 amino acid repeats, which are identical except that the two residues at positions 12 and 13 of each repeat are repeat variable di-residues (RVDs). Each RVD determines the binding of the repeat to the nucleotides 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, the entire contents of which are incorporated herein by reference. 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.

[0626] In some embodiments, modification of nucleic acids encoding CD3-TCR complex polypeptides according to the present disclosure (e.g., endogenous nucleic acids) employs a site-specific nuclease (SSN) system targeting nucleic acids encoding the CD3-TCR complex polypeptides. 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.

[0627] In some embodiments, methods for producing cells according to the present disclosure include introducing nucleic acids encoding a CRISPR / Cas9 system targeting CD3E into cells. In some embodiments, the nucleic acid encodes a CRISPR RNA (crRNA) targeting 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.

[0628] In some embodiments, methods for producing cells according to the present disclosure include introducing into cells nucleic acids encoding a CRISPR / Cas9 system targeting TRAC, TRBC1, and / or TRBC2 (e.g., TRAC and TRBC1). In some embodiments, nucleic acids encode CRISPR RNA (crRNA) targeting TRAC, TRBC1, and / or TRBC2 (e.g., TRAC and TRBC1; e.g., exons of TRAC, TRBC1, and / or TRBC2 (e.g., TRAC and TRBC1)) and trans-activating crRNA (tracrRNA) for processing crRNA into its mature form.

[0629] Compositions

[0630] The present disclosure also provides compositions comprising the polypeptides, polypeptide complexes, nucleic acids, expression vectors and cells described herein.

[0631] The polypeptides, polypeptide complexes, nucleic acids, expression vectors and cells described herein (and in particular the nucleic acids, expression vectors and cells described herein) can be formulated as pharmaceutical compositions or drugs for clinical use, and may contain pharmaceutical carriers, diluents, excipients or adjuvants. In preferred aspects and embodiments, the present disclosure provides pharmaceutical compositions or drugs comprising cells according to the present disclosure. Therefore, the present disclosure also provides pharmaceutical compositions / drugs comprising polypeptides, polypeptide complexes, nucleic acids / multiple nucleic acids, expression vectors / multiple expression vectors or cells described herein. In preferred embodiments, pharmaceutical compositions / drugs according to the present disclosure comprise nucleic acids / multiple nucleic acids, expression vectors / multiple expression vectors or cells described herein.

[0632] The pharmaceutical compositions / medicaments of the present disclosure may comprise one or more pharmaceutically acceptable carriers (e.g., liposomes, micelles, microspheres, nanoparticles), diluents / excipients (e.g., starch, cellulose, cellulose derivatives, polyols, glucose, maltodextrin, magnesium stearate), adjuvants, fillers, buffers, preservatives (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium citrate, methylparaben, propylparaben), antioxidants (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium), lubricants (e.g., magnesium stearate, talc, silicon dioxide, stearic acid, vegetable stearin), binders (e.g., sucrose, lactose, starch, cellulose, gelatin, polyethylene glycol (PEG), polyvinyl pyrrolidone (PVP), xylitol, sorbitol, mannitol), stabilizers, solubilizers, surfactants (e.g., wetting agents), masking agents, or colorants (e.g., titanium oxide).

[0633] As used herein, the term "pharmaceutical" refers to compounds, ingredients, materials, compositions, dosage forms, etc., which are suitable for contacting tissues of relevant subjects (e.g., human subjects) within the scope of reasonable medical judgment without excessive toxicity, irritation, allergic reactions or other problems or complications, and are commensurate with reasonable benefit / risk ratios. According to each carrier, diluent, excipient, adjuvant, filler, buffer, preservative, antioxidant, lubricant, adhesive, stabilizer, solubilizer, surfactant, masking agent, colorant, flavoring agent or sweetener of the composition disclosed herein, it must also be "acceptable" in the sense of being compatible with other ingredients of the preparation. Suitable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, adhesives, stabilizers, solubilizers, surfactants, masking agents, colorants, flavoring agents or sweeteners can be found in standard pharmacy textbooks such as Remington's "The Science and Practice of Pharmacy" (A. Adejare, ed.), 23rd edition (2020), Academic Press.

[0634] 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 routes of administration. In some embodiments, the pharmaceutical composition / medicament can be formulated for administration by injection or infusion, or by ingestion.

[0635] Suitable preparations may comprise cells provided in a sterile or isotonic medium. Medications and pharmaceutical compositions may be formulated as fluid forms, including gel forms. Fluid preparations may be formulated for administration to selected areas of the human or animal body by injection or infusion (e.g., via a catheter).

[0636] In some embodiments, the pharmaceutical composition / drug is formulated for injection or infusion into, for example, a blood vessel, a tissue / organ of interest, or a tumor.

[0637] The present disclosure also provides methods for producing pharmaceutically useful compositions, such production methods may include one or more steps selected from the following:

[0638] producing the cells described herein;

[0639] Isolating / purifying the cells described herein; and / or

[0640] The cells described herein are mixed with a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.

[0641] For example, another aspect of the present disclosure relates to a method for formulating or producing a medicament or pharmaceutical composition for treating a disease / disorder (e.g., a disease / disorder described herein), the method comprising formulating a pharmaceutical composition or medicament by mixing the cells described herein with a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.

[0642] Therapeutic and prophylactic applications

[0643] The articles of the present disclosure can be used in therapeutic and preventive methods. Specifically, cells according to the present disclosure, such as cells containing / expressing a CD3-TCR polypeptide complex containing a recombinant CD3-TCR complex polypeptide according to the present disclosure, can be used in therapeutic and preventive methods. Similarly, compositions according to the present disclosure, such as pharmaceutical compositions comprising cells according to the present disclosure (e.g., cells containing / expressing a CD3-TCR polypeptide complex containing a recombinant CD3-TCR complex polypeptide according to the present disclosure), can be used in such methods.

[0644] Therefore, the present disclosure provides cells or compositions as described herein for use in methods of medical treatment or prevention. Also provided are cells or compositions as described herein for use in methods of treating or preventing diseases or conditions as described herein. Also provided are uses of cells or compositions as described herein in the manufacture of medicaments for treating or preventing diseases or conditions as described herein. Also provided are methods of treating or preventing diseases or conditions as described herein, comprising administering to a subject a therapeutically or prophylactically effective amount of cells or compositions as described herein.

[0645] The intervention described in the previous paragraph can effectively reduce the development or progress of the disease / illness, alleviate the symptoms of the disease / illness or reduce the pathology of the disease / illness. The intervention can effectively prevent the progress of the disease / illness, such as preventing the disease / illness from worsening or slowing down the development speed of the disease / illness. In certain embodiments, the intervention can lead to the improvement of the disease / illness, such as. The symptoms of the disease / illness reduce or the reduction of some other related events of the severity / activity of the disease / illness. In certain embodiments, the intervention can prevent the progression / development of the disease / illness later stage (such as chronic phase or transfer).

[0646] Therapeutic or prophylactic interventions according to the present disclosure generally comprise administering a cell or pharmaceutical composition according to the present disclosure to a subject to which an antigen binding molecule has been or will be administered, the 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.

[0647] It should be understood that, according to such intervention, the cell (or cell of the composition) comprises / expresses a CD3-TCR complex comprising an antigen binding portion bound to a variant Fc domain of an antigen binding molecule. For example, the intervention may include administering a T cell expressing a CD3-TCR polypeptide complex comprising a recombinant CD3-TCR complex polypeptide according to SEQ ID NO: 121 to a subject to whom an antibody comprising an Fc domain comprising a CH2-CH3 region according to SEQ ID NO: 213 has been or will be administered. For example, the intervention may include administering a T cell expressing a CD3-TCR polypeptide complex comprising (i) a recombinant CD3-TCR complex polypeptide according to SEQ ID NO: 137 and (ii) a recombinant CD3-TCR complex polypeptide according to SEQ ID NO: 165 to a subject to whom an antibody comprising an Fc domain comprising a CH2-CH3 region according to SEQ ID NO: 213 has been or will be administered. By further example, the intervention may comprise administering T cells expressing a CD3-TCR polypeptide complex comprising a recombinant CD3-TCR complex polypeptide according to 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 according to SEQ ID NO: 212.

[0648] In the therapeutic / preventive intervention of the present disclosure, the antigen binding molecules with variant Fc domains act as adapter molecules and guide the activity of cells according to the present disclosure against the antigen to which the antigen binding molecules bind. That is, in embodiments where the cells are immune cells (e.g., T cells), the antigen binding molecules with variant Fc domains guide a cell-mediated immune response (e.g., a T cell-mediated immune response) against cells expressing the antigen to which the antigen binding molecules bind.

[0649] For example, in an example of the present disclosure, a T cell expressing a CD3-TCR polypeptide complex comprising a recombinant CD3-TCR complex polypeptide according to SEQ ID NO: 121 is used together with an anti-FolR1 antibody comprising an Fc domain containing P329G, such that the T cell is directed against cells expressing FolR1. By further example, in an example of the present disclosure, a T cell expressing a CD3-TCR polypeptide complex comprising a recombinant CD3-TCR complex polypeptide according to SEQ ID NO: 121 is used together with an anti-CD19 antibody comprising an Fc domain containing P329G, such that the T cell is directed against cells expressing CD19. By further example, in an example of the present disclosure, a T cell expressing a CD3-TCR polypeptide complex comprising (i) a recombinant CD3-TCR complex polypeptide according to SEQ ID NO: 137 and (ii) a recombinant CD3-TCR complex polypeptide according to SEQ ID NO: 165 is used together with an anti-FolR1 antibody comprising an Fc domain containing P329G, such that the T cell is directed against cells expressing FolR1. By further example, in an example of the present disclosure, T cells expressing a CD3-TCR polypeptide complex comprising (i) a recombinant CD3-TCR complex polypeptide according to SEQ ID NO: 137 and (ii) a recombinant CD3-TCR complex polypeptide according to SEQ ID NO: 165 are used together with an anti-CD19 antibody comprising an Fc domain containing P329G, such that the T cells are directed against cells expressing CD19.

[0650] Antigen binding molecules with variant Fc domains used with cells or compositions according to the present disclosure can bind to any given target antigen.

[0651] The target antigen can be any target antigen expressed by cells that are desired to be killed / consumed to obtain a therapeutic / preventive effect. In some embodiments, the target antigen is an antigen whose expression / activity or its upregulated expression / activity is positively correlated with a disease / disorder (e.g., cancer, infectious disease, or autoimmune disease). The target antigen is preferably expressed at the cell surface of cells expressing the target antigen.

[0652] In some embodiments, the target antigen may be a cancer cell antigen. Cancer cell antigens are antigens expressed or overexpressed by cancer cells. Cancer cell antigens may be any peptide / polypeptide, glycoprotein, lipoprotein, polysaccharide, glycolipid, lipid or fragment thereof. The expression of cancer cell antigens may be associated with cancer. Cancer cell antigens may be abnormally expressed by cancer cells (e.g., cancer cell antigens may be expressed with abnormal positioning), or may be expressed by cancer cells with abnormal structures. Cancer cell antigens may be able to trigger an immune response. In some embodiments, antigens are expressed at the cell surface of cancer cells (i.e., cancer cell antigens are cancer cell surface antigens). In some embodiments, the portion of the antigen bound by the antigen binding molecules described herein is displayed on the outer surface of the cancer cell (i.e., outside the cell). Cancer cell antigens may be cancer-associated antigens. In some embodiments, cancer cell antigens are antigens whose expression is associated with the development, progression or severity of cancer symptoms. Cancer-associated antigens may be associated with the etiology or pathology of cancer, or may be abnormally expressed due to cancer. In some embodiments, cancer cell antigens are antigens whose expression is upregulated by cancer cells (e.g., at RNA and / or protein levels), for example, compared to the level expressed by comparable non-cancer cells (e.g., non-cancer cells from the same tissue / cell type). In some embodiments, cancer-associated antigens may be preferentially expressed by cancer cells and not expressed by comparable non-cancerous cells (e.g., non-cancerous cells derived from the same tissue / cell type). In some embodiments, cancer-associated antigens may be products of mutated oncogenes or mutated tumor suppressor genes. In some embodiments, cancer-associated antigens may be products of overexpressed cellular proteins, cancer antigens produced by oncogenic viruses, carcinoembryonic antigens, or cell surface glycolipids or glycoproteins.

[0653] Cancer cell antigens are reviewed by the following literature: Zarour HM, DeLeo A, Finn OJ, et al. Categories of Tumor Antigens. In: Kufe DW, Pollock RE, Weichselbaum RR, et al., eds. Holland-Frei Cancer Medicine. 6th edition Hamilton (ON): BC Decker; 2003. Cancer cell antigens include carcinoembryonic antigens: CEA, immature laminin receptor, TAG-72; tumor virus antigens, such as HPV E6 and E7; overexpressed proteins: BING-4, calcium-activated chloride channel 2, cyclin B1, 9D7, Ep-CAM, EphA3, HER2 / neu, telomerase, mesothelin, SAP-1, survivin; cancer testis antigens: BAGE, CAGE, GAGE, MAGE, SAGE, XAGE, CT9, CT10, NY-ESO-1, PRAME, SSX-2; lineage-restricted antigens: MART1, Gp100, tyrosinase, TRP-1 / 2, MC1R, prostate-specific antigen; mutant antigens: β-catenin, BRCA1 / 2, CDK4, CML66, fibronectin, MART-2, p53, Ras, TGF-βRII; post-translationally altered antigens: MUC1; idiotypic antigens: immunoglobulins, TCR. Other cancer cell antigens include heat shock protein 70 (HSP70), heat shock protein 90 (HSP90), glucose-regulated protein 78 (GRP78), vimentin, nucleolar protein, fetal pancreatic acinar protein (FAPP), alkaline phosphatase placental-like protein 2 (ALPPL-2), siglec-5, stress-induced 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, the entire contents of which are incorporated herein by reference.

[0654] In some embodiments, the target antigen is selected from the group consisting of: 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 (FOLR 1), human 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 peptides that bind to molecules of the human major histocompatibility complex (MHC). In some embodiments, the target antigen is CD19. In some embodiments, the target antigen is FOLR1.

[0655] It will be appreciated that the cells and compositions of the present disclosure may be used in the treatment / prevention of any disease / disorder that would derive therapeutic or prophylactic benefit from a reduction in the level / activity of a given target antigen, or a reduction in the number / proportion / activity of cells containing / expressing a given target antigen.

[0656] For example, the disease / disorder can be a disease / disorder in which the target antigen or cells containing / expressing the target antigen are pathologically involved, such as a disease / disorder in which an increase in the level / activity of the target antigen or an increase in the number / ratio / activity of cells containing / expressing the target antigen is positively correlated with the onset, development or progression of the disease / disorder and / or the severity of one or more symptoms of the disease / disorder. In some embodiments, an increase in the level / activity of the target antigen or an increase in the number / ratio / activity of cells containing / expressing the target antigen can be a risk factor for the onset, development or progression of the disease / disorder.

[0657] In some embodiments, the disease / disorder to be treated / prevented according to the present disclosure is a disease / disorder characterized by an increase in the expression or activity level of the target antigen, for example, compared to the expression / activity level in the absence of the disease / disorder. In some embodiments, the disease / disorder to be treated / prevented is a disease / disorder characterized by an increase in the number / proportion / activity of cells expressing the target antigen, for example, compared to the level / number / proportion / activity in the absence of the disease / disorder (e.g., in a healthy subject or in an equivalent non-diseased tissue). When the disease / disorder is cancer, the level of expression or activity of the target antigen may be higher than the level of expression or activity of the target antigen in an equivalent non-cancerous cell / non-tumor tissue. Cancer / its cells may contain one or more mutations (e.g., relative to an equivalent non-cancerous cell / non-tumor tissue), which cause an upregulation of the expression or activity of the target antigen.

[0658] Therapeutic / prophylactic interventions 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 an appropriate 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.

[0659] In particular, cells and compositions according to the present disclosure are contemplated for use in methods of treating / preventing diseases / disorders by adoptive cell transfer (ACT).

[0660] Adoptive cell transfer generally refers to the process of obtaining cells (such as immune cells) from a subject, usually by extracting a blood sample from which the cells are separated. Then, the cells are usually modified and / or amplified, and then applied to the same subject (in the case of adoptive transfer of autologous homologous / autologous cells) or to different subjects (in the case of adoptive transfer of allogeneic cells). Treatment is generally intended to provide a cell population with certain desired characteristics to a subject, or to increase the frequency of such cells with such characteristics in the subject. Adoptive transfer can be carried out for the purpose of introducing cells or cell populations into a subject and / or increasing the frequency of cells or cell populations in a subject.

[0661] 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, the entire contents of which are incorporated herein by reference. A skilled person can determine appropriate reagents and procedures for adoptive transfer of cells based on the present disclosure, for example, with reference to Dai et al., 2016 J Nat Cancer Inst 108 (7): djv439, the entire contents of which are incorporated by reference.

[0662] The cells and compositions according to the present disclosure can be used to treat / prevent diseases / disorders by allogeneic transplantation or autologous transplantation.

[0663] As used herein, "allogeneic transplantation" refers to the transplantation of cells, tissues or organs that are genetically different from the recipient subject to the recipient subject. The cells, tissues or organs may come from or may be derived from cells, tissues or organs of a donor subject that is genetically different from the recipient subject. Allogeneic transplantation is different from autologous transplantation, which refers to the transplantation of cells, tissues or organs (i.e., autologous materials) from / derived from donor subjects that are genetically identical to the recipient subject. It should be understood that adoptive transfer of allogeneic immune cells is a form of allogeneic transplantation, and adoptive transfer of autologous homologous immune cells is a form of autologous transplantation.

[0664] The present disclosure provides methods comprising administering to a subject the cells and compositions according to the present disclosure.

[0665] In some embodiments, the method comprises modifying an immune cell to contain / express a polypeptide according to the present disclosure (eg, a recombinant CD3-TCR complex polypeptide, a composite polypeptide).

[0666] In some embodiments, the method comprises:

[0667] modifying an immune cell to express or comprise a CD3-TCR complex polypeptide comprising one or more recombinant CD3-TCR complex polypeptides according to the present disclosure (e.g., as described herein), and

[0668] The modified immune cells are administered to a subject.

[0669] In some embodiments, the method further comprises:

[0670] An antigen binding molecule comprising a variant Fc domain according to the present disclosure is administered to a subject, wherein the modified immune CD3-TCR complex polypeptide comprises an antigen binding portion that binds to the variant Fc domain of the antigen binding molecule.

[0671] In some embodiments, the method further comprises:

[0672] Modifying an immune cell 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 association domain of a recombinant CD3-TCR complex polypeptide of the CD3-TCR complex is derived.

[0673] It will be appreciated that the method steps described in the preceding three paragraphs may be performed in any suitable order.

[0674] In some embodiments, the method comprises:

[0675] Immune cells modified to express or comprise a CD3-TCR complex comprising one or more recombinant CD3-TCR complex polypeptides according to the present disclosure are administered to a subject.

[0676] In some embodiments, the method comprises:

[0677] administering to a subject an immune cell modified to (i) express or comprise a CD3-TCR complex comprising one or more recombinant CD3-TCR complex polypeptides according to the present disclosure and (ii) reduce / prevent expression of a CD3-TCR complex polypeptide;

[0678] The recombinant CD3-TCR complex polypeptide of the CD3-TCR complex of (i) comprises a CD3-TCR complex association domain derived from the CD3-TCR complex polypeptide of (ii).

[0679] In some embodiments, according to the previous two paragraphs, the subject is a subject to whom an antigen binding molecule comprising a variant Fc domain according to the present disclosure has been or will be administered, wherein the CD3-TCR complex comprises an antigen binding portion that binds to the variant Fc domain of the antigen binding molecule.

[0680] In some embodiments, the method comprises:

[0681] (a) modifying an immune cell to express or comprise a CD3-TCR complex polypeptide comprising one or more recombinant CD3-TCR complex polypeptides according to the present disclosure (e.g., as described herein); and

[0682] (b) administering to a subject an antigen binding molecule comprising a variant Fc domain according to the present disclosure; and

[0683] (c) administering the modified immune cells to a subject;

[0684] The CD3-TCR complex of (a) comprises an antigen-binding portion bound to a variant Fc domain of the antigen-binding molecule of (b).

[0685] In some embodiments of the method according to the preceding paragraph, step (c) may be performed before step (b).

[0686] In some embodiments, the method comprises:

[0687] (a) modifying immune cells to reduce / prevent the expression of CD3-TCR complex polypeptides;

[0688] (b) modifying an immune cell to express or comprise a CD3-TCR complex polypeptide comprising one or more recombinant CD3-TCR complex polypeptides according to the present disclosure (e.g., as described herein); and

[0689] (c) administering to a subject an antigen binding molecule comprising a variant Fc domain according to the present disclosure; and

[0690] (d) administering the modified immune cells to a subject;

[0691] wherein the recombinant CD3-TCR complex polypeptide of the CD3-TCR complex of (b) comprises a CD3-TCR complex association domain derived from the CD3-TCR complex polypeptide of (a); and

[0692] The CD3-TCR complex of (b) comprises an antigen-binding portion bound to the variant Fc domain of the antigen-binding molecule of (c).

[0693] In some embodiments of the method according to the preceding paragraph, step (d) may be performed before step (c).

[0694] 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 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 allogeneic cells).

[0695] In some embodiments, the method may further include one or more of the following:

[0696] obtaining a blood sample from the subject;

[0697] isolating immune cells (e.g., PBMCs) from a blood sample that has been obtained from a subject;

[0698] Generate / expand immune cell populations;

[0699] growing immune cells in vitro or ex vivo cell culture;

[0700] Cultivating immune cells expressing / comprising a CD3-TCR complex comprising one or more recombinant CD3-TCR complex polypeptides according to the present disclosure in in vitro or ex vivo cell culture;

[0701] Collecting / isolating immune cells expressing / containing CD3-TCR complexes containing one or more recombinant CD3-TCR complex polypeptides according to the present invention;

[0702] Immune cells expressing / comprising a CD3-TCR complex comprising one or more recombinant CD3-TCR complex polypeptides according to the present disclosure are formulated into a pharmaceutical composition, for example, by mixing the cells with a pharmaceutically acceptable adjuvant, diluent or carrier.

[0703] The administration of the articles disclosed herein is preferably carried out in a "therapeutically effective" or "preventively effective" amount, which is sufficient to show a therapeutic or preventive benefit to the subject. The actual amount applied, as well as the rate and time course of administration, will depend on the nature and severity of the disease / disorder and the specific article administered. Prescribing treatment, such as determining dosage, etc., is the responsibility of general practitioners and other doctors, and generally takes into account the disease / disorder to be treated, the condition of the individual subject, treatment site delivery, administration method, and other factors known to practitioners. Examples of the above techniques and protocols can be found in Remington's The Science and Practice of Pharmacy (A. Adejare, ed.), 23rd edition (2020), Academic Press.

[0704] The administration of the articles 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 carried out by injection or infusion. Administration of the articles of the present disclosure can be intratumoral. In some cases, the articles of the present disclosure can be formulated for targeted delivery to specific cells, tissues, organs and / or tumors.

[0705] Multiple doses of the articles of the present disclosure may be provided. Multiple doses may be separated by a predetermined time interval, which may be selected to be 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 one of 1, 2, 3, 4, 5, or 6 months.

[0706] Administration of a cell or composition according to the present disclosure and an antigen binding molecule described herein to a subject according to the therapeutic and prophylactic interventions described herein can be simultaneous or sequential.

[0707] Simultaneous administration refers to 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 immediately sequentially, and optionally via the same route of administration, e.g., to the same artery, vein or other blood vessel.

[0708] Sequential administration refers to administration of (i) a cell or composition according to the present disclosure and (ii) one of the antigen binding molecules described herein, followed by separate administration of the other agent after a given time interval. It is not necessary to administer the two agents by the same route, although in some embodiments the same route is used. The time interval can be any time interval.

[0709] The present disclosure also provides a method for depleting or killing a cell comprising or expressing a target antigen, the method comprising contacting a cell comprising / expressing the target antigen with:

[0710] (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

[0711] (ii) an immune cell comprising / expressing a CD3-TCR complex comprising one or more recombinant CD3-TCR complex polypeptides according to the present disclosure;

[0712] wherein the CD3-TCR complex of (ii) comprises an antigen-binding portion bound to a variant Fc domain of the antigen-binding molecule of (i).

[0713] Subject

[0714] According to various aspects of the present disclosure, the subject can be any animal or human. Therapeutic and prophylactic applications can be in humans or in animals (veterinary use).

[0715] The subject to whom the articles of the present disclosure are to be administered (e.g., pursuant to 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 more preferably a human. The subject may be male or female. The subject may be a patient.

[0716] The subject may be suffering from (e.g., may 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 / infecting such a disease / condition. In embodiments according to the present disclosure, a subject may be selected for treatment based on a method based on the characterization of one or more markers for such a disease / condition.

[0717] In some embodiments, a subject may be selected for a therapeutic or prophylactic intervention as described herein based on, for example, detecting cells / tissues expressing a target antigen (i.e., the target antigen of an antigen binding molecule to be used in conjunction with a cell or composition according to the present disclosure) or cells / tissues overexpressing the target antigen in a sample obtained from the subject.

[0718] According to the present disclosure, a subject can be an allogeneic or non-autologous subject. As used herein, when a subject is referred to as "allogeneic" or "non-autologous" with respect to an intervention, the subject is a subject other than the subject from which the intervention cells (i.e., the cells to be administered, or the cells of the pharmaceutical composition / drug to be administered) are derived. The subject to be treated / prevented according to the present disclosure may be genetically different from the subject from which the cells (such as the cells of the pharmaceutical composition / drug to be administered) to be administered to the subject are derived. The subject to be treated / prevented according to the present disclosure may include MHC / HLA genes encoding MHC / HLA molecules (such as MHC class I α and / or MHC class II molecules) that are different from the MHC / HLA molecules (such as MHC class I α and / or MHC class II molecules) encoded by the cells (such as the cells of the pharmaceutical composition / drug to be administered) to be administered to the subject. The subject to be treated / prevented according to the present disclosure can be HLA-mismatched relative to the subject from which the cells (such as the cells of the pharmaceutical composition / drug to be administered) to be administered to the subject are derived.

[0719] Relative to the source from which the cells (such as the cells of the pharmaceutical composition / drug to be administered) to be administered to the subject are derived, the subject to which cells are administered according to the present disclosure can be allogeneic / non-autologous. The subject to which cells are administered can be a different subject from the subject from which cells are obtained for production of the cells (such as the cells of the pharmaceutical composition / drug to be administered) to be administered. The subject to which cells are administered can be genetically different from the subject from which cells are obtained for production of the cells (such as the cells of the pharmaceutical composition / drug to be administered) to be administered to the subject.

[0720] According to the present disclosure, a subject can be an autologous / autologous subject. As used herein, when a subject is referred to as "autologous" or "autologous" with respect to an intervention, the subject is the same subject from which the intervention cells (i.e., the cells to be administered, or the cells of the pharmaceutical composition / drug to be administered) are derived. The subject to be treated / prevented according to the present disclosure may be genetically the same as the subject from which the cells (such as the cells of the pharmaceutical composition / drug to be administered) to be administered to the subject are derived. The subject to be treated / prevented according to the present disclosure may include MHC / HLA genes encoding MHC / HLA molecules (such as MHC class I α and / or MHC class II molecules) that are the same as the MHC / HLA molecules (such as MHC class I α and / or MHC class II molecules) encoded by the cells (such as the cells of the pharmaceutical composition / drug to be administered) to be administered to the subject. The subject to be treated / prevented according to the present disclosure can be HLA-matched relative to the subject from which the cells (such as the cells of the pharmaceutical composition / drug to be administered) to be administered to the subject are derived.

[0721] The subject to which the cells are administered according to the present disclosure may be autologous / autologous relative to the source from which the cells (e.g., cells of a pharmaceutical composition / drug) to be administered to the subject are derived. The subject to which the cells are administered may be the same subject as the subject from which the cells are obtained for the production of the cells to be administered (e.g., cells of a pharmaceutical composition / drug). The subject to which the cells are administered may be genetically identical to the subject from which the cells are obtained for the production of the cells to be administered to the subject (e.g., cells of a pharmaceutical composition / drug).

[0722] Kits

[0723] The present disclosure also provides kits of parts.

[0724] In some aspects and embodiments, a kit of parts according to the present disclosure comprises (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 should 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).

[0725] In some aspects and embodiments, a kit of parts according to the present disclosure comprises (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 should be understood that according to such aspects and embodiments, the composition of (i) comprises a cell comprising / expressing a CD3-TCR complex comprising an antigen binding portion that binds to a variant Fc domain of an antigen binding molecule of (ii).

[0726] In some aspects and embodiments, a multi-component kit according to the present disclosure comprises (i) a nucleic acid / multiple nucleic acids or expression vector / multiple 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 according to the present disclosure. It should be understood that according to such aspects and embodiments, the nucleic acid / multiple nucleic acids or expression vector / multiple expression vectors of (i) encode a polypeptide for engineering a cell to comprise / express a CD3-TCR complex comprising an antigen binding portion that binds to a variant Fc domain of an antigen binding molecule of (ii).

[0727] A multi-component kit according to the present disclosure may include a predetermined number of articles according to (i) and / or (ii), as described in the previous three paragraphs. In some embodiments, the articles according to (i) and / or (ii) are provided in a container (e.g., a vial or bottle). The kit may provide the articles according to (i) and / or (ii) together with instructions (e.g., a protocol) on how to use them according to a therapeutic or preventive intervention as described herein.

[0728] In some embodiments, the multi-component kit includes materials for producing polypeptides according to the present disclosure, such as recombinant CD3-TCR complex polypeptides according to the present disclosure. In some embodiments, the multi-component kit includes materials for producing polypeptides according to the present disclosure, such as CD3-TCR polypeptide complexes containing recombinant CD3-TCR complex polypeptides according to the present disclosure. In some embodiments, the multi-component kit includes materials for producing cells according to the present disclosure, such as cells containing / expressing CD3-TCR polypeptide complexes containing recombinant CD3-TCR complex polypeptides according to the present disclosure. In some embodiments, the multi-component kit includes materials for producing compositions according to the present disclosure, such as pharmaceutical compositions containing cells according to the present disclosure (e.g., cells containing / expressing CD3-TCR polypeptide complexes containing recombinant CD3-TCR complex polypeptides according to the present disclosure).

[0729] In some embodiments, a multi-component kit may include a nucleic acid / multiple nucleic acids or expression vector / multiple expression vectors according to the present disclosure, and optionally materials for introducing the nucleic acid / multiple nucleic acids or expression vector / multiple expression vectors into cells. In some embodiments, a multi-component kit may include a system for producing cells according to the present disclosure under GMP conditions. In some embodiments, a multi-component kit may include a (closed) bag cell incubation system, wherein a nucleic acid / multiple nucleic acids or expression vector / multiple vectors according to the present disclosure may be introduced into cells and subsequently cultured under GMP conditions.

[0730] In some embodiments, a 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.

[0731] The manufacture of the multi-component kit according to the present disclosure preferably follows standard procedures known to those skilled in the art.

[0732] Sequence identity

[0733] 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 percentage of sequence identity between the sequences. Paired and multiple sequence alignments for the purpose of determining the percentage of sequence identity between two or more amino acid or nucleic acid sequences can be achieved in various ways known to those skilled in the art, for example, using publicly available computer software such as ClustalOmega ( 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 preferred to use the default parameters, for example, for gap penalties and extension penalties.

[0734] Numbered paragraphs

[0735] The following numbered paragraphs provide further description of features and combinations of features contemplated to be relevant to the present invention:

[0736] 1. A recombinant CD3-TCR complex polypeptide comprising:

[0737] (i) an antigen binding portion or a component thereof, 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; and

[0738] (ii) a CD3-TCR complex association domain having an amino acid sequence derived from a CD3-TCR complex polypeptide.

[0739] 2. The 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 through its CD3-TCR complex association domain to form a CD3-TCR complex.

[0740] 3. The recombinant CD3-TCR complex polypeptide according to paragraph 1 or paragraph 2, wherein the amino acid sequence derived from the CD3-TCR complex polypeptide is derived from CD3ε, TCRα or TCRβ.

[0741] 4. A recombinant CD3-TCR complex polypeptide according to 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 with one of SEQ ID NO: 30, 52, 1, 53, 5, 54 or 9.

[0742] 5. The 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.

[0743] 6. The 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.

[0744] 7. The 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.

[0745] 8. A recombinant CD3-TCR complex polypeptide according to 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 NO: 52, 1, 53, 5, 54 or 9.

[0746] 9. A recombinant CD3-TCR complex polypeptide according to paragraph 7 or paragraph 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 the position corresponding to position 47 numbered according to SEQ ID NO:1.

[0747] 10. A recombinant CD3-TCR complex polypeptide according to any one of paragraphs 7 to 9, or 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 NO: 53, 5, 54 or 9, and comprises a cysteine ​​residue at the position corresponding to position 56 numbered according to SEQ ID NO: 5.

[0748] 11. The 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 the antibody that binds to the variant Fc domain.

[0749] 12. The recombinant CD3-TCR complex polypeptide according to any one of paragraphs 1 to 11, wherein the antigen binding portion is or comprises Fv, scFv, Fab, Fab', Fab'-SH, F(ab') 2 , crossFab, scFab or dAb portion.

[0750] 13. The recombinant CD3-TCR complex polypeptide according to any one of paragraphs 1 to 12, wherein the antigen binding portion is or comprises a scFv.

[0751] 14. The recombinant CD3-TCR complex polypeptide according to any one of paragraphs 1 to 13, wherein the component of the antigen binding portion is or comprises the VH region or VL region of an antibody bound to a variant Fc domain.

[0752] 15. The 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.

[0753] 16. The recombinant CD3-TCR complex polypeptide of any one of paragraphs 1 to 15, wherein the variant Fc domain binds to the Fc receptor with a lower affinity than the reference Fc domain binds to the Fc receptor, optionally wherein the Fc receptor is an Fcγ receptor or a neonatal Fc receptor (FcRn).

[0754] 17. The recombinant CD3-TCR complex polypeptide according to any one of paragraphs 1 to 16, wherein the variant Fc domain comprises a CH2-CH3 region comprising an amino acid difference relative to the amino acid sequence of the CH2-CH3 region of the reference Fc domain at one or more of the following positions according to EU numbering: L234, L235, 1253, N297, S298, H310, P329, E333, K334 or H435.

[0755] 18. The recombinant CD3-TCR complex polypeptide according to any one of paragraphs 1 to 17, wherein the variant Fc domain comprises a CH2-CH3 region comprising G329 according to EU numbering.

[0756] 19. The recombinant CD3-TCR complex polypeptide according to any one of paragraphs 1 to 18, wherein the antigen binding portion comprises a VL region incorporating the following CDRs:

[0757] LC-CDR1 having the amino acid sequence of SEQ ID NO:69;

[0758] LC-CDR2 having the amino acid sequence of SEQ ID NO:70; and

[0759] LC-CDR3 having the amino acid sequence of SEQ ID NO:71.

[0760] 20. The recombinant CD3-TCR complex polypeptide according to any one of paragraphs 1 to 19, wherein the antigen binding portion comprises a VH incorporating:

[0761] (i) the following CDRs:

[0762] HC-CDR1 having the amino acid sequence of SEQ ID NO:56;

[0763] an HC-CDR2 having the amino acid sequence of SEQ ID NO: 64; and

[0764] an HC-CDR3 having the amino acid sequence of SEQ ID NO: 58;

[0765] or

[0766] (ii) the following CDRs:

[0767] HC-CDR1 having the amino acid sequence of SEQ ID NO:56;

[0768] an HC-CDR2 having the amino acid sequence of SEQ ID NO: 57; and

[0769] HC-CDR3 having the amino acid sequence of SEQ ID NO:58.

[0770] 21. The recombinant CD3-TCR complex polypeptide according to any one of paragraphs 1 to 20, wherein the antigen binding portion comprises:

[0771] (a)(i) A VH region incorporating the following CDRs:

[0772] HC-CDR1 having the amino acid sequence of SEQ ID NO:56;

[0773] an HC-CDR2 having the amino acid sequence of SEQ ID NO: 64; and

[0774] an HC-CDR3 having the amino acid sequence of SEQ ID NO: 68;

[0775] and

[0776] (ii) a VL region incorporating the following CDRs:

[0777] LC-CDR1 having the amino acid sequence of SEQ ID NO:69;

[0778] LC-CDR2 having the amino acid sequence of SEQ ID NO:70; and

[0779] a LC-CDR3 having the amino acid sequence of SEQ ID NO: 71;

[0780] or (b)(i) a VH region incorporating the following CDRs:

[0781] HC-CDR1 having the amino acid sequence of SEQ ID NO:56;

[0782] an HC-CDR2 having the amino acid sequence of SEQ ID NO: 57; and

[0783] an HC-CDR3 having the amino acid sequence of SEQ ID NO: 58;

[0784] and

[0785] (ii) a VL region incorporating the following CDRs:

[0786] LC-CDR1 having the amino acid sequence of SEQ ID NO:69;

[0787] LC-CDR2 having the amino acid sequence of SEQ ID NO:70; and

[0788] LC-CDR3 having the amino acid sequence of SEQ ID NO:71.

[0789] 22. The recombinant CD3-TCR complex polypeptide according to 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 with SEQ ID NO: 68.

[0790] 23. The recombinant CD3-TCR complex polypeptide according to 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 with SEQ ID NO: 65, 63 or 55.

[0791] 24. The recombinant CD3-TCR complex polypeptide according to any one of paragraphs 1 to 23, wherein the antigen binding portion comprises:

[0792] (a)(i) a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:65; and

[0793] (ii) a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 68;

[0794] or

[0795] (b)(i) a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:63; and

[0796] (ii) a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 68;

[0797] or

[0798] (c)(i) a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:55; and

[0799] (ii) a VL having an amino acid sequence that has at least 70% amino acid sequence identity to SEQ ID NO:68.

[0800] 25. The recombinant CD3-TCR complex polypeptide according to 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.

[0801] 26. The recombinant CD3-TCR complex polypeptide according to any one of paragraphs 1 to 17 or paragraph 25, wherein the antigen binding portion comprises a VH region incorporating the following CDRs:

[0802] HC-CDR1 having the amino acid sequence of SEQ ID NO: 77;

[0803] an HC-CDR2 having the amino acid sequence of SEQ ID NO: 78; and

[0804] HC-CDR3 having the amino acid sequence of SEQ ID NO:79.

[0805] 27. The recombinant CD3-TCR complex polypeptide according to any one of paragraphs 1 to 17, paragraph 25 or paragraph 26, wherein the antigen binding portion comprises a VL region incorporating the following CDRs:

[0806] LC-CDR1 having the amino acid sequence of SEQ ID NO:85;

[0807] LC-CDR2 having the amino acid sequence of SEQ ID NO:86; and

[0808] LC-CDR3 having the amino acid sequence of SEQ ID NO:87.

[0809] 28. The recombinant CD3-TCR complex polypeptide according to any one of paragraphs 1 to 17 or 25 to 27, wherein the antigen binding portion comprises:

[0810] (i) a VH region incorporating the following CDRs:

[0811] HC-CDR1 having the amino acid sequence of SEQ ID NO: 77;

[0812] an HC-CDR2 having the amino acid sequence of SEQ ID NO: 78; and

[0813] an HC-CDR3 having the amino acid sequence of SEQ ID NO: 79;

[0814] and

[0815] (ii) a VL region incorporating the following CDRs:

[0816] LC-CDR1 having the amino acid sequence of SEQ ID NO:85;

[0817] LC-CDR2 having the amino acid sequence of SEQ ID NO:86; and

[0818] LC-CDR3 having the amino acid sequence of SEQ ID NO:87.

[0819] 29. The recombinant CD3-TCR complex polypeptide according to any one of paragraphs 1 to 17 or 25 to 28, wherein the antigen binding portion comprises a VH having an amino acid sequence having at least 70% amino acid sequence identity with SEQ ID NO: 76.

[0820] 30. The recombinant CD3-TCR complex polypeptide according to any one of paragraphs 1 to 17 or 25 to 29, wherein the antigen binding portion comprises a VL having an amino acid sequence having at least 70% amino acid sequence identity with SEQ ID NO: 84.

[0821] 31. The recombinant CD3-TCR complex polypeptide according to any one of paragraphs 1 to 17 or 25 to 30, wherein the antigen binding portion comprises:

[0822] (i) having an amino acid sequence that has at least 70% amino acid sequence identity to SEQ ID NO: 76

[0823] VH of the column; and

[0824] (ii) a VL having an amino acid sequence that has at least 70% amino acid sequence identity to SEQ ID NO:84.

[0825] 32. A recombinant CD3-TCR complex polypeptide comprising:

[0826] (i) an antigen binding portion which is a scFv, 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

[0827] (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.

[0828] 33. The 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 through its CD3-TCR complex association domain to form a CD3-TCR complex.

[0829] 34. The 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.

[0830] 35. The 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.

[0831] 36. The recombinant CD3-TCR complex polypeptide according to any one of paragraphs 32 to 35, wherein the antigen binding portion comprises a VL region incorporating the following CDRs:

[0832] LC-CDR1 having the amino acid sequence of SEQ ID NO:69;

[0833] LC-CDR2 having the amino acid sequence of SEQ ID NO:70; and

[0834] LC-CDR3 having the amino acid sequence of SEQ ID NO:71.

[0835] 37. The recombinant CD3-TCR complex polypeptide according to any one of paragraphs 32 to 36, wherein the antigen binding portion comprises a VH incorporating:

[0836] (i) the following CDRs:

[0837] HC-CDR1 having the amino acid sequence of SEQ ID NO:56;

[0838] an HC-CDR2 having the amino acid sequence of SEQ ID NO: 64; and

[0839] an HC-CDR3 having the amino acid sequence of SEQ ID NO: 58;

[0840] or

[0841] (ii) the following CDRs:

[0842] HC-CDR1 having the amino acid sequence of SEQ ID NO:56;

[0843] an HC-CDR2 having the amino acid sequence of SEQ ID NO: 57; and

[0844] HC-CDR3 having the amino acid sequence of SEQ ID NO:58.

[0845] 38. The recombinant CD3-TCR complex polypeptide according to any one of paragraphs 32 to 37, wherein the antigen binding portion comprises:

[0846] (a)(i) A VH region incorporating the following CDRs:

[0847] HC-CDR1 having the amino acid sequence of SEQ ID NO:56;

[0848] an HC-CDR2 having the amino acid sequence of SEQ ID NO: 64; and

[0849] an HC-CDR3 having the amino acid sequence of SEQ ID NO: 68;

[0850] and

[0851] (ii) a VL region incorporating the following CDRs:

[0852] LC-CDR1 having the amino acid sequence of SEQ ID NO:69;

[0853] LC-CDR2 having the amino acid sequence of SEQ ID NO:70; and

[0854] a LC-CDR3 having the amino acid sequence of SEQ ID NO: 71;

[0855] or (b)(i) a VH region incorporating the following CDRs:

[0856] HC-CDR1 having the amino acid sequence of SEQ ID NO:56;

[0857] an HC-CDR2 having the amino acid sequence of SEQ ID NO: 57; and

[0858] an HC-CDR3 having the amino acid sequence of SEQ ID NO: 58;

[0859] and

[0860] (ii) a VL region incorporating the following CDRs:

[0861] LC-CDR1 having the amino acid sequence of SEQ ID NO:69;

[0862] LC-CDR2 having the amino acid sequence of SEQ ID NO:70; and

[0863] LC-CDR3 having the amino acid sequence of SEQ ID NO:71.

[0864] 39. The recombinant CD3-TCR complex polypeptide according to 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 with SEQ ID NO: 68.

[0865] 40. The recombinant CD3-TCR complex polypeptide according to 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.

[0866] 41. The recombinant CD3-TCR complex polypeptide according to any one of paragraphs 32 to 40, wherein the antigen binding portion comprises:

[0867] (a)(i) a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:65; and

[0868] (ii) a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 68;

[0869] or

[0870] (b)(i) a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:63; and

[0871] (ii) a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 68;

[0872] or

[0873] (c)(i) a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:55; and

[0874] (ii) a VL having an amino acid sequence that has at least 70% amino acid sequence identity to SEQ ID NO:68.

[0875] 42. A polypeptide complex comprising an antigen binding portion, wherein the antigen binding portion is bound to a variant Fc domain, the 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 is not bound, and wherein the polypeptide complex comprises a first recombinant CD3-TCR complex polypeptide and a second recombinant CD3-TCR complex polypeptide, wherein the first CD3-TCR complex polypeptide and the second recombinant CD3-TCR complex polypeptide each comprise: (i) a CD3-TCR complex association domain having an amino acid sequence derived from a CD3-TCR complex polypeptide, and (ii) a component of the antigen binding portion.

[0876] 43. A polypeptide complex comprising:

[0877] (a) a first recombinant CD3-TCR complex polypeptide comprising:

[0878] (i) a first component of an antigen binding moiety, wherein the antigen binding moiety 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 moiety does not bind; and

[0879] (ii) a CD3-TCR complex association domain having an amino acid sequence derived from a CD3-TCR complex polypeptide; and

[0880] (b) a second recombinant CD3-TCR complex polypeptide comprising:

[0881] (i) a second component of the antigen binding portion of (a) (i); and

[0882] (ii) a CD3-TCR complex association domain having an amino acid sequence derived from a CD3-TCR complex polypeptide;

[0883] The first recombinant CD3-TCR complex polypeptide and the second recombinant CD3-TCR complex polypeptide are capable of associating via their CD3-TCR complex association domains to form the antigen binding portion.

[0884] 44. The polypeptide complex according to paragraph 42 or paragraph 43, wherein the first component of the antigen-binding portion is or comprises the heavy-chain variable (VH) region of an antibody that binds to the variant Fc domain, and wherein the second component of the antigen-binding portion is or comprises the light-chain variable (VL) region of an antibody that binds to the variant Fc domain.

[0885] 45. The polypeptide complex according to any one of paragraphs 42 to 44, wherein:

[0886] (i) the CD3-TCR complex association domain of the first recombinant CD3-TCR complex polypeptide is derived from TCRα, and the CD3-TCR complex association domain of the second recombinant CD3-TCR complex polypeptide is derived from TCRβ, or

[0887] (ii) 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.

[0888] 46. The 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 with SEQ ID NO:52 or 1.

[0889] 47. The polypeptide complex according to paragraph 46, wherein the CD3-TCR complex association domain derived from TCRα comprises a cysteine residue at the position corresponding to position 47 numbered according to SEQ ID NO:1.

[0890] 48. The 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 with one of SEQ ID NO:53, 5, 54 or 9.

[0891] 49. The polypeptide complex according to paragraph 48, wherein the CD3-TCR complex association domain derived from TCRβ comprises a cysteine residue at the position corresponding to position 56 numbered according to SEQ ID NO:5.

[0892] 50. The polypeptide complex according to any one of paragraphs 42 to 49, wherein:

[0893] (i) in the first recombinant CD3-TCR complex polypeptide, the first component of the antigen binding moiety is linked at its C-terminus to the N-terminus of the CD3-TCR complex association domain of the first recombinant CD3-TCR complex polypeptide, optionally via a linker sequence; and / or

[0894] (ii) in a second recombinant CD3-TCR complex polypeptide, the second component of the antigen binding moiety 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.

[0895] 51. The polypeptide complex of any of paragraphs 42 to 50, wherein the variant Fc domain binds to an Fc receptor with a lower affinity than the reference Fc domain binds to the Fc receptor, optionally wherein the Fc receptor is an Fcγ receptor or a neonatal Fc receptor (FcRn).

[0896] 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 relative to the amino acid sequence of the CH2-CH3 region of the reference Fc domain at one or more of the following positions according to EU numbering: L234, L235, 1253, N297, S298, H310, P329, E333, K334 or H435.

[0897] 53. The polypeptide complex of any one of paragraphs 42 to 52, wherein the variant Fc domain comprises a CH2-CH3 region comprising G329 according to EU numbering.

[0898] 54. The polypeptide complex of any one of paragraphs 42 to 53, wherein the antigen binding portion comprises a VL region incorporating the following CDRs:

[0899] LC-CDR1 having the amino acid sequence of SEQ ID NO:69;

[0900] LC-CDR2 having the amino acid sequence of SEQ ID NO:70; and

[0901] LC-CDR3 having the amino acid sequence of SEQ ID NO:71.

[0902] 55. The polypeptide complex according to any one of paragraphs 42 to 54, wherein the antigen binding portion comprises a VH incorporating:

[0903] (i) the following CDRs:

[0904] HC-CDR1 having the amino acid sequence of SEQ ID NO:56;

[0905] HC-CDR2 having the amino acid sequence of SEQ ID NO:64; and

[0906] HC-CDR3 having the amino acid sequence of SEQ ID NO:58;

[0907] or

[0908] (ii) the following CDRs:

[0909] HC-CDR1 having the amino acid sequence of SEQ ID NO:56;

[0910] HC-CDR2 having the amino acid sequence of SEQ ID NO:57; and

[0911] HC-CDR3 having the amino acid sequence of SEQ ID NO:58.

[0912] 56. The polypeptide complex according to any one of paragraphs 42 to 55, wherein the antigen-binding portion comprises:

[0913] (a) (i) a VH region incorporating the following CDRs:

[0914] HC-CDR1 having the amino acid sequence of SEQ ID NO:56;

[0915] HC-CDR2 having the amino acid sequence of SEQ ID NO:64; and

[0916] HC-CDR3 having the amino acid sequence of SEQ ID NO:68;

[0917] and

[0918] (ii) a VL region incorporating the following CDRs:

[0919] LC-CDR1 having the amino acid sequence of SEQ ID NO:69;

[0920] LC-CDR2 having the amino acid sequence of SEQ ID NO:70; and

[0921] LC-CDR3 having the amino acid sequence of SEQ ID NO:71;

[0922] or (b) (i) a VH region incorporating the following CDRs:

[0923] HC-CDR1 having the amino acid sequence of SEQ ID NO:56;

[0924] an HC-CDR2 having the amino acid sequence of SEQ ID NO: 57; and

[0925] an HC-CDR3 having the amino acid sequence of SEQ ID NO: 58;

[0926] and

[0927] (ii) a VL region incorporating the following CDRs:

[0928] LC-CDR1 having the amino acid sequence of SEQ ID NO:69;

[0929] LC-CDR2 having the amino acid sequence of SEQ ID NO:70; and

[0930] LC-CDR3 having the amino acid sequence of SEQ ID NO:71.

[0931] 57. The polypeptide complex 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.

[0932] 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.

[0933] 59. The polypeptide complex according to any one of paragraphs 42 to 58, wherein the antigen binding portion comprises:

[0934] (a)(i) a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:65; and

[0935] (ii) a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 68;

[0936] or

[0937] (b)(i) a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:63; and

[0938] (ii) a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 68;

[0939] or

[0940] (c)(i) a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:55; and

[0941] (ii) a VL having an amino acid sequence that has at least 70% amino acid sequence identity to SEQ ID NO:68.

[0942] 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.

[0943] 61. The polypeptide complex of any one of paragraphs 42 to 52 or paragraph 60, wherein the antigen binding portion comprises a VH region incorporating the following CDRs:

[0944] HC-CDR1 having the amino acid sequence of SEQ ID NO: 77;

[0945] an HC-CDR2 having the amino acid sequence of SEQ ID NO: 78; and

[0946] HC-CDR3 having the amino acid sequence of SEQ ID NO:79.

[0947] 62. The polypeptide complex of any one of paragraphs 42 to 52, paragraph 60 or paragraph 61, wherein the antigen binding portion comprises a VL region incorporating the following CDRs:

[0948] LC-CDR1 having the amino acid sequence of SEQ ID NO:85;

[0949] LC-CDR2 having the amino acid sequence of SEQ ID NO:86; and

[0950] LC-CDR3 having the amino acid sequence of SEQ ID NO:87.

[0951] 63. The polypeptide complex of any one of paragraphs 42 to 52 or 60 to 62, wherein the antigen binding portion comprises:

[0952] (i) a VH region incorporating the following CDRs:

[0953] HC-CDR1 having the amino acid sequence of SEQ ID NO: 77;

[0954] an HC-CDR2 having the amino acid sequence of SEQ ID NO: 78; and

[0955] an HC-CDR3 having the amino acid sequence of SEQ ID NO: 79;

[0956] and

[0957] (ii) a VL region incorporating the following CDRs:

[0958] LC-CDR1 having the amino acid sequence of SEQ ID NO:85;

[0959] LC-CDR2 having the amino acid sequence of SEQ ID NO:86; and

[0960] LC-CDR3 having the amino acid sequence of SEQ ID NO:87.

[0961] 64. The polypeptide complex of any one of paragraphs 42 to 52 or 60 to 63, wherein the antigen binding portion comprises a VH having an amino acid sequence having at least 70% amino acid sequence identity with SEQ ID NO: 76.

[0962] 65. The polypeptide complex of any one of paragraphs 42 to 52 or 60 to 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.

[0963] 66. The polypeptide complex according to any one of paragraphs 42 to 52 or 60 to 65, wherein the antigen binding portion comprises:

[0964] (i) a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 76; and

[0965] (ii) a VL having an amino acid sequence that has at least 70% amino acid sequence identity to SEQ ID NO:84.

[0966] 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, wherein:

[0967] (1)(a) a first recombinant CD3-TCR complex polypeptide comprises: (i) a first component of an antigen binding moiety, wherein the first component of the antigen binding moiety comprises or consists of a heavy chain variable (VH) region of an antibody bound to a variant Fc domain, and (ii) a CD3-TCR complex association domain having an amino acid sequence derived from TCRα, and

[0968] (b) a second recombinant CD3-TCR complex polypeptide comprising: (i) a second component of an antigen binding moiety, wherein the second component of the antigen binding moiety comprises or consists of a light chain variable (VL) region of an antibody bound to a variant Fc domain, and (ii) a CD3-TCR complex association domain having an amino acid sequence derived from TCRβ;

[0969] or

[0970] (2) (a) a first recombinant CD3-TCR complex polypeptide comprises: (i) a first component of an antigen binding moiety, wherein the first component of the antigen binding moiety comprises or consists of a heavy chain variable (VH) region of an antibody bound to a variant Fc domain, and (ii) a CD3-TCR complex associating domain having an amino acid sequence derived from TCRβ, and

[0971] (b) the second recombinant CD3-TCR complex polypeptide comprises: (i) a second component of the antigen binding portion, wherein the second component of the antigen binding portion comprises or consists of a light chain variable (VL) region of an antibody bound to a variant Fc domain, and (ii) a CD3-TCR complex association domain having an amino acid sequence derived from TCRα.

[0972] 68. A polypeptide complex comprising:

[0973] (a) a first recombinant CD3-TCR complex polypeptide comprising:

[0974] (i) a first component of an antigen binding moiety, wherein the antigen binding moiety 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 moiety does not bind, wherein the variant Fc domain comprises a CH2-CH3 region comprising G329 according to EU numbering; and

[0975] (ii) a CD3-TCR complex association domain having an amino acid sequence derived from a CD3-TCR complex polypeptide; and

[0976] (b) a second recombinant CD3-TCR complex polypeptide comprising:

[0977] (i) a second component of the antigen binding portion of (a) (i); and

[0978] (ii) a CD3-TCR complex association domain having an amino acid sequence derived from a CD3-TCR complex polypeptide;

[0979] wherein the first recombinant CD3-TCR complex polypeptide and the second recombinant CD3-TCR complex polypeptide are capable of associating via their CD3-TCR complex association domains to form the antigen binding portion;

[0980] wherein the first component of the antigen binding moiety is or comprises a heavy chain variable (VH) region of an antibody that binds to the variant Fc domain, and wherein the second component of the antigen binding moiety is or comprises a light chain variable (VL) region of an antibody that binds to the variant Fc domain;

[0981] And among them:

[0982] (i) the CD3-TCR complex association domain of the first recombinant CD3-TCR complex polypeptide is derived from TCRα, and the CD3-TCR complex association domain of the second recombinant CD3-TCR complex polypeptide is derived from TCRβ, or

[0983] (ii) 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.

[0984] 69. The 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 with SEQ ID NO: 52 or 1.

[0985] 70. The polypeptide complex according to paragraph 69, wherein the CD3-TCR complex association domain derived from TCRα comprises a cysteine ​​residue at the position corresponding to position 47 according to SEQ ID NO:1.

[0986] 71. The polypeptide complex of 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 with one of SEQ ID NO: 53, 5, 54 or 9.

[0987] 72. The polypeptide complex according to paragraph 71, wherein the CD3-TCR complex association domain derived from TCRα comprises a cysteine ​​residue at the position corresponding to position 56 numbered according to SEQ ID NO:5.

[0988] 73. The polypeptide complex according to any one of paragraphs 67 to 72, wherein:

[0989] (i) in the first recombinant CD3-TCR complex polypeptide, the first component of the antigen binding moiety is linked at its C-terminus to the N-terminus of the CD3-TCR complex association domain of the first recombinant CD3-TCR complex polypeptide, optionally via a linker sequence; and / or

[0990] (ii) in a second recombinant CD3-TCR complex polypeptide, the second component of the antigen binding moiety 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.

[0991] 74. The polypeptide complex of any one of paragraphs 67 to 73, wherein the antigen binding portion comprises a VL region incorporating the following CDRs:

[0992] LC-CDR1 having the amino acid sequence of SEQ ID NO:69;

[0993] LC-CDR2 having the amino acid sequence of SEQ ID NO:70; and

[0994] LC-CDR3 having the amino acid sequence of SEQ ID NO:71.

[0995] 75. The polypeptide complex of any one of paragraphs 67 to 74, wherein the antigen binding portion comprises a VH incorporating:

[0996] (i) the following CDRs:

[0997] HC-CDR1 having the amino acid sequence of SEQ ID NO:56;

[0998] an HC-CDR2 having the amino acid sequence of SEQ ID NO: 64; and

[0999] an HC-CDR3 having the amino acid sequence of SEQ ID NO: 58;

[1000] or

[1001] (ii) the following CDRs:

[1002] HC-CDR1 having the amino acid sequence of SEQ ID NO:56;

[1003] an HC-CDR2 having the amino acid sequence of SEQ ID NO: 57; and

[1004] HC-CDR3 having the amino acid sequence of SEQ ID NO:58.

[1005] 76. The polypeptide complex of any one of paragraphs 67 to 75, wherein the antigen binding portion comprises:

[1006] (a)(i) a VH region incorporating the following CDRs:

[1007] HC-CDR1 having the amino acid sequence of SEQ ID NO:56;

[1008] an HC-CDR2 having the amino acid sequence of SEQ ID NO: 64; and

[1009] an HC-CDR3 having the amino acid sequence of SEQ ID NO: 68;

[1010] and

[1011] (ii) a VL region incorporating the following CDRs:

[1012] LC-CDR1 having the amino acid sequence of SEQ ID NO:69;

[1013] LC-CDR2 having the amino acid sequence of SEQ ID NO:70; and

[1014] a LC-CDR3 having the amino acid sequence of SEQ ID NO: 71;

[1015] or (b)(i) a VH region incorporating the following CDRs:

[1016] HC-CDR1 having the amino acid sequence of SEQ ID NO:56;

[1017] an HC-CDR2 having the amino acid sequence of SEQ ID NO: 57; and

[1018] an HC-CDR3 having the amino acid sequence of SEQ ID NO: 58;

[1019] and

[1020] (ii) a VL region incorporating the following CDRs:

[1021] LC-CDR1 having the amino acid sequence of SEQ ID NO:69;

[1022] LC-CDR2 having the amino acid sequence of SEQ ID NO:70; and

[1023] LC-CDR3 having the amino acid sequence of SEQ ID NO:71.

[1024] 77. The polypeptide complex of any 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.

[1025] 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.

[1026] 79. The polypeptide complex of any one of paragraphs 67 to 78, wherein the antigen binding portion comprises:

[1027] (a)(i) a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:65; and

[1028] (ii) a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 68;

[1029] or

[1030] (b)(i) a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:63; and

[1031] (ii) a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 68;

[1032] or

[1033] (c)(i) a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:55; and

[1034] (ii) a VL having an amino acid sequence that has at least 70% amino acid sequence identity to SEQ ID NO:68.

[1035] 80. A CD3-TCR polypeptide complex, wherein the CD3-TCR polypeptide complex comprises 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.

[1036] 81. A composite polypeptide comprising:

[1037] (a) an amino acid sequence encoding a first recombinant CD3-TCR complex polypeptide, wherein the first recombinant CD3-TCR complex polypeptide comprises:

[1038] (i) a first component of an antigen binding moiety, wherein the antigen binding moiety 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 moiety does not bind; and

[1039] (ii) a CD3-TCR complex association domain having an amino acid sequence derived from a CD3-TCR complex polypeptide; and

[1040] (b) an amino acid sequence encoding a second recombinant CD3-TCR complex polypeptide, wherein the second recombinant CD3-TCR complex polypeptide comprises:

[1041] (i) a second component of the antigen binding portion of (a) (i); and

[1042] (ii) a CD3-TCR complex association domain having an amino acid sequence derived from a CD3-TCR complex polypeptide;

[1043] wherein the first recombinant CD3-TCR complex polypeptide and the second recombinant CD3-TCR complex polypeptide are capable of associating via their CD3-TCR complex association domains to form a CD3-TCR complex comprising the antigen binding portion;

[1044] And wherein the composite polypeptide further comprises a cleavage site between the amino acid sequences of (a) and (b).

[1045] 82. A composite polypeptide according to paragraph 81, wherein the first component of the antigen binding portion is or comprises a heavy chain variable (VH) region of an antibody bound to a variant Fc domain, and wherein the second component of the antigen binding portion is or comprises a light chain variable (VL) region of an antibody bound to a variant Fc domain.

[1046] 83. The composite polypeptide according to paragraph 81 or paragraph 82, wherein:

[1047] (i) the CD3-TCR complex association domain of the first recombinant CD3-TCR complex polypeptide is derived from TCRα, and the CD3-TCR complex association domain of the second recombinant CD3-TCR complex polypeptide is derived from TCRβ, or

[1048] (ii) 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.

[1049] 84. The composite polypeptide according to paragraph 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 with SEQ ID NO: 52 or 1.

[1050] 85. The composite polypeptide according to paragraph 84, wherein the CD3-TCR complex association domain derived from TCRα comprises a cysteine ​​residue at the position corresponding to position 47 numbered according to SEQ ID NO:1.

[1051] 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 with one of SEQ ID NO: 53, 5, 54 or 9.

[1052] 87. The composite polypeptide according to paragraph 86, wherein the CD3-TCR complex association domain derived from TCRβ comprises a cysteine ​​residue at the position corresponding to position 56 numbered according to SEQ ID NO:5.

[1053] 88. The composite polypeptide according to any one of paragraphs 81 to 87, wherein:

[1054] (i) in the first recombinant CD3-TCR complex polypeptide, the first component of the antigen binding moiety is linked at its C-terminus to the N-terminus of the CD3-TCR complex association domain of the first recombinant CD3-TCR complex polypeptide, optionally via a linker sequence; and / or

[1055] (ii) in a second recombinant CD3-TCR complex polypeptide, the second component of the antigen binding moiety 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.

[1056] 89. The composite polypeptide of any of paragraphs 81 to 88, wherein the variant Fc domain binds to an Fc receptor with a lower affinity than the reference Fc domain binds to the Fc receptor, optionally wherein the Fc receptor is an Fcγ receptor or a neonatal Fc receptor (FcRn).

[1057] 90. The composite polypeptide of any of paragraphs 81 to 89, wherein the variant Fc domain comprises a CH2-CH3 region comprising an amino acid difference relative to the amino acid sequence of the CH2-CH3 region of a reference Fc domain at one or more of the following positions according to EU numbering: L234, L235, 1253, N297, S298, H310, P329, E333, K334 or H435.

[1058] 91. The composite polypeptide of any one of paragraphs 81 to 90, wherein the variant Fc domain comprises a CH2-CH3 region comprising G329 according to EU numbering.

[1059] 92. The composite polypeptide of any one of paragraphs 81 to 91, wherein the antigen binding portion comprises a VL region incorporating the following CDRs:

[1060] LC-CDR1 having the amino acid sequence of SEQ ID NO:69;

[1061] LC-CDR2 having the amino acid sequence of SEQ ID NO:70; and

[1062] LC-CDR3 having the amino acid sequence of SEQ ID NO:71.

[1063] 93. The composite polypeptide according to any one of paragraphs 81 to 92, wherein the antigen binding portion comprises a VH incorporating:

[1064] (i) the following CDRs:

[1065] HC-CDR1 having the amino acid sequence of SEQ ID NO:56;

[1066] an HC-CDR2 having the amino acid sequence of SEQ ID NO: 64; and

[1067] an HC-CDR3 having the amino acid sequence of SEQ ID NO: 58;

[1068] or

[1069] (ii) the following CDRs:

[1070] HC-CDR1 having the amino acid sequence of SEQ ID NO:56;

[1071] an HC-CDR2 having the amino acid sequence of SEQ ID NO: 57; and

[1072] HC-CDR3 having the amino acid sequence of SEQ ID NO:58.

[1073] 94. The composite polypeptide according to any one of paragraphs 81 to 93, wherein the antigen binding portion comprises:

[1074] (a)(i) a VH region incorporating the following CDRs:

[1075] HC-CDR1 having the amino acid sequence of SEQ ID NO:56;

[1076] an HC-CDR2 having the amino acid sequence of SEQ ID NO: 64; and

[1077] an HC-CDR3 having the amino acid sequence of SEQ ID NO: 68;

[1078] and

[1079] (ii) a VL region incorporating the following CDRs:

[1080] LC-CDR1 having the amino acid sequence of SEQ ID NO:69;

[1081] LC-CDR2 having the amino acid sequence of SEQ ID NO:70; and

[1082] a LC-CDR3 having the amino acid sequence of SEQ ID NO: 71;

[1083] or (b)(i) a VH region incorporating the following CDRs:

[1084] HC-CDR1 having the amino acid sequence of SEQ ID NO:56;

[1085] an HC-CDR2 having the amino acid sequence of SEQ ID NO: 57; and

[1086] an HC-CDR3 having the amino acid sequence of SEQ ID NO: 58;

[1087] and

[1088] (ii) a...

Claims

1. A recombinant CD3-TCR complex polypeptide comprising: (i) an antigen binding portion or a component thereof, 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; and (ii) a CD3-TCR complex association domain having an amino acid sequence derived from a CD3-TCR complex polypeptide.

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

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

4. The recombinant CD3-TCR complex polypeptide according to any one of claims 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 with one of SEQ ID NO: 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 the CD3-TCR complex polypeptide is derived from CD3ε.

6. The recombinant CD3-TCR complex polypeptide according to any one of claims 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 with 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 the CD3-TCR complex polypeptide is derived from TCRα or TCRβ.

8. A recombinant CD3-TCR complex polypeptide according to any one of claims 1 to 4 or claim 7, wherein the CD3-TCR complex association domain comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity with one of SEQ ID NO: 52, 1, 53, 5, 54 or 9.

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

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

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

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

13. The recombinant CD3-TCR complex polypeptide according to 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 association domain, optionally via a linker sequence.

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

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

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

17. The recombinant CD3-TCR complex polypeptide according to any one of claims 1 to 16, wherein the antigen binding portion comprises 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.

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

19. The recombinant CD3-TCR complex polypeptide according to any one of claims 1 to 18, wherein 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; an HC-CDR2 having the amino acid sequence of SEQ ID NO: 64; and an 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 a 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; an HC-CDR2 having the amino acid sequence of SEQ ID NO: 57; and an 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.

20. The recombinant CD3-TCR complex polypeptide according to 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 with SEQ ID NO:

68.

21. The recombinant CD3-TCR complex polypeptide according to 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 with SEQ ID NO: 65, 63 or 55.

22. The recombinant CD3-TCR complex polypeptide according to any one of claims 1 to 21, wherein 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 that has at least 70% amino acid sequence identity to SEQ ID NO:

68.

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

25. The recombinant CD3-TCR complex polypeptide according to any one of claims 1 to 15, claim 23 or claim 24, wherein the antigen binding portion comprises 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 according to any one of claims 1 to 15 or 23 to 25, wherein 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; an HC-CDR2 having the amino acid sequence of SEQ ID NO: 78; and an 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.

27. The recombinant CD3-TCR complex polypeptide according to 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 with SEQ ID NO:

76.

28. The recombinant CD3-TCR complex polypeptide according to 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 with SEQ ID NO:

84.

29. The recombinant CD3-TCR complex polypeptide according to any one of claims 1 to 15 or 23 to 28, wherein 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 that has at least 70% amino acid sequence identity to SEQ ID NO:

84.

30. A recombinant CD3-TCR complex polypeptide comprising: (i) an antigen binding portion which is a scFv, 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 (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.

31. A polypeptide complex comprising: (a) a first recombinant CD3-TCR complex polypeptide comprising: (i) a first component of an antigen binding moiety, wherein the antigen binding moiety 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 moiety does not bind; and (ii) a CD3-TCR complex association 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)(i) a second component of the antigen binding portion; and (ii) a CD3-TCR complex association domain having an amino acid sequence derived from a CD3-TCR complex polypeptide; Wherein the first recombinant CD3-TCR complex polypeptide and the second recombinant CD3-TCR complex polypeptide are capable of associating through their CD3-TCR complex association domains to form the antigen binding portion.

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

33. The polypeptide complex according to claim 31 or claim 32, in: (i) the CD3-TCR complex association domain of the first recombinant CD3-TCR complex polypeptide is derived from TCRα, and the CD3-TCR complex association domain of the second recombinant CD3-TCR complex polypeptide is derived from TCRβ, or (ii) 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.

34. The polypeptide complex according to claim 33, 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 with SEQ ID NO: 52 or 1.

35. The polypeptide complex of claim 33 or claim 34, 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 with one of SEQ ID NO: 53, 5, 54 or 9.

36. A polypeptide complex comprising: (a) a first recombinant CD3-TCR complex polypeptide comprising: (i) a first component of an antigen binding moiety, wherein the antigen binding moiety 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 moiety does not bind, wherein the variant Fc domain comprises a CH2-CH3 region comprising 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; and (b) a second recombinant CD3-TCR complex polypeptide comprising: (i)(a)(i) a second component of the antigen binding portion; and (ii) a CD3-TCR complex association domain having an amino acid sequence derived from a CD3-TCR complex polypeptide; wherein the first recombinant CD3-TCR complex polypeptide and the second recombinant CD3-TCR complex polypeptide are capable of associating via their CD3-TCR complex association domains to form the antigen binding portion; wherein said first component of the antigen binding moiety is or comprises a heavy chain variable (VH) region of an antibody that binds to said variant Fc domain, and wherein said second component of the antigen binding moiety is or comprises a light chain variable (VL) region of said antibody that binds to said variant Fc domain; and in: (i) the CD3-TCR complex association domain of the first recombinant CD3-TCR complex polypeptide is derived from TCRα, and the CD3-TCR complex association domain of the second recombinant CD3-TCR complex polypeptide is derived from TCRβ, or (ii) 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.

37. A CD3-TCR polypeptide complex, wherein the CD3-TCR polypeptide complex comprises the recombinant CD3-TCR complex polypeptide according to any one of claims 1 to 30 or the 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, wherein the first recombinant CD3-TCR complex polypeptide comprises: (i) a first component of an antigen binding moiety, wherein the antigen binding moiety 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 moiety does not bind; and (ii) a CD3-TCR complex association 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, wherein the second recombinant CD3-TCR complex polypeptide comprises: (i)(a)(i) a second component of the antigen binding portion; and (ii) a CD3-TCR complex association domain having an amino acid sequence derived from a CD3-TCR complex polypeptide; wherein the first recombinant CD3-TCR complex polypeptide and the second recombinant CD3-TCR complex polypeptide are capable of associating via their CD3-TCR complex association domains to form a CD3-TCR complex comprising the antigen binding portion; And wherein the composite polypeptide further comprises a cleavage site between the amino acid sequences of (a) and (b).

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

40. The composite polypeptide according to claim 38 or claim 39, in: (i) the CD3-TCR complex association domain of the first recombinant CD3-TCR complex polypeptide is derived from TCRα, and the CD3-TCR complex association domain of the second recombinant CD3-TCR complex polypeptide is derived from TCRβ, or (ii) 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.

41. The composite polypeptide according to claim 40, 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 with SEQ ID NO: 52 or 1.

42. A composite polypeptide according to claim 40 or claim 41, 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 with one of SEQ ID NO: 53, 5, 54 or 9.

43. A nucleic acid or multiple 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 composite polypeptide according to any one of claims 38 to 42.

44. A nucleic acid or nucleic acids encoding: (a) a first recombinant CD3-TCR complex polypeptide comprising: (i) a first component of an antigen binding moiety, wherein the antigen binding moiety 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 moiety does not bind; and (ii) a CD3-TCR complex association 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)(i) a second component of the antigen binding portion; and (ii) a CD3-TCR complex association domain having an amino acid sequence derived from a CD3-TCR complex polypeptide; The first recombinant CD3-TCR complex polypeptide and the second recombinant CD3-TCR complex polypeptide are capable of associating through their CD3-TCR complex association domains to form a CD3-TCR complex comprising the antigen binding portion.

45. An expression vector or multiple expression vectors comprising a nucleic acid or multiple nucleic acids according to 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 cell according to claim 46.

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. The cell according to claim 46 or the pharmaceutical composition according to claim 47, Its use in a method for treating or preventing a disease in which cells containing or expressing a target antigen are pathologically affected, wherein the method comprises administering the cell or the pharmaceutical composition to a subject to whom an antigen-binding molecule has been or is to be administered; wherein 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 comprising at least one amino acid difference relative to a reference Fc domain; And 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 according to claim 46 or in the cell contained in the pharmaceutical composition according to 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 or multiple nucleic acids or expression vector or multiple expression vectors contained in the cell according to claim 46 or in the cell contained in the pharmaceutical composition according to claim 47, binds to the variant Fc domain.

50. A method for depleting or killing cells containing or expressing a target antigen, the method comprising contacting the cells containing / expressing the target antigen with: (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 comprising at least one amino acid difference relative to a reference Fc domain; 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 according to claim 46 or in the cell contained in the pharmaceutical composition according to claim 47, or 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 multiple nucleic acids or expression vector or multiple expression vectors contained in the cell according to claim 46 or in the cell contained in the pharmaceutical composition according to claim 47, binds to the variant Fc domain.

51. A kit, include: (i) the cell according to claim 46 or the 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 comprising at least one amino acid difference relative to a reference Fc domain; 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 according to claim 46 or in the cell contained in the pharmaceutical composition according to claim 47, or 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 multiple nucleic acids or expression vector or multiple expression vectors contained in the cell according to claim 46 or in the cell contained in the pharmaceutical composition according to claim 47, binds to the variant Fc domain.

52. A kit, include: (i) a nucleic acid or nucleic acids according to claim 43 or claim 44, or an expression vector or expression 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 comprising at least one amino acid difference relative to a reference Fc domain; wherein the antigen binding portion of the CD3-TCR complex polypeptide, polypeptide complex, CD3-TCR polypeptide complex or composite polypeptide encoded by the nucleic acid or multiple nucleic acids according to claim 43 or claim 44, or encoded by the nucleic acid or multiple nucleic acids contained in the expression vector or multiple expression vectors according to claim 45, is bound to the variant Fc domain.

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