WO2021190580A1

WO2021190580A1 - Bispecific polypeptide complexes, compositions, and methods of preparation and use

Info

Publication number: WO2021190580A1
Application number: PCT/CN2021/082853
Authority: WO
Inventors: Jianqing Xu; Zhuozhi Wang; Jing Li
Original assignee: Wuxi Biologics (Shanghai) Co., Ltd.; WuXi Biologics Ireland Limited
Priority date: 2020-03-26
Filing date: 2021-03-25
Publication date: 2021-09-30

Abstract

The present disclosure provides a polypeptide complex comprising antibody variable regions of the heavy chain and light chain respectively fused to TCR constant regions, more specifically related to bispecific polypeptide complexes with desired stability and/or expression levels. Also provided herein include a bispecific antigen binding polypeptide complex that contains a first antigen-binding moiety of the polypeptide complex and a second antigen-binding moiety, methods of producing the polypeptide complex or the bispecific antigen binding polypeptide complex, methods of treating disease or disorder using the polypeptide complex or the bispecific antigen binding polypeptide complex, polypeptides encoding the polypeptide complex and/or the bispecific antigen binding polypeptide complex, vectors and host cells containing the polypeptides, compositions and pharmaceutical compositions comprising the polypeptide complex and/or the bispecific antigen binding polypeptide complex.

Description

Bispecific Polypeptide Complexes, Compositions, and Methods of Preparation and Use

PRIORITY INFORMATION

The present application claims the benefits of PCT Application No. PCT/CN2020/081353 filed on Mach 26, 2020, which is incorporated herein by reference as an entirety.

BACKGROUND

Bispecific antibodies are growing to be a new category of therapeutic antibodies. They can bind two different targets or two different epitopes on a target, creating additive or synergistic effect superior to the effect of individual antibodies. A lot of antibody engineering efforts have been put into designing new bispecific formats, such as DVD-Ig, CrossMab, BiTE etc. (Spiess et al., Molecular Immunology, 67 (2) , pp. 95–106 (2015) ) . However, these formats may potentially have various limitations in stability, solubility, short half-life, and immunogenicity.

Among these bispecific antibody formats, an IgG-like bispecific antibody is a common format: one arm binding to target A and another arm binding to target B. Structurally, it is made from half of antibody A and half of antibody B, with the similar size and shape as natural IgG. In order to facilitate downstream development, it is desired that such bispecific molecules can be easily produced like a normal IgG from a single host cell with high expression level and correctly assembled form. Unfortunately, the pairing of cognate light-heavy chains as well as the assembly of two different half antibodies cannot be automatically controlled. All kinds of mispairings in a random manner could result in significant product heterogeneity.

There is a great need to design bispecific molecules with desirable expression level, stability, and/or affinity to antigens. Certain polypeptide complexes comprising antibody variable regions fused to TCR constant regions, as well as bispecific polypeptide complexes comprising the same, are described in PCT/CN2018/106766 (WO 2019/057122) , the full content of which is herein incorporated by reference. The present disclosure describes certain bispecific polypeptide complexes with desired stability and/or protein expression levels.

BRIEF SUMMARY OF THE DISCLOSURE

In one aspect, the present disclosure provides a polypeptide complex comprising a first polypeptide comprising, from N-terminus to C-terminus, a first heavy chain variable domain (VH) of a first antibody operably linked to a first T cell receptor (TCR) constant region (C1) , and a second polypeptide comprising, from N-terminus to C-terminus, a first light chain variable domain (VL) of the first antibody operably linked to a second TCR constant region (C2) , wherein C1 and C2 are capable of forming a dimer comprising at least one non-native interchain bond between C1 and C2, and the non-native interchain bond is capable of stabilizing the dimer, and the first antibody has a first antigenic specificity.

In one aspect, the present disclosure provides a bispecific polypeptide complex, comprising a first antigen-binding moiety associated with a second antigen-binding moiety, wherein the first antigen-binding moiety comprising a first polypeptide comprising, from N-terminal to C-terminal, a first heavy chain variable domain (VH) of a first antibody operably linked to a first T cell receptor (TCR) constant region (C1) , and a second polypeptide comprising, from N-terminal to C-terminal, a first light chain variable domain (VL) of the first antibody operably linked to a second TCR constant region (C2) , wherein C1 and C2 are capable of forming a dimer comprising at least one non-native interchain bond between a first mutated residue comprised in C1 and a second mutated residue comprised in C2, and the non-native interchain bond is capable of stabilizing the dimer, and the first antibody has a first antigenic specificity, a second antigen-binding moiety has a second antigenic specificity which is different from the first antigenic specificity, and the first antigen-binding moiety and the second antigen-binding moiety are less prone to mispair than otherwise would have been if both the first and the second antigen-binding moieties are counterparts of natural Fab.

In one aspect, the present disclosure provides herein a bispecific polypeptide complex, comprising a first antigen binding moiety comprising the polypeptide complex provided herein having a first antigenic specificity, associated with a second antigen binding moiety having a second antigenic specificity which is different from the first antigenic specificity, and the first antigen-binding moiety and the second antigen-binding moiety are less prone to mispair than otherwise would have been if both the first and the second antigen-binding moieties are counterparts of natural Fab.

In one aspect, the present disclosure provides a bispecific fragment of the bispecific polypeptide complex provided herein. In one aspect, the present disclosure provides herein a conjugate comprising the polypeptide complex provided herein, or the bispecific polypeptide complex provided herein conjugated to a moiety.

In one aspect, the present disclosure provides herein an isolated polynucleotide encoding the polypeptide complex provided herein, or the bispecific polypeptide complex provided herein. In one aspect, the present disclosure provides herein an isolated vector comprising the polynucleotide provided herein. In one aspect, the present disclosure provides herein a host cell comprising the isolated polynucleotide provided herein or the isolated vector provided herein. In one aspect, the present disclosure provides herein a method of expressing the polypeptide complex provided herein, or the bispecific polypeptide complex provided herein, comprising culturing the host cell provided herein under the condition at which the polypeptide complex, or the bispecific polypeptide complex is expressed.

In one aspect, the present disclosure provides herein a method of producing the polypeptide complex provided herein, comprising a) introducing to a host cell a first polynucleotide encoding a first polypeptide comprising, from N-terminal to C-terminal, a first heavy chain variable domain (VH) of a first antibody operably linked to a first TCR constant domain (C1) , and a second polynucleotide encoding a second polypeptide comprising, from N-terminal to C-terminal, a first light chain variable domain (VL) of the first antibody operably linked to a second TCR constant domain (C2) , wherein C1 and C2 are capable of forming a dimer comprising at least one non-native interchain bond between a first mutated residue comprised in C1 and a second mutated residue comprised in C2, and the non-native interchain bond is capable of stabilizing the dimer of C1 and C2, and the first antibody has a first antigenic specificity; b) allowing the host cell to express the polypeptide complex.

In one aspect, the present disclosure provides herein a method of producing the bispecific polypeptide complex provided herein, comprising a) introducing to a host cell a first polynucleotide encoding a first polypeptide comprising, from N-terminal to C-terminal, a first heavy chain variable domain (VH) of a first antibody operably linked to a first TCR constant region (C1) , a second polynucleotide encoding a second polypeptide comprising, from N-terminal to C-terminal, a first light chain variable domain (VL) of the first antibody operably linked to a second TCR constant region (C2) , a third polynucleotide encoding a third polypeptide comprising VH of a second antibody, and a fourth polynucleotide encoding a fourth polypeptide comprising VL of the second antibody, wherein C1 and C2 are capable of forming a dimer comprising at least one non-native interchain bond between C1 and C2, and the non-native interchain bond is capable of stabilizing the dimer, and the first antibody has a first antigenic specificity and the second antibody has a second antigenic specificity; b) allowing the host cell to express the bispecific polypeptide complex. In certain embodiments, the method of producing the bispecific polypeptide complex provided herein further comprising isolating the polypeptide complex.

In one aspect, the present disclosure provides a composition comprising the polypeptide complex provided herein, or the bispecific polypeptide complex provided herein. In one aspect, the present disclosure provides herein a pharmaceutical composition comprising the polypeptide complex provided herein, or the bispecific polypeptide complex provided herein and a pharmaceutically acceptable carrier.

In one aspect, the present disclosure provides herein a method of treating a condition in a subject in need thereof, comprising administrating to the subject a therapeutically effective amount of the polypeptide complex provided herein, or the bispecific polypeptide complex provided herein. In certain embodiments, the condition can be alleviated, eliminated, treated, or prevented when the first antigen and the second antigen are both modulated.

In certain embodiments, the non-native interchain bond is formed between a first mutated residue comprised in C1 and a second mutated residue comprised in C2. In certain embodiments, at least one of the first and the second mutated residues is a cysteine residue. In certain embodiments, the non-native interchain bond is a disulphide bond. In certain embodiments, the first mutated residue is comprised within a contact interface of C1, and/or the second mutated residue is comprised within a contact interface of C2. In certain embodiments, at least one native cysteine residue is absent or present in C1 and/or C2. In certain embodiments, the native cysteine residue at position C74 of engineered CBeta is absent or present. In certain embodiments, the native C74 is absent in CBeta.

In certain embodiments, at least one native N-glycosylation site is absent or present in C1 and/or C2. In certain embodiments, the native N-glycosylation sites are absent in C1 and/or C2.

In certain embodiments, the dimer comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more non-native interchain bonds. In certain embodiments, at least one of the non-native interchain bonds is disulphide bond. In certain embodiments, the dimer comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more disulphide bonds.

In certain embodiments, a) C1 comprises an engineered CBeta, and C2 comprises an engineered CAlpha; b) C1 comprises an engineered CAlpha, and C2 comprises an engineered CBeta; c) C1 comprises an engineered CBeta, and C2 comprises an engineered CPre-Alpha; d) C1 comprises an engineered CPre-Alpha, and C2 comprises an engineered CBeta; e) C1 comprises an engineered CGamma, and C2 comprises an engineered CDelta; or f) C1 comprises an engineered CDelta, and C2 comprises an engineered CGamma.

In certain embodiments, the first VH is operably linked to C1 at a first conjunction domain, and the first VL is operably linked to C2 at a second conjunction domain. In certain embodiments, the first VH associates to C1 at a first conjunction domain via a connector, the first VL associates to C2 at a second conjunction domain via a connector.

In certain embodiments, the first and/or the second conjunction domain comprises a proper length (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues) of the C terminal fragment of antibody V/C conjunction, and a proper length (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues) of the N terminal fragment of TCR V/C conjunction.

In certain embodiments, the engineered CBeta comprises a mutated cysteine residue within a contact interface selected from the group consisting of amino acid residues 9-35, 52-66, 71-86, and 122-127; and/or the engineered CAlpha comprises a mutated cysteine residue within a contact interface selected from a group consisting of amino acid residues 6-29, 37-67, and 86-95.

In certain embodiments, the engineered CBeta comprises a mutated cysteine residue that substitutes for an amino acid residue at a position selected from: S56C, S16C, F13C, V12C, E14C, L62C, D58C, S76C, and R78C, and/or the engineered CAlpha comprises a mutated cysteine residue that substitutes for an amino acid residue at a position selected from: T49C, Y11C, L13C, S16C, V23C, Y44C, T46C, L51C, and S62C.

In certain embodiments, the engineered CBeta and the engineered CAlpha comprise a pair of mutated cysteine residues that substitute for a pair of amino acid residues selected from the group consisting of: S16C in CBeta and Y11C in CAlpha, F13C in CBeta and L13C in CAlpha, S16C in CBeta and L13C in CAlpha, V12C in CBeta and S16C in CAlpha, E14C in CBeta and S16C in CAlpha, F13C in CBeta and V23C in CAlpha, L62C in CBeta and Y44C in CAlpha, D58C in CBeta and T46C in CAlpha, S76C in CBeta and T46C in CAlpha, S56C in CBeta and T49C in CAlpha, S56C in CBeta and L51C in CAlpha, S56C in CBeta and S62C in CAlpha, and R78C in CBeta and S62C in CAlpha, and wherein the pair of cysteine residues are capable of forming a non-native interchain disulphide bond.

In certain embodiments, at least one native glycosylation site is absent or present in the engineered CBeta and/or in the engineered CAlpha. In certain embodiments, the native glycosylation site in the engineered CBeta is N69, and/or the native glycosylation site (s) in the engineered CAlpha is/are selected from N34, N68, N79, and any combination thereof.

In certain embodiments, the engineered CBeta lacks or retains an FG loop encompassing amino acid residues 101-117 of the native CBeta and/or a DE loop encompassing amino acid residues 66-71 of the native CBeta.

In certain embodiments, the engineered CAlpha comprises any one of SEQ ID NOs: 43-48, and/or the engineered CBeta comprises any one of SEQ ID NOs: 33-41 and 306.

In certain embodiments, C1 comprises the engineered CBeta, and C2 comprises the engineered CAlpha; and wherein the first conjunction domain comprises or is SEQ ID NO: 49 or 50, and/or the second conjunction domain comprises or is SEQ ID NO: 51 or 52.

In certain embodiments, the C1 comprises the engineered CAlpha, and the C2 comprises the engineered CBeta; and wherein the first conjunction domain comprises or is SEQ ID NO: 129 or 130, and/or the second conjunction domain comprises or is SEQ ID NO: 49 or 50.

In certain embodiments, the engineered CBeta comprises a mutated cysteine residue within a contact interface selected from the group consisting of: amino acid residues 9-35, 52-66, 71-86 and 122-127; and/or the engineered CPre-Alpha comprises a mutated cysteine residue within a contact interface selected from a group consisting of: amino acid residues 7-19, 26-34, 56-75 and 103-106. In certain embodiments, the engineered CBeta comprises a mutated cysteine residue that substitutes for an amino acid residue at a position selected from: S16C, A18C, E19C, F13C, A11C, S56C, and S76C, and/or the engineered CPre-Alpha comprises a mutated cysteine residue that substitutes for an amino acid residue at a position selected from S11C, A13C, I16C, S62C, T65C, and Y59C.

In certain embodiments, the engineered CBeta and the engineered CPre-Alpha comprise a pair of mutated cysteine residues that substitute for a pair of amino acid residues selected from the group consisting of: S16C in CBeta and S11C in CPre-Alpha, A18C in CBeta and S11C in CPre-Alpha, E19C in CBeta and S11C in CPre-Alpha, F13C in CBeta and A13C in CPre-Alpha, S16C in CBeta and A13C in CPre-Alpha, A11C in CBeta and I16C in CPre-Alpha, S56C in CBeta and S62C in CPre-Alpha, S56C in CBeta and T65C in CPre-Alpha, and S76C in CBeta, and Y59C in CPre-Alpha, and wherein the pair of mutated cysteine residues are capable of forming a non-native interchain disulphide bond.

In certain embodiments, at least one native glycosylation site is absent in the engineered CBeta and/or in the engineered CPre-Alpha. In certain embodiments, the absent or present glycosylation site in the engineered CBeta is N69, and/or the absent glycosylation site in the engineered CPre-Alpha is N50.

In certain embodiments, the engineered CBeta lacks or retains an FG loop encompassing the amino acid residues 101-107 of the native CBeta and/or a DE loop at position encompassing the amino acid residues 66-71 of the native CBeta.

In certain embodiments, the engineered CPre-Alpha comprises any one of SEQ ID NOs: 82, 83, and 311-318; and/or the engineered CBeta comprises any one of SEQ ID NOs: 84, 33-41, and 319-324.

In certain embodiments, C1 comprises the engineered CBeta, and C2 comprises the engineered CPre-Alpha; and wherein the first conjunction domain comprises SEQ ID NO: 49 or 50, and/or the second conjunction domain comprises SEQ ID NO: 81 or 131.

In certain embodiments, C1 comprises the engineered CPre-Alpha, and C2 comprises the engineered CBeta; and wherein the first conjunction domain comprises SEQ ID NO: 132 or 133, and/or the second conjunction domain comprises SEQ ID NO: 49 or 50.

In certain embodiments, the engineered CDelta comprises a mutated cysteine residue within a contact interface selected from the group consisting of: amino acid residues 8-26, 43-64, and 84-88; and/or the engineered CGamma comprises a mutated cysteine residue within a contact interface selected from a group consisting of: amino acid residues 11-35 and 55-76.

In certain embodiments, the engineered CGamma comprises a mutated cysteine residue that substitutes for an amino acid residue at a position selected from: S17C, E20C, F14C, T12C, M62C, Q57C, and A19C, and/or the engineered CDelta comprises a mutated cysteine residue that substitutes for an amino acid residue at a position selected from: F12C, M14C, N16C, D46C, V50C, F87C, and E88C.

In certain embodiments, the engineered CGamma and the engineered CDelta comprise a pair of mutated cysteine residues that substitute for a pair of amino acid residues selected from the group consisting of: S17C in CGamma and F12C in CDelta, E20C in CGamma and F12C in CDelta, F14C in CGamma and M14C in CDelta, T12C in CGamma and N16C in CDelta, M62C in CGamma and D46C in CDelta, Q57C in CGamma and V50C in CDelta, A19C in CGamma and F87C in CDelta, and A19C in CGamma and E88C in CDelta, and wherein the introduced pair of cysteine residues are capable of forming an interchain disulphide bond.

In certain embodiments, at least one native glycosylation site is absent or present in the engineered CGamma and/or in the engineered CDelta.

In certain embodiments, the native glycosylation site in the engineered CGamma is N65, and/or the native glycosylation site (s) in the engineered CDelta is/are one or both of N16 and N79.

In certain embodiments, the engineered CGamma comprises SEQ ID NO: 113, 114, 333, 334, 335, 336, 337, 338, 339, or 340, and/or the engineered CDelta comprises SEQ ID NO: 115, 116, 310, 325, 326, 327, 328, 329, 330, 331, or 332.

In certain embodiments, C1 comprises the engineered CGamma, and C2 comprises the engineered CDelta; and wherein the first conjunction domain comprises SEQ ID NO: 117 or 118, and/or the second conjunction domain comprises SEQ ID NO: 119 or 120.

In certain embodiments, C1 comprises the engineered CDelta, and C2 comprises the engineered CGamma; and wherein the first conjunction domain comprises SEQ ID NO: 123 or 124, and/or the second conjunction domain comprises SEQ ID NO: 125 or 126.

In certain embodiments, the first polypeptide further comprises an antibody CH2 domain, and/or an antibody CH3 domain.

In certain embodiments, the first antigenic specificity and the second antigenic specificity are directed to two different antigens, or are directed to two different epitopes on one antigen.

In certain embodiments, the first antigen-binding moiety binds to CD3. In certain embodiments, the second antigen-binding moiety binds to CD19. In certain embodiments, the first antigen-binding moiety binds to CD19. In certain embodiments, the second antigen-binding moiety binds to CD3. In certain embodiments, the first antigen-binding moiety binds to CTLA-4. In certain embodiments, the second antigen-binding moiety binds to PD-1. In certain embodiments, the first antigen-binding moiety binds to PD-1. In certain embodiments, the second antigen-binding moiety binds to CTLA-4.

In certain embodiments, the association is via a connecter, a disulphide bond, a hydrogen bond, electrostatic interaction, a salt bridge, or hydrophobic-hydrophilic interaction, or the combination thereof.

In certain embodiments, the second antigen-binding moiety comprises a heavy chain variable domain and a light chain variable domain of a second antibody having the second antigenic specificity. In certain embodiments, the second antigen-binding moiety comprises a Fab. In certain embodiments, the first antigenic specificity and the second antigenic specificity are directed to two different antigens, or are directed to two different epitopes on one antigen.

In certain embodiments, one of the first and the second antigenic specificities is directed to a T-cell specific receptor molecule and/or a natural killer cell (NK cell) specific receptor molecule, and the other is directed to a tumor associated antigen. In certain embodiments, one of the first and the second antigenic specificities is directed to CD3, and the other is directed to a tumor associated antigen. In certain embodiments, one of the first and the second antigenic specificities is directed to CD3, and the other is directed to CD19.

In certain embodiments, the first antigen-binding moiety further comprises a first dimerization domain, and the second antigen-binding moiety further comprises a second dimerization domain, wherein the first and the second dimerization domains are associated.

In certain embodiments, the first and/or the second dimerization domain comprises at least a portion of an antibody hinge region, optionally derived from IgG1, IgG2 or IgG4.

In certain embodiments, the first and/or the second dimerization domain further comprises a dimerization domain. In certain embodiments, the dimerization domain comprises at least a portion of an antibody hinge region, an antibody CH2 domain, and/or an antibody CH3 domain. In certain embodiments, the first dimerization domain is operably linked to the first TCR constant region (C1) at a third conjunction domain.

In certain embodiments, a) C1 comprises an engineered CBeta, and the third conjunction domain is comprised in SEQ ID NO: 53 or 54; b) C1 comprises an engineered CAlpha, and the third conjunction domain is comprised in SEQ ID NO: 134, 135, 140, or 141; c) C1 comprises an engineered CPre-Alpha, and the third conjunction domain is comprised in SEQ ID NO: 134, 135, 140, or 141; d) C1 comprises an engineered CGamma, and the third conjunction domain is comprised in SEQ ID NO: 121 or 122; or e) C1 comprises an engineered CDelta, and the third conjunction domain is comprised in SEQ ID NO: 127 or 128.

In certain embodiments, the second dimerization domain is operably linked to the heavy chain variable domain of the second antigen-binding moiety.

In certain embodiments, the first and the second dimerization domains are different and associate in a way that discourages homodimerization and/or favors heterodimerization.

In certain embodiments, the first and the second dimerization domains are capable of associating into heterodimers via knobs-into-holes, hydrophobic interaction, electrostatic interaction, hydrophilic interaction, or increased flexibility.

In certain embodiments, the first antigen-binding moiety comprising the first polypeptide comprising VH operably linked to a chimeric constant region, and the second polypeptide comprises VL operably linked to C2, wherein the chimeric constant region and C2 comprises a pair of sequences selected from the group consisting of: SEQ ID NOs: 177/176, 179/178, 184/183, 185/183, 180/176, 181/178, 182/178, 184/186, 185/186, 188/187, 196/187, 190/189, 192/191, 192/193, 195/194, 198/197, 200/199, 202/201, 203/201, 203/204, 205/204, 206/204, 208/207, 208/209, 211/210, 213/212, 213/215, 213/151, 214/212, 214/151, 232/231, 216/215, 218/217, 220/219, 222/221, 224/223, 226/225, 227/223, 229/228, 229/230, 236/235, and 238/237.

In certain embodiments, the first antigenicity is directed to CD3, and the first polypeptide and the second polypeptide comprise a pair of sequences selected from the group consisting of: SEQ ID NOs: 2/1, 3/4/, 5/1, 6/3, 7/3, 9/8, 10/8, 9/11, 10/11, 13/12, 15/14, 17/16, 17/18, 20/19, 21/12, 65/64, 67/66, 69/68, 70/68, 70/71, 72/71, 73/71, 75/74, 75/76, 78/77, 86/85, 90/89, 91/92/, 94/93, 96/95, 98/97, 99/95, 101/100, 101/102, 106/105, 108/107, 110/109, 112/111, 137/136, 138/136, 137/139, and 138/139.

In certain embodiments, the first antigen-binding moiety and the second antigen-binding moiety comprise a four-sequence combination selected from the group consisting of: SEQ ID NOs: 22/12/24/23, 25/12/26/23, and 25/12/27/23, wherein the first antigen-binding moiety is capable of binding to CD3, and the second antigen-binding moiety is capable of binding to CD19.

In certain embodiments, the stability and/or expression level of the polypeptide complex as disclosed herein can be further improved via improving the stability of the one or more TCR constant regions. For example, the stability of the polypeptide complex can be further improved via improving the stability of CAlpha, the stability of CBeta, and/or the CAlpha-CBeta interfacial stability.

In certain embodiment, a polypeptide complex as disclosed herein comprises an engineered CAlpha and an engineered CBeta, wherein the engineered CAlpha comprises one or more modifications that improve the stability of the engineered CAlpha (the stability of an engineered CAlpha with the one or more modifications is further improved as compared to a corresponding engineered CAlpha without the one or more modifications) , the engineered CBeta comprises one or more modifications that improve the stability of the engineered CBeta (the stability of an engineered CBeta with the one or more modifications is further improved as compared to a corresponding engineered CBeta without the one or more modifications) , and/or the interface of the engineered CBeta and the engineered CAlpha comprise one or more modifications that improve the stability of the engineered CBeta and the engineered CAlpha, and wherein the one or more modifications are capable of stabilizing and/or increasing expression level of the polypeptide complex.

In certain embodiments, the engineered CAlpha and CBeta comprise at least one residue substituted with at least one corresponding amino acid residue that contributes to the stability of murine TCR, to improve the stability and/or expression level of the polypeptide complexes disclosed herein.

In certain embodiments, one or more modifications of CAlpha, for example its C-terminus, may improve the stability and/or expression level of the polypeptide complexes disclosed herein. In certain embodiments, replacing the CAlpha c-terminus residues (residues at the c-terminal region of CAlpha, e.g., amino acid residues at positions 81-96, 84-95, or 92-95 wherein the amino acid numbering is shown in Figure 10) by a human IgG1 sequence segment may improve the stability of soluble TCR in Fc form, and may improve stability and/or expression level of the polypeptide complexes disclosed herein.

In certain embodiments, modifications of at least one amino acid residue on CBeta, for example, the residues that are structurally or spatially close to the C-terminus of CAlpha, may also improve the stability and/or expression level of the polypeptide complexes disclosed herein.

In certain embodiments, the engineered CBeta lacks an FG loop that is present in the native CBeta. In certain embodiments of the engineered CBeta, the FG loop is replaced with 1 to 6 amino acid residues, e.g., 1, 2, 3, 4, 5, or 6 amino acid residues, including, for example, amino acid residues “YPSN” , “PS” , “NG” , or “GN” . In certain embodiments, the amino acid residues “YGLSENDEWTQDRAKPVT” of the native CBeta are replaced with amino acid residues “YPSN” , “PS” , “NG” , or “GN” , respectively.

In certain embodiments, the engineered CAlpha and CBeta comprise one or more mutated residues with corresponding one or more amino acid residues from murine TCR to improve stability and/or expression levels of the polypeptide complexes disclosed herein. For example, the engineered CAlpha comprises at least one mutated residue selected from substitutions P92S, E93D, S94V, and S95P (see amino acid residue numbering in Figure 10) , and/or the engineered CBeta comprises at least one mutated residue selected from substitutions E17K and S21A (see amino acid residue numbering in Figure 11) . In certain embodiments, the engineered CAlpha comprises mutated residues P92S, E93D, S94V, and S95P, and the engineered CBeta comprises mutated residues E17K and S21A.

In certain embodiments, the engineered CAlpha comprises one or more mutated residues, selected from T33V, P84R, V10I, F26A, T33W, F78H, P84G, A65S, F72Y, P8E, V10I, F26R, F29T, A63T, and P84G. In certain embodiments, the engineered CAlpha comprises mutated residues T33V and P84R. In certain embodiments, the engineered CAlpha comprise mutated residues V10I, F26A, T33W, F78H, and P84G. In certain embodiments, the engineered CAlpha comprise mutated residues A65S and F72Y. In certain embodiments, the engineered CAlpha comprise mutated residues P8E, V10I, F26R, F29T, A63T, and P84G.

In certain embodiments, at least one native cysteine residue can be absent or present in the engineered CAlpha and CBeta. In certain embodiments, the native residues A128, D129 and C130 are absent in CBeta and/or the native residue C96 is absent in CAlpha. In certain embodiments, the native residues A128, D129 and C130 are present in CBeta and/or the native residue C96 is present in CAlpha, such that the original TCR native disulfide bond formed by the two Cys residues is present. In certain embodiments, one or more mutated amino acid residues as disclosed herein forming non-native disulfide bond are absent in the engineered CAlpha and CBeta. For example, one or more mutated amino acid residues such as T46C on the CAlpha and S76C on CBeta are not present in the engineered CAlpha and CBeta and the native TCR amino acid residues are present in the corresponding positions in the engineered CAlpha and CBeta. In another example, one or more mutations such as T46C on the CAlpha and S76C on CBeta are present in the engineered CAlpha and CBeta, respectively.

In certain embodiments, the engineered CAlpha and CBeta comprise one or more mutated residues to form one or more non-native disulfide bonds, selected from: T46C on CAlpha, S76C on CBeta; S62C on CAlpha; L51C on CAlpha; Y11C on CAlpha, S16C on CBeta; S16C on CAlpha, V12C on CBeta; S16C on CAlpha, E14C on CBeta; T46C on CAlpha, D58C on CBeta; L13C on CAlpha, S16C on CBeta; S62C on CAlpha, R78C on CBeta; L13C on CAlpha, F13C on CBeta; V23C on CAlpha, F13C on CBeta; and Y44C on CAlpha, L62C on CBeta.

In certain embodiments, the engineered CAlpha and CBeta comprise one or more mutated residues at the CAlpha-CBeta interface, for example, S62R on CAlpha and R78G on CBeta.

In certain embodiments, the engineered CAlpha comprises one or more mutated residues, e.g., 1, 2, 3, 4, 5, or 6 mutated residues, at the C-terminus, wherein mutated residues comprise a fragment derived from a human IgG1 hinge sequence, a human kappa light c-terminal sequence, a PDB-5DK3 (human IgG4) CH1 region sequence, a human lambda light chain c-terminus region sequence, a PDB-1IGT (mouse IgG2a) CH1 region sequence, a PDB-1IGT (mouse IgG2a) kappa light chain c-terminus region sequence, a human IgG2 hinge region sequence (e.g., ERKCC-ERKSC) , PDB-5E8E (human IgA) CH1 region sequence, a PDB-5E8E (human IgA) kappa light chain c-terminus region sequence, a PDB-1DEE (human IgM) CH1 region sequence, a human IgE CH1 region sequence, or a human IgD CH1 region sequence.

In certain embodiments, the 1 to 6 amino acid residues at the C-terminus of the engineered CAlpha, e.g., the amino acid residues “PESS, ” are replaced by, for example, “VEPKS” wherein “VEPKS” is derived from a human IgG1 hinge sequence; “EPKS” , wherein “EPKS” is derived from a human IgG1 hinge sequence; “NRGE” , wherein “NRGE” is derived from human kappa light c-terminal residues; “PLAPC” , wherein “PLAPC” is derived from PDB-5DK3 (human IgG4) CH1 region sequence; “APTEC” , wherein “APTEC” is derived from human lambda light chain c-terminus region sequence; “LAPVC” , wherein “LAPVC” is derived from the PDB-1IGT (mouse IgG2a) CH1 region sequence; “NRNEC” , wherein “NRNEC” is derived from the PDB-1IGT (mouse IgG2a) kappa light chain c-terminus region sequence; “ERKSC” , wherein “ERKSC” is derived from human IgG2 hinge region sequence (ERKCC-ERKSC) ; “PLSLC” , wherein PLSLC is derived from PDB-5E8E (human IgA) CH1 region sequence, “LRGEC” , wherein “LRGEC” is derived from the PDB-5E8E (human IgA) kappa light chain c-terminus region sequence; “PLVSC” , wherein “PLVSC” is derived from the PDB-1DEE (human IgM) CH1 region sequence; “PLTRC” , wherein “PLTRC” is derived from human IgE CH1 region sequence; or “IISGC” , wherein “IISGC” is derived from human IgD CH1 region sequence.

In certain embodiments, the engineered CBeta comprises one or more mutated residues, e.g., 1, 2, 3, 4, 5, or 6 mutated residues, at the C-terminus, wherein the mutated residues comprise a fragment derived from a human IgG1 hinge sequence, a human kappa light c-terminal sequence, a PDB-5DK3 (human IgG4) CH1 region sequence, a human lambda light chain c-terminus region sequence, a PDB-1IGT (mouse IgG2a) CH1 region sequence, a PDB-1IGT (mouse IgG2a) kappa light chain c-terminus region sequence, a human IgG2 hinge region sequence (e.g., ERKCC-ERKSC) , PDB-5E8E (human IgA) CH1 region sequence, a PDB-5E8E (human IgA) kappa light chain c-terminus region sequence, a PDB-1DEE (human IgM) CH1 region sequence, a human IgE CH1 region sequence, or a human IgD CH1 region sequence. In certain embodiments, the 1 to 6 amino acid residues at the C-terminus of the engineered CBeta, e.g., the amino acid residues “WGR, ” are replaced by, for example, “EPKS” or “NRGE” .

In certain embodiments, the engineered CBeta comprises a Gly amino acid residue at the C-terminal end of CBeta such that the Gly amino acid residue is adjacent to or forms part of the hinge region.

In certain embodiments, the engineered CAlpha comprises a deletion of 1 to 22 amino acid residues, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22, at or around its C-terminus. In certain embodiments, the engineered CAlpha comprises a deletion of 8 amino acid residues at or around its C-terminus, such as amino acid residues “FFPSPESS. ”

In certain embodiments, the engineered CAlpha and CBeta comprise one or more mutations, for example, S91A on the CAlpha, to remove one or more glycosylation sites. In certain embodiments, certain native glycosylation sites are present in the engineered CAlpha and CBeta, for example, one or more residues selected from N34, N68, and N79 on CAlpha, and N69 on CBeta are present in the engineered CAlpha and CBeta, respectively. In certain embodiments, native glycosylation sites N34, N68, and N79 are present in the engineered CAlpha, and N69 is present in the engineered CBeta.

In certain embodiments, the engineered C2 comprises any one of SEQ ID NOs: 418-431 and 444-457, and/or the engineered C1 comprises any one of SEQ ID NOs: 432-443 and 458-466. In certain embodiments, the engineered C1 comprises any one of SEQ ID NOs: 418-431 and 444-457, and/or the engineered C2 comprises any one of SEQ ID NOs: 432-443 and 458-466. In certain embodiments, the engineered C1 and the engineered C2 respectively comprise a pair of sequences selected from the group consisting of 418/432, 420/438, 421/439, and 423/441. In certain embodiments, the engineered C2 and the engineered C1 respectively comprise a pair of sequences selected from the group consisting of 418/432, 420/438, 421/439, and 423/441.

In certain embodiments, the polypeptide complex provided herein can be made into a Fab, a (Fab) ₂, a bibody, a tribody, a triFabs, tandem linked Fabs, a Fab-Fv, tandem linked V domains, tandem linked scFvs, and among other formats.

In another aspect, the present disclosure provides a kit comprising the polypeptide complex provided herein for detection, diagnosis, prognosis, or treatment of a disease or condition.

The foregoing and other features and advantages of the disclosure will become more apparent from the following detailed description of several embodiments which proceeds with reference to the accompanying figures.

BRIEF DESCFRIPTION OF FIGURES

Figure 1A presents schematic representations of antibody formats as disclosed herein. Both anti-CD3 antibody T3 and anti-CD19 antibody U4 were developed. The constant region (CL and CH1) of T3 was replaced by the constant domains of TCR to design unique light-heavy chain interface that is orthogonal to regular antibody. The TCR-modified T3 and native U4 in conjunction with “knobs-into-holes” mutations in Fc domain were used to design bispecific antibody formats E17 and F16.

Figure 1B shows schematic description of four symmetric WuXiBody formats G19, G19R, G25 and G25R. For formats G19 and G25, two TCR-containing chimeric Fab-like domains were grafted at the C-terminus and N-terminus of a normal antibody, respectively. The rectangles indicate TCR constant domains, and the ovals indicate variable and constant domains of an antibody. The difference between formats G19 and G19R or G25 and G25R is the switched position of normal Fab and chimeric Fab. These formats can accommodate different variable regions from different antibody pairs and usually have a molecular weight around 240-250 kD.

Figure 1C shows schematic description of three symmetric formats G26, G27, and G26R with light-heavy switched chimeric Fab-like domains.

Figure 2A shows the sequence of native TCR alpha chain and its counterpart sequence with mutated cysteine residues. TRAC_Human is a natural sequence of alpha chain constant region. 4L4T_Alpha_Crystal is the sequence of a crystal structure (PDB code 4L4T) with S55C mutations that can form inter-chain disulphide bond. The gray region is the constant region used as backbone of chimeric protein in this disclosure.

Figure 2B shows the sequence of native TCR beta chain and its counterpart sequence with mutated cysteine residues. TRBC1_Human and TRBC2_Human are natural sequences of beta constant region.

Figure 2C shows the sequences of native TCR pre-alpha chain. PTCRA_Human is a natural sequence of pre-alpha chain constant region (pre-alpha chain only has no variable region) . 3OF6_PreAlpha_Crystal is the sequence of a crystal structure (PDB code 3OF6) . The gray region is the constant region used above to define the numbering.

Figure 2D shows the sequences of native TCR delta chain. TRA@_Human is the natural sequences of delta constant region. 4LFH_Delta_Crystal is the constant region of a delta chain sequence of a crystal structure (PDB code 4LFH) . The gray region is the constant region used above to define the numbering.

Figure 2E shows the sequences of native TCR gamma chain. TRGC1_Human and TRGC2_Human are natural sequences of gamma constant region. 4LFH_Gamma_Crystal is the constant region of a gamma chain sequence of a crystal structure (PDB code 4LFH) . The gray region is the constant region used above to define the numbering.

Figures 3A-3E show the sequences and numbering of the TCR constant regions. Figure 3A shows the sequences and numbering of the TCR Alpha constant region. Figure 3B shows the sequences and numbering of the TCR Beta constant region. Figure 3C shows the sequences and numbering of the TCR Pre-Alpha constant region. Figure 3D shows the sequences and numbering of the TCR Delta constant region. Figure 3E shows the sequences and numbering of the TCR Gamma constant region.

Figures 4A-4D show the sequences and numbering of the IgG1 and IgG4 knobs-into-holes. Figure 4A shows the sequences and numbering of the IgG1 “knob” mutations. Figure 4B shows the sequences and numbering of the IgG4 “knob” mutations. Figure 4C shows the sequences and numbering of the IgG1 “hole” mutations. Figure 4D shows the sequences and numbering of the IgG4 “hole” mutations.

Figures 5A-5B show (A) SDS-page and (B) SEC-HPLC characterization of one batch of engineered WuXiBody molecules after purification.

Figures 6A-6B show (A) SDS-page and (B) SEC-HPLC characterization of another batch of engineered bispecific antibody molecules after purification.

Figures 7A-7B show (A) SDS-page and (B) SEC-HPLC characterization of yet another batch of engineered bispecific antibody molecules after purification.

Figure 8 shows DSC analysis of W3248-U6T1. G25R-1. uIgG4. SP, W3248-U6T1. G25R-8. uIgG4. SP, and W3248-U6T1. G25R-23. uIgG4. SP.

Figure 9 shows pharmacokinetic profiles of certain bispecific antibody molecules in rats after 10 mg/kg intravenous administration.

Figure 10 shows the sequences and numbering, from N-terminus to the C-terminus, of the TCR Alpha constant region of TRAC_Human, TRAC_Design_2_QQQQ, and TRAC_Mouse. TRAC_Human is a natural sequence of the alpha chain constant region of human TCR. TRAC_Design_2_QQQQ shows the relevant amino acid sequence of a WuXiBody CAlpha construct described in PCT/CN2018/l06766. TRAC_Mouse is a natural sequence of the alpha chain constant region of mouse TCR.

Figure 11 shows the sequences and numbering, from N-terminus to the C-terminus, of the TCR Beta constant region: TRBC2_Human, TRBC_Design_2_QQQQ, TRBC2_Mouse. TRBC_Design_2_QQQQ shows the relevant amino acid sequence of a WuXiBody CBeta construct described in PCT/CN2018/l06766. TRBC2_Human is a natural sequence of the beta chain constant region of human TCR. TRBC2_Mouse is a natural sequence of the beta chain constant region of mouse TCR.

DETAILED DESCRIPTION

The following description of the disclosure is merely intended to illustrate various embodiments of the disclosure. As such, the specific modifications discussed are not to be construed as limitations on the scope of the disclosure. It will be apparent to one skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of the disclosure, and it is understood that such equivalent embodiments are to be included herein. All references cited herein, including publications, patents and patent applications are incorporated herein by reference in their entirety. If certain content of a reference cited herein contradicts or is inconsistent with the present disclosure, the present disclosure controls.

For instance, certain terms as used herein are defined and/or explained in PCT/CN2018/l06766. Those definitions and/or explanations are incorporated herein and apply to this disclosure. As stated in PCT/CN2018/l06766, “WuXiBody” is a bispecific antibody comprising soluble chimeric protein with variable domains of an antibody and the constant domains of TCR, wherein the subunits (such as alpha and beta domains) of TCR constant domains are linked by engineered disulfide bond. As also stated in PCT/CN2018/l06766, the term “spacer” refers to an artificial amino acid sequence having 1, 2, 3, 4 or 5 amino acid residues, or a length of between 5 and 15, 20, 30, 50 or more amino acid residues, joined by peptide bonds and are used to link one or more polypeptides. A spacer may or may not have a secondary structure. Spacer sequences are known in the art, see, for example, Holliger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993) ; Poljak et al. Structure 2: 1121-1123 (1994) . Any suitable spacers known in the art can be used. For example, a useful spacer in the present disclosure may be rich in glycine and proline residues. Examples include spacers having a single or repeated sequences composed of threonine/serine and glycine, such as TGGGG (SEQ ID NO: 266) , GGGGS (SEQ ID NO: 267) or SGGGG (SEQ ID NO: 268) or its tandem repeats (e.g. 2, 3, 4, or more repeats) . Alternatively, a spacer may be a long peptide chain containing one or more sequential or tandem repeats of the amino acid sequence of GAPGGGGGAAAAAGGGGG (SEQ ID NO: 269) . In certain embodiment, the spacer comprises 1, 2, 3, 4 or more sequential or tandem repeats of SEQ ID NO: 269. As also stated in PCT/CN2018/l06766, a “homologue sequence” and “homologous sequence” are used interchangeably and refer to polynucleotide sequences (or its complementary strand) or amino acid sequences that have sequences identity of at least 80% (e.g. at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) to another sequences when optionally aligned. Such definitions apply to the present disclosure as well.

A. Polypeptide complex

Provided herein are novel polypeptide complexes that comprise an antibody heavy chain variable domain operably linked to a first T cell receptor (TCR) constant region, and an antibody light chain variable domain operably linked to a second TCR constant region, wherein the first TCR constant region and the second TCR constant region are associated via at least one non-native interchain bond. The polypeptide complex comprises at least two polypeptide chains, each of which comprises a variable domain derived from an antibody and a constant region derived from a TCR. The two polypeptide chains of the polypeptide complexes comprise a pair of heavy chain variable domain and a light chain variable domain, which are operably linked to a pair of TCR constant regions respectively. Examples of pairs of TCR constant regions include, for example, alpha/beta, pre-alpha/beta, and gamma/delta TCR constant regions. The TCR constant regions in the polypeptide complexes provided herein can be in full length or in a fragment, and can be engineered, as long as the pair of TCR constant regions are capable of associating with each other to form a dimer.

It is surprisingly found that the polypeptide complexes provided herein with at least one non-native interchain bond (in particular a non-native disulphide bond) can be recombinantly expressed and assembled into the desired conformation, which stabilizes the TCR constant region dimer while providing for good antigen-binding activity of the antibody variable regions. Moreover, the polypeptide complexes are found to well tolerate routine antibody engineering, for example, modification of glycosylation sites, and removal of some natural sequences. Furthermore, the polypeptide complexes provided herein can be incorporated into a bispecific format which can be readily expressed and assembled with minimal or substantially no mispairing of the antigen-binding sequences due to the presence of the TCR constant regions in the polypeptide complexes. Additional advantages of the polypeptide complexes and constructs provided herein will become more evident in the following disclosure below.

In one aspect, the present disclosure provides polypeptide complexes, comprising a first polypeptide comprising, from N-terminus to C-terminus, a first heavy chain variable domain (VH) of a first antibody operably linked to a first T cell receptor (TCR) constant region (C1) , and a second polypeptide comprising, from N-terminus to C-terminus, a first light chain variable domain (VL) of the first antibody operably linked to a second TCR constant region (C2) , wherein: C1 and C2 are capable of forming a dimer comprising at least one non-native interchain bond between C1 and C2, and the non-native interchain bond is capable of stabilizing the dimer, and the first antibody has a first antigenic specificity.

i. TCR constant region

The polypeptide complexes provided herein comprise constant regions derived from a TCR. Native TCR consists of two polypeptide chains, and has in general two types: one consists of alpha and beta chains (i.e. alpha/beta TCR) , and the other consists of gamma and delta chains (i.e.gamma/delta TCR) . These two types are structurally similar but have distinct locations and functions. About 95%human T cells have alpha/beta TCRs, whereas the rest 5%have gamma/delta TCRs. A precursor of alpha chain is also found and named as pre-alpha chain. Each of the two TCR polypeptide chains comprises an immunoglobulin domain and a membrane proximal region. The immunoglobulin region comprises a variable region and a constant region, and is characterized by the presence of an immunoglobulin-type fold. Each TCR polypeptide chain has a cysteine residue (e.g. at C terminal of the constant domain or at N terminal of the membrane proximal region) which together can form a disulphide bond that tethers the two TCR chains together.

Figures 2A-2E set forth the amino acid sequences of native TCR constant regions of TCR alpha, pre-alpha, beta, gamma and delta chains. For clarity and consistency, each of the amino acid residues in these sequences are numbered in Figures 3A-3E, and such numbering is used throughout the present disclosure to refer to a particular amino acid residue on a particular TCR constant region.

Human TCR alpha chain constant region is known as TRAC, with the NCBI accession number of P01848, or an amino acid sequence of SEQ ID NO: 254.

Human TCR beta chain constant region has two different variants, known as TRBC1 and TRBC2 (IMGT nomenclature) , with corresponding sequences set forth in SEQ ID NO: 256 and SEQ ID NO: 257, respectively (see also Toyonaga B, et al., PNAs, Vol. 82, pp. 8624-8628, Immunology (1985) ) . These two beta constant domains are different in the 4 ^th, 5 ^th and 37 ^th amino acid residues of exon 1. Specifically, TRBC1 has 4N, 5K and 37F in exon 1, and TRBC2 has 4K, 5N and 37Y in exon 1.

Specifically, the native TCR beta chain contains a native cysteine residue at position 74 (see Figure 3B) , which is unpaired and therefore does not form a disulphide bond in a native alpha/beta TCR. In certain embodiments, in the polypeptide complexes provided herein, this native cysteine residue is absent or mutated to another residue. This may be useful to avoid incorrect intrachain or interchain pairing. In certain embodiments, the native cysteine residue is substituted for another residue, for example serine or alanine. In certain embodiments, the substitution in certain embodiments can improve the TCR refolding efficiencies in vitro.

Human TCR gamma chain constant regions have two variants, known as TRGC1 and TRGC2 (see Lefranc et al., Eur. J. Immunol. 19: 989-994 (1989) ) , with the NCBI accession number of A26659 and P03986, respectively, or amino acid sequences of SEQ ID NO: 263 and SEQ ID NO: 265, respectively.

Human TCR delta chain constant region is known as TRDC, with the NCBI accession number of A35591, or an amino acid sequence of SEQ ID NO: 261.

The constant region of TCR in the polypeptide complexes provided herein may also be derived from pre-T-cell antigen receptor (pre-TCR) . Pre-TCR is expressed by immature thymocytes, which has a pivotal role in early T-cell development. Pre-TCR has a regular beta chain, but a special pre-alpha chain with only constant region available, with sequence and structure distinct from those of regular alpha chain (see Harald von Boehmer, Nat Rev Immunol, Jul; 5 (7) : 571-7 (2005) ) . The sequence of human pre-alpha chain constant region (PTCRA) has the NCBI accession number of AAF89556.1, or an amino acid sequence of SEQ ID NO: 259.

In the present disclosure, the first and the second TCR constant regions of the polypeptide complexes provided herein are capable of forming a dimer comprising, between the TCR constant regions, at least one non-native interchain bond that is capable of stabilizing the dimer.

An interchain bond is formed between one amino acid residue on one TCR constant region and another amino acid residue on the other TCR constant region. In certain embodiments, the non-native interchain bond can be any bond or interaction that is capable of associating two TCR constant regions into a dimer. Examples of suitable non-native interchain bond include, a disulphide bond, a hydrogen bond, electrostatic interaction, a salt bridge, or hydrophobic-hydrophilic interaction, a knobs-into-holes or the combination thereof. Examples of non-native interchain bonds are described in PCT/CN2018/106766 and are incorporated herein by reference.

In certain embodiments, the TCR constant region dimer comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 non-native interchain bonds. Optionally, at least one of the 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 non-native interchain bonds are disulphide bonds, hydrogen bonds, electrostatic interaction, salt bridge, or hydrophobic-hydrophilic interaction, or any combination thereof.

The TCR constant region comprising a mutated residue is also referred to herein as an “engineered” TCR constant region. In certain embodiments, the first TCR constant region (C1) of the polypeptide complex comprises an engineered TCR Alpha chain (CAlpha) , and the second TCR constant region (C2) comprises an engineered TCR Beta chain (CBeta) . In certain embodiments, C1 comprises an engineered CBeta, and C2 comprises an engineered CAlpha. In certain embodiments, C1 comprises an engineered TCR Pre-Alpha chain (CPre-Alpha) , and C2 comprises an engineered CBeta. In certain embodiments, C1 comprises an engineered CBeta, and C2 comprises an engineered CPre-Alpha. In certain embodiments, C1 comprises an engineered TCR Gamma chain (CGamma) , and C2 comprises an engineered TCR Delta chain (CDelta) . In certain embodiments, C1 comprises an engineered CDelta, and C2 comprises an engineered CGamma.

In certain embodiments, the engineered TCR constant region comprises one or more mutated cysteine residue. In certain embodiments, the one or more mutated residue is comprised within a contact interface of the first and/or the second engineered TCR constant regions.

The term “contact interface” as used herein refers to the particular region (s) on the polypeptides where the polypeptides interact/associate with each other. A contact interface comprises one or more amino acid residues that are capable of interacting with the corresponding amino acid residue (s) that comes into contact or association when interaction occurs. The amino acid residues in a contact interface may or may not be in a consecutive sequence. For example, when the interface is three-dimensional, the amino acid residues within the interface may be separated at different positions on the linear sequence.

In certain embodiments, the engineered CBeta comprises a mutated cysteine residue within a contact interface selected from the group consisting of: amino acid residues 9-35, 52-66, 71-86, and 122-127. In certain embodiments, the engineered CAlpha comprises a mutated cysteine residue within a contact interface selected from a group consisting of: amino acid residues 6-29, 37-67, and 86-95. Unless specified, the numbering of amino acid residues in the TCR constant region in the present disclosure is as set forth in Figures 3A-3E.

In certain embodiments, one or more disulphide bonds can be formed between the engineered CAlpha and the engineered CBeta. The mutated cysteine residue in CBeta can be a substitution selected from the group consisting of: S56C, S16C, F13C, V12C, E14C, F13C, L62C, D58C, S76C, and R78C, and/or the mutated cysteine residues in CAlpha can be a substitution selected from the group consisting of: T49C, Y11C, L13C, S16C, V23C, Y44C, T46C, L51C, and S62C. In certain embodiments, the pair of mutated cysteine residues can be a pair of substitutions selected from the group consisting of: S16C in CBeta and Y11C in CAlpha, F13C in CBeta and L13C in CAlpha, S16C in CBeta and L13C in CAlpha, V12C in CBeta and S16C in CAlpha, E14C in CBeta and S16C in CAlpha, F13C in CBeta and V23C in CAlpha, L62C in CBeta and Y44C in CAlpha, D58C in CBeta and T46C in CAlpha, S76C in CBeta and T46C in CAlpha, S56C in CBeta and T49C in CAlpha, S56C in CBeta and L51C in CAlpha, S56C in CBeta and S62C in CAlpha, and R78C in CBeta and S62C in CAlpha, and wherein the pair of cysteine residues are capable of forming a non-native interchain disulphide bond.

As used herein throughout the application, “XnY” with respect to a TCR constant region is intended to mean that the n ^th amino acid residue X on the TCR constant region (based on the numbering in Figures 3A-3E as provided herein) is replaced by amino acid residue Y, where X and Y are respectively the one-letter abbreviation of a particular amino acid residue. It should be noted that the number n is solely based on the numbering provided in Figures 3A-3E, and it could appear different from its actual position. To illustrate, the sequence of CBeta (S56C) (N69Q) shown in SEQ ID NO: 34 is used as an example. While the substitution of S to C occurs at the 48 ^th residue in SEQ ID NO: 34, the very residue is designated as the 56 ^th residue based on the numbering system in Figures 3A-3E, and therefore that substitution of S to C is designated as S56C, but not S48C. Similarly, the substitution of N to Q is also designated as N69Q based on the numbering system in Figures 3A-3E. This designation rule of amino acid residue substitution applies to all TCR constant region in the present disclosure, unless otherwise specified. Similarly, “XnY” when used with respect to an Fc region, is intended to mean that the n ^th amino acid residue X on the Fc constant region (based on the numbering in Figures 4A-4D as provided herein) is replaced by amino acid residue Y.

In certain embodiments, the engineered CBeta comprises or is any one of SEQ ID NOs: 33-41, and the engineered CAlpha comprises or is any one of SEQ ID NOs: 43-48.

In certain embodiments, one or more non-native disulphide bonds can be formed within the contact interfaces between CPre-Alpha and CBeta. In certain embodiments, the contact interface on CPre-Alpha is selected from substitutions at position amino acid residues 7-19, 26-34, 56-75 and 103-106. In certain embodiments, the contact interface on CBeta is selected from substitutions at position amino acid residues 9-35, 52-66, 71-86 and 122-127.

In certain embodiments, one or more disulphide bonds can be formed between the engineered Pre-TCR alpha constant region (CPre-Alpha) and beta chain constant region (CBeta) . The mutated cysteine residues in CBeta can be a substitution selected from the group consisting of: S16C, A18C, E19C, F13C, A11C, S56C, and S76C, and/or the mutated cysteine residues in CPre-Alpha can be a substitution selected from the group consisting of: S11C, A13C, I16C, S62C, T65C, and Y59. In certain embodiments, the pair of mutated cysteine residues can be a pair of substitutions selected from the group consisting of: S16C in CBeta and S11C in CPre-Alpha, A18C in CBeta and S11C in CPre-Alpha, E19C in CBeta and S11C in CPre-Alpha, F13C in CBeta and A13C in CPre-Alpha, S16C in CBeta and A13C in CPre-Alpha, A11C in CBeta and I16C in CPre-Alpha, S56C in CBeta and S62C in CPre-Alpha, S56C in CBeta and T65C in CPre-Alpha, and S76C in CBeta, and Y59C in CPre-Alpha, and wherein the pair of mutated cysteine residues are capable of forming a non-native interchain disulphide bond.

In certain embodiments, the engineered CBeta comprises or is any one of SEQ ID NOs: 33-41, and the engineered CPre-Alpha comprises or is any one of SEQ ID NOs: 82 and 83.

In certain embodiments, one or more non-native disulphide bonds can be formed within the contact interfaces between CGamma and CDelta. In certain embodiments, the contact interface on CGamma is selected from substitutions at position amino acid residues 11-35 and 55-76. In certain embodiments, the contact interface on CDelta is selected from substitutions at position amino acid residues 8-26, 43-64, and 84-88.

In certain embodiments, one or more disulphide bonds can be formed between the engineered CGamma and CDelta. The mutated cysteine residue in CGamma can be a substitution selected from the group consisting of: S17C, E20C, F14C, T12C, M62C, Q57C, and A19C, and/or the mutated cysteine residues in CDelta can be a substitution selected from the group consisting of: F12C, M14C, N16C, D46C, V50C, F87C, and E88C. In certain embodiments, the pair of mutated cysteine residues can be a pair of substitutions selected from the group consisting of: S17C in CGamma and F12C in CDelta, E20C in CGamma and F12C in CDelta, F14C in CGamma and M14C in CDelta, T12C in CGamma and N16C in CDelta, M62C in CGamma and D46C in CDelta, Q57C in CGamma and V50C in CDelta, A19C in CGamma and F87C in CDelta, and A19C in CGamma and E88C in CDelta, and wherein the introduced pair of cysteine residues are capable of forming an interchain disulphide bond.

In certain embodiments, the engineered CGamma comprises or is any one of SEQ ID NOs: 113 and 114, and the engineered CDelta comprises or is any one of SEQ ID NOs: 115 and 116.

In addition to the non-native amino acid residue, the engineered TCR constant region in certain embodiments may further comprise an additional modification to one or more native residues in the wild-type TCR constant region sequence. Examples of such additional modification include, such as modification to a native cysteine residue, modification to a native glycosylation site, and/or modification to a native loop.

Certain native TCR constant regions (such as CBeta) comprise a native cysteine residue which, in some embodiments of the present disclosure could be modified (e.g. removed) , or alternatively could be kept in some other embodiments. In certain embodiments, a native disulphide bond on the alpha/beta heterodimeric TCR between the TRAC and TRBC1 or TRBC2 constant domain, i.e. between Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC1 or TRBC2, according to IMGT TCR nomenclature, may be present or absent.

In certain embodiments, at least one native cysteine residue is absent or present in the engineered CBeta. For example, the native cysteine residue at position C74 of CBeta may be present or absent in the engineered CBeta. In certain embodiments, the engineered CBeta in which the native cysteine residue C74 is absent comprises or is any one of SEQ ID NOs: 32-41.

Without wishing to be bound by any theory, but it is believed that the polypeptide complex provided herein is advantageous in that it tolerates both presence and absence of the native cysteine residue on the CBeta. Although it was suggested (see, for example, U.S. Patent No. 7,666,604) that presence of the native cysteine residues on soluble TCR heterodimers is detrimental to the ligand binding ability of the TCR, the polypeptide complex provided herein can tolerate presence of this native cysteine residue without negatively affecting its antigen-binding activity. Furthermore, the polypeptide complex provided herein in the absence of the native cysteine residue expressed at high level, despite of the contrary teachings by Wu et al. mAbs, 7 (2) , pp. 364–376 (2005) that native disulphide bond in the TCR heterodimer is good for stabilizing the TCR heterodimer.

In certain embodiments, one or more native glycosylation site present in the native TCR constant regions may be modified (e.g. removed) or kept in the polypeptide complex provided in the present disclosure. The term “glycosylation site” as used herein with respect to a polypeptide sequence refers to an amino acid residue with a side chain to which a carbohydrate moiety (e.g. an oligosaccharide structure) can be attached. Glycosylation of polypeptides like antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue, for example, an asparagine residue in a tripeptide sequence such as asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline. O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly to serine or threonine. Removal of native glycosylation sites can be conveniently accomplished by altering the amino acid sequence such that one or more of the above-described tripeptide sequences (for N-linked glycosylation sites) or one or more serine or threonine residues (for O-linked glycosylation sites) are substituted.

In certain embodiments, in the polypeptide complex provided herein, at least one native glycosylation site is absent or present in the engineered TCR constant regions, for example, in the first and/or the second TCR constant regions. Without wishing to be bound by any theory, but it is believed that the polypeptide complex provided herein can tolerate removal of all or part of the glycosylation sites without affecting the protein expression and stability, in contrast to existing teachings that presence of N-linked glycosylation sites on TCR constant region, such as CAlpha (i.e. N34, N68, and N79) and CBeta (i.e. N69) are necessary for protein expression and stability (see Wu et al., Mabs, 7: 2, 364-376, 2015) .

In certain embodiments, in the polypeptide complex provided herein, at least one of the N-glycosylation sites in the engineered CAlpha, e.g. N34, N68, N79 and N61 are absent or present. In certain embodiments, the engineered CAlpha sequences absent of a glycosylation site comprises or is any one of SEQ ID NOs: 44-48. In certain embodiments, at least one of the N-glycosylation sites in the engineered CBeta, e.g. N69, is absent or present. The engineered CBeta sequences (TRBC1) absent of glycosylation site comprises or is any one of SEQ ID NOs: 34-36. The engineered CBeta sequences (TRBC2) absent of a glycosylation site comprises or is any one of SEQ ID NOs: 38-40.

In certain embodiments, in the polypeptide complex provided herein, at least one of the N-glycosylation sites in the engineered CPre-Alpha, e.g. N50, is absent or present. The engineered CPre-Alpha sequence absent of a glycosylation site comprises or is SEQ ID NO: 83.

In certain embodiments, in the polypeptide complex provided herein, at least one of the N-glycosylation sites in the engineered CGamma, e.g. N65, is absent or present. In certain embodiments, the engineered CGamma sequence absent of a glycosylation site comprises or is SEQ ID NO: 114. In certain embodiments, at least one of the N-glycosylation sites in the engineered CDelta, e.g. N16 and N79 is absent or present. The engineered CDelta sequence absent of glycosylation site comprises or is SEQ ID NO: 116.

In certain embodiments, one or more native secondary structure present in the native TCR constant regions may be modified (e.g. removed) or kept in the polypeptide complex provided in the present disclosure. In certain embodiments, a native loop (such as FG loop and/or DE loop of native CBeta) is modified (e.g. removed) or kept in the polypeptide complex provided herein. The term “FG loop” and “DE loop” are structures mainly found in the TCR beta chain constant domain. The FG loop encompasses amino acid residues 101-117 of the native CBeta and is an unusually elongated, solvent-exposed structural element that forms one component of an alpha/beta TCR cavity against CD3. The DE loop encompasses amino acid residues 66-71 of the native CBeta. Alignment of sequence of TCR beta chain constant region with that of an immunoglobulin CH1 constant region revealed that the FG loop of TCR beta chain constant region are significantly longer. Figure 3 in PCT/CN2018/106766 shows the differences of constant regions between T cell beta chain and antibody heavy chain. In certain embodiments, the sequence at FG loop (YGLSENDEWTQDRAKPVT, SEQ ID NO: 79) is absent and/or replaced with YPSN (SEQ ID NO: 80) . In certain embodiments, the sequence at native DE loop (QPALNDSR, SEQ ID NO: 88) is absent and/or replaced with QSGR (SEQ ID NO: 87) . In certain embodiments, the CBeta sequences absent of native FG loop comprises or is any one of SEQ ID NOs: 37-40. In certain embodiments, the CBeta sequence absent of both native FG loop and native DE loop comprises or is SEQ ID NO: 41.

In the polypeptide complex provided herein, the constant regions derived from a TCR are operably linked to the variable regions derived from an antibody. The heavy chain or light chain variable region of an antibody can be operably linked to a TCR constant region, with or without a spacer.

In certain embodiments, the first antibody variable domain (VH) is fused to the first TCR constant region (C1) at a first conjunction domain, the first antibody variable domain (VL) is fused to the second TCR constant region (C2) at a second conjunction domain.

In certain embodiments, the first conjunction domain comprises at least a portion of the C terminal fragment of an antibody V/C conjunction, and at least a portion of the N-terminal fragment of a TCR V/C conjunction.

The term “antibody V/C conjunction” as used herein refers to the boundary of antibody variable domain and constant domain, for example, the boundary between heavy chain variable domain and the CH1 domain, or between light chain variable domain and the light chain constant domain. Similarly, the term “TCR V/C conjunction” refers to the boundary of TCR variable domain and constant domain, for example, the boundary between TCR Alpha variable domain and constant domain, or between TCR Beta variable domain and constant domain.

If the Fv region of an immunoglobulin is aligned with a TCR immunoglobulin-like domain, the antibody V/C conjunction and the TCR V/C conjunction would also be aligned. An example is given in Table 1 below, where antibody heavy chain V/C conjunction (SEQ ID NO: 270) is aligned to TCR Beta V/C conjunction (SEQ ID NO: 271) , and antibody light chain V/C conjunction (SEQ ID NO: 272) is aligned to TCR Beta V/C conjunction (SEQ ID NO: 273) .

The first and/or the second conjunction domains of the polypeptide complex as provided herein can be selected such that it comprises a proper length (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues) of the C terminal fragment of antibody V/C conjunction, and a proper length (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues) of the N terminal fragment of TCR V/C conjunction. For example, as shown in Table 1, the conjunction domain may be selected to have all sequence from the TCR V/C conjunction (see, e.g. SEQ ID NO: 145) , or most sequence (see, e.g. SEQ ID NO: 147) , or some sequence (see, e.g. SEQ ID NO: 146) from the TCR V/C conjunction. Still using Table 1 as an example, the conjunction domain may comprise more residues from TCR V/C conjunction than from antibody V/C conjunction (see, e.g. SEQ ID NO: 147) , or vice versa (see, e.g. SEQ ID NO: 146) .

In certain embodiments, the first and/or the second conjunction domains of the polypeptide complex as provided herein has a total length comparable to that of the antibody V/C conjunction or that of the TCR V/C conjunction.

A proper conjunction domain can be determined on a structural basis. For example, the three-dimensional structures of antibody and TCR may be superimposed, and overlappings of the antibody V/C conjunction and the TCR V/C conjunction on the superimposed structure may be determined and considered when determining the length or proportion of sequences from antibody or TCR V/C conjunction.

In certain embodiments, the first and/or the second conjunction domain comprises a spacer in between the fragments from antibody V/C conjunction and TCR V/C conjunction. Any suitable sequences or length of spacer sequences can be used, as long as it does not negatively affect the antigen binding or stability of the polypeptide complex.

Exemplary sequences of antibody variable/constant domain boundary, and TCR variable/constant domain boundary, and the antibody variable /TCR constant region boundary are provided in the below Tables 1-6.

In certain embodiments, C1 comprises an engineered CBeta and C2 comprises an engineered CAlpha. To illustrate, Table 1 shows the exemplary designs for the conjunction domains useful for antibody VH fused to TCR CBeta, or for antibody VL fused to TCR CAlpha. The antibody VH/constant domain boundary is aligned to TCR variable/CBeta boundary, and antibody VL/constant domain boundary is aligned to TCR variable/CAlpha boundary. Exemplary designs of the conjunction domains are also provided in an alignment form (see, e.g., SEQ ID NO: 144, 145, 146, or 147) , with the first or the second conjunction domain shown with underline. In such embodiments, the first conjunction domain comprises or is SEQ ID NO: 49 or 50.In such embodiments, the second conjunction domain comprises or is SEQ ID NO: 51 or 52.

Table 1. First and second conjunction domain designs for VH-CBeta/VL-CAlpha

In certain embodiments, C1 comprises an engineered CAlpha and C2 comprises an engineered CBeta. Table 2 shows the exemplary designs for the conjunction domains useful for antibody VH fused to TCR CAlpha, or for antibody VL fused to TCR CBeta. The antibody VH/constant domain boundary is aligned to TCR variable/CAlpha boundary, and antibody VL/constant domain boundary is aligned to TCR variable/CBeta boundary. Exemplary designs of the conjunction domains are also provided in an alignment form (see, e.g., SEQ ID NO: 148, 149, or 150) , with the first or the second conjunction domain shown with underline. In such embodiments, the first conjunction domain comprises or is SEQ ID NO: 129 or 130. In such embodiments, the second conjunction domain comprises or is SEQ ID NO: 49 or 50.

Table 2. First and second conjunction domain designs for VH-CAlpha/VL-CBeta

In certain embodiments, C1 comprises an engineered CBeta and C2 comprises an engineered CPre-Alpha. Table 3 shows the exemplary designs for the conjunction domains useful for antibody VH fused to TCR CBeta, or for antibody VL fused to TCR CPre-Alpha. The antibody VH/constant domain boundary is aligned to TCR variable/CBeta boundary, and antibody VL/constant domain boundary is aligned to TCR variable/CPre-Alpha boundary. Exemplary designs of the conjunction domains are also provided in an alignment form (see, e.g., SEQ ID NO: 170, 171, 169, or 156) , with the first or the second conjunction domain shown with underline. In such embodiments, the first conjunction domain comprises or is SEQ ID NO: 49 or 50.In such embodiments, the second conjunction domain comprises or is SEQ ID NO: 81 or 131.

Table 3. First and second conjunction domain designs for VH-CBeta/VL-CPre-Alpha

In certain embodiments, C1 comprises an engineered CPre-Alpha and C2 comprises an engineered CBeta. Table 4 shows the exemplary designs for the conjunction domains useful for antibody VH fused to TCR CPre-Alpha, or for antibody VL fused to TCR CBeta. The antibody VH/constant domain boundary is aligned to TCR variable/CPre-Alpha boundary, and antibody VL/constant domain boundary is aligned to TCR variable/CBeta boundary. Exemplary designs of the conjunction domains are also provided in an alignment form (see, e.g., SEQ ID NO: 172, 173, 174, or 175) , with the first or the second conjunction domain shown with underline. In such embodiments, the first conjunction domain comprises or is SEQ ID NO: 81, 131, 132, or 133. In such embodiments, the second conjunction domain comprises or is SEQ ID NO: 49 or 50.

Table 4. First and second conjunction domain designs for VH-CPre-Alpha/VL-CBeta

In certain embodiments, C1 comprises an engineered CGamma and C2 comprises an engineered CDelta. Table 5 shows the exemplary designs for the conjunction domains useful for antibody VH fused to TCR CGamma, or for antibody VL fused to TCR CDelta. The antibody VH/constant domain boundary is aligned to TCR variable/CGamma boundary, and antibody VL/constant domain boundary is aligned to TCR variable/CDelta boundary. Exemplary designs of the conjunction domains are also provided in an alignment form (see, e.g., SEQ ID NO: 157, 158, 159, or 160) , with the first or the second conjunction domain shown with underline. In such embodiments, the first conjunction domain comprises or is SEQ ID NO: 117 or 118. In such embodiments, the second conjunction domain comprises or is SEQ ID NO: 119 or 120.

Table 5. First and second conjunction domain designs for VH-CGamma/VL-CDelta

In certain embodiments, C1 comprises an engineered CDelta and C2 comprises an engineered CGamma. Table 6 shows the exemplary designs for the conjunction domains useful for antibody VH fused to TCR CDelta, or for antibody VL fused to TCR CGamma. The antibody VH/constant domain boundary is aligned to TCR variable/CDelta boundary, and antibody VL/constant domain boundary is aligned to TCR variable/CGamma boundary. Exemplary designs of the conjunction domains are also provided in an alignment form (see, e.g., SEQ ID NO: 161, 162, 163, or 164) , with the first or the second conjunction domain shown with underline. In such embodiments, the first conjunction domain comprises or is SEQ ID NO: 123 or 124. In such embodiments, the second conjunction domain comprises or is SEQ ID NO: 125 or 126.

Table 6. First and second conjunction domain designs for VH-CDelta/VL-CGamma

In certain embodiments, the first polypeptide comprises a sequence comprising domains operably linked as in formula (I) : VH-HCJ-C1, and the second polypeptide comprises a sequence comprising domains operably linked as in formula (II) : VL-LCJ-C2, wherein:

VH is a heavy chain variable domain of an antibody;

HCJ is a first conjunction domain as defined supra;

C1 is a first TCR constant domain as defined supra;

VL is a light chain variable domain of an antibody;

LCJ is a second conjunction domain as defined supra;

C2 is a second TCR constant domain as defined supra.

In certain embodiments, C1 is an engineered CAlpha which comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 42-48, and C2 is an engineered CBeta which comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 32- 41, the HCJ comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 49 and 50; LCJ comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 51 and 52.

In certain embodiments, C1 is an engineered CBeta which comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 32-41, and C2 is an engineered CAlpha which comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 42-48, the HCJ comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 129 and 130; LCJ comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 49 and 50.

In certain embodiments, C1 is an engineered CBeta which comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 32-41, 84, 319, 320, 321, 322, 323, and 324, and C2 is an engineered CPre-Alpha which comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 311, 312, 313, 314, 315, 316, 317, and 318, the HCJ comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 49 and 50; LCJ comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 81 and 131.

In certain embodiments, C1 is an engineered CPre-Alpha which comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 311, 312, 313, 314, 315, 316, 317, and 318, and C2 is an engineered CBeta which comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 32-41, the HCJ comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 81, 131, 132, and 133; LCJ comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 49 and 50.

In certain embodiments, C1 is an engineered CGamma which comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 113, 114, 333, 334, 335, 336, 337, 338, 339, and 340, and C2 is an engineered CDelta which comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 325, 326, 327, 328, 329, 330, 331, and 332, the HCJ comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 117 and 118; LCJ comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 119 and 120.

In certain embodiments, C1 is an engineered CDelta which comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 325, 326, 327, 328, 329, 330, 331, and 332, and C2 is an engineered CGamma which comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 113, 114, 333, 334, 335, 336, 337, 338, 339, and 340, the HCJ comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 123 and 124; LCJ comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 125 and 126.

U.S. Patent No. 9,683,052 discloses that certain residues within the contact interface between TCR constant regions can be engineered into an Fc region to facilitate the hetero-dimeric pairing of two heavy chains. Such residues and/or corresponding residues within the contact interface between TCR constant regions disclosed herein can also be engineered into a Fab region, e.g., the CH1 and CL domains, to facilitate the pairing between a light chain and a heavy chain.

ii. Antibody variable region

The polypeptide complex provided herein comprises a first heavy chain variable domain (VH) and a first light chain variable domain (VL) of the first antibody. In a conventional native antibody, a variable region comprises three CDR regions interposed by flanking framework (FR) regions, for example, as set forth in the following formula: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, from N-terminus to C-terminus. The polypeptide complex provided herein can comprise but is not limited to such a conventional antibody variable region. For example, the variable domain may comprise all three or less than three of the CDRs, with all four or less than four of the FRs of the antibody heavy or light chain, as long as the variable domain is capable of specifically binding to an antigen.

The first antibody has a first antigenic specificity. In other words, VH and VL form an antigen-binding site which can specifically bind to an antigen or an epitope. The antigenic specificity can be directed to any suitable antigen or epitope, for example, one that is exogenous antigen, endogenous antigen, autoantigen, neoantigen, viral antigen or tumor antigen. An exogenous antigen enters a body by inhalation, ingestion or injection, and can be presented by the antigen-presenting cells (APCs) by endocytosis or phagocytosis and form MHC II complex. An endogenous antigen is generated within normal cells as a result of cell metabolism, intracellular viral or bacterial infection, which can form MHC I complex. An autoantigen (e.g. peptide, DNA or RNA, etc. ) is recognized by the immune system of a patient suffering from autoimmune diseases, whereas under normal condition, this antigen should not be the target of the immune system. A neoantigen is entirely absent from the normal body, and is generated because of a certain disease, such as tumor or cancer. In certain embodiments, the antigen is associated with a certain disease (e.g. tumor or cancer, autoimmune diseases, infectious and parasitic diseases, cardiovascular diseases, neuropathies, neuropsychiatric conditions, injuries, inflammations, coagulation disorder) . In certain embodiments, the antigen is associated with immune system (e.g. immunological cells such as B cell, T cell, NK cells, macrophages, etc. ) .

In certain embodiments, the first antigenic specificity is directed to an immune-related antigen or an epitope thereof. Examples of an immune-related antigen include a T-cell specific receptor molecule and/or a natural killer cell (NK cell) specific receptor molecule.

The T-cell specific receptor molecule allows a T cell to bind to and, if additional signals are present, to be activated by and respond to an epitope/antigen presented by another cell called the antigen-presenting cell or APC. The T-cell specific receptor molecule can be TCR, CD3, CD28, CD134 (also termed OX40) , 4-1 BB, CD5, and CD95 (also known as the Fas receptor) .

Examples of a NK cell specific receptor molecule include CD16, a low affinity Fc receptor and NKG2D, and CD2.

In certain embodiments, the first antigenic specificity is directed to a tumor-associated antigen or an epitope thereof. The term “tumor associated antigen” refers to an antigen that is or can be presented on a tumor cell surface and that is located on or within tumor cells. In some embodiments, the tumor associated antigens can be presented only by tumor cells and not by normal, i.e. non-tumor cells. In some other embodiments, the tumor associated antigens can be exclusively expressed on tumor cells or may represent a tumor specific mutation compared to non-tumor cells. In some other embodiments, the tumor associated antigens can be found in both tumor cells and non-tumor cells, but is overexpressed on tumor cells when compared to non-tumor cells or are accessible for antibody binding in tumor cells due to the less compact structure of the tumor tissue compared to non-tumor tissue. In some embodiments the tumor associated antigen is located on the vasculature of a tumor. Illustrative examples of a tumor associated surface antigen are described in PCT/CN2018/106766 and are incorporated herein by reference.

In certain embodiments, the first antigenic specificity is directed to an antigen or an epitope thereof, selected from the group consisting of: CD3, 4.1BB (CD137) , OX40 (CD134) , CD16, CD47, CD19, CD20, CD22, CD33, CD38, CD123, CD133, CEA, cdH3, EpCAM, epidermal growth factor receptor (EGFR) , EGFRvIII (amutant form of EGFR) , HER2, HER3, dLL3, BCMA, Sialyl-Lea, 5T4, ROR1, melanoma-associated chondroitin sulfate proteoglycan, mesothelin, folate receptor 1, VEGF receptor, EpCAM, HER2/neu, HER3/neu, G250, CEA, MAGE, proteoglycans, VEGF, FGFR, alphaVbeta3-integrin, HLA, HLA-DR, ASC, CD1, CD2, CD4, CD5, CD6, CD7, CD8, CD11, CD13, CD14, CD21, CD23, CD24, CD28, CD30, CD37, CD40, CD41, CD44, CD52, CD64, c-erb-2, CALLA, MHCII, CD44v3, CD44v6, p97, ganglioside GM1, GM2, GM3, GD1a, GD1b, GD2, GD3, GT1b, GT3, GQ1, NY-ESO-1, NFX2, SSX2, SSX4 Trp2, gp100, tyrosinase, Muc-1, telomerase, survivin, G250, p53, CA125 MUC, Wue antigen, Lewis Y antigen, HSP-27, HSP-70, HSP-72, HSP-90, Pgp, MCSP, EpHA2 and cell surface targets GC182, GT468 or GT512.

The antibody variable domains can be derived from a parent antibody. A parent antibody can be any type of antibody, including for example, a fully human antibody, a humanized antibody, or an animal antibody (e.g. mouse, rat, rabbit, sheep, cow, dog, etc. ) . The parent antibody can be a monoclonal antibody or a polyclonal antibody.

In certain embodiments, the parent antibody is a monoclonal antibody. A monoclonal antibody can be produced by various methods known in the art, for example, hybridoma technology, recombinant method, phage display, or any combination thereof. Exemplary methods of producing antibodies are described in PCT/CN2018/106766 and are incorporated herein by reference.

The parent antibodies described herein can be further modified, for example, to graft the CDR sequences to a different framework or scaffold, to substitute one or more amino acid residues in one or more framework regions, to replace one or more residues in one or more CDR regions for affinity maturation, and so on. These can be accomplished by a person skilled in the art using conventional techniques.

The parent antibody can also be a therapeutic antibody known in the art, for example those approved by FDA for therapeutic or diagnostic use, or those under clinical trial for treating a condition, or those in research and development. Polynucleotide sequences and protein sequences for the variable regions of known antibodies can be obtained from public databases such as, for example, e.g., www. ncbi. nlm. nih. gov/entrez/query. fcgi; www. atcc. org/phage/hdb. html; www. sciquest. com/; www. abcam. com/; www. antibodyresource. com/onlinecomp. html. Examples of therapeutic antibodies include, but are not limited to the therapeutic antibodies disclosed in PCT/CN2018/106766 and are incorporated herein by reference.

a) Anti-CD3 binding moiety

In certain embodiments, the first antigen-binding moiety or the second antigen-binding moiety is an anti-CD3 binding moiety derived from an anti-CD3 antibody comprising 1, 2, or 3 heavy chain CDR sequences selected from the group consisting of: SEQ ID NOs: 342-344 and/or 1, 2, or 3 light chain CDR sequences selected from SEQ ID NOs: 345-347.

These CDR sequences are derived from the anti-CD3 antibody shown in Table A below. The CDR sequences of the WBP3311_2.306.4 antibody are provided below.

Table A.

Heavy and kappa light chain variable region sequences of the WBP3311_2.306.4 antibody are provided below.

WBP3311_2.306.4-VH

Amino acid sequence (SEQ ID NO: 348) :

Nucleic acid sequence (SEQ ID NO: 349) :

WBP3311_2.306.4-VL

Amino acid sequence (SEQ ID NO: 350) :

Nucleic acid sequence (SEQ ID NO: 351) :

CDRs are known to be responsible for antigen binding, however, it has been found that not all of the 6 CDRs are indispensable or unchangeable. In other words, it is possible to replace or change or modify one or more CDRs in the anti-CD3 binding moiety derived from WBP3311_2.306.4, yet substantially retain the specific binding affinity to CD3.

In certain embodiments, the anti-CD3 binding moiety provided herein comprises a heavy chain CDR3 sequence of one of the anti-CD3 antibodies WBP3311_2.306.4. In certain embodiments, the anti-CD3 binding moiety provided herein comprises a heavy chain CDR3 comprising SEQ ID NO: 344. Heavy chain CDR3 regions are located at the center of the antigen-binding site, and therefore are believed to make the most contact with antigen and provide the most free energy to the affinity of antibody to antigen. It is also believed that the heavy chain CDR3 is by far the most diverse CDR of the antigen-binding site in terms of length, amino acid composition and conformation by multiple diversification mechanisms (Tonegawa S., Nature. 302: 575-81 (1983) ) . The diversity in the heavy chain CDR3 is sufficient to produce most antibody specificities (Xu JL, Davis MM., Immunity. 13: 37-45 (2000) ) as well as desirable antigen-binding affinity (Schier R, et al., J Mol Biol. 263: 551-67 (1996) ) .

In certain embodiments, the anti-CD3 binding moiety provided herein comprises suitable framework region (FR) sequences, as long as the anti-CD3 binding moiety can specifically bind to CD3. The CDR sequences provided in Table A are obtained from mouse antibodies, but they can be grafted to any suitable FR sequences of any suitable species such as mouse, human, rat, rabbit, among others, using suitable methods known in the art such as recombinant techniques.

In certain embodiments, the anti-CD3 binding moiety provided herein is humanized.

In certain embodiments, the humanized antigen binding moiety provided herein is composed of substantially all human sequences except for the CDR sequences which are non-human. In some embodiments, the variable region FRs, and constant regions if present, are entirely or substantially from human immunoglobulin sequences. The human FR sequences and human constant region sequences may be derived different human immunoglobulin genes, for example, FR sequences derived from one human antibody and constant region from another human antibody. In some embodiments, the humanized antigen binding moiety comprises human FR1-4.

The heavy chain and light chain variable region sequences of the anti-CD3 humanized antibody WBP3311_2.306.4-z1 are provided below.

WBP3311_2.306.4-z1-VH

Amino acid sequence (SEQ ID NO: 352) :

Nucleic acid sequence (SEQ ID NO: 353) :

WBP3311_2.306.4-z1-VL

Amino acid sequence (SEQ ID NO: 354) :

Nucleic acid sequence (SEQ ID NO: 355) :

b) Anti-CD19 antibody

In certain embodiments, the first antigen-binding moiety or the second antigen-binding moiety is an anti-CD19 binding moiety derived from an anti-CD19 antibody comprising 1, 2, or 3 heavy chain CDR sequences selected from the group consisting of SEQ ID NOs: 356-359, and/or 1, 2, or 3 kappa light chain CDR sequences selected from the group consisting of: SEQ ID NOs: 360-362.

These CDR sequences are derived from the antibodies shown in Table B below. The CDR sequences of these anti-CD19 antibodies are provided below.

Table B.

Heavy and kappa light chain variable region sequences of the WBP7011_4.155.8 antibody are provided below.

WBP7011-4.155.8-VH

Amino acid sequence (SEQ ID NO: 363) :

Nucleic acid sequence (SEQ ID NO: 364) :

WBP7011-4.155.8-VL

Amino acid sequence (SEQ ID NO: 365) :

Nucleic acid sequence (SEQ ID NO: 366) :

In certain embodiments, the anti-CD19 binding moiety disclosed herein comprises a heavy chain CDR3 sequence of the anti-CD19 antibody WBP7011_4.155.8 or W7011-4.155.8-z1-P15. In certain embodiments, the anti-CD19 binding moiety provided herein comprises a heavy chain CDR3 sequence comprising SEQ ID NO: 358. Heavy chain CDR3 regions are located at the center of the antigen-binding site, and therefore are believed to make the most contact with antigen and provide the most free energy to the affinity of antibody to antigen. It is also believed that the heavy chain CDR3 is by far the most diverse CDR of the antigen-binding site in terms of length, amino acid composition and conformation by multiple diversification mechanisms (Tonegawa S., Nature. 302: 575-81 (1983) ) . The diversity in the heavy chain CDR3 is sufficient to produce most antibody specificities (Xu JL, Davis MM. Immunity. 13: 37-45 (2000) ) as well as desirable antigen-binding affinity (Schier R, etc., J Mol Biol. 263: 551-67 (1996) ) .

In certain embodiments, the anti-CD19 antibodies disclosed herein are humanized. The heavy chain and light chain variable region sequences for the anti-CD19 humanized antibody W7011-4.155.8-z1-P15 are provided below.

W7011-4.155.8-z1-P15-VH

Amino acid sequence (SEQ ID NO: 367) :

Nucleic acid sequence (SEQ ID NO: 368) :

W7011-4.155.8-z1-P15-VL

Amino acid sequence (SEQ ID NO: 369) :

Nucleic acid sequence (SEQ ID NO: 370) :

Bispecific Polypeptide Complexes

In one aspect, the present disclosure provides herein a bispecific polypeptide complex. The term “bispecific” as used herein means that, there are two antigen-binding moieties, each of which is capable of specifically binding to a different antigen or a different epitope on the same antigen. The bispecific polypeptide complex provided herein comprises a first antigen-binding moiety comprising a first heavy chain variable domain operably linked to a first TCR constant region (C1) and a first light chain variable domain operably linked to a second TCR constant region (C2) , wherein C1 and C2 are capable of forming a dimer comprising at least one non-native and stabilizing interchain bond between C1 and C2. The bispecific polypeptide complex provided herein further comprises a second antigen-binding moiety comprising a second antigen-binding site but does not contain a sequence derived from a TCR constant region.

In certain embodiments, the present disclosure provides a bispecific polypeptide complex, comprising a first antigen-binding moiety associated with a second antigen-binding moiety, wherein:

the first antigen-binding moiety comprising:

a first polypeptide comprising, from N-terminus to C-terminus, a first heavy chain variable domain (VH) of a first antibody operably linked to a first T cell receptor (TCR) constant region (C1) , and

a second polypeptide comprising, from N-terminus to C-terminus, a first light chain variable domain (VL) of the first antibody operably linked to a second TCR constant region (C2) ,

wherein:

C1 and C2 are capable of forming a dimer comprising at least one non-native interchain bond between a first mutated residue comprised in C1 and a second mutated residue comprised in C2, and the non-native interchain bond is capable of stabilizing the dimer, and

the first antibody has a first antigenic specificity,

a second antigen-binding moiety has a second antigenic specificity which is different from the first antigenic specificity, and

the first antigen-binding moiety and the second antigen-binding moiety are less prone to mispair than otherwise would have been if both the first and the second antigen-binding moieties are counterparts of natural Fab.

The bispecific polypeptide complex provided herein is significantly less prone to have mispaired heavy chain and light chain variable domains. Without wishing to be bound by any theory, it is believed that the stabilized TCR constant regions in the first antigen-binding moiety can specifically associate with each other and therefore contribute to the highly specific pairing of the intended VH1 and VL1, while discouraging unwanted mispairings of VH1 or VL1 with other variable regions that do not provide for the intended antigen-binding sites.

The bispecific polypeptide complexes in WuXiBody formats have longer in vivo half-life and are relatively easier to manufacture when compared to bispecific polypeptide complexes in other formats.

In certain embodiments, the second antigen-binding moiety of the bispecific polypeptide complex provided herein comprises a second heavy chain variable domain (VH2) and a second light chain variable domain (VL2) of a second antibody. In certain embodiments, at least one of VH2 and VL2 is operably linked to an antibody constant region, or both VH2 and VL2 are operably linked to antibody heavy chain and light chain constant regions respectively. In certain embodiments, the second antigen-binding moiety further comprises an antibody constant CH1 domain operably linked to VH2, and an antibody light chain constant domain operably linked to VL2. For example, the second antigen-binding moiety comprises a Fab.

Where a first, second, third, and fourth variable domains (e.g. VH1, VH2, VL1 and VL2) are expressed in one cell, it is highly desired that VH1 specifically pairs with VL1, and VH2 specifically pairs with VL2, such that the resulting bispecific protein product would have the correct antigen-binding specificities. However, in existing technologies such as hybrid-hybridoma (or quadroma) , random pairing of VH1, VH2, VL1 and VL2 occurs and consequently results in generation of up to ten different species, of which only one is the functional bispecific antigen-binding molecule. This not only reduces production yields but also complicates the purification of the target product.

The bispecific polypeptide complexes provided herein are exceptional in that the variable domains are less prone to mispair than otherwise would have been if both the first and the second antigen-binding moieties are counterparts of natural Fab. In an illustrative example, the first antigen-binding domain comprises VH1-C1 paired with VL1-C2, and the second antigen-binding domain comprises VH2-CH1 paired with VL2-CL. It has been surprisingly found that C1 and C2 preferentially associates with each other, and are less prone to associate with CL or CH1, thereby formation of unwanted pairs such as C1-CH, C1-CL, C2-CH, and C2-CL are discouraged and significantly reduced. As a result of specific association of C1-C2, VH1 specifically pairs with VL1 and thereby rendering the first antigen binding site, and CH1 specifically pairs with CL, thereby allowing specific pairing of VH2-VL2 which provides for the second antigen binding site. Accordingly, the first antigen binding moiety and the second antigen binding moiety are less prone to mismatch, and mispairings between for example VH1-VL2, VH2-VL1, VH1-VH2, VL1-VL2 would be significantly reduced otherwise could have been if both the first and the second antigen-binding moieties are counterparts of natural Fabs, e.g. in the form of VH1-CH1, VL1-CL, VH2-CH1, and VL2-CL.

In certain embodiments, the bispecific polypeptide complex provided herein, when expressed from a cell, would have significantly less mispairing products (e.g., at least 1, 2, 3, 4, 5 or more mispairing products less) and/or significantly higher production yield (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%or more higher yield) , than a reference molecule expressed under comparable conditions, wherein the reference molecule is otherwise identical to the bispecific polypeptide complex except having a native CH1 in the place of C1 and a native CL in the place of C2.

In certain embodiments, the first and/or the second antigen binding moiety is multivalent, such as bivalent, trivalent, tetravalent. The term “valent” as used herein refers to the presence of a specified number of antigen binding sites in a given molecule. As such, the terms “bivalent” , “tetravalent” , and “hexavalent” denote the presence of two binding site, four binding sites, and six binding sites, respectively, in an antigen-binding molecule. A bivalent molecule can be monospecific if the two binding sites are both for specific binding of the same antigen or the same epitope. Similarly, a trivalent molecule can be bispecific, for example, when two binding sites are monospecific for a first antigen (or epitope) and the third binding site is specific for a second antigen (or epitope) . In certain embodiments, the first and/or the second antigen-binding moiety in the bispecific polypeptide complex provided herein can be bivalent, trivalent, or tetravalent, with at least two binding sites specific for the same antigen or epitope. This, in certain embodiments, provides for stronger binding to the antigen or the epitope than a monovalent counterpart. In certain embodiments, in a bivalent antigen-binding moiety, the first valent of binding site and the second valent of binding site are structurally identical (i.e. having the same sequences) , or structurally different (i.e. having different sequences albeit with the same specificity) .

In certain embodiments, the first and/or the second antigen binding moiety is multivalent and comprises two or more antigen binding sites operably linked together, with or without a spacer.

In certain embodiments, the first and/or the second antigen binding moiety comprises one or more Fab, Fab', Fab'-SH, F (ab') ₂, Fd, Fv, and scFv fragments, and other fragments described in Spiess et al., 2015, supra and Brinkmann et al., 2017, supra, or the combination thereof, which are linked with or without a spacer at the heavy chain and/or the light chain and forms at least one are capable of binding to a second antibody.

In certain embodiments, the second antigen binding moiety comprises two or more Fab of the second antibody. The two Fabs can be operably linked to each other, for example the first Fab can be covalently attached to the second Fab via heavy chain, with or without a spacer in between.

In certain embodiments, the first antigen-binding moiety further comprises a first dimerization domain, and the second antigen-binding moiety further comprises a second dimerization domain. The term “dimerization domain” as used herein refers to the peptide domain which is capable of associating with each other to form a dimer, or in some examples, enables spontaneous dimerization of two peptides.

In certain embodiments, the first dimerization domain can be associated with the second dimerization domain. The association can be via any suitable interaction or linkage or bonding, for example, via a connecter, a disulphide bond, a hydrogen bond, electrostatic interaction, a salt bridge, or hydrophobic-hydrophilic interaction, or the combination thereof. Exemplary dimerization domains include, without limitation, antibody hinge region, an antibody CH2 domain, an antibody CH3 domain, and other suitable protein monomers capable of dimerizing and associating with each other. Hinge region, CH2 and/or CH3 domain can be derived from any antibody isotypes, such as IgG1, IgG2, and IgG4.

In certain embodiments, the first and/or the second dimerization domain comprises at least a portion of an antibody hinge region. In certain embodiments, the first and/or the second dimerization domain may further comprise an antibody CH2 domain, and/or an antibody CH3 domain. In certain embodiments, the first and/or the second dimerization domain comprises at least a portion of Hinge-Fc region, i.e. Hinge-CH2-CH3 domain. In certain embodiments, the first dimerization domain can be operably linked to the C terminal of the first TCR constant region. In certain embodiments, the second dimerization domain can be operably linked to the C terminal of the antibody CH1 constant region of the second antigen-binding moiety.

In certain embodiments, the first dimerization domain is operably linked to (with or without a spacer in between) the first TCR constant region (C1) at a third conjunction domain.

If the Fv region of an immunoglobulin is aligned with a TCR immunoglobulin-like domain, the antibody Hinge N terminal and the TCR Hinge N terminal would also be aligned. An example is given in Table 7 below, where antibody Hinge N terminal (SEQ ID NO: 278 or 279) is aligned to TCR Beta Hinge N terminal (SEQ ID NO: 280) .

The third conjunction domain of the bispecific polypeptide complex as provided herein can be selected such that it comprises a proper length (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues) of the antibody Hinge N terminal, and a proper length (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues) of the TCR Hinge N terminal. The term “Hinge N terminal” as used herein refers to the most N terminal fragment of the hinge region. For example, the conjunction domain may be selected to have all, or most, or some sequences from the antibody Hinge N terminal or from the TCR Hinge N terminal, or may comprise more residues from antibody Hinge N terminal than from TCR Hinge N terminal, or vice versa.

In certain embodiments, the third conjunction domains of the polypeptide complex as provided herein have a total length comparable to that of the antibody Hinge N terminal or that of the TCR Hinge N terminal.

Similarly, a proper third conjunction domain can be determined on a structural basis. For example, the three-dimensional structures of antibody and TCR may be superimposed, and overlappings of the antibody Hinge N terminal and the TCR Hinge N terminal on the superimposed structure may be determined and considered when determining the length or proportion of sequences from antibody or TCR Hinge N terminal.

In certain embodiments, the third conjunction domain comprises a spacer in between the fragments from antibody Hinge N terminal and TCR Hinge N terminal. Any suitable sequences or length of spacer sequences can be used, as long as it does not negatively affect the antigen binding or stability of the polypeptide complex.

Exemplary sequences of antibody Hinge N terminal, TCR Hinge N terminal, and the third conjunction domains are provided in the below Tables 7, 8, 9 and 10.

In certain embodiments, C1 comprises an engineered CBeta and the first dimerization domain comprises hinge and Fc of IgG1 or IgG4. Table 7 shows the exemplary designs for the conjunction domains useful for TCR CBeta fused to antibody Hinge. The antibody Hinge N terminal is aligned to TCR Beta Hinge N terminal. Exemplary designs of the conjunction domains are also provided in an alignment form (see, e.g., SEQ ID NO: 152 or 153) . In such embodiments, the third conjunction domain is comprised in SEQ ID NO: 53 or 54 (which encompass the third conjunction domain and the Hinge C terminal) .

Table 7. Third conjunction domain designs for VH-CBeta-Hinge

In certain embodiments, C1 comprises an engineered CAlpha or CPre-Alpha and the first dimerization domain comprises hinge and Fc of IgG1 or IgG4. Table 8 shows the exemplary designs for the conjunction domains useful for TCR CAlpha or CPre-Alpha fused to antibody Hinge. The antibody Hinge N terminal is aligned to TCR Alpha or CPre-Alpha Hinge N terminal. In such embodiments, the third conjunction domain is comprised in SEQ ID NO: 134, 135, 140, or 141 (which encompass the third conjunction domain and the Hinge C terminal) .

Table 8. Third conjunction domain designs for VH-CAlpha-Hinge

In certain embodiments, C1 comprises an engineered CGamma and the first dimerization domain comprises hinge and Fc of IgG1 or IgG4. Table 9 shows the exemplary designs for the conjunction domains useful for TCR CGamma fused to antibody Hinge. The antibody Hinge N terminal is aligned to TCR Gamma Hinge N terminal. Exemplary designs of the conjunction domains are also provided in an alignment form (see, e.g., SEQ ID NO: 165 or 166) . In such embodiments, the third conjunction domain is comprised in SEQ ID NO: 121 or 122 (which encompass the third conjunction domain and the Hinge C terminal) .

Table 9. Third conjunction domain designs for VH-CGamma-Hinge

In certain embodiments, C1 comprises an engineered CDelta and the first dimerization domain comprises hinge and Fc of IgG1 or IgG4. Table 10 shows the exemplary designs for the conjunction domains useful for TCR CDelta fused to antibody Hinge. The antibody Hinge N terminal is aligned to TCR Delta Hinge N terminal. Exemplary designs of the conjunction domains are also provided in an alignment form. In such embodiments, the third conjunction domain is comprised in SEQ ID NO: 127, or 128 (which encompass the third conjunction domain and the Hinge C terminal) .

Table 10. Third conjunction domain designs for VH-CDelta-Hinge-Fc

In certain embodiments, the first dimerization domain is operably linked to the C-terminal of an engineered TCR constant region, and together forms a chimeric constant region. In other words, the chimeric constant region comprises the first dimerization domain operably linked with the engineered TCR constant region.

In certain embodiments, the chimeric constant region comprises an engineered CBeta attached to the first hinge-Fc region derived from IgG1, IgG2 or IgG4. Exemplary sequences of such a chimeric constant region are provided in Tables 11, 12, 13 and 14.

In certain embodiments, the chimeric constant region comprises an engineered CAlpha attached to the first hinge derived from IgG1, IgG2 or IgG4. Exemplary sequences of such chimeric constant region are provided in Tables 11, 12, and 13.

In certain embodiments, the chimeric constant region comprises an engineered CPre-Alpha attached to the first hinge derived from IgG1, IgG2 or IgG4, at the third conjunction domain comprising or being SEQ ID NO: 134, 135, 140 or 141. Exemplary sequences of such a chimeric constant region are provided in Tables 15 and 16.

In certain embodiments, the chimeric constant region comprises an engineered CGamma attached to the first hinge derived from IgG1, IgG2 or IgG4. Exemplary sequences of such a chimeric constant region are provided in Tables 17 and 18.

In certain embodiments, the chimeric constant region comprises an engineered CDelta attached to the first hinge derived from IgG1, IgG2 or IgG4. Exemplary sequences of such a chimeric constant region are provided in Tables 17 and 18.

In certain embodiments, the chimeric constant region further comprises a first antibody CH2 domain, and/or a first antibody CH3 domain. For example, the chimeric constant region further comprises a first antibody CH2-CH3 domain attached to the C-terminus of the third conjunction domain. Exemplary sequences of such chimeric constant region are provided in Table 19.

In certain embodiments, the first chimeric constant region and the second TCR constant domain comprises a pair of sequences selected from the group consisting of SEQ ID NOs: 177/176, 179/178, 184/183, 185/183, 180/176, 181/178, 182/178, 184/186, 185/186, 188/187, 196/187, 190/189, 192/191, 192/193, 195/194, 198/197, 200/199, 202/201, 203/201, 203/204, 205/204, 206/204, 208/207, 208/209, 211/210, 213/212, 213/151, 214/212, 214/151, 234/233, 232/231, 216/215, 218/217, 220/219, 222/221, 224/223, 226/225, 227/223, 229/228, 229/230, 236/235 and 238/237, as shown in Table 19.

These pairs of chimeric constant regions and second TCR constant domains are useful in that they can be manipulated to fuse to a desired antibody variable region, so as to provide for the polypeptide complex as disclosed herein. For example, an antibody heavy chain variable region can be fused to the chimeric constant region (comprising C1) , thereby rendering the first polypeptide chain of the polypeptide complex provided herein; and similarly, an antibody light chain variable region can be fused to the second TCR constant domain (comprising C2) , thereby rendering the second polypeptide chain of the polypeptide complex provided herein.

These pairs of chimeric constant regions and second TCR constant domains can be used as a platform for generating the first antigen-binding moiety of the bispecific polypeptide complexes provided herein. For example, variable regions of a first antibody can be fused at the N-terminus of the platform sequences (e.g. fusing the VH to the chimeric constant domain and the VL to the TCR constant domain, respectively) . To produce the bispecific polypeptide complex, the second antigen-binding moiety can be designed and produced, so as to associate into the bispecific polypeptide complex provided herein.

In certain embodiments, the second dimerization domain comprises a hinge region. The hinge region may be derived from an antibody, such as IgG1, IgG2, or IgG4. In certain embodiments, the second dimerization domain may optionally further comprise an antibody CH2 domain, and/or an antibody CH3 domain, for example such as a hinge-Fc region. The hinge region may be attached to the antibody heavy chain of the second antigen binding site (e.g. Fab) .

In the bispecific polypeptide complex, the first and the second dimerization domain are capable of associating into a dimer. In certain embodiments, the first and the second dimerization domains are different and associate in a way that discourages homodimerization and/or favors heterodimerization. For example, the first and the second dimerization domains can be selected so that they are not identical and that they preferentially form heterodimers between each other rather than to form homodimers within themselves. In certain embodiments, the first and the second dimerization domains are capable of associating into heterodimers via formation of knob-into-hole, hydrophobic interaction, electrostatic interaction, hydrophilic interaction, or increased flexibility.

In certain embodiments, the first and the second dimerization domains comprise CH2 and/or CH3 domains which are respectively mutated to be capable of forming a knobs-into-holes. A knob can be obtained by replacement of a small amino acid residue with a larger one in the first CH2/CH3 polypeptide, and a hole can be obtained by replacement of a large residue with a smaller one. For details of the mutation sites for knobs into holes please see Ridgway et al., 1996, supra, Spiess et al., 2015, supra and Brinkmann et al., 2017, supra.

In certain embodiments, the first and the second dimerization domains comprise a first CH3 domain of the IgG1 isotype containing S139C and T151W substitution (SEQ ID NO: 295, knob) and a second CH3 domain of the IgG1 isotype containing Y134C, T151S, L153A and Y192V substitution (SEQ ID NO: 296, hole) . In another embodiments, the first and the second dimerization domains comprise a first CH3 domain of the IgG4 isotype containing S136C and T148W substitution (SEQ ID NO: 298, knob) and a second CH3 domain of the IgG4 isotype containing Y131C, T148S, L150A and Y189V substitution (SEQ ID NO: 299, hole) . The sequences and numberings of wild type Fc IgG1 (SEQ ID NO: 294) and Fc IgG4 (SEQ ID NO: 297) are shown in Figures 4A-4D. As noted above, XnY when referring to Fc region (e.g. CH3 domain of the Fc region) , the numbering of the amino acid residue is based on the numbering shown in Figures 4A-4D.

In certain embodiments, the first and the second dimerization domains further comprise a first hinge region and a second hinge region. For example, charge pairs of substitution can be introduced into the hinge region of IgG1 and IgG2 to promote heterodimerization. For details see Brinkmann et al., 2017, supra.

Bispecific format

The bispecific polypeptide complex provided herein can be in any suitable bispecific format known in the art. In certain embodiments, the bispecific polypeptide complex is based on a reference bispecific antibody format. “Based on” as used herein with respect to a bispecific format means that the bispecific polypeptide complex provided herein takes the same bispecific format of a reference bispecific antibody, except that one of the antigen-binding moiety has been modified to comprise a VH operably linked to C1 and a VL operably linked to C2 wherein C1 and C2 are associated with at least one non-native interchain bond, as defined above. Examples of reference bispecific antibody formats known in the art include, without limitation, (i) a bispecific antibody with symmetric Fc, (ii) a bispecific antibody with asymmetric Fc, (iii) a regular antibody appended with an additional antigen-binding moiety, (iv) a bispecific antibody fragment, (v) a regular antibody fragment appended with an additional antigen-binding moiety, (vi) a bispecific antibody appended with human albumin or human albumin-binding peptide.

BsIgG is monovalent for each antigen and can be produced by co-expression of the two light and two heavy chains in a single host cell. An appending IgG is engineered to form bispecific IgG by appending either the amino or carboxy termini of either light or heavy chains with additional antigen-binding units. The additional antigen-binding units can be single domain antibodies (unpaired VL or VH) , such as DVD-Ig, paired antibody variable domains (e.g. Fv or scFv) or engineered protein scaffolds. Any of the antigen-binding units in BsIgG, in particular paired VH-CH1/VL-CL, can be modified to replace the CH1 to C1 and CL to C2 as disclosed herein, to render the bispecific polypeptide complex as provided herein.

Bispecific antibody fragments are antigen-binding fragments that are derived from an antibody but lack some or all of the antibody constant domains. Examples of such a bispecific antibody fragment include, for example, such as single domain antibody, Fv, Fab and diabody etc. To render the bispecific polypeptide complex as provided herein, an antigen-binding site (e.g. particular paired VH-CH1/VL-CL) in a bispecific antibody fragment, can be modified to comprise the polypeptide complex as disclosed herein (e.g. VH-C1/CL-C2) .

In certain embodiments, the bispecific polypeptide complex as provided herein is based on the format of a “whole” antibody, such as whole IgG or IgG-like molecules, and small recombinant formats, such as tandem single chain variable fragment molecules (taFvs) , diabodies (Dbs) , single chain diabodies (scDbs) and various other derivatives of these (cf. bispecific antibody formats as described by Byrne H. et al. (2013) Trends Biotech, 31 (11) : 621-632. Examples of bispecific antibody is based on a format which include, but is not limited to, quadroma, chemically coupled Fab (fragment antigen binding) , and BiTE (bispecific T cell engager) .

In certain embodiments, the bispecific polypeptide complex as provided herein is based on a bispecific format selected from Triomabs; hybrid hybridoma (quadroma) ; Multispecific anticalin platform (Pieris) ; Diabodies; Single chain diabodies; Tandem single chain Fv fragments; TandAbs, Trispecific Abs (Affimed) ; Darts (dual affinity retargeting; Macrogenics) ; Bispecific Xmabs (Xencor) ; Bispecific T cell engagers (Bites; Amgen; 55 kDa) ; Triplebodies; Tribody (Fab-scFv) Fusion Protein (CreativeBiolabs) multifunctional recombinant antibody derivates; Duobody platform (Genmab) ; Dock and lock platform; Knob into hole (KIH) platform; Humanized bispecific IgG antibody (REGN1979) (Regeneron) ; Mab ₂ bispecific antibodies (F-Star) ; DVD-Ig (dual variable domain immunoglobulin) (Abbvie) ; kappa-lambda bodies; TBTI (tetravalent bispecific tandem Ig) ; and CrossMab.

In certain embodiments, the bispecific polypeptide complex as provided herein is based on a bispecific format selected from bispecific IgG-like antibodies (BsIgG) comprising CrossMab; DAF (two-in-one) ; DAF (four-in-one) ; DutaMab; DT-IgG; Knobs-in-holes common LC; Knobs-in-holes assembly; Charge pair; Fab-arm exchange; SEEDbody; Triomab; LUZ-Y; Fcab; kappa-lamda-body; and Orthogonal Fab. For detailed description of the bispecific antibody formats please see Spiess C., Zhai Q. and Carter P. J. (2015) Molecular Immunology 67: 95-106, which is incorporated herein by reference in its entirety.

In certain embodiments, the bispecific polypeptide complex as provided herein is based on a bispecific format selected from IgG-appended antibodies with an additional antigen-binding moiety comprising DVD-IgG; IgG (H) -scFv; scFv- (H) IgG; IgG (L) -scFv; scFV- (L) IgG; IgG (L, H) -Fv; IgG (H) -V; V (H) -IgG; IgG (L) -V; V (L) -IgG; KIH IgG-scFab; 2scFv-IgG; IgG-2scFv; scFv4-Ig; scFv4-Ig; Zybody; and DVI-IgG (four-in-one) (see Id. ) .

In certain embodiments, the bispecific polypeptide complex as provided herein is based on a format selected from bispecific antibody fragments comprising Nanobody; Nanobody-HAS; BiTE; Diabody; DART; TandAb; scDiabody; sc-Diabody-CH3; Diabody-CH3; Triple Body; Miniantibody; Minibody; TriBi minibody; scFv-CH3 KIH; Fab-scFv; scFv-CH-CL-scFv; F (ab') 2; F (ab') 2-scFv2; scFv-KIH; Fab-scFv-Fc; Tetravalent HCAb; scDiabody-Fc; Diabody-Fc; Tandem scFv-Fc; and Intrabody (see Id. ) .

In certain embodiments, the bispecific polypeptide complex as provided herein is based on a bispecific format such as Dock and Lock; ImmTAC; HSAbody; scDiabody-HAS; and Tandem scFv-Toxin (see Id. ) .

In certain embodiments, the bispecific polypeptide complex as provided herein is based on a format selected from bispecific antibody conjugates comprising IgG-IgG; Cov-X-Body; and scFv1-PEG-scFv2 (see Id. ) .

In certain embodiments, the first antigen-binding moiety and the second binding moiety can be associated into an Ig-like structure. An Ig-like structure is like a natural antibody having a Y shaped construct, with two arms for antigen-binding and one stem for association and stabilization. The resemblance to natural antibody can provide for various advantages such as good in vivo pharmacokinetics, desired immunological response and stability etc. It has been found that the Ig-like structure comprising the first antigen-binding moiety provided herein associated with the second antigen-binding moiety provided herein has thermal stability which is comparable to that of an Ig (e.g. an IgG) . In certain embodiments, the Ig-like structure provided herein is at least 70%, 80%, 90%, 95%or 100%of that of a natural IgG.

In certain embodiments, the bispecific polypeptide complex comprises four polypeptide chains: i) VH1-C1-Hinge-CH2-CH3; ii) VL1-C2; iii) VH2-CH1-Hinge-CH2-CH3, and iv) VL2-CL, wherein the C1 and C2 are capable of forming a dimer comprising at least one non-native interchain bond, and the two hinge regions and/or the two CH3 domains are capable of forming one or more interchain bond that can facilitate dimerization.

Antigenic Specificities of the Bispecific Complex

The bispecific complex provided herein have two antigenic specificities. The first and the second antigen-binding moieties are directed to the first and the second antigenic specificities respectively.

The first and the second antigenic specificities may be identical, in other words, the first and the second antigen-binding moieties binds to the same antigen molecule, or to the same epitope of the same antigen molecule.

Alternatively, the first and the second antigenic specificities may be distinct. For example, the first and the second antigen-binding moieties can bind to different antigens. Such a bispecific polypeptide complex could be useful in, for example, bringing the two different antigens into close proximity and thereby promoting their interactions (e.g. bringing immunological cells in close proximity to a tumor antigen or a pathogen antigen and hence promoting recognition or elimination of such an antigen by the immune system) . For another example, the first and the second antigen-binding moieties can bind to different (and optionally non-overlapping) epitopes of one antigen. This may be helpful in enhancing the recognition of or binding to a target antigen, in particular one which is susceptible to mutation (e.g. a viral antigen) .

In some embodiments, one of the antigenic specificity of the bispecific complex provided herein is directed to a T-cell specific receptor molecule and/or a natural killer cell (NK cell) specific receptor molecule. In some embodiments, one of the first and second antigen-binding moiety is capable of specifically binding to CD3, TCR, CD28, CD16, NKG2D, Ox40, 4-1BB, CD2, CD5 or CD95, and the other is capable of specifically binding to a tumor associated antigen.

In certain embodiments, one of the antigenic specificity of the bispecific complex provided herein is directed to CD3. In certain embodiments, the first antigen-binding moiety of the bispecific complex is capable of specifically binding to CD3. In certain embodiments, the second antigen-binding moiety of the bispecific complex is capable of specifically binding to CD3.

In certain embodiments, the antigen-binding moiety of the bispecific complex comprises a VH1 and a VL1 both derived from an anti-CD3 antibody. In certain embodiments, the polypeptide complex or the bispecific polypeptide complex provided herein, wherein the first polypeptide and the second polypeptide comprise a pair of sequences selected from the group consisting of SEQ ID NOs: 2/1, 3/4/, 5/1, 6/3, 7/3, 9/8, 10/8, 9/11, 10/11, 13/12, 15/14, 17/16, 17/18, 20/19, 21/12, 28/3, 29/3, 30/12, 31/12, 65/64, 67/66, 69/68, 70/68, 70/71, 72/71, 73/71, 75/74, 75/76, 78/77, 86/85, 90/89, 91/92/, 94/93, 96/95, 98/97, 99/95, 101/100, 101/102, 106/105, 108/107, 110/109, 112/111, 137/136, 138/136, 137/139 and 138/139, wherein the variable regions of anti-CD3 antibody (T3) are fused to the TCR constant region as shown in Table 20.

In some embodiments, one of the antigenic specificity of the bispecific complex provided herein is directed to a T-cell specific receptor molecule and/or a natural killer cell (NK cell) specific receptor molecule, and the other antigenic specificity is directed to a tumor associated surface antigen. In certain embodiments, the first antigen-binding moiety of the bispecific complex is capable of specifically binding to T-cell specific receptor molecule and/or a natural killer cell (NK cell) specific receptor molecule (such as CD3) , and the second antigen-binding moiety is capable of specifically binding to a tumor associated antigen (such as CD19) , or vice versa.

In certain embodiments, the bispecific polypeptide complex comprises a four-sequence combination selected from the group consisting of: SEQ ID NOs: 22/12/24/23 (E17, IgG1) , 25/12/26/23 (E17, IgG4) , and 25/12/27/23 (F16) , as shown in Example 8 and Table 20, wherein the first antigen binding moiety binds to CD3, and the second antigen binding moiety binds to CD19. The design of E17 is a bispecific, bivalent antibody, and the design of F16 is a bispecific and trivalent antigen-binding complex, with two repeats of anti-CD19 antibody Fab.

In certain embodiments, the bispecific polypeptide complex comprises a first antigen binding moiety that binds to CTLA-4, and a second antigen binding moiety that binds to PD-1, or vice versa.

In certain embodiments, the bispecific polypeptide complex comprises four polypeptide chains comprising: i) VH1 operably linked to a first chimeric constant region; ii) VL1 operably linked to a second chimeric constant region; iii) VH2 operably linked to conventional antibody heavy chain constant region, and iv) VL2 operably linked to conventional antibody light chain constant region. In certain embodiments, the first chimeric constant region can comprise C1-Hinge-CH2-CH3, each as defined supra. In certain embodiments, the second chimeric constant region can comprise C2, as defined supra. In certain embodiments, the conventional antibody heavy chain constant region can comprise CH1-Hinge-CH2-CH3, each as defined supra. In certain embodiments, the conventional antibody light chain constant region can comprise CL, as defined supra.

The following construct names are used interchangeably in this disclosure: E17-Design_2-QQQQ and W3438-T3U4. E17-1. uIgG4. SP; F16-Design-2-QQQQ and W3438-T3U4. F16-1. uIgG4. SP; U6T5. G25. IgG4 and W3248-U6T5. G25-1. uIgG4. SP; and U6T1. G25R. IgG4 and W3248-U6T1. G25R-1. uIgG4. SP.

Method of preparation

The present disclosure provides isolated nucleic acids or polynucleotides that encode the polypeptide complex, and the bispecific polypeptide complex provided herein.

The term “nucleic acid” or “polynucleotide” as used herein refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single-or double-stranded form. Unless specifically limited, the term encompasses polynucleotides containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular polynucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) , alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (see Batzer et al., Nucleic Acid Res. 19: 5081 (1991) ; Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985) ; and Rossolini et al., Mol. Cell. Probes 8: 91-98 (1994) ) .

The nucleic acids or polynucleotides encoding the polypeptide complex and the bispecific polypeptide complex provided herein can be constructed using recombinant techniques. To this end, DNA encoding an antigen-binding moiety of a parent antibody (such as CDR or variable region) can be isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody) . Likewise, DNA encoding a TCR constant region can also be obtained. As an example, the polynucleotide sequence encoding the variable domain (VH) and the polynucleotide sequence encoding the first TCR constant region (C1) are obtained and operably linked to allow transcription and expression in a host cell to produce the first polypeptide. Similarly, polynucleotide sequence encoding VL are operably linked to polynucleotide sequence encoding C1, so as to allow expression of the second polypeptide in the host cell. If needed, encoding polynucleotide sequences for one or more spacers are also operably linked to the other encoding sequences to allow expression of the desired product.

The encoding polynucleotide sequences can be further operably linked to one or more regulatory sequences, optionally in an expression vector, such that the expression or production of the first and the second polypeptides is feasible and under proper control.

The encoding polynucleotide sequence (s) can be inserted into a vector for further cloning (amplification of the DNA) or for expression, using recombinant techniques known in the art. In another embodiment, the polypeptide complex and the bispecific polypeptide complex provided herein may be produced by homologous recombination known in the art. Many vectors are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1α) , and a transcription termination sequence.

The term “vector” as used herein refers to a vehicle into which a polynucleotide encoding a protein may be operably inserted so as to bring about the expression of that protein. Typically, the construct also includes appropriate regulatory sequences. For example, the polynucleotide molecule can include regulatory sequences located in the 5’-flanking region of the nucleotide sequence encoding the guide RNA and/or the nucleotide sequence encoding a site-directed modifying polypeptide, operably linked to the coding sequences in a manner capable of expressing the desired transcript/gene in a host cell. A vector may be used to transform, transduce, or transfect a host cell so as to bring about expression of the genetic element it carries within the host cell. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC) , bacterial artificial chromosome (BAC) , or P1-derived artificial chromosome (PAC) , bacteriophages such as lambda phage or M13 phage, and animal viruses. Categories of animal viruses used as vectors include retrovirus (including lentivirus) , adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus) , poxvirus, baculovirus, papillomavirus, and papovavirus (e.g., SV40) . A vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. In addition, the vector may contain an origin of replication. A vector may also include materials to aid in its entry into the cell, including but not limited to a viral particle, a liposome, or a protein coating.

In some embodiments, the vector system includes mammalian, bacterial, yeast systems, etc., and comprises plasmids such as, but not limited to, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pCMV, pEGFP, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS420, pLexA, pACT2.2 etc., and other laboratorial and commercially available vectors. Suitable vectors may include, plasmid, or viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses) .

Vectors comprising the polynucleotide sequence (s) provided herein can be introduced to a host cell for cloning or gene expression. The phrase “host cell” as used herein refers to a cell into which an exogenous polynucleotide and/or a vector has been introduced.

Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells described above. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis, Pseudomonas such as P. aeruginosa, and Streptomyces.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for the vectors encoding the polypeptide complex and the bispecific polypeptide complex. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12, 424) , K. bulgaricus (ATCC 16, 045) , K. wickeramii (ATCC 24, 178) , K. waltii (ATCC 56, 500) , K.drosophilarum (ATCC 36, 906) , K. thermotolerans, and K. marxianus; yarrowia (EP 402, 226) ; Pichia pastoris (EP 183, 070) ; Candida; Trichoderma reesia (EP 244, 234) ; Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.

Suitable host cells for the expression of glycosylated polypeptide complex, the bispecific polypeptide complex provided herein are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar) , Aedes aegypti (mosquito) , Aedes albopictus (mosquito) , Drosophila melanogaster (fruiffly) , and Bombyx mori have been identified. A variety of viral strains for transfection are publicly available, e.g., the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present disclosure, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts.

However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651) ; human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36: 59 (1977) ) , such as Expi293; baby hamster kidney cells (BHK, ATCC CCL 10) ; Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980) ) ; mouse sertoli cells (TM4, Mather, Biol. Reprod. 23: 243-251 (1980) ) ; monkey kidney cells (CV1 ATCC CCL 70) ; African green monkey kidney cells (VERO-76, ATCC CRL-1587) ; human cervical carcinoma cells (HELA, ATCC CCL 2) ; canine kidney cells (MDCK, ATCC CCL 34) ; buffalo rat liver cells (BRL 3A, ATCC CRL 1442) ; human lung cells (W138, ATCC CCL 75) ; human liver cells (Hep G2, HB 8065) ; mouse mammary tumor (MMT 060562, ATCC CCL51) ; TRI cells (Mather et al., Annals N. Y. Acad. Sci. 383: 44-68 (1982) ) ; MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2) .

Host cells are transformed with the above-described expression or cloning vectors can be cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the cloning vectors.

For production of the polypeptide complex and the bispecific polypeptide complex provided herein, the host cells transformed with the expression vector may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma) , Minimal Essential Medium (MEM) , (Sigma) , RPMI-1640 (Sigma) , and Dulbecco's Modified Eagle's Medium (DMEM) , Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58: 44 (1979) , Barnes et al., Anal. Biochem. 102: 255 (1980) , U.S. Pat. No. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re. 30,985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor) , salts (such as sodium chloride, calcium, magnesium, and phosphate) , buffers (such as HEPES) , nucleotides (such as adenosine and thymidine) , antibiotics (such as GENTAMYCIN ^TM drug) , trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range) , and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

In one aspect, the present disclosure provides a method of expressing the polypeptide complex and the bispecific polypeptide complex provided herein, comprising culturing the host cell provided herein under the condition at which the polypeptide complex, or the bispecific polypeptide complex is expressed.

In certain embodiments, the present disclosure provides a method of producing the polypeptide complex provided herein, comprising a) introducing to a host cell: a first polynucleotide encoding a first polypeptide comprising, from N-terminus to C-terminus, a first heavy chain variable domain (VH) of a first antibody operably linked to a first TCR constant region (C1) , and a second polynucleotide encoding a second polypeptide comprising, from N-terminus to C-terminus, a first light chain variable domain (VL) of the first antibody operably linked to a second TCR constant region (C2) , wherein: C1 and C2 are capable of forming a dimer comprising at least one non-native interchain bond between C1 and C2, and the non-native interchain bond is capable of stabilizing the dimer of C1 and C2, and the first antibody has a first antigenic specificity; b) allowing the host cell to express the polypeptide complex.

In certain embodiments, the present disclosure provides a method of producing the bispecific polypeptide complex provided herein, comprising a) introducing to a host cell: a first polynucleotide encoding a first polypeptide comprising, from N-terminus to C-terminus, a first heavy chain variable domain (VH) of a first antibody operably linked to a first TCR constant region (C1) , a second polynucleotide encoding a second polypeptide comprising, from N-terminus to C-terminus, a first light chain variable domain (VL) of the first antibody operably linked to a second TCR constant region (C2) , and one or more additional polynucleotides encoding a second antigen-binding moiety, wherein: C1 and C2 are capable of forming a dimer comprising at least one non-native interchain bond between a first mutated residue comprised in C1 and a second mutated residue comprised in C2, and the non-native interchain bond is capable of stabilizing the dimer of C1 and C2, the first antigen-binding moiety and the second antigen-binding moiety have reduced mispairing than otherwise would have been if the first antigen-binding moiety was a natural Fab counterpart, and the first antibody has a first antigenic specificity and the second antibody has a second antigenic specificity, b) allowing the host cell to express the bispecific polypeptide complex.

In certain embodiments, the method further comprises isolating the polypeptide complex.

When using recombinant techniques, the polypeptide complex, the bispecific polypeptide complex provided herein can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the product is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10: 163-167 (1992) describe a procedure for isolating antibodies which are secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5) , EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min. Cell debris can be removed by centrifugation. Where the product is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.

The polypeptide complex and the bispecific polypeptide complex provided herein prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being the preferred purification technique.

Where the polypeptide complex or the bispecific polypeptide complex provided herein comprises immunoglobulin Fc domain, then protein A can be used as an affinity ligand, depending on the species and isotype of the Fc domain that is present in the polypeptide complex. Protein A can be used for purification of polypeptide complexes based on human γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983) ) . Protein G is recommended for all mouse isotypes and for human γ3 (Guss et al., EMBO J. 5: 1567 1575 (1986) ) . The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly (styrenedivinyl) benzene allow for faster flow rates and shorter processing times than can be achieved with agarose.

Where the polypeptide complex or the bispecific polypeptide complex provided herein comprises a CH3 domain, the Bakerbond ABX resin (J. T. Baker, Phillipsburg, N. J. ) is useful for purification. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE ^TM chromatography on an anion or cation exchange resin (such as a polyaspartic acid column) , chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.

Following any preliminary purification step (s) , the mixture comprising the polypeptide complex of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations (e.g., from about 0-0.25M salt) .

In certain embodiments, the bispecific polypeptide complex provided herein can be readily purified with high yields using conventional methods. One of the advantages of the bispecific polypeptide complex is the significantly reduced mispairing between heavy chain and light chain variable domain sequences. This reduces production of unwanted byproducts and make it possible to obtain high purity product in high yields using relatively simple purification processes.

Derivatives

In certain embodiments, the polypeptide complex or the bispecific polypeptide complex can be used as the base of conjugation with desired conjugates.

It is contemplated that a variety of conjugates may be linked to the polypeptide complex or the bispecific polypeptide complex provided herein (see, for example, “Conjugate Vaccines” , Contributions to Microbiology and Immunology, J. M. Cruse and R. E. Lewis, Jr. (eds. ) , Carger Press, New York, (1989) ) . These conjugates may be linked to the polypeptide complex or the bispecific polypeptide complex by covalent binding, affinity binding, intercalation, coordinate binding, complexation, association, blending, or addition, among other methods.

In certain embodiments, the polypeptide complex or the bispecific polypeptide complex provided herein may be engineered to contain specific sites outside the epitope binding portion that may be utilized for binding to one or more conjugates. For example, such a site may include one or more reactive amino acid residues, such as for example cysteine or histidine residues, to facilitate covalent linkage to a conjugate.

In certain embodiments, the polypeptide complex or the bispecific polypeptide complex may be linked to a conjugate indirectly, or indirectly for example through another conjugate or through a linker.

For example, the polypeptide complex or the bispecific polypeptide complex having a reactive residue such as cysteine may be linked to a thiol-reactive agent in which the reactive group is, for example, a maleimide, an iodoacetamide, a pyridyl disulphide, or other thiol-reactive conjugation partner (Haugland, 2003, Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Inc.; Brinkley, 1992, Bioconjugate Chem. 3: 2; Garman, 1997, Non-Radioactive Labelling: A Practical Approach, Academic Press, London; Means (1990) Bioconjugate Chem. 1: 2; Hermanson, G. in Bioconjugate Techniques (1996) Academic Press, San Diego, pp. 40-55, 643-671) .

For another example, the polypeptide complex or the bispecific polypeptide complex may be conjugated to biotin, then indirectly conjugated to a second conjugate that is conjugated to avidin. For still another example, the polypeptide complex or the bispecific polypeptide complex may be linked to a linker which further links to the conjugate. Examples of linkers include bifunctional coupling agents such as N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP) , succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) , iminothiolane (IT) , bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl) , active esters (such as disuccinimidyl suherate) , aldehydes (such as glutaraldehyde) , bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine) , bis-diazonium derivatives (such as bis- (p-diazoniumbenzoyl) -ethylenediamine) , diisocyanates (such as toluene 2, 6-diisocyanate) , and his-active fluorine compounds (such as 1, 5-difluoro-2, 4-dinitrobenzene) . Particularly preferred coupling agents include N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP) (Carlsson et al., Biochem. J. 173: 723-737 (1978) ) and N-succinimidyl-4- (2-pyridylthio) pentanoate (SPP) to provide for a disulphide linkage.

The conjugate can be a detectable label, a pharmacokinetic modifying moiety, a purification moiety, or a cytotoxic moiety. Examples of detectable label may include a fluorescent labels (e.g. fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red) , enzyme-substrate labels (e.g. horseradish peroxidase, alkaline phosphatase, luceriferases, glucoamylase, lysozyme, saccharide oxidases or β-D-galactosidase) , radioisotopes (e.g. ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ³⁵S, ³H, ¹¹¹In, ¹¹²In, ¹⁴C, ⁶⁴Cu, ⁶⁷Cu, ⁸⁶Y, ⁸⁸Y, ⁹⁰Y, ¹⁷⁷Lu, ²¹¹At, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ²¹²Bi, and ³²P, other lanthanides, luminescent labels) , chromophoric moiety, digoxigenin, biotin/avidin, a DNA molecule or gold for detection. In certain embodiments, the conjugate can be a pharmacokinetic modifying moiety such as PEG which helps increase half-life of the antibody. Other suitable polymers include, such as, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, copolymers of ethylene glycol/propylene glycol, and the like. In certain embodiments, the conjugate can be a purification moiety such as a magnetic bead. A “cytotoxic moiety” can be any agent that is detrimental to cells or that can damage or kill cells. Examples of cytotoxic moiety include, without limitation, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and analogs thereof, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine) , alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU) , cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin) , anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin) , antibiotics (e.g., dactinomycin (formerly actinomycin) , bleomycin, mithramycin, and anthramycin (AMC) ) , and anti-mitotic agents (e.g., vincristine and vinblastine) .

Methods for the conjugation of conjugates to proteins such as antibodies, immunoglobulins or fragments thereof are found, for example, in U.S. Pat. No. 5,208,020; U.S. Pat. No. 6,441,163; WO2005037992; WO2005081711; and WO2006/034488, which are incorporated herein by reference to the entirety.

Pharmaceutical composition

The present disclosure also provides a pharmaceutical composition comprising the polypeptide complex or the bispecific polypeptide complex provided herein and a pharmaceutically acceptable carrier.

The term “pharmaceutically acceptable” indicates that the designated carrier, vehicle, diluent, excipient (s) , and/or salt is generally chemically and/or physically compatible with the other ingredients comprising the formulation, and physiologically compatible with the recipient thereof.

A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is bioactivity acceptable and nontoxic to a subject. Pharmaceutical acceptable carriers for use in the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending/dispending agents, sequestering or chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.

Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, coloring agents, emulsifiers or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxanisol, butylated hydroxytoluene, and/or propyl gallate. As disclosed herein, inclusion of one or more antioxidants such as methionine in a pharmaceutical composition provided herein decreases oxidation of the polypeptide complex or the bispecific polypeptide complex. This reduction in oxidation prevents or reduces loss of binding affinity, thereby improving protein stability and maximizing shelf-life. Therefore, in certain embodiments, compositions are provided that comprise the polypeptide complex or the bispecific polypeptide complex disclosed herein and one or more antioxidants such as methionine.

To further illustrate, pharmaceutical acceptable carriers may include, for example, aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection, nonaqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil, antimicrobial agents at bacteriostatic or fungistatic concentrations, isotonic agents such as sodium chloride or dextrose, buffers such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcelluose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone, emulsifying agents such as Polysorbate 80 (TWEEN-80) , sequestering or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid) , ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Antimicrobial agents utilized as carriers may be added to pharmaceutical compositions in multiple-dose containers that include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride. Suitable excipients may include, for example, water, saline, dextrose, glycerol, or ethanol. Suitable non-toxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.

The pharmaceutical compositions can be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained release formulation, or powder. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrollidone, sodium saccharine, cellulose, magnesium carbonate, etc.

In certain embodiments, the pharmaceutical compositions are formulated into an injectable composition. The injectable pharmaceutical compositions may be prepared in any conventional form, such as for example liquid solution, suspension, emulsion, or solid forms suitable for generating liquid solution, suspension, or emulsion. Preparations for injection may include sterile and/or non-pyretic solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just prior to use, and sterile and/or non-pyretic emulsions. The solutions may be either aqueous or nonaqueous.

In certain embodiments, unit-dose parenteral preparations are packaged in an ampoule, a vial or a syringe with a needle. All preparations for parenteral administration should be sterile and not pyretic, as is known and practiced in the art.

In certain embodiments, a sterile, lyophilized powder is prepared by dissolving the polypeptide complex or the bispecific polypeptide complex as disclosed herein in a suitable solvent. The solvent may contain an excipient which improves the stability or other pharmacological components of the powder or reconstituted solution, prepared from the powder. Excipients that may be used include, but are not limited to, water, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agent. The solvent may contain a buffer, such as citrate, sodium or potassium phosphate or other such buffer known to those of skill in the art at, in one embodiment, about neutral pH. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those of skill in the art provides a desirable formulation. In one embodiment, the resulting solution will be apportioned into vials for lyophilization. Each vial can contain a single dosage or multiple dosages of the polypeptide complex, the bispecific polypeptide complex provided herein or composition thereof. Overfilling vials with a small amount above that needed for a dose or set of doses (e.g., about 10%) is acceptable so as to facilitate accurate sample withdrawal and accurate dosing. The lyophilized powder can be stored under appropriate conditions, such as at about 4 ℃ to room temperature.

Reconstitution of a lyophilized powder with water for injection provides a formulation for use in parenteral administration. In one embodiment, for reconstitution the sterile and/or non-pyretic water or other liquid suitable carrier is added to lyophilized powder. The precise amount depends upon the selected therapy being given, and can be empirically determined.

Method of treatment

Therapeutic methods are also provided, comprising: administering a therapeutically effective amount of the polypeptide complex or the bispecific polypeptide complex provided herein to a subject in need thereof, thereby treating or preventing a condition or a disorder. In certain embodiments, the subject has been identified as having a disorder or condition likely to respond to the polypeptide complex or the bispecific polypeptide complex provided herein.

“Treating” or “treatment” of a condition as used herein includes preventing or alleviating a condition, slowing the onset or rate of development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or ending symptoms associated with a condition, generating a complete or partial regression of a condition, curing a condition, or some combination thereof.

The therapeutically effective amount of the polypeptide complex and the bispecific polypeptide complex provided herein will depend on various factors known in the art, such as for example body weight, age, past medical history, present medications, state of health of the subject and potential for cross-reaction, allergies, sensitivities and adverse side-effects, as well as the administration route and extent of disease development. Dosages may be proportionally reduced or increased by one of ordinary skill in the art (e.g., physician or veterinarian) as indicated by these and other circumstances or requirements.

In certain embodiments, the polypeptide complex or the bispecific polypeptide complex provided herein may be administered at a therapeutically effective dosage of about 0.01 mg/kg to about 100 mg/kg (e.g., about 0.01 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, or about 100 mg/kg) . In certain of these embodiments, the polypeptide complex or the bispecific polypeptide complex provided herein is administered at a dosage of about 50 mg/kg or less, and in certain of these embodiments the dosage is 10 mg/kg or less, 5 mg/kg or less, 1 mg/kg or less, 0.5 mg/kg or less, or 0.1 mg/kg or less. In certain embodiments, the administration dosage may change over the course of treatment. For example, in certain embodiments the initial administration dosage may be higher than subsequent administration dosages. In certain embodiments, the administration dosage may vary over the course of treatment depending on the reaction of the subject.

Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response) . For example, a single dose may be administered, or several divided doses may be administered over time.

The polypeptide complex or the bispecific polypeptide complex provided herein may be administered by any route known in the art, such as for example parenteral (e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular, or intradermal injection) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal, or topical) routes.

In certain embodiments, the condition or disorder treated by the polypeptide complex or the bispecific polypeptide complex provided herein is cancer or a cancerous condition, autoimmune diseases, infectious and parasitic diseases, cardiovascular diseases, neuropathies, neuropsychiatric conditions, injuries, inflammations, or coagulation disorder.

With regard to cancer, “treating” or “treatment” may refer to inhibiting or slowing neoplastic or malignant cell growth, proliferation, or metastasis, preventing or delaying the development of neoplastic or malignant cell growth, proliferation, or metastasis, or some combination thereof. With regard to a tumor, “treating” or “treatment” includes eradicating all or part of a tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of a tumor, or some combination thereof.

For example, with regard to the use of the polypeptide complex or bispecific polypeptide complex disclosed herein to treat cancer, a therapeutically effective amount is the dosage or concentration of the polypeptide complex capable of eradicating all or part of a tumor, inhibiting or slowing tumor growth, inhibiting growth or proliferation of cells mediating a cancerous condition, inhibiting tumor cell metastasis, ameliorating any symptom or marker associated with a tumor or cancerous condition, preventing or delaying the development of a tumor or cancerous condition, or some combination thereof.

In certain embodiments, the conditions and disorders include tumors and cancers, for example, non-small cell lung cancer, small cell lung cancer, renal cell cancer, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric carcinoma, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, leukemia, lymphomas, myelomas, mycoses fungoids, merkel cell cancer, and other hematologic malignancies, such as classical Hodgkin lymphoma (CHL) , primary mediastinal large B-cell lymphoma, T-cell/histiocyte-rich B-cell lymphoma, EBV-positive and -negative PTLD, and EBV-associated diffuse large B-cell lymphoma (DLBCL) , plasmablastic lymphoma, extranodal NK/T-cell lymphoma, nasopharyngeal carcinoma, and HHV8-associated primary effusion lymphoma, Hodgkin's lymphoma, neoplasm of the central nervous system (CNS) , such as primary CNS lymphoma, spinal axis tumor, brain stem glioma.

In certain embodiments, the conditions and disorders include a CD19-related disease or condition, such as, B cell lymphoma, optionally Hodgkin lymphoma or non-Hodgkin lymphoma, wherein the non-Hodgkin lymphoma comprises: Diffuse large B-cell lymphoma (DLBCL) , Follicular lymphoma, Marginal zone B-cell lymphoma (MZL) , Mucosa-Associated Lymphatic Tissue lymphoma (MALT) , Small lymphocytic lymphoma (chronic lymphocytic leukemia, CLL) , or Mantle cell lymphoma (MCL) , Acute Lymphoblastic Leukemia (ALL) , or Waldenstrom's Macroglobulinemia (WM) .

In certain embodiments, the conditions and disorders include hyperproliferative conditions or infectious diseases that can be treated via regulation of immune responses by CTLA-4 and/or PD-1. Examples of hyperproliferative conditions include, but are not limited to, solid tumors, hematological cancers, soft tissue tumors, and metastatic lesions.

The polypeptide complex or the bispecific polypeptide complex may be administered alone or in combination with one or more additional therapeutic means or agents.

In certain embodiments, when used for treating cancer or tumor or proliferative disease, the polypeptide complex or the bispecific polypeptide complex provided herein may be administered in combination with chemotherapy, radiation therapy, surgery for the treatment of cancer (e.g., tumorectomy) , one or more anti-emetics or other treatments for complications arising from chemotherapy, or any other therapeutic agent for use in the treatment of cancer or any medical disorder that related. “Administered in combination” as used herein includes administration simultaneously as part of the same pharmaceutical composition, simultaneously as separate compositions, or at different timings as separate compositions. A composition administered prior to or after another agent is considered to be administered “in combination” with that agent as the phrase is used herein, even if the composition and the second agent are administered via different routes. Where possible, additional therapeutic agents administered in combination with the polypeptide complex or the bispecific polypeptide complex provided herein are administered according to the schedule listed in the product information sheet of the additional therapeutic agent, or according to the Physicians'Desk Reference (Physicians'Desk Reference, 70th Ed (2016) ) or protocols well known in the art.

In certain embodiments, the therapeutic agents can induce or boost immune response against cancer. For example, a tumor vaccine can be used to induce immune response to certain tumor or cancer. Cytokine therapy can also be used to enhance tumor antigen presentation to the immune system. Examples of cytokine therapy include, without limitation, interferons such as interferon-α, -β, and –γ, colony stimulating factors such as macrophage-CSF, granulocyte macrophage CSF, and granulocyte-CSF, interleukins such IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, and IL-12, tumor necrosis factors such as TNF-α and TNF-β. Agents that inactivate immunosuppressive targets can also be used, for example, TGF-beta inhibitors, IL-10 inhibitors, and Fas ligand inhibitors. Another group of agents include those that activate immune responsiveness to tumor or cancer cells, for example, those enhance T cell activation (e.g. agonist of T cell costimulatory molecules such as CTLA-4, ICOS and OX-40) , and those enhance dendritic cell function and antigen presentation.

Kits

The present disclosure further provides kits comprising the polypeptide complex or the bispecific polypeptide complex provided herein. In some embodiments, the kits are useful for detecting the presence or level of, or capturing or enriching one or more target of interest in a biological sample. The biological sample can comprise a cell or a tissue.

In some embodiments, the kit comprises the polypeptide complex or the bispecific polypeptide complex provided herein which is conjugated with a detectable label. In certain other embodiments, the kit comprises an unlabeled polypeptide complex or the bispecific polypeptide complex provided herein, and further comprises a secondary labeled antibody which is capable of binding to the unlabeled polypeptide complex or the bispecific polypeptide complex provided herein. The kit may further comprise an instruction of use, and a package that separates each of the components in the kit.

In certain embodiments, the polypeptide complex or the bispecific polypeptide complex provided herein are associated with a substrate or a device. Useful substrate or device can be, for example, magnetic beads, microtiter plate, or test strip. Such can be useful for a binding assay (such as ELISA) , an immunographic assay, capturing or enriching of a target molecule in a biological sample.

The following examples are provided to better illustrate the present disclosure and are not to be interpreted as limiting the scope of the invention. All specific compositions, materials, and methods described below, in whole or in part, fall within the scope of the present disclosure. These specific compositions, materials, and methods are not intended to limit the invention, but merely to illustrate specific embodiments falling within the scope of the invention. One skilled in the art may develop equivalent compositions, materials, and methods without the exercise of inventive capacity and without departing from the scope of the invention. It will be understood that many variations can be made in the procedures herein described while still remaining within the bounds of the present disclosure. It is the intention of the inventors that such variations are included within the scope of the disclosure.

EXAMPLES

The design, generation, characterization, and testing results of exemplary bispecific antibodies are described in Examples 1-18 of PCT/CN2018/106766 and are incorporated herein by reference.

Table 11. Designs and names of chimeric constant regions (CBeta/CAlpha)

Table 12. Domains and SEQ ID NOS of chimeric constant regions (CBeta/CAlpha)

Table 13. Designs and names of Design_2 (CBeta/CAlpha) without glycosylation sites

Table 14. Domains and SEQ ID NOs of Design_2 (CBeta/CAlpha) without glycosylation sites

Table 15. Designs and names of chimeric constant regions (CBeta/Cpre-Alpha)

Table 16. Domains and SEQ ID NOs of chimeric constant regions (CBeta/Cpre-Alpha)

Table 17. Designs and names of chimeric constant regions (CGamma/CDelta)

Table 18. Domains and SEQ ID NOs of chimeric constant regions (CGamma/CDelta)

Certain sequences of bispecific CTLA-4 x PD-1 antibodies are provided below:

Variable heavy chain and light chain sequences of anti-CD3 and anti-CD19 binding moieties from W3438-T3U4. E17-1. uIgG4. SP and W3438-T3U4. F16-1. uIgG4. SP are provided below:

Full-length W3438-T3U4. E17-1. uIgG4. SP and W3438-T3U4. F16-1. uIgG4. SP sequences are provided below:

Relevant sequences of W3248-U6T1. G25R-1. uIgG4. SP are provided below:

Relevant sequences of W3248-U6T5. G25-1. uIgG4. SP are provided below:

Example: Bispecific Anti-CD13 x CD19 WuXiBody and Bispecific Anti-CTLA-4 x PD-1 WuXiBody

The structure of CAlpha of TCR is not a typical Ig domain. CAlpha comprises certain loop structures, especially at the c-terminus region. Murine TCR is relatively more stable than human TCR. The sequence identity between human and murine TCR constant regions is around 72%. Certain residues in murine TCR, e.g., four residues SDVP (at positions 92-95, as shown in Figure 10) located at the CAlpha c-terminus region and two residues K17 and A21 (as shown in Figure 11) on CBeta, may affect the stability of murine TCR. Engineered CAlpha and CBeta that comprise mutations with corresponding amino acid residues from murine TCR may improve stability and/or expression levels of certain polypeptide complexes disclosed herein.

Replacing the CAlpha c-terminus residues by certain human IgG1 sequence segment may improve the stability of soluble TCR in Fc form. CAlpha, particularly at the CAlpha c-terminus, may be a fragile region, and modifications of CAlpha, for example its C-terminus, may improve the stability of the polypeptide complexes disclosed herein.

Modifications of certain amino acid residues on CBeta, in particular the residues that are structurally or spatially close to the C-terminus of CAlpha, may also improve the stability of the polypeptide complexes disclosed herein. Not wishing to be bound by any theory, amino acid residues on CBeta that are structurally or spatially located close to CAlpha c-terminus, could be mutated to form stronger interaction (s) with CAlpha c-terminus so as to stabilize it, and thus the stability of the TCR constant region (CAlpha-CBeta pairing) could be improved.

Generation of Proteins Listed in Tables 21 and 22

The VL, VH, C kappa, CH1 genes were amplified by PCR from existing in-house DNA templates. CAlpha and CBeta genes were synthesized by Genewiz Inc. Anti-CD19 native or anti-CD3 chimeric light chain genes were inserted into a linearized vector containing a CMV promoter and a kappa signal peptide. The DNA fragments of Anti-CD3 VH-CBeta were inserted into a linearized vector containing human IgG4 (with S228P mutation) constant region CH2-CH3 with a knob mutation. The DNA fragments of Anti-CD19 VH-CH1 were inserted into a linearized vector containing human IgG4 (with S228P mutation) constant region CH2-CH3 with a hole mutation. The vector contains a CMV promoter and a human antibody heavy chain signal peptide.

DNA sequence encoding anti-PD-1 heavy chain variable region, constant region 1, anti-CTLA-4 heavy chain variable region, TCR beta constant region, and IgG4 (S228P)

constant region

2 and 3, linked from 5’end to 3’end, were cloned into a modified pcDNA3.3 expression vector. DNA sequence encoding anti-CTLA-4 antibody light chain variable region on the 5’of TCR alpha constant region was cloned into another modified pcDNA3.3 expression vector. Anti-PD-1 light chain was cloned into the third modified pcDNA3.3 expression vector.

Expression and Purification

Heavy chain and light chain expression plasmids were co-transfected into Expi293 cells using Expi293 expression system kit (ThermoFisher-A14635) according to the manufacturer's instructions. Five days after transfection, the supernatants were collected and the protein was purified using Protein A column (GE Healthcare-17543 802) and further size exclusion column (GE Healthcare-17104301) . Antibody concentration was measured by Nano Drop. The purity of proteins was evaluated by SDS-PAGE and HPLC-SEC.

Differential Scanning Fluorimetry (DSF) Characterization

The T _m of antibodies was investigated using QuantStudio ^TM 7 Flex Real-Time PCR system (Applied Biosystems) . 19 μL of antibody solution was mixed with 1 μL of 62.5 X SYPRO Orange solution (Invitrogen) and transferred to a 96 well plate (Biosystems) . The plate was heated from 26 ℃ to 95 ℃ at a rate of 0.9 ℃/min, and the resulting fluorescence data was collected. The negative derivatives of the fluorescence changes with respect to different temperatures were calculated, and the maximal value was defined as melting temperature T _m. If a protein has multiple unfolding transitions, the first two T _m were reported, named as T _m1 and T _m2. Data collection and T _m calculation were conducted automatically by the operation software (QuantStudio ^TM Real Time PCR software v1.3) .

Differential Scanning Calorimetry (DSC) Characterization

The DSC analysis was performed by a Malvern DSC System. The protein sample was first diluted to 1 mg/mL with formulation buffer before analysis. 400 μL respective formulation buffer was added to a 96-well plate as reference and 400 μL protein sample was added. The samples were heated from 10℃ to 95℃ at a heating rate of 90℃/h in the DSC system. The DSC results (Tm Onset and Tm values) were analyzed by vendor’s software (MicroCal PEAQ DSC Software 1.30 and Malvern) .

Acceleration Thermostability Test

Incubated the antibodies at 40 ℃ using Eppendorf Constant temperature mixer, and measure the absorption value of protein solution at 280 nm by Nanodrop 2000, record the appearance and detect the purity by SEC-HPLC after Day 0, Day 1, Day 3, Day 7, Day 14 and Day 28.

Rat PK &ADA test

Five animals as one group were used in this study. Animals were administered with antibodies at 10 mg/kg once in 10 minutes intravenous infusion, individually. Baseline samples (pre dose) were collected on Study Day-1. PK samples were collected at 0.5h, 2h, 6h, 24h, 48h, 72h, 120h, 168h, 240h, 288h, 336h and 504h after dosing.

Anti-drug antibody (ADA) samples were collected at pre-dose (Day-1) , and post-dose at 312h and 648h. Serum concentrations of antibodies and ADA in serum samples were determined by ELISA.

Results

Various design strategies were conducted in this Example to optimize the stability of WuXiBody format, including, for example, modifications of the CAlpha-CBeta interface, one or more interface disulfide bond, the CBeta FG loop, and one or more N-glycosylation and O-glycosylation sites. In particular, the modifications were made to CBeta and the c-terminus of CAlpha, as well as the residues that are not at the c-terminus in the amino acid sequences but structurally or spatially close to the c-terminus.

Various mutations, as detailed in Tables 21 and 22, were carried out on an asymmetric WuXiBody format [Anti-CD3 x CD19] , and one symmetric WuXiBody format [Anti-CTLA-4 x PD-1] to carry out the engineering. Such modifications or similar modifications may be carried out on other WuXiBody formats disclosed herein.

Protein Production

All the proteins listed in Table 21 and Table 22 were constructed and produced. Most of the proteins listed in Tables 21 and 22 had comparable expression level to W3438-T3U4. E17-1. uIgG4. SP and W3248-U6T1. G25R-1. uIgG4. SP, respectively, and some of them had better yield than the corresponding reference protein. Protein data of certain designs are listed in Tables 23-25. SDS-PAGE and SEC-HPLC characterizations of certain proteins are depicted in Figures 5-7.

Table 23. Parameters of one batch of engineered WuXiBody molecules after purification.

Table 24. Parameters of one batch of engineered WuXiBody molecules after purification.

Table 25. Parameters of one batch of engineered WuXiBody molecules after purification.

Thermostability Characterization

W3248-U6T1. G25R-1. uIgG4. SP, as well as its further engineered versions (W3248-U6T1. G25R-8. uIgG4. SP and W3248-U6T1. G25R-23. uIgG4. SP) were selected for further characterization using DSC. Figure 8 shows that the main differences between these three proteins were located in the curves before the first peak. The two further engineered molecules had curves shifted to the right, indicating they had relatively stronger resistance to temperature increase. This result further confirmed that CAlpha c-terminus region is a relatively unstable region in TCR constant domain, but the stabilities can be improved by proper design and engineering.

Accelerated Thermostability Test

The effectiveness of the modifications on CAlpha and CBeta was further inspected in thermo-stressed condition. A number of proteins, as shown in Table 26, were prepared at 5 mg/ml and >90%purity, and were incubated at 40 ℃ for more thab one month. The purities of these samples were monitored at Day 1, Day 3, Day 7, Day 14, Day 21, Day 28 and Day 34. Table 26 and Table 27 listed the purity change of WuXiBody molecules, respectively.

Table 26. Concentration and purity changes of molecules in asymmetric WuXiBody format. All samples were in buffer: 20 mM Citrate buffer, 8% (w/v) Sucrose, 0.02% (w/v) PS80, pH6.0

Table 27. Concentration and purity changes of molecules in asymmetric WuXiBody format. All samples were in buffer: 20mM Phosphate, 120mM NaCl, 0.02%PS80, pH 7.0

For asymmetric WuXiBody format, the purity of the reference WuXiBody molecule reduced around 7.7%after 34 days, while the purity of the best performed design declined 3.8%. This is the design adopting the six residues from murine TCR that were proved helpful to stabilize human TCR (see Table 21 and Table 22 above) . In symmetric WuXiBody format, the same design still displayed the best resistance to the thermo-stress, although the symmetric WuXiBody format molecules overall were less stable due to more complex molecular structure. It had 11.4%purity loss while the reference molecule had 23.1%drop. These data confirmed that engineering the TCR c-terminus region can effectively stabilize WuXiBody molecules.

Rat PK Study

Since the thermostability of WuXiBody platform has been improved, it is likely that other properties of the WuXiBody bispecific molecules may also benefit from this engineering. The PK of the designed molecules in rats using intravenous injections were evaluated. Figure 9 displayed the PK profiles of both the reference and one engineered molecule (i.e., W3248-U6T1. G25R-8. uIgG. SP) , in which the optimized molecule clearly improved the elimination phase and extended the protein half-life from 143 hours to 173 hours. Other parameters like clearance was also improved from 143 ml/day/kg to 173 ml/day/kg, shown in Table 28. This result confirmed that the engineering at the c-terminus of TCR constant region can effectively improve the rat PK of WuXiBody bispecific molecules.

Table 28. Pharmacokinetic parameters of symmetric WuXiBody proteins in rats

Compound	W3248-U6T1. G25R-1. uIgG. SP	W3248-U6T1. G25R-8. uIgG4V1
Dose
	10 mg/kg, iv, n=5	10 mg/kg, iv, n=5
t _1/2 (h)	143	173
C _max (μg/mL)	169	137
AUC _0-t (h*μg/ml)	11375	14214
Cl_obs (ml/day/kg)	20.3	14.9
MRTINF_obs (h)	161	227
Vss_obs (ml/kg)	129	141

While the disclosure has been particularly shown and described with reference to specific embodiments, it should be understood by those having skill in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the present disclosure as disclosed herein.

Claims

A polypeptide complex comprising a first antigen-binding moiety and a second antigen-binding moiety, wherein:

the first antigen-binding moiety comprises

a first polypeptide comprising, from N-terminus to C-terminus, a first heavy chain variable domain (VH) of a first antibody operably linked to a first T cell receptor (TCR) constant region (C1) , and

a second polypeptide comprising, from N-terminus to C-terminus, a first light chain variable domain (VL) of the first antibody operably linked to a second TCR constant region (C2) ,

wherein:

C1 comprises an engineered CBeta or an engineered CAlpha,

C2 comprises an engineered CBeta or an engineered CAlpha,

C1 and C2 are capable of forming a dimer comprising at least one non-native interchain bond between C1 and C2, and the non-native interchain bond is capable of stabilizing the polypeptide complex, and

the first antibody has a first antigenic specificity; and

the second antigen-binding moiety has a second antigenic specificity which is different from the first antigenic specificity.
The polypeptide complex of claim 1, wherein the engineered CAlpha comprises one or more modifications that improve the stability of the engineered CAlpha, the engineered CBeta comprises one or more modifications that improve the stability of the engineered CBeta, or the interface of the engineered CBeta and the engineered CAlpha comprise one or more modifications that improve the stability of the engineered CBeta and the engineered CAlpha, and wherein the one or more modifications are capable of stabilizing the polypeptide complex.
The polypeptide complex of claim 2, the engineered CAlpha and/or engineered CBeta comprise at least one residue substituted with at least one corresponding amino acid residue that contributes to the stability of murine TCR to improve stability and/or expression level of the polypeptide complex.
The polypeptide complex of claim 3, the engineered CAlpha comprises at least one mutated residue selected from P92S, E93D, S94V, and S95P, and/or the engineered CBeta comprises at least one mutated residue selected from E17K and S21A.
The polypeptide complex of claim 2, wherein an FG loop is absent in the engineered CBeta and the FG loop is replaced with amino acid residues selected from “YPSN” , “PS” , “NG” , or “GN. ”
The polypeptide complex of claim 2, wherein the amino acid residues “YGLSENDEWTQDRAKPVT” is absent in the engineered CBeta, and the engineered CBeta comprises amino acid residues “YPSN” , “PS” , “NG” , or “GN” in place of “YGLSENDEWTQDRAKPVT” .
The polypeptide complex of claim 2, the engineered CAlpha comprises at least one mutated residue selected from T33V, P84R, V10I, F26A, T33W, F78H, P84G, A65S, F72Y, P8E, V10I, F26R, F29T, A63T, and P84G.
The polypeptide complex of claim 2, wherein at least one native residue is present in the engineered CAlpha and/or CBeta, wherein the at least one native residue is C96 in CAlpha, A128 in CBeta, D129 in CBeta, and C130 in CBeta.
The polypeptide complex of claim 2, the engineered CAlpha and/or CBeta comprise at least one mutated residue to form one or more non-native disulfide bonds, wherein the at least one mutated residue is selected from T46C on CAlpha, S76C on CBeta; S62C on CAlpha; L51C on CAlpha; Y11C on CAlpha, S16C on CBeta; S16C on CAlpha, V12C on CBeta; S16C on CAlpha, E14C on CBeta; T46C on CAlpha, D58C on CBeta; L13C on CAlpha, S16C on CBeta; S62C on CAlpha, R78C on CBeta; L13C on CAlpha, F13C on CBeta; V23C on CAlpha, F13C on CBeta; and Y44C on CAlpha, L62C on CBeta.
The polypeptide complex of claim 2, wherein the engineered CAlpha and/or CBeta comprise at least one mutated residue at the CAlpha-CBeta interface selected from S62R on CAlpha and R78G on CBeta.
The polypeptide complex of claim 2, wherein the engineered CAlpha comprises one or more mutated residues that comprise a fragment derived from a human IgG1 hinge sequence, a human kappa light c-terminal sequence, a PDB-5DK3 (human IgG4) CH1 region sequence, a human lambda light chain c-terminus region sequence, a PDB-1IGT (mouse IgG2a) CH1 region sequence, a PDB-1IGT (mouse IgG2a) kappa light chain c-terminus region sequence, a human IgG2 hinge region sequence, PDB-5E8E (human IgA) CH1 region sequence, a PDB-5E8E (human IgA) kappa light chain c-terminus region sequence, a PDB-1DEE (human IgM) CH1 region sequence, a human IgE CH1 region sequence, or a human IgD CH1 region sequence.
The polypeptide complex of claim 11, wherein the amino acid residues “PESS” is absent at the C-terminus of the engineered CAlpha and are replaced by amino acid residues “VEPKS, ” “EPKS” , “NRGE” , “PLAPC” , “APTEC” , “LAPVC” , “NRNEC” , “ERKSC” , “PLSLC” , “LRGEC” , “PLVSC” , “PLTRC” , or “IISGC” .
The polypeptide complex of claim 2, wherein the engineered CBeta comprises one or more mutated residues that comprise a fragment derived from a human IgG1 hinge sequence, a human kappa light c-terminal sequence, a PDB-5DK3 (human IgG4) CH1 region sequence, a human lambda light chain c-terminus region sequence, a PDB-1IGT (mouse IgG2a) CH1 region sequence, a PDB-1IGT (mouse IgG2a) kappa light chain c-terminus region sequence, a human IgG2 hinge region sequence, PDB-5E8E (human IgA) CH1 region sequence, a PDB-5E8E (human IgA) kappa light chain c-terminus region sequence, a PDB-1DEE (human IgM) CH1 region sequence, a human IgE CH1 region sequence, or a human IgD CH1 region sequence.
The polypeptide complex of claim 13, wherein the amino acid residues “WGR” is absent at the C-terminus of the engineered CBeta and are replaced by amino acid residues “EPKS” or “NRGE” .
The polypeptide complex of claim 2, the engineered CBeta comprises a Gly amino acid residue at the C-terminal end of the engineered CBeta such that the Gly amino acid residue is adjacent to or forms part of the hinge region.
The polypeptide complex of claim 2, the engineered CAlpha comprises a deletion of 1 to 22 amino acid residues at or around its C-terminus.
The polypeptide complex of claim 16, the engineered CAlpha comprises a deletion of amino acid residues “FFPSPESS” at the C-terminus of the engineered CAlpha.
The polypeptide complex of claim 2, the engineered CAlpha and CBeta comprise a mutation S91A on the CAlpha.
The polypeptide complex of claim 2, the engineered CAlpha and/or CBeta comprise at least one more native glycosylation site selected from N34, N68, and N79 on CAlpha, and N69 on CBeta.
The polypeptide complex of claim 2, the engineered C2 comprises any one of SEQ ID NOs: 418-431 and 444-457, and/or the engineered C1 comprises any one of SEQ ID NOs: 432- 443 and 458-466; or the engineered C1 comprises any one of SEQ ID NOs: 418-431 and 444-457, and/or the engineered C2 comprises any one of SEQ ID NOs: 432-443 and 458-466.
The polypeptide complex of any of claims 1-20 and 37, wherein the first antigenic specificity is directed to an exogenous antigen, an endogenous antigen, an autoantigen, a neoantigen, a viral antigen or a tumor antigen.
The bispecific polypeptide complex of any of claims 1-21 and 37, wherein one of the first and the second antigenic specificities is directed to a T-cell specific receptor molecule and/or a natural killer cell (NK cell) specific receptor molecule, and the other is directed to a tumor associated antigen.
The bispecific polypeptide complex of any of claims 1-22 and 37, wherein one of the first and the second antigenic specificities is directed to CD3, and the other is directed to CD19.
The bispecific polypeptide complex of any of claims 1-22 and 37, wherein one of the first and the second antigenic specificities is directed to PD1, and the other is directed to CTLA-4.
A conjugate comprising the polypeptide complex of any of claims 1-24 and 37 conjugated to a moiety.
An isolated polynucleotide encoding the polypeptide complex of any of claims 1-24 and 37.
An isolated vector comprising the polynucleotide of claim 26.
A host cell comprising the isolated polynucleotide of claim 26 or the isolated vector of claim 27.
A method of expressing the polypeptide complex of any of preceding claims, comprising culturing the host cell of claim 28 under the condition at which the polypeptide complex, or the bispecific polypeptide complex is expressed.
A method of producing the polypeptide complex of any of claims 1-24 and 37 comprising:

a) introducing to a host cell:

a first polynucleotide encoding a first polypeptide comprising, from N-terminus to C-terminus, a first heavy chain variable region (VH) of a first antibody operably linked to a first TCR constant region (C1) , and

a second polynucleotide encoding a second polypeptide comprising, from N-terminus to C-terminus, a first light chain variable domain (VL) of the first antibody operably linked to a second TCR constant region (C2) ,

wherein:

C1 and C2 are capable of forming a dimer comprising at least one non-native interchain bond between C1 and C2, and the non-native interchain bond is capable of stabilizing the dimer, and

the first antibody has a first antigenic specificity,

b) allowing the host cell to express the polypeptide complex.
The method of claim 30, further comprising:

a) introducing to the host cell

one or more additional polynucleotides encoding a second antigen-binding moiety,

wherein the second antigen-binding moiety has a second antigenic specificity different from the first antigenic specificity,

b) allowing the host cell to express the bispecific polypeptide complex.
The method of any of claims 30-31, further comprising isolating the polypeptide complex or the bispecific polypeptide complex.
A composition comprising the polypeptide complex of any of claims 1-24 and 37.
A pharmaceutical composition comprising the polypeptide complex of any of claims 1-24 and 37 and a pharmaceutically acceptable carrier.
A method of treating a condition in a subject in need thereof, comprising administrating to the subject a therapeutically effective amount of the polypeptide complex of any of claims 1-24 and 37.
The method of claim 35, wherein the condition can be alleviated, eliminated, treated, or prevented when the first antigen and the second antigen are both modulated.
The polypeptide complex of claim 20, wherein the engineered C2 comprises SEQ ID NO: 421, and the engineered C1 comprises SEQ ID NO: 439; or the engineered C1 comprises SEQ ID NO: 421, and the engineered C2 comprises SEQ ID NO: 439.