WO2020057610A1

WO2020057610A1 - Novel bispecific anti-ctla-4/pd-1 polypeptide complexes

Info

Publication number: WO2020057610A1
Application number: PCT/CN2019/106730
Authority: WO
Inventors: Zhuozhi Wang; Jianqing Xu; Jing Li
Original assignee: Wuxi Biologics (Shanghai) Co., Ltd.; WuXi Biologics Ireland Limited
Priority date: 2018-09-20
Filing date: 2019-09-19
Publication date: 2020-03-26

Abstract

The present disclosure provides bispecific anti-CTLA-4 x PD-1 polypeptide complexes that contain antibody variable regions fused to the TCR constant regions, methods of producing the bispecific anti-CTLA-4 x PD-1polypeptide complexes, methods of treating diseases or conditions using the bispecific anti-CTLA-4 x PD-1 polypeptide complexes, host cells expressing the bispecific anti-CTLA-4 x PD-1 polypeptide complexes, and compositions and pharmaceutical compositions comprising the bispecific anti-CTLA-4 x PD-1 polypeptide complexes.

Description

Novel bispecific Anti-CTLA-4/PD-1 polypeptide complexes

SEQUENCE LISTING

The present application is filed with a Sequence Listing in electronic form. The entire contents of the Sequence Listing are hereby incorporated by reference.

FIELD OF THE INVENTION

The present disclosure generally relates to bispecific anti-CTLA-4 x PD-1 polypeptide complexes comprising antibody variable regions fused to TCR constant regions.

BACKGROUND

Bispecific antibodies are growing to be the new category of therapeutic antibodies. They can bind two different targets or two different epitopes on a target, creating additive or synergistic effects superior to the effects of individual antibodies. A lot of antibody engineering efforts have been put into designing new bispecific formats, such as DVD-Ig, CrossMab, and BiTE (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 isa 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 ofa natural IgG. In order to facilitate downstream development, it is desired that such bispecific molecules can be easily produced like normal IgG from a single host cell with a 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.

By introducing mutations in the Fc region, such as “knobs-into-holes” (Ridgway et al., Protein Engineering, 9 (7) , pp. 617–21 (1996) ; Merchant et al., Nature Biotechnology, 16 (7) , pp. 677–681 (1998) ) , electrostatics (Gunasekaran et al., Journal of Biological Chemistry, 285 (25) , pp. 19637–19646 (2010) ) or negative state designs (Kreudenstein et al., mAbs, 5 (5) , pp. 646–654 (2013) ; Leaver-Fay et al., Structure, 24 (4) , pp. 641–651 (2016) ) , the preferred heterodimeric assembly of two different heavy chains has been accomplished. However, the selective pairing of light-heavy chains of each individual antibody remains challenging. The interface between light-heavy chains includes the variable domain (VH-VL) and the constant domain (CH1-CL) . Several strategies have been applied to design orthogonal interfaces to facilitate cognate pairing. Roche swapped the domains of CH1 and CL and created the CrossMab platform (Schaefer et al., Proceedings of the National Academy of Sciences of the United States of America, 108 (27) , pp. 11187–11192 (2011) ) . MedImmune introduced alternatively disulphide bonds (Mazor et al., mAbs, 7 (2) , pp. 377–389 (2015) ) . Amgen made further electrostatic interactions in the CH1-CL region (Liu et al., Journal of Biological Chemistry, 290 (12) , pp. 7535–7562 (2015) ) . Lilly (Lewis et al., Nature Biotechnology, 32 (2) , pp. 191–198 (2014) ) and Genentech (Dillon et al., mAbs, 9 (2) , pp. 213–230 (2017) ) introduced mutations in both variable and constant domains.

Cancer immunotherapy has become a hot research area for treating cancer. Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is one of the validated targets of immune checkpoints. After T cell activation, CTLA-4 quickly expresses on those T cells, generally within one hour of antigen engagement with TCR. CTLA-4 can inhibit T cell signaling through competition with CD28, which mediates a well-characterized T cell co-stimulatory signal. CD28 binding to its ligands CD80 (B7-1) and CD86 (B7-2) on antigen presenting cells leads to T cell proliferation by inducing production of interleukin-2 and anti-apoptotic factors. Due to much higher binding affinity of CTLA-4 to CD80 and CD86 than that of CD28, CTLA-4 can out-compete CD28 for binding toCD80 and CD86, leading to suppression of T cell activation. In addition to induced expression on activated T cells, CTLA-4 is constitutively expressed on the surface of regulatory T cells (Treg) , suggesting that CTLA-4 may be required for contact-mediated suppression and is associated with Treg production of immunosuppressive cytokines such as transforming growth factor beta and iterleukin-10.

CTLA-4 blockade can induce tumor regression, as demonstrated in a number of preclinical and clinical studies. Two antibodies against CTLA-4 are in clinical development. Ipilimumab (MDX-010, BMS-734016) , a fully human anti-CTLA-4 monoclonal antibody of IgG1-kappa isotype, is an immunomodulatory agent that has been approved as monotherapy for treatment of advanced melanoma. The proposed mechanism of action for Ipilimumab is interference in the interaction of CTLA-4, which is expressed on a subset of activated T cells, with CD80/CD86 molecules on professional antigen presenting cells. This results in T-cell potentiation due to blockade of the inhibitory modulation of T-cell activation promoted by the CTLA-4 and CD80/CD86 interaction. The resulting T-cell activation, proliferation and lymphocyte infiltration into tumors, leads to tumor cell death. The commercial dosage form is a 5mg/ml concentrate solution for infusion. Ipilimumab is also under clinical investigation for other tumor types, including prostate and lung cancers. The second anti-CTLA-4 antibody in clinical development, Tremelimumab, was evaluated as monotherapy in melanoma and malignant mesothelioma.

Programmed Death-1 (PD-1, CD279) is a member of CD28 family expressed on activated T cells and other immune cells. Engagement of PD-1 inhibits function in these immune cells. PD-1 has two known ligands, PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273) , both belonging to the B7 family. PD-L1 expression is inducible on a variety of cell types in lymphoid and peripheral tissues, whereas PD-L2 is more restricted to myeloid cells including dendritic cells. The major role of the PD-1 pathway is to reduce inflammatory immune response in tissues and organs.

There is great need to design bispecific molecules, with desirable expression level and in vivo half-life, to both CTLA-4 and PD-1 antigens. Such bispecific anti-CTLA-4 x PD-1 polypeptide complexes can induce antitumor immunity through simultaneous blockade of both checkpoint molecules and are useful for treating various diseases or conditions including cancer. BRIEF SUMMARY OF THE INVENTION

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 comprises:

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 first light chain variable domain (VL) of the first antibody operably linked to a second TCR constant region (C2) , and

the second antigen-binding moiety comprises:

a second VH of a second antibody operably linked to an antibody heavy chain CH1 domain, and a second VL of the second antibody operably linked to an antibody light chain constant (CL) domain,

wherein: (a) one of the first and the second antigen-binding moiety is an anti-CTLA-4 binding moiety, and the other one is an anti-PD-1 binding moiety,

(b) the anti-CTLA-4 binding moiety is derived from an anti-CTLA-4 antibody comprising:

(i) a a heavy chain complementarity determining region (CDRH) 1consisting of SEQ ID NO: 5, a CDRH2 consisting of SEQ ID NO: 6, a CDRH3 consisting of SEQ ID NO: 7, a light chain complementarity determining region (CDRL) 1consisting of SEQ ID NO: 8, a CDRL2 consisting of SEQ ID NO: 9, and a CDRL3 consisting of SEQ ID NO: 10;

or (ii) a CDRH1 consisting of SEQ ID NO: 11, a CDRH2 consisting of SEQ ID NO: 12, a CDRH3 consisting of SEQ ID NO: 13, a CDRL1 consisting of SEQ ID NO: 14, a CDRL2 consisting of SEQ ID NO: 15, and a CDRL3 consisting of SEQ ID NO: 16,

(c) the anti-PD-1 binding moiety is derived from an anti-PD-1 antibody comprising:

a CDRH1 consisting of SEQ ID NO: 21, a CDRH2 consisting of SEQ ID NO: 22, a CDRH3 consisting of SEQ ID NO: 23, a CDRL1 consisting of SEQ ID NO: 24, a CDRL2 consisting of SEQ ID NO: 25, and a CDRL3 consisting of SEQ ID NO: 26.

In certain embodiments, the C1 described hereincomprises an engineered TCR beta constant region comprising one or more mutated residues selected from the group consisting of K9E, S56C, N69Q and C74A relative to a native human TCR beta constant region comprising the amino acid sequence of SEQ ID NO: 37; and/or

the C2 described herein comprises an engineered TCR alpha constant region comprising one or more mutated residues selected from the group consisting of N32Q, T47C, N66Q, and N77Q relative to a native human TCR alpha constant region comprising the amino acid sequence of SEQ ID NO: 35.

In certain embodiments, C1 comprises a S56C mutation and C2 comprises a T47C mutation to form a non-native interchain disulphide bond.

In one aspect, the present disclosure provides abispecific polypeptide complex comprising a first antigen-binding moiety associated with a second antigen-binding moiety, wherein:

the first antigen-binding moiety comprises:

a first heavy chain variable domain (VH) of a first antibody operably linked to a first Tcell receptor (TCR) constant region (C1) , and 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 comprising SEQ ID NO: 1and C2 comprises an engineered CAlpha comprising SEQ ID NO: 2, wherein amino acid C48 in SEQ ID NO: 1 and amino acid C41 in SEQ ID NO: 2 are capable of forming a non-native interchain disulphide bond, C1 and C2 are capable of forming a dimer, and the non-native interchain disulphide bond is capable of stabilizing the dimer, and

the second antigen-binding moiety comprises:

wherein: one of the first and the second antigen-binding moiety is an anti-CTLA-4 binding moiety, and the other antigen-binding moietyis an anti-PD-1 binding moiety,

the anti-CTLA-4 binding moiety is derived from an anti-CTLA-4 antibody comprising:

(i) a heavy chain CDR1 comprising SEQ ID NO: 5, a heavy chain CDR2 comprising SEQ ID NO: 6, aheavy chain CDR3 comprising SEQ ID NO: 7, a light chain CDR1 comprising SEQ ID NO: 8, a light chain CDR2 comprising SEQ ID NO: 9, and alight chain CDR3 comprising SEQ ID NO: 10; or (ii) a heavy chain CDR1 comprising SEQ ID NO: 11, a heavy chain CDR2 comprising SEQ ID NO: 12, a heavy chain CDR3 comprising SEQ ID NO: 13, a light chain CDR1 comprising SEQ ID NO: 14, a light chain CDR2 comprising SEQ ID NO: 15, and a light chain CDR3 comprising SEQ ID NO: 16,

the anti-PD-1 binding moiety is derived from an anti-PD-1 antibody comprising:

a heavy chain CDR1 comprising SEQ ID NO: 21, a heavy chain CDR2 comprising SEQ ID NO: 22, a heavy chain CDR3 comprising SEQ ID NO: 23, a light chain CDR1 comprising SEQ ID NO: 24, a light chain CDR2 comprising SEQ ID NO: 25, and a light chain CDR3 comprising SEQ ID NO: 26,

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 certain embodiments, the anti-CTLA-4 binding moiety of the bispecific polypeptide complex is derived from an CTLA-4 antibody comprising (i) a heavy chain variable domain sequence comprising SEQ ID NO: 17 and a light chain variable domain sequence comprising SEQ ID NO: 18, or (ii) a heavy chain variable domain sequence comprising SEQ ID NO: 19 and a light chain variable domain sequence comprising SEQ ID NO: 20.

In certain embodiments, the anti-PD-1 binding moiety of the bispecific polypeptide complex is derived from an anti-PD-1antibody comprising a heavy chain variable domain sequence comprising SEQ ID NO: 27 and a light chain variable domain sequence comprising SEQ ID NO: 28.

In certain embodiments, the bispecific polypeptide complex disclosed herein has a G25R WuXiBody structure (see Figure 1) and comprises a combination of three polypeptide sequences, wherein:

the first polypeptide sequence comprises a VL of an anti-PD-1 binding moiety operably linked to a CL domain of the anti-PD-1 binding moiety,

the second polypeptide sequence comprisesa VL of an anti-CTLA-4 binding moiety operably linked to an engineered CAlpha at a second conjunction domain, and

the third polypeptide sequence comprises (i) a VH of the anti-PD-1 binding moiety operably linked to a CH1 domain of the anti-PD-1 binding moiety, and (ii) a VH of the anti-CTLA-4 binding moiety operably linked to an engineered CBeta at a first conjunction domain, wherein the CH1 domain in (i) and the VH in (ii) are operably linked via a linker, and wherein:

the anti-CTLA-4 binding moiety is derived from an anti-CTLA-4 antibody comprising a heavy chain CDR1 comprising SEQ ID NO: 5, a heavy chain CDR2 comprising SEQ ID NO: 6, a heavy chain CDR3 comprising SEQ ID NO: 7, a light chain CDR1 comprising SEQ ID NO: 8, a light chain CDR2 comprising SEQ ID NO: 9, and a light chain CDR3 comprising SEQ ID NO: 10; and

the anti-PD-1 binding moiety is derived from an anti-PD-1 antibody comprising a heavy chain CDR1 comprising SEQ ID NO: 21, a heavy chain CDR2 comprising SEQ ID NO: 22, a heavy chain CDR3 comprising SEQ ID NO: 23, a light chain CDR1 comprising SEQ ID NO: 24, a light chain CDR2 comprising SEQ ID NO: 25, and a light chain CDR3 comprising SEQ ID NO: 26.

In certain embodiments, the bispecific polypeptide complex disclosed herein has a G25 WuXiBody structure (see Figure 1) and comprises a combination of three polypeptide sequences, wherein:

the first polypeptide sequence comprises a VL of an anti-PD-1 binding moiety operably linked to an engineered CAlpha at a second conjunction domain,

the second polypeptide sequence comprisesa VL of an anti-CTLA-4 binding moiety operably linked to aCL domain of the anti-CTLA-4 binding moiety, and

the third polypeptide sequence comprises (i) a VH of the anti-PD-1 binding moiety operably linked to an engineered CBeta at a first conjunction domain, and (ii) a VH of the anti-CTLA-4 binding moiety operably linked to a CH1 domain of the anti-CTLA-4 binding moiety, wherein the engineered CBeta in (i) and the VH in (ii) are operably linked via a linker, and wherein:

the anti-CTLA-4 binding moiety is derived from an anti-CTLA-4 antibody comprising a heavy chain CDR1 comprising SEQ ID NO: 11, a heavy chain CDR2 comprising SEQ ID NO: 12, a heavy chain CDR3 comprising SEQ ID NO: 13, a light chain CDR1 comprising SEQ ID NO: 14, a light chain CDR2 comprising SEQ ID NO: 15, and a light chain CDR3 comprising SEQ ID NO: 16; and

In certain embodiments, the bispecific polypeptide complex comprises a combination of three polypeptide sequences: SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31.

In certain embodiments, the bispecific polypeptide complex comprises a combination of three polypeptide sequences: SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34.

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

In one aspect, the present disclosure provides a host cell expressing the bispecific polypeptide complex provided herein.

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

In one aspect, the present disclosure provides a composition comprising the bispecific polypeptide complex provided herein.

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

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

In one aspect, the present disclosure provides a method of modulating an immune response in a subject in need thereof, comprising administrating to the subject a therapeutically effective amount of the bispecific polypeptide complex provided herein.

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 invention 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 1 shows a schematic description of symmetric WuXiBody formatsG25 and G25R. Both formats containTCR-constant domainsfused withvariable region of an antibody. The rectangles indicate TCR constant domains, and the ovals indicate variable and constant domains of an antibody. The difference between G25 and G25R is the switched position of the normal Fab and the chimeric Fab-like fragments. These formats can accommodate different variable regions from different antibody pairs and usually havea molecular weight around 240-250 kD.

Figures 2A-2D present superimposed poses of antibody Fv and TCR structures providing guidance in fusing antibody Fv and TCR constant regions. Figure 2A presents an antibody Fv structure. Figure 2B presents the TCR structure from PDB 4L4T. Figure 2C presents an antibody Fv structural model superimposed on the TCR variable region in different orientations. Rough chimeric proteins were created by removing the TCR variable domain in the superimposed poses, as shown in Figure 2D. The overlapped residues in the conjunction area helped design conjunction regions. The antibody VL chain and the TCR alpha chain were colored in white. The VH and beta chains were colored in black.

Figures 3A-3B show SDS-PAGE characterizations of W3248-U6T5. G25-1. uIgG4. SP and W3248-U6T1. G25R-1. uIgG4. SP. M: Protein marker. PC: a positive control of a bispecific antibody at around 250 kDa (Figure 3A) and SEC-HPLC characterizations of W3248-U6T1. G25R-1. uIgG4. SP and W3248-U6T5. G25-1. uIgG4. SP (Figure 3B) .

Figure 4 shows melting temperatures of W3248-U6T1. G25R-1. uIgG4. SP, W3248-U6T5. G25-1. uIgG4. SP, and a benchmark bispecific anti-CTLA-4 x PD-1antibody WBP324-BMK1. uIgG1. KDL.

Figure 5 shows FACS bindings of W3248-U6T5. G25-1. uIgG4. SP and W3248-U6T1. G25R-1. uIgG4. SP to human PD-1 engineered cells. WBP324-BMK1. uIgG1. KDL, W324-BMK2. uIgG4, and W324-BMK3. uIgG4 are different benchmark bispecific anti-CTLA-4 x PD-1antibodies. WBP305-BMK1. IgG4 is an anti-PD-1antibody. An IgG4 antibody was used as the negative control.

Figure 6 shows FACS bindings of W3248-U6T5. G25-1. uIgG4. SP and W3248-U6T1. G25R-1. uIgG4. SP to cynomolgus PD-1 engineered cells. WBP3055_1.153.7. hAb and WBP305-BMK1. IgG4 are anti-PD-1 antibodies. An IgG4 antibody was used as the negative control.

Figure 7 shows FACS bindings of W3248-U6T5. G25-1. uIgG4. SP and W3248-U6T1. G25R-1. uIgG4. SPto human CTLA-4 engineered cells. WBP324-BMK1. uIgG1. KDL, W324-BMK2. uIgG4, and W324-BMK3. uIgG4 are different benchmark bispecific anti-CTLA-4 x PD-1 antibodies. WBP316-BMK1. IgG4 is an anti-CTLA-4-1antibody. An IgG4 antibody was used as the negative control.

Figure 8 shows FACS bindings of W3248-U6T5. G25-1. uIgG4. SP and W3248-U6T1. G25R-1. uIgG4. SP to cynomolgus CTLA-4 engineered cells. WBP324-BMK1. uIgG1. KDL is abenchmark bispecific anti-CTLA-4 x PD-1antibody. W3162_1.154.8-z35-IgG1K and

WBP316-BMK1. IgG4 are anti-CTLA-4 antibodies. An IgG4 antibody was used as the negative control.

Figure 9 summarizes binding affinities of W3248-U6T5. G25-1. uIgG4. SP and W3248-U6T1. G25R-1. uIgG4. SP to CTLA-4 and PD-1, as measured by SPR. WBP316-BMK1. IgG4 is an anti-CTLA-4-1antibody. A parent antibody of anti-PD-1 was used as a control.

Figure 10 shows FACS competition assays of W3248-U6T5. G25-1. uIgG4. SP and W3248-U6T1. G25R-1. uIgG4. SP to block human PD-L1 protein binding to PD-1 engineered cells. WBP324-BMK1. uIgG1. KDL is a benchmark bispecific anti-CTLA-4 x PD-1antibody. WBP3055_1.153.7. hAb and WBP305-BMK1. IgG4 are anti-PD-1 antibodies. An IgG4 antibody was used as the negative control.

Figure 11 shows FACS competition assays of W3248-U6T5. G25-1. uIgG4. SP and W3248-U6T1. G25R-1. uIgG4. SP to block human CTLA-4 protein binding to CD80 engineered cells. WBP324-BMK1. uIgG1. KDL is abenchmark bispecific anti-CTLA-4 x PD-1antibody. W3162_1.154.8-z35-IgG1K and WBP316-BMK1. IgG4 are anti-CTLA-4 antibodies. An IgG4 antibody was used as the negative control.

Figure 12 shows FACS competition assays of W3248-U6T5. G25-1. uIgG4. SP and W3248-U6T1. G25R-1. uIgG4. SP to block cynomolgus CTLA-4 protein binding to CD80 engineered cells. WBP324-BMK1. uIgG1. KDL is abenchmark bispecific anti-CTLA-4 x PD-1antibody. W3162-1.154.8-z35-IgG1K and WBP316-BMK1. IgG4 are anti-CTLA-4 antibodies. An IgG4 antibody was used as the negative control.

Figure 13 shows an ELISA dual binding assay of W3248-U6T5. G25-1. uIgG4. SP and W3248-U6T1. G25R-1. uIgG4. SP. WBP324-BMK1. uIgG1. KDL is abenchmark bispecific anti-CTLA-4 x PD-1antibody. An IgG4 antibody was used as the negative control.

Figure 14 shows FACS dual binding of W3248-U6T5. G25-1. uIgG4. SP and W3248-U6T1. G25R-1. uIgG4. SP to CTLA-4 and PD-1. An IgG4 antibody was used as the negative control.

Figures 15A-15B show stability ofW3248-U6T5. G25-1. uIgG4. SP in serum for 14 days, as measured by ELISA dual binding to human CTLA-4 and PD-1 (Figure 15A) and stability ofW3248-U6T1. G25R-1. uIgG4. SP in serum for 14 days, as measured by ELISA dual binding to human CTLA-4 and PD-1 (Figure 15B) .

Figure 16 shows the result of pharmacokinetics study of W3248-U6T5. G25-1. uIgG4. SP and W3248-U6T1. G25R-1. uIgG4. SP in mouse.

DETAILED DESCRIPTION OF THE INVENTION

The following description of the disclosure is merely intended to illustrate various embodiments of the disclosure.

Definitions

The articles “a, ” “an, ” and “the” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “apolypeptide complex” means one polypeptide complex or more than one polypeptide complex.

As used herein, the term “about” or “approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by as much as 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In particular embodiments, the terms “about” or “approximately” when preceding a numerical value indicates the value plus or minus a range of 15%, 10%, 5%, or 1%.

Throughout this disclosure, unless the context requires otherwise, the words “comprise, ” “comprises, ” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By “consisting of” is meant including, and limited to, whatever follows the phrase “consisting of. ” Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of” is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of” indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

Reference throughout this disclosure to “one embodiment, ” “an embodiment, ” “aparticular embodiment, ” “a related embodiment, ” “a certain embodiment, ” “an additional embodiment, ” or “a further embodiment, ” or combinations thereof means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

The terms “polypeptide, ” “peptide, ” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues, or an assembly of multiple polymers of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an alpha-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, or methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. An alpha-carbon refers to the firstcarbonatomthat attaches to afunctional group, such as acarbonyl. A beta-carbon refers to the second carbon atom linked to the alpha-carbon, and the system continues naming the carbons in alphabetical order withGreek letters. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid. The term “protein” typically refers to large polypeptides. The term “peptide” typically refers to short polypeptides. The left-hand end of a polypeptide sequence is usually described as the amino-terminus (N-terminus) ; and the right-hand end of a polypeptide sequence is usually described as the carboxyl-terminus (C-terminus) . “Polypeptide complex” as used herein refers to a complex comprising one or more polypeptides that are associated to perform certain functions. In certain embodiments, the polypeptides are immune-related.

The term “antibody” as used herein encompasses any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody, or bispecific (bivalent) antibody that binds to a specific antigen. A nativeintact antibody comprises two heavy chains and two light chains. Each heavy chain consists of a variable region ( “HCVR” ) and a first, second, and third constant region (CH1, CH2 and CH3) , while each light chain consists of a variable region ( “LCVR” ) and a constant region (CL) . Mammalian heavy chains are classified as α, δ, ε, γ, and μ, and mammalian light chains are classified as λ or κ. The antibody has a “Y” shape, with the stem of the Y consisting of the second and third constant regions of two heavy chains bound together via disulphide bonding. Each arm of the Y includes the variable region and first constant region of a single heavy chain bound to the variable and constant regions of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in both chains generally contain three highly variable loops called the complementarity determining regions (CDRs) (light (L) chain CDRs including LCDR1, LCDR2, and LCDR3, heavy (H) chain CDRs including HCDR1, HCDR2, HCDR3) . CDR boundaries for antibodies may be defined or identified by the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, A.M., J. Mol. Biol., 273 (4) , 927 (1997) ; Chothia, C. et al., J Mol Biol. Dec 5; 186 (3) : 651-63 (1985) ; Chothia, C. and Lesk, A.M., J. Mol. Biol., 196, 901 (1987) ; Chothia, C. et al., Nature. Dec 21-28; 342 (6252) : 877-83 (1989) ; Kabat E.A. et al., National Institutes of Health, Bethesda, Md. (1991) ) . The three CDRs are interposed between flanking stretches known as framework regions (FRs) , which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops. Each HCVR and LCVR comprises four FRs, and the CDRs and FRs are arranged from amino terminus to carboxy terminus in the order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant regions of the heavy and light chains are not involved in antigen binding, but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chain. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several of the major antibody classes are divided into subclasses such as IgG1 (γ1 heavy chain) , IgG2 (γ2 heavy chain) , IgG3 (γ3 heavy chain) , IgG4 (γ4 heavy chain) , IgA1 (α1 heavy chain) , or IgA2 (α2 heavy chain) .

The term “variable domain” with respect to an antibody as used herein refers to an antibody variable region or a fragment thereof comprising one or more CDRs. Although a variable domain may comprise an intact variable region (such as HCVR or LCVR) , it is also possible to comprise less than an intact variable region yet still retain the capability of binding to an antigen or forming an antigen-binding site.

The term “antigen-binding moiety” as used herein refers to an antibody fragmentformed from a portion of an antibody comprising one or more CDRs, or any other antibody fragment that binds to an antigen but does not comprise an intact native antibody structure. Examples of antigen-binding moiety include, without limitation, a variable domain, a variable region, a diabody, a Fab, a Fab', a F (ab') ₂, an Fv fragment, a disulphide stabilized Fv fragment (dsFv) , a (dsFv) ₂, a bispecific dsFv (dsFv-dsFv') , a disulphide stabilized diabody (ds diabody) , a multispecific antibody, a camelized single domain antibody, a nanobody, a domain antibody, and a bivalent domain antibody. An antigen-binding moiety is capable of binding to the same antigen to which the parent antibody binds. In certain embodiments, an antigen-binding moiety may comprise one or more CDRs from a particular human antibody grafted to a framework region from one or more different human antibodies. For more and detailed formats of antigen-binding moiety are described in Spiess et al, 2015 (Supra) , and Brinkman et al., mAbs, 9 (2) , pp. 182–212 (2017) , which are incorporated herein by their entirety.

“Fab” with regard to an antibody refers to that portion of the antibody consisting of a single light chain (both variable and constant regions) associating to the variable region and first constant region of a single heavy chain by a disulphide bond. In certain embodiments, the constant regions of both the light chain and heavy chain are replaced with TCR constant regions.

“Fab'” refers to a Fab fragment that includes a portion of the hinge region.

“F (ab') ₂” refers to a dimer of Fab’.

“Fc” with regard to an antibody refers to that portion of the antibody consisting of the second (CH2) and third (CH3) constant regions of a first heavy chain bound to the second and third constant regions of a second heavy chain via disulphide bonding. The Fc portion of the antibody is responsible for various effector functions such as ADCC, and CDC, but does not function in antigen binding.

“Hinge region” in terms of an antibody includes the portion of a heavy chain molecule that joins the CH1 domain to the CH2 domain. This hinge region comprises approximately 25 amino acid residues and is flexible, thus allowing the two N-terminus antigen binding regions to move independently.

“CH2 domain” as used herein refers to the portion of a heavy chain molecule that extends, e.g., from about amino acid 244 to amino acid 360 of an IgG antibody using conventional numbering schemes (amino acids 244 to 360, Kabat numbering system; and amino acids 231-340, EU numbering system; see Kabat, E., et al., U.S. Department of Health and Human Services, (1983) ) .

The “CH3 domain” extends from the CH2 domain to the C-terminus of the IgG molecule and comprises approximately 108 amino acids. Certain immunoglobulin classes, e.g., IgM, further include a CH4 region.

“Fv” with regard to an antibody refers to the smallest fragment of the antibody to bear the complete antigen binding site. An Fv fragment consists of the variable domain of a single light chain bound to the variable domain of a single heavy chain. A number of Fv designs have been provided, including dsFvs, in which the association between the two domains is enhanced by an introduced disulphide bond; and scFvs can be formed using a peptide linker to bind the two domains together as a single polypeptide. Fv constructs containing a variable domain of a heavy or light immunoglobulin chain associated to the variable and constant domain of the corresponding immunoglobulin heavy or light chain have also been produced. Fvs have also been multimerised to form diabodies and triabodies (Maynard et al., Annu Rev Biomed Eng 2 339-376 (2000) ) .

“DVD-Ig” refers to a dual-variable-domain antibody that is formed by fusion of an additional HCVR domain and LCVR domain of a second specificity to an IgG heavy chain and light chain. “CODV-Ig” refers to a related format where the two HCVR and two LCVR domains are linked in a way that allows crossover pairing of the variable HCVR-LCVR domains, which are arranged either (from N-to C-terminus) in the order HCVRA-HCVRB and LCVRB-LCVRA, or in the order HCVRB-HCVRA and LCVRA-LCVRB.

A “CrossMab” refers to a technology of pairing of unmodified light chain with the corresponding unmodified heavy chain and pairing of the modified light chain with the corresponding modified heavy chain, thus resulting in an antibody with reduced mispairing in the light chain.

A “BiTE” is a bispecific T-cell engager molecule, comprising a first scFv with a first antigen specificity in the LCVR-HCVR orientation linked to a second scFv with a second specificity in the HCVR-LCVR orientation.

A “WuXiBody” is a bispecific antibody comprising a soluble chimeric protein with the variable domains of an antibody and the constant domains of a TCR, wherein the subunits (such as alpha and beta domains) of the TCR constant domains are linked by an engineered disulfide bond.

An “antigen” or “Ag” as used herein refers to a compound, composition, peptide, polypeptide, protein, or substance that can stimulate the production of antibodies or a T cell response in cell culture or in an animal, including compositions (such as one that includes a cancer-specific protein) that are added to a cell culture (such as a hybridoma) , or injected or absorbed into an animal. An antigen reacts with the products of specific humoral or cellular immunity (such as an antibody) , including those induced by heterologous antigens.

An “epitope” or “antigenic determinant” refers to the region of an antigen to which a binding agent (such as an antibody) binds. Epitopes can be formed both from contiguous amino acids (also called linear or sequential epitopes) or noncontiguous amino acids juxtaposed by tertiary folding of a protein (also called configurational or conformational epitopes) . Epitopes formed from contiguous amino acids are typically arranged linearly along the primary amino acid residues on the protein and the small segments of the contiguous amino acids can be digested from an antigen binding with major histocompatibility complex (MHC) molecules or retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5, about 7, or about 8-10 amino acids in a unique spatial conformation.

The term “specific binding” or “specifically binds” as used herein refers to a non-random binding reaction between two molecules, such as for example between an antibody and an antigen. In certain embodiments, the polypeptide complex and the bispecific polypeptide complex provided herein specifically bind anantigen with a binding affinity (K _D) of ≤ 10 ^-6 M (e.g., ≤ 5x10 ^-7 M, ≤ 2x10 ^-7 M, ≤ 10 ^-7 M, ≤ 5x10 ^-8 M, ≤ 2x10 ^-8 M, ≤ 10 ^-8 M, ≤ 5x10 ^-9 M, ≤ 2x10 ^-9 M, ≤ 10 ^-9 M, or ≤ 10 ^-10 M) . K _D as used herein refers to the ratio of the dissociation rate to the association rate (k _off/k _on) , andmay be determined using surface plasmon resonance methods for example using instrument such as Biacore.

The terms “operably link” and “operably linked” refer to a juxtaposition, with or without a spacer or linker, of two or more biological sequences of interest in such a way that they are in a relationship permitting them to function in an intended manner. When used with respect to polypeptides, it is intended to mean that the polypeptide sequences are linked in such a way that permits the linked product to have the intended biological function. For example, an antibody variable region may be operably linked to a constant region so as to provide for a stable product with antigen-binding activity. The term may also be used with respect to polynucleotides. For one instance, when a polynucleotide encoding a polypeptide is operably linked to a regulatory sequence (e.g., promoter, enhancer, silencer sequence, etc. ) , it is intended to mean that the polynucleotide sequences are linked in such a way that permits regulated expression of the polypeptide from the polynucleotide.

The term “fusion” or “fused” when used with respect to amino acid sequences (e.g. peptide, polypeptide, or protein) refers to combination of two or more amino acid sequences, for example by chemical bonding or recombinant means, into a single amino acid sequence thatdoes not exist naturally. A fusion amino acid sequence may be produced by genetic recombination of two encoding polynucleotide sequences, and can be expressed by a method of introducing a construct containing the recombinant polynucleotides into a host cell.

The term “spacer” or “linker” as used herein 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 or linker 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) .

The term “antigenic specificity” refers to a particular antigen or an epitope thereof that is selectively recognized by an antigen-binding molecule.

The term “substitution” with regard to amino acid residue as used herein refers to naturally occurring or induced replacement of one or more amino acids with another in a peptide, polypeptide, or protein. Substitution in a polypeptide may result in diminishment, enhancement, or elimination of the polypeptide’s function.

The term “mutation” or “mutated” with regard to an amino acid residue as used herein refers to substitution, insertion, or addition of an amino acid residue.

“T cell” as used herein refers to a type of lymphocyte that plays a critical role in the cell-mediated immunity, including helper T cells (e.g. CD4 ⁺ T cells, T helper 1 type T cells, T helper 2 type T cells, T helper 3 type T cells, T helper 17 type T cells) , cytotoxic T cells (e.g. CD8 ⁺ T cells) , memory T cells (e.g. central memory T cells (TCM cells) , effector memory T cells (TEMcells and TEMRAcells) , and resident memory T cells (TRM) that are either CD8+ or CD4+) , natural killer T (NKT) cells, and inhibitory T cells.

A native “T cell receptor” or a native “TCR” is a heterodimeric T cell surface protein which is associated with invariant CD3 chains to form a complex capable of mediating signal transduction. TCR belongs to the immunoglobulin superfamily, and is similar to a half antibody with a single heavy chain and a single light chain. a native TCR has an extracellular portion, a transmembrane portion, and an intracellular portion. The extracellular domain of a TCR has a membrane-proximal constant region and a membrane-distal variable region.

The term “subject” or “individual” or “animal” or “patient” as used herein refers to a human or non-human animal, including a mammal or a primate, in need of diagnosis, prognosis, amelioration, prevention, and/or treatment of a disease or condition. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, swine, cows, bears, and so on.

Antigen-binding moiety comprising engineered CAlpha and CBeta

The first antigen-binding moiety provided herein comprises a first antibody heavy chain variable domain operably linked to a first T cell receptor (TCR) constant region, and a first 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 a non-native interchain disulphide bond. The first antigen-binding moiety 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. Thus, the first antigen-binding moiety comprises a heavy chain variable domain and a light chain variable domain, which are operably linked to a pair of TCR constant regions, respectively. The pair of TCR constant regions in the first antigen-binding moiety includesTCR alpha and beta constant regions. The TCR constant regions in the polypeptide complexes provided herein are capable of associating with each other to form a dimer through a non-native disulphide bond.

It is surprisingly found that the first antigen-binding moiety provided herein with at least one 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 first antigen-binding moiety is 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 first antigen-binding moiety. Additional advantages of the first antigen-binding moiety provided herein will become more evident in the following disclosure below.

i) TCR constant region

The first antigen-binding moiety provided herein comprisesan alpha anda beta constant region derived from a TCR.

Human TCR alpha chain constant region is known as TRAC, with the NCBI accession number of P01848 (https: //www. uniprot. org/uniprot/P01848) , the sequence of wild type TCR alpha domain is: IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESS (SEQ ID NO: 35) ; the engineered TCR alpha chain constant region in the invention comprises one or more mutated sites selected from the group consisting of N32Q, T47C, N66Q, and N77Q.

Human TCR beta chain constant region has two different variants, known as TRBC1 and TRBC2 (IMGT nomenclature) . In the present disclosure, the sequence of wild type TCR beta domain is DLKNVFPPKVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGR (SEQ ID NO: 37) , with the NCBI accession number of A0A5B9 (https: //www. uniprot. org/uniprot/A0A5B9) and the engineered TCR beta domain comprises one or more mutated sites selected from the group consisting of K9E, S56C, N69Q, and C74A.

Specifically, the native TCR beta chain contains a native cysteine residue at position 74, which is unpaired and therefore does not form a disulphide bond in a native alpha/beta TCR. In the polypeptide complexes provided herein, this native cysteine residue at position 74 of TCR beta chain is mutated to an alanine residue. This may be useful to avoid incorrect intrachain or interchain pairing. In certain embodiments, the substitution in certain embodiments can improve the TCR refolding efficiencies in vitro.

In the present disclosure, the first and the second TCR constant regions of the first antigen-binding moiety provided herein are capable of forming a dimer comprising, between the TCR constant regions (i.e., CAlpha and CBeta) , at least one non-native interchain disulphide bond that is capable of stabilizing the dimer.

The term “dimer” as used herein refers to an associated structure formed by two molecules, such as polypeptides or proteins, via covalent or non-covalent interactions. A homodimer is formed by two identical molecules (homodimerization) , and a heterodimer is formed by two different molecules (heterodimerization) . The dimer formed by the first and the second TCR constant regions is a heterodimer.

A “mutated” amino acid residue refers to one which is substituted, inserted, or added and is different from its native counterpart residue in a corresponding native TCR constant region. For example, if an amino acid residue at a particular position in the wild-type TCR constant region is referred to as the “native” residue, then its mutated counterpart is any residue that is different from the native residue but resides at the same position on the TCR constant region. A mutated residue can be a different residue which substitutes the native residue at the same position.

In the polypeptide complexes provided herein, the first and/or the second TCR constant regions have been engineered to comprise one or more mutated amino acid residues that are responsible for forming the non-native interchain disulphide bond. To introduce such a mutated residue to the TCR constant region, an encoding sequence of a TCR region can be manipulated to for example, substitute a codon encoding a native residue for the codon encoding the mutated residue.

In the polypeptide complexes provided herein, the first and/or the second TCR constant regions have been engineered to comprise one or more mutated cysteine residues such that, after replacement to cysteine residues, a non-native interchain disulphide bond could be formed between the two TCR constant regions (i.e., CAlpha and CBeta) .

The non-native interchain disulphide bond is capable of stabilizing the first antigen-binding moiety. Such effects in stablization can be embodied in various ways. For example, the presence of the mutated amino acid residue or the non-native interchain disulphide bond can enable the polypeptide complex to stably express, and/or to express in a high level, and/or to associate into a stable complex having the desired biological activity (e.g. antigen binding activity) , and/or to express and assemble into a high level of desired stable complex having the desired biological activity. The capability of the interchain disulphide bond to stabilize the first and the second TCR constant regions can be assessed using proper methods known in the art, such as the molecular weight displayed on SDS-PAGE, or thermostability measured by differential scanning calorimetry (DSC) or differential scanning fluorimetry (DSF) . In an illustrative example, formation of a stable first antigen-binding moiety provided herein can be confirmed by SDS-PAGE, if a product shows a molecular weight comparable to the combined molecular weight of the first and the second polypeptides. In certain embodiments, the first antigen-binding moiety provided herein is stable in that its thermal stability is no less than 50%, 60%, 70%, 80%, or 90%of that of a natural Fab. In certain embodiments, the first antigen-binding moiety provided herein is stable in that its thermal stability is comparable to that of a natural Fab.

Without wishing to be bound by any theory, it is believed that the non-native interchain disulphide bond formed between the first and the second TCR constant regions in the first antigen-binding moiety are capable of stabilizing the heterodimer of TCR constant regions, thereby enhancing the level of correct folding, the structural stability, and/or the expression level of the heterodimer and of the first antigen-binding moiety. Unlike a native TCR anchored on the membrane of T cell surface, heterodimers of native TCR extracellular domains are found to be much less stable, despite their similarity to antibody Fab in 3D structure. As a matter of fact, the instability of a native TCR in soluble conditions used to be a significant obstacle that prevented elucidation of its crystal structure (see Wang, Protein Cell, 5 (9) , pp. 649–652 (2014) ) . By introducing a pair of cysteine (Cys) mutations in the TCR constant regions and thereby enabling formation of an interchain non-native disulphide bond, the first antigen-binding moiety can be stably expressed while in the meantime the antigen-binding capabilities of the antibody variable region are retained.

The TCR constant region comprising a mutated residue is also referred to herein as an “engineered” TCR constant region. In the polypeptide complexes provided herein, C1 comprises an engineered CBeta, and C2 comprises an engineered CAlpha.

In the polypeptide complexes provided herein, the engineered TCR constant region comprises one or more mutated cysteine residue 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, one or more disulphide bonds can be formed between the engineered CAlpha and the engineered CBeta. In certain embodiments, the mutated cysteine residue in CBeta is S56C (corresponding to amino acid C48 in SEQ ID NO: 1) , and the mutated cysteine residues in CAlpha is T47C (corresponding to amino acid C41 in SEQ ID NO: 2) , 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 SEQ ID NOs: 35 and 37, the starting amino acid is denoted as position 1) is replaced by amino acid residue Y, where X and Y are respectively the one-letter abbreviation of a particular amino acid residue.

In the polypeptide complexes provided herein, the engineered CBeta comprises SEQ ID NO: 1, and the engineered CAlpha comprises SEQ ID NO: 2. The amino acid sequences of SEQ ID NOs: 1 and 2 are provided below, with the introduced cysteines annotated in bold and underline.

In the peptide complexes provided herein, one or more native glycosylation sites present in the native TCR constant regions have beenmodified (e.g., removed) in the first antigen-binding moiety 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 the first antigen-binding moiety provided herein, at least one native glycosylation site is absent 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, it is believed that the first antigen-binding moiety 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 a 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, e.g., Wu et al., Mabs, 7: 2, 364-376, 2015) .

In the first antigen-binding moiety provided herein, the N-glycosylation sites in the engineered CAlpha at N32, N66, and N77 are absent. The engineered CAlpha sequence absent of a glycosylation site comprises SEQ ID NO: 2. In the first antigen-binding moiety provided herein, the N-glycosylation site in the engineered CBeta at N69 is absent. The engineered CBeta sequence (TRBC1) absent of a glycosylation site comprises SEQ ID NO: 1.

In the first antigen-binding moiety provided herein, the constant regions derived from a TCR are operably linked to the variable regions derived from an antibody.

In certain embodiments, the first antibody heavy chain variable domain (VH) is fused to the enginnered CBeta at a first conjunction domain, and the first antibody light chain variable domain (VL) is fused to the engineered CAlpha at a second conjunction domain, wherein the first conjunction domain comprises SEQ ID NO: 3 (LEDLKNVFPP) , and the second conjunction domain comprises SEQ ID NO: 4 (PDIQNPDP) .

“Conjunction domain” as used herein refers to a boundary or border region where two amino acid sequences are fused or combined. In certain embodiments, the first conjunction domain comprises at least a portion of the C terminal fragment of an antibody V/C conjunction, and the second conjunction domain comprises 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 an antibody variable domain and constant domain, for example, the boundary between a heavy chain variable domain and the CH1 domain, or between a light chain variable domain and the light chain constant domain. Similarly, the term “TCR V/C conjunction” refers to the boundary of a TCR variable domain and constant domain, for example, the boundary between a TCRAlpha variable domain and the constant domain, or between a TCRBeta variable domain and the constant domain.

In certain embodiments, a first polypeptide comprises a sequence comprising domains operably linked as in formula (I) : VH-HCJ-C1, and a 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; and

C2 is a second TCR constant domain as defined supra.

In such embodiments, C1 is the engineered CBeta comprisingSEQ ID NO: 1, C2 is the engineered CAlpha compriseing SEQ ID NO: 2, HCJ comprises SEQ ID NO: 3, and LCJ comprises SEQ ID NO: 4.

In summary, the first antigen-binding moiety provided herein 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 comprising SEQ ID NO: 1 and C2 comprises an engineered CAlpha comprising SEQ ID NO: 2, amino acid C48 in SEQ ID NO: 1 and amino acid C41 in SEQ ID NO: 2 are capable of forming a non-native interchain disulphide bond, C1 and C2 are capable of forming a dimer, and the non-native interchain disulphide bond between C1 and C2 is capable of stabilizing the dimer.

ii) Antibody variable region

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 bispecific polypeptide complex provided herein comprises a first antigen-binding moiety associated with a second antigen-binding moiety, and one of them specifically binds to CTLA-4, while the other specifically binds to PD-1. In the polypeptide complex provided herein, the first antigen-binding moiety comprises a first heavy chain variable domain (VH1) and a first light chain variable domain (VL1) of a first antibody, and the second antigen-binding moiety comprises a second heavy chain variable domain (VH2) and a second light chain variable domain (VL2) of a second antibody, wherein the first antibody and the second antibody are different and are selected from the group consisting of an anti-CTLA-4 antibody and an anti-PD-1 antibody. In certain embodiments, the first antibody is an anti-CTLA-4antibody, and the second antibody is an anti-PD-1 antibody. In certain other embodiments, the first antibody is an anti-PD-1 antibody, and the second antibody is an anti-CTLA-4 antibody.

a) Anti-CTLA-4 binding moiety

In the polypeptide complex provided herein, the first antigen-binding moiety or the second antigen-binding moiety is an anti-CTLA-4 binding moiety. In certain embodiments, the anti-CTLA-4binding moiety is derived from two anti-CTLA-4 antibodiesshown in Table 1 below. The CDR sequences of these two antibodies are provided below.

Table 1. CDR sequences of anti-CTLA-4 antibodies

Heavy and light chain variable region sequences ofthese two anti-CTLA-4 antibodiesare provided below with CDR sequences annotated in bold and underline.

Anti-CTLA-4 #1-VH

Anti-CTLA-4 #1-VL

Anti-CTLA-4 #2-VH

Anti-CTLA-4 #2-VL

CDRs are known to be responsible for antigen binding.

In certain embodiments, the anti-CTLA-4 binding moiety comprisesa heavy chain CDR3 sequence of the anti-CTLA-4 antibodies disclosed herein. In certain embodiments, the anti-CTLA-4 binding moiety provided herein comprises a heavy chain CDR3 comprising SEQ ID NO: 7 or SEQ ID NO: 13. Heavy chain CDR3 regions are located at the center of the antigen-binding site, and therefore are believed to make the most contact with the 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) ) .

The anti-CTLA-4binding moiety provided herein further comprises suitable framework region (FR) sequences, as long as the anti-CTLA-4 binding moiety can specifically bind to CTLA-4.

In certain embodiments, the anti-CTLA-4 binding moiety provided herein comprisesa heavy chain variable domain sequence comprising SEQ ID NO: 17 and a light chain variable domain sequence comprising SEQ ID NO: 18. In certain embodiments, the anti-CTLA-4 binding moiety provided herein comprisesa heavy chain variable domain sequence comprising SEQ ID NO: 19 and a light chain variable domain sequence comprising SEQ ID NO: 20.

The binding affinity of the anti-CTLA-4 binding moiety provided herein can be represented by K _D value, which represents the ratio of dissociation rate to association rate (k _off/k _on) when the binding between the antigen and antigen-binding molecule reaches equilibrium. The antigen-binding affinity (e.g. K _D) can be appropriately determined using suitable methods known in the art, including, for example, a flow cytometry assay. In some embodiments, binding of the antibody to the antigen at different concentrations can be determined by flow cytometry, the determined mean fluorescence intensity (MFI) can be firstly plotted against antibody concentration, K _D value can then be calculated by fitting the dependence of specific binding fluorescence intensity (Y) and the concentration of antibodies (X) into the one site saturation equation: Y=B _max*X/ (K _D + X) using Prism version 5 (GraphPad Software, San Diego, CA) , wherein B _max refers to the maximum specific binding of the tested antibody to the antigen.

In certain embodiments, the anti-CTLA-4 binding moiety provided herein is capable of specifically binding to human CTLA-4 expressed on a cell surface, or a recombinant human CTLA-4. CTLA-4 is a cell surface receptor. A recombinant CTLA-4 is a soluble CTLA-4 which is recombinantly expressed and is not associated with a cell membrane. A recombinant CTLA-4 can be prepared by various recombinant technologies known in the art (see, e.g., Example 2) .

In some embodiments, the anti-CTLA-4 binding moiety provided herein is capable of specifically binding to human CTLA-4 expressed on the surface of cells with a binding affinity (K _D) of no more than 5x10 ^-9M, no more than 4x10 ^-9M, no more than 3x10 ^-9M, no more than 2x10 ^-9M, no more than 10 ^-9M, no more than 5x10 ^-10M, no more than 4x10 ^-10M, no more than 3x10 ^-10M, no more than 2x10 ^-10M, no more than 10 ^-10M, no more than 5x10 ^-11 M, no more than 4x10 ^-11 M, no more than 3x10 ^-11 M, no more than 2x10 ^-11 M, or no more than 10 ^-11 M as measured by flow cytometry assay.

In certain embodiments, the anti-CTLA-4 binding moiety provided herein cross-reacts with cynomolgus monkey CTLA-4, for example, cynomolgus monkey CTLA-4 expressed on a cell surface, or a soluble recombinant cynomolgus monkey CTLA-4.

Binding of the anti-CTLA-4 binding moiety to recombinant CTLA-4 or CTLA-4expressed on the surface of cells can be measured by methods known in the art, for example, a sandwich assay such as ELISA, Western Blot, flow cytometry assay, and other binding assays. In certain embodiments, the anti-CTLA-4 binding moiety provided herein specifically binds to recombinant human CTLA-4 at an EC ₅₀ (i.e. 50%binding concentration) of no more than 0.01 nM, no more than 0.02 nM, no more than 0.03 nM, no more than 0.04 nM, no more than 0.05 nM, no more than 0.06 nM, no more than 0.07 nM, or no more than 0.08 nM by ELISA. In certain embodiments, the anti-CTLA-4 binding moiety provided herein specifically binds to human CTLA-4 expressed on surface of cells at an EC ₅₀ of no more than 0.5 nM, no more than 0.6 nM, no more than 0.7 nM, no more than 0.8 nM, no more than 0.9 nM, no more than 1 nM, no more than 2 nM, no more than 3 nM, no more than 4 nM, no more than 5 nM, no more than 6 nM, no more than 7 nM, no more than 8 nM, no more than 9 nM, or no more than 10 nM by flow cytometry assay.

In certain embodiments, the anti-CTLA-4 binding moiety binds to cynomolgus monkey CTLA-4 with a binding affinity similar to that of human CTLA-4. For example, binding of the exemplary anti-CTLA-4 antibodies to cynomolgus monkey CTLA-4 is at a similar affinity or EC ₅₀ value to that of human CTLA-4.

In certain embodiments, the anti-CTLA-4 binding moiety provided herein specifically binds to recombinant cynomolgus monkey CTLA-4 with an EC ₅₀of no more than 0.001 nM, no more than 0.005 nM, no more than 0.01 nM, no more than 0.02 nM, no more than 0.03 nM, no more than 0.04 nM, no more than 0.05 nM, no more than 0.1 nM, or no more than 0.5 nM by ELISA.

In certain embodiments, the anti-CTLA-4 binding moiety provided herein has a specific binding affinity to human CTLA-4 which is sufficient to provide for diagnostic and/or therapeutic use.

b) Anti-PD-1 antibody

In the polypeptide complex provided herien, the first antigen-binding moiety or the second antigen-binding moiety is an anti-PD-1 binding moiety. In certain embodiments, the anti-PD-1 binding moiety is derived from the anti-PD-1 antibody shown in Table 2 below. The CDR sequences of the anti-PD-1antibody are provided below.

Table 2. CDR sequences of anti-PD-1 antibody

Heavy andlight chain variable region sequences of the anti-PD-1antibody are provided below with the CDR sequences annotated in bold and underline.

Anti-PD-1-VH

Anti-PD-1-VL

CDRs are known to be responsible for antigen binding.

In certain embodiments, the anti-PD-1 binding moiety comprises a heavy chain CDR3 sequence of the anti-PD-1antibody disclosed herein. In certain embodiments, the anti-PD-1 binding moiety provided herein comprises a heavy chain CDR3 sequence comprising SEQ ID NO: 23. Heavy chain CDR3 regions are located at the center of the antigen-binding site, and therefore are believed to make the most contact with the 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) ) .

The anti-PD-1 binding moiety provided herein further comprises suitable framework region (FR) sequences, as long as the anti-PD-1 binding moiety can specifically bind to PD-1.

In certain embodiments, the anti-PD-1 binding moiety provided herein comprises a heavy chain variable domain sequence comprising SEQ ID NO: 27 and a light chain variable domain sequence comprising SEQ ID NO: 28.

In some embodiments, the anti-PD-1 binding moiety provided herein is capable of specifically binding to human PD-1 expressed on surface of cells with a binding affinity (K _D) of no more than 5x10 ^-9M, no more than 1x10 ^-9M, no more than 9x10 ¹⁰M, no more than 8x10 ^-10M, no more than 7x10 ^-10M, no more than 6x10 ^-10M, no more than 5x10 ^-10M, no more than 4x10 ^-10M, no more than 3x10 ^-10M, no more than 2x10 ^-10M, or no more than 1x10 ^-10M as measured by flow cytometry assay.

In certain embodiments, the anti-PD-1 binding moiety provided herein cross-reacts with cynomolgus monkey PD-1, for example, cynomolgus monkey PD-1 expressed on a cell surface, or a soluble recombinant cynomolgus monkey PD-1.

Binding of the anti-PD-1 binding moiety to PD-1 expressed on a cell can be measured by methods known in the art, for example, by a sandwich assay such as ELISA, Western Blot, flow cytometry assay, and other binding assays. In certain embodiments, the anti-PD-1 binding moiety provided herein specifically binds to human PD-1 expressed on a cell with an EC ₅₀ of no more than 0.01 nM, no more than 0.02 nM, no more than 0.03 nM, no more than 0.04 nM, no more than 0.05 nM, no more than 0.1 nM, no more than 0.2 nM, no more than 0.3 nM, no more than 0.4 nM, no more than 0.5 nM, no more than 0.5 nM, no more than 0.6 nM, no more than 0.7 nM, no more than 0.8 nM, no more than 0.9 nM, or no more than 1 nM by flow cytometry assay.

In certain embodiments, the anti-PD-1 binding moiety binds to cynomolgus monkey PD-1 with a binding affinity similar to that of human PD-1. In certain embodiments, the anti-PD-1 binding moietyprovided herein specifically binds to cynomolgus monkey PD-1 expressed on a cell at an EC ₅₀ of no more than 0.2 nM, no more than 0.5 nM, no more than 0.8 nM, no more than 1 nM, no more than 2 nM, or no more than 3 nM by flow cytometry assay.

In certain embodiments, the anti-PD-1 binding moiety provided herein has a specific binding affinity to human PD-1 which is sufficient to provide for diagnostic and/or therapeutic use.

Bispecific polypeptide complex

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. The bispecific polypeptide complex provided herein comprises a first antigen-binding moiety associated with a second antigen-binding moiety, and one of them specifically binds to CTLA-4, and the other specifically binds to PD-1. In other words, the first antigen-binding moiety may specifically bind to CTLA-4 and the second antigen-binding moiety may specifically bind to PD-1. Alternatively, the first antigen-binding moiety may specifically bind to PD-1 and the second antigen-binding moiety may specifically bind to CTLA-4.

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 comprises:

C1 comprises an engineered CBeta comprising SEQ ID NO: 1and C2 comprises an engineered CAlpha comprising SEQ ID NO: 2, amino acid C48 in SEQ ID NO: 1 and amino acid C41 in SEQ ID NO: 2are capable of forming a non-native interchain disulphide bond, C1 and C2 are capable of forming a dimer, and the non-native interchain disulphide bond is capable of stabilizing the dimer, and

the second antigen-binding moiety comprises:

wherein: one of the first and the second antigen-binding moiety is an anti-CTLA-4 binding moiety, and the other one is an anti-PD-1 binding moiety,

the anti-PD-1 binding moiety is derived from an anti-PD-1 antibody comprising:

In certain embodiments, the bispecific polypeptide complex provided herein comprises a first antigen-binding moiety containing a sequence derived from a TCR constant region but the second antigen-binding moiety does not contain a sequence derived from a TCR constant region.

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.

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. Thus, the second antigen-binding moiety comprises a Fab.

Where the 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, 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, and VL1-VL2 are significantly reduced than 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, has 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.

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

a) Bispecific G25R WuXiBody

In certain embodiments, the first and the second antigen binding moiety can be operably linked together via a linker sequence.

In certain embodiments, the bispecific polypeptide complex has a G25R WuXiBody structure (see Figure 1) wherein the first and the second antigen binding moiety is operably linked together via a linker sequence. In such embodiments, the bispecific polypeptide complex comprises a combination of three differentpolypeptide sequences, whereinthe first polypeptide sequence comprises a VL of an anti-PD-1 binding moietyoperably linked to a CL domain of the anti-PD-1 binding moiety; the second polypeptide sequence comprises a VL of an anti-CTLA-4 binding moiety operably linked to an engineered CAlpha at a second conjunction domain; andthe third polypeptide sequence comprises (i) a VH of the anti-PD-1 binding moiety operably linked to a CH1 domain of the anti-PD-1 binding moiety, and (ii) a VH of the anti-CTLA-4 binding moiety operably linked to an engineered CBeta at a first conjunction domain, wherein the CH1 domain in (i) and the VH in (ii) are operably linked via a linker.

In the bispecific U6T1. G25R-1 WuXiBody disclosed herein (see Example 2) , the linker comprises SEQ ID NO: 45 (GGGGSGGGGS) . The first conjunction domain comprises SEQ ID NO: 3. The second conjunction domain comprises SEQ ID NO: 4.

In certain embodiments, the bispecific polypeptide complex having the G25R WuXiBody structure comprises an antibody CH2 domain, and/or an antibody CH3 domain, wherein the engineered CBeta in the third polypeptide sequence is operably linked to the antibody CH2 domain at a third conjunction domain. In such embodiments, the third conjunction domain comprises SEQ ID NO: 46 (YGPPCPPCPAPEFLGGP) . Exemplary sequences of such bispecific polypeptide complex are provided in Example 2.

In certain embodiments, the engineered CBeta in the anti-CTLA-4 binding moiety is operably linked to a dimerization domain, such that two copies of the third polypeptide of the bispecific polypeptide complex having the G25R WuXiBody structure can asscoiate with each other to form a dimer. In the bispecific G25R WuXiBody provided herein, at least one dimerization domain is operably linked to the engineered CBeta at a third conjunction domain, which comprises SEQ ID NO: 46.

The term “dimerization domain” as used herein refers to peptide domains which arecapable of associating with each other to form a dimer, or in some examples, enables spontaneous dimerization of two peptides. The association can be via any suitable interaction or linkage or bonding, for example, via a connecter, a disulphide bond, a hydrogen bond, an electrostatic interaction, a salt bridge, or a hydrophobic-hydrophilic interaction, or the combination thereof. Exemplary dimerization domains include, without limitation, an 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 domains can be derived from any antibody isotypes, such as IgG1, IgG2, and IgG4.

A “disulphide bond” refers to a covalent bond with the structure R-S-S-R’. The amino acid cysteine comprises a thiol group that can form a disulphide bond with a second thiol group, for example from another cysteine residue. The disulphide bond can be formed between the thiol groups of two cysteine residues residing respectively on the two polypeptide chains, thereby forming an interchain bridge or interchain bond.

A hydrogen bond is formed by electrostatic attraction between two polar groups when a hydrogen atom is covalently bound to a highly electronegative atom such as nitrogen, oxygen, or fluorine. A hydrogen bond can be formed in a polypeptide between the backbone oxygens (e.g. chalcogen groups) and amide hydrogens (nitrogen group) of two residues, respectively, such as a nitrogen group in Asn and an oxygen group in His, or an oxygen group in Asn and a nitrogen group in Lys. Ahydrogen bond is stronger than a Van der Waals interaction, but weaker than covalent or ionic bonds, and is critical in maintaining the secondary structure and tertiary structure of a polypeptide. For example, an alpha helix is formed when the spacing of amino acid residues occurs regularly between positions i and i+4, and a beta sheet is a stretch of peptide chain 3-10 amino acids long formed when two peptides joined by at least two or three backbone hydrogen bonds, form a twisted, pleated sheet.

Electrostatic interactionsarenon-covalent interactions and areimportant in protein folding, stability, flexibility, and function, including ionic interactions, hydrogen bonding, and halogen bonding. Electrostatic interactions can be formed in a polypeptide, for example, between Lys and Asp, between Lys and Glu, between Glu and Arg, or between Glu, Trp on the first chain and Arg, Val or Thr on the second chain.

A salt bridge is a close-range electrostatic interaction that mainly arises from the anionic carboxylate of either Asp or Glu and the cationic ammonium from Lys or the guanidinium of Arg, which are spatially proximal pairs of oppositely charged residues in native protein structures. Charged and polar residues in largely hydrophobic interfaces may act as hot spots for binding. Among others, residues with ionizable side chains such as His, Tyr, and Ser can also participate the formation of a salt bridge.

A hydrophobic interaction can be formed between one or more of Val, Tyr, and Ala on the first chain and one or more Val, Leu, and Trp on the second chain, or His and Ala on the first chain and Thr and Phe on the second chain (see Brinkmann, et al., 2017) .

b) Bispecific G25 WuXiBody

In certain embodiments, the bispecific polypeptide complex has a G25 WuXiBody structure (see Figure 1) wherein the first and the second antigen binding moiety is operably linked together via a linker sequence. In such embodiments, the bispecific polypeptide complex comprises a combination of three different polypeptide sequences, wherein the first polypeptide sequence comprises a VL of an anti-PD-1 binding moiety operably linked to an engineered CAlpha at a second conjunction domain; the second polypeptide sequence comprises a VL of an anti-CTLA-4 binding moiety operably linked to a CL domain of the anti-CTLA-4 binding moiety; and the third polypeptide sequence comprises (i) a VH of the anti-PD-1 binding moiety operably linked to an engineered CBeta at a first conjunction domain, and (ii) a VH of the anti-CTLA-4 binding moiety operably linked to a CH1 domain of the anti-CTLA-4 binding moiety, wherein the engineered CBeta in (i) and the VH in (ii) are operably linked via a linker.

In the bispecific U6T5. G25-1 WuXiBody disclosed herein (see Example 2) , the linker comprises SEQ ID NO: 46. The first conjunction domain comprises SEQ ID NO: 3. The second conjunction domain comprises SEQ ID NO: 4.

In certain embodiments, the bispecific polypeptide complex having the G25 WuXiBody structure comprises an antibody CH2 domain and/or an antibody CH3 domain. Exemplary sequences of such bispecific polypeptide complex are provided in Example 2.

In certain embodiments, the CH1 domain in the anti-CTLA-4 binding moiety is operably linked to a dimerization domain, such that two copies of the third polypeptide of the bispecific polypeptide complex having the G25 WuXiBody structure can asscoiate with each other to form a dimer. The association can be via any suitable interaction or linkage or bonding, for example, via a connecter, a disulphide bond, a hydrogen bond, an electrostatic interaction, a salt bridge, or a hydrophobic-hydrophilic interaction, or the combination thereof as disclosed herein. Exemplary dimerization domains include, without limitation, an antibody hinge region, an antibody CH2 domain, an antibody CH3 domain, and other suitable protein monomers capable of dimerizing and associating with each other. A hinge region, CH2, and/or CH3 domain can be derived from any antibody isotypes, such as IgG1, IgG2, and IgG4. In the bispecific G25 WuXiBody provided herein, at least one dimerization domain is within the antibody hinge region.

Bispecific complex sequences

In certain embodiments, the bispecific polypeptide complex comprises a combination of three polypeptide sequences: SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31 (G25R) , as shown in Example 2. In certain embodiments, the bispecific polypeptide complex comprises a combination of three polypeptide sequences: SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34 (G25) , as shown in Example 2. Both G25R and G25 formats have two copies of the first and the second antigen-binding moiety. In such embodiments, the first antigen-binding moiety binds to PD-1, and the second antigen binding moiety binds to CTLA-4.

Method of preparation

In one aspect, the present disclosure provides a method for preparing the bispecific polypeptide complex in a host cell.

The phrase “host cell” as used herein refers to a cell into which an exogenous polynucleotide and/or a vector has been introduced, such that the cell expressesthe bispecific polypeptide complex disclosed herein.

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 of the invention. 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 fungisuch as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hostssuch as A. nidulansand A. niger.

Suitable host cells for the expression of the glycosylated polypeptide complex and 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 invention, 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 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 bispecific polypeptide complex provided herein, comprising culturing the host cell provided herein under the condition at which the bispecific polypeptide complex is expressed.

In certain embodiments, the present disclosure provides a method of producing the bispecific polypeptide complex provided herein, comprising a) introducing to a host cell one or more polynucleotides encoding the bispecific polypeptide complex disclosed herein, and b) allowing the host cell to express the bispecific polypeptide complex.

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

When using recombinant techniques, 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 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 bispecific polypeptide complex provided herein comprises an 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 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 makes it possible to obtain high purity product in high yields using relatively simple purification processes.

Derivatives

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

For example, 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 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 ofdetectable labels mayincludefluorescent 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) , a chromophoricmoiety, 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 moieties 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 in their entirety.

Pharmaceutical composition

The present disclosure also provides a pharmaceutical composition comprising 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. Pharmaceutically 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, pharmaceutically 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 a 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 anampoule, 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 disease or a condition. In certain embodiments, the subject has been identified as having a disease 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 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 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 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, thedisease or condition treated by 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.

“Cancer” or “cancerous condition” as used herein refers to any medical condition mediated by neoplastic or malignant cell growth, proliferation, or metastasis, and includes both solid cancers and non-solid cancers such as leukemia. “Tumor” as used herein refers to a solid mass of neoplastic and/or malignant cells.

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 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 diseases or conditions include tumors and cancers, for example, lymphoma, lung cancer, liver cancer, cervical cancer, colon cancer, breast cancer, ovarian cancer, pancreatic cancer, melanoma, glioblastoma, prostate cancer, esophageal cancer, and gastric cancer.

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 prolierative disease, 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 related medical disorder. “Administered in combination” as used herein includes administeration 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 known in the art.

In certain embodiments, thetherapeutic agents can induce or boost immune response against cancer. For example, a tumor vaccine can be used to induce an immune response to a 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-αand 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 ICOS and OX-40) , and those enhance dendritic cell function and antigen presentation.

Kits

The present disclosure further provides kits comprising the bispecific polypeptide complex provided herein. In some embodiments, the kits are useful for detecting the presence or level of, or capturing or enrichingone 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 bispecific polypeptide complex provided herein which is conjugated with a detectable label. In certain other embodiments, the kit comprises an unlabeled bispecific polypeptide complex provided herein, and further comprises a secondary labeled antibody which is capable of binding to the unlabeled 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 bispecific polypeptide complex provided hereinisassociated with a substrate or a device. Auseful substrate or device can be, for example, magnetic beads, a microtiter plate, or a test strip. Such can be useful for a binding assay (such as ELISA) , an immunographic assay, and capturing or enriching of a target molecule in a biological sample.

The following examples are provided to better illustrate the claimed invention 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 invention. These specific compositions, materials, and methods are not intended to limit the invention, but merely to illustrate specific embodiments of the invention.

EXAMPLES

Example 1: Design and engineeringof antibody and TCR chimeric proteins

TCR sequences

TCRs are heterodimeric proteins made up of two chains. About 95%of human T cells have TCRs consisting of alpha and beta chains. Considering that more crystal structures are available for beta chain TRBC1, TRBC1 sequences were chosen as the major backbone to design the polypeptide complex disclosed herein ( “WuXiBody” ) . A typical amino acid sequence of TRBC1 can be found in Protein Data Bank (PDB) structure 4L4T.

Interchain disulphide-bond of TCR

TCR crystal structures were used to guide our WuXiBody design. Unlike native TCR anchored on the membrane of T cell surface, soluble TCR molecules are less stable, although its 3D structure is very similar to antibody Fab. As a matter of fact, the instability of TCRs in soluble conditions used to be a big obstacle that prevented the elucidation of its crystal structure (Wang 2014, supra) . We adopted a strategy of introducing a pair of Cys mutations in the TCR constant region and found it can significantly improve chain assembly and enhance expression. In the present disclosure, the TCR constant regions comprise an engineered CAlpha and an engineered CBeta. The mutated cysteine residue in the engineered CBeta is S56C (corresponding to amino acid C48 in SEQ ID NO: 1) , and the mutated cysteine residue in the engineered CAlpha is T47C (corresponding to amino acid C41 in SEQ ID NO: 2) . This pair of cysteine residues iscapable of forming a non-native interchain disulphide bond between the engineered CAlpha and the engineered CBeta.

The conjunctions connecting the antibody variable and TCR constant domains, their relative fusion orientations, as well as the Fc-connecting conjunctions were all carefully fine-tuned to make a stable and functional WuXiBody. As the TCR structure is very similar to an antibody Fab, we superimposed the antibody Fv homology model on the TCR variable region (PDB 4L4T, Figure 2B) . The superimposed structure indicates that an antibody Fv is structurally compatible with the TCR constant domain. Based on this structural alignment and corresponding sequences, all the relevant engineering parameters were designed. Suitable conjunction regions are disclosed in Example 2.

EXAMPLE 2: Bispecific Anti-CTLA-4 x PD-1 WuXiBody

Background

A bispecific anti-CTLA-4 x PD-1 WuXiBody was developed to induce antitumor immunity through simultaneous blockade ofimmunomodulatory checkpoint molecules CTLA-4 and PD-1.

Materials and Methods

General Materials

General research materials and their sources are listed in Table below.

Generation of Soluble Antigens

DNA sequences encoding the extracellular domain sequence of human PD-1 (Uniport No.: Q15116) were synthesized in Sangon Biotech (Shanghai, China) , and then subcloned into modified pcDNA3.3 expression vectors with 6xhis in the C-terminus. Proteins of human, cynomolgus, and mouse CTLA-4 and mouse and cynomolgus PD-1 were purchased from Sino Biological.

Expi293 cells (Invitrogen-A14527) were transfected with the purified expression vector pcDNA3.3. Cells were cultured for 5 days and supernatant was collected for protein purification using a Ni-NTA column (GE Healthcare, 175248) . The obtained human PD-1 was QC’ed by SDS-PAGE and SEC, and then stored at -80 ℃.

Generation of Reference Antibodies

DNA sequences encoding the variable region of an anti-CTLA-4 antibody (WBP316-BMK1, Ipilimumab) , and an anti-PD-1 antibody (WBP305-BMK1, nivolumab) weresynthesized in Sangon Biothech (Shanghai, China) , and then subcloned into modified pcDNA3.4 expression vectors with the constant region of human IgG1 or human IgG4 (S228P) . Anti-PD-1 WBP3055-1.153.7. uIgG4k and WBP3055-1.103.11. uIgG4k antibodies were generated after immunizing rats with human PD-1 and mouse PD-1, and were converted to an IgG4 (S228P) format. Anti-CTLA-4 antibody W3162_1.154.8-z35-IgG1k as disclosed in WO2018209701A was prepared in house. DNA sequences encoding three benchmark bispecific anti-CTLA-4 x PD-1 antibodies, WBP324-BMK1. IgG1. KDL ( “BiAb004” of patent publication CN106967172A) , WBP324-BMK2. uIgG4 (XENP20717 of WO2017218707A2) and WBP324-BMK3. uIgG4 ( “MGD019” of WO2017106061A1) were synthesized.

The plasmids containing the VH and VL genes were co-transfected into Expi293 cells. Cells were cultured for 5 days and supernatant was collected for protein purification using a Protein A column (GE Healthcare, 175438) or a Protein G column (GE Healthcare, 170618) . The obtained antibodies were tested by SDS-PAGE and SEC, and then stored at -80 ℃.

Generation of Target-expressing Cell Lines

Using Lipofectamine 2000, CHO-Sor 293F cells were transfected with the expression vectors containing the genes encoding full length human PD-1 or mouse PD-1. The cells were cultured in medium containing proper selection markers. The human PD-1 high expression stable cell line (WBP305. CHO-S. hPro1. C6) and mouse PD-1 high expression stable cell line (WBP305.293F. mPro1. B4) were obtained by limiting dilution.

Generation of Bispecific Anti-CTLA-4/PD-1 Bispecific Antibodies

Construction of W3248-U6T1. G25R-1. uIgG4. SP: 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 sequences encoding the anti- CTLA-4 antibody light chain variable region on the 5’ of TCR alpha constant region werecloned into another modified pcDNA3.3 expression vector. The anti-PD-1 light chain was cloned into the third modified pcDNA3.3 expression vector.

Construction of W3248-U6T5. G25-1. uIgG4. SP: DNA sequences encoding anti-PD-1 heavy chain variable region, constant region of TCR beta chain, anti-CTLA-4 heavy chain variable region, and IgG4 (S228P) constant region, linked from 5’ end to 3’ end, were cloned into a modified pcDNA3.3 expression vector. DNA sequences encoding the anti-PD-1 antibody light chain variable region on the 5’ end of the TCR alpha constant region werecloned into another modified pcDNA3.3 expression vector. The anti-CTLA-4 light chain was cloned into the third modified pcDNA3.3 expression vector.

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

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

For both bispecific antibodies, one heavy chain expression vector and two light chain expression vectors were co-transfected into Expi293 cells (ThermoFisher-A14527) according to the manufacturer’s instructions. Five days after transfection, the supernatants were harvested and purified using Protein A column (GE Healthcare-17543802) and further size-exclusion chromatography (GE Healthcare-17104301) . Antibody concentration was measured by Nano Drop. The low endotoxin level was confirmed by using endotoxin detection kit (GenScript-L00350) , and the endotoxin level of two bispecific antibodies was less than 10 EU/mg. The purity of proteins was evaluated by SDS-PAGE and HPLC-SEC.

Differential scanning fluorimetry (DSF)

A DSF assay was performed using a 7500 Fast Real-Time PCR system (Applied Biosystems) . Briefly, 19 μL of bispecific antibody solution was mixed with 1 μL of 62.5x SYPRO Orange solution (TheromFisher-S6650) and added to a 96 well plate. The plate was heated from 26 ℃ to 95 ℃ at a rate of 2 ℃/min and the resulting fluorescence data was collected. The data was analyzed automatically by its operation software and Th was calculated by taking the maximal value of the negative derivative of the resulting fluorescence data with respect to temperature. T _on can be roughly determined as the temperature of negative derivative plot beginning to decrease from a pre-transition baseline.

Human PD-1-binding by FACS

Engineered human PD-1 expressing cells W305-CHO-S. hPro1. C6 were seeded at 1×10 ⁵ cells/well in U-bottom 96-well plates (COSTAR 3799) . Antibodies with 3.16-fold titration in 1%BSA DPBS from 200 nM to 0.002 nM were added to the cells. The plates were incubated at 4 ℃ for 1 hour. After wash, 100 μL of 1: 125 diluted PE-labeled goat anti-human antibody (Jackson 109-115-098) was added to each well and the plates were incubated at 4 ℃ for 1 hour. The binding of the antibodies onto the cells was tested by flow cytometry and the mean fluorescence intensity (MFI) was analyzed by FlowJo.

Cynomolgus PD-1-binding by FACS

Engineered cynomolgus PD-1 expressing cells W305-293F. cynoPro1. FL. pool were seeded at 1×10 ⁵ cells/well in U-bottom 96-well plates (COSTAR 3799) . 4.0-fold titrated Abs with 1%BSA DPBS from 40 μg/ml to 0.0001526 μg/ml were added to the cells. Plates were incubated at 4 ℃ for 1 hour. After wash, 100 μL of 1: 150 diluted PE-labeled goat anti-human antibody (Jackson 109-115-098) was added to each well and the plates were incubated at 4 ℃ for 1 hour. The binding of the antibodies onto the cells was tested by flow cytometry and the mean fluorescence intensity (MFI) was analyzed by FlowJo.

Human CTLA-4-binding by FACS

Engineered human CTLA-4 expressing cells W316-293F. hPro1. FL were seeded at 1×10 ⁵ cells/well in U-bottom 96-well plates (COSTAR 3799) . 3.16-fold titrated Abs with 1%BSA DPBS from 200 nM to 0.002 nM were added to the cells. Plates were incubated at 4 ℃ for 1 hour. After wash, 100 μL of 1: 150 diluted PE-labeled goat anti-human antibody (Jackson 109-115-098) was added to each well and the plates were incubated at 4 ℃ for 1 hour. The binding of the antibodies onto the cells was tested by flow cytometry and the mean fluorescence intensity (MFI) was analyzed by FlowJo.

Cynomolgus CTLA-4-binding by FACS

Engineered human CTLA-4 expressing cells W316-293F. cynoPro1. F1. Pool were seeded at 1×10 ⁵ cells/well in U-bottom 96-well plates (COSTAR 3799) . 4-fold titrated Abs with 1%BSA DPBS from 40 μg/ml to 0.00004 μg/ml were added to the cells. Plates were incubated at 4 ℃ for 1 hour. After wash, 100 μL of 1: 150 diluted PE-labeled goat anti-human antibody (Jackson 109-115-098) was added to each well and the plates were incubated at 4 ℃ for 1 hour. The binding of the antibodies onto the cells was tested by flow cytometry and the mean fluorescence intensity (MFI) was analyzed by FlowJo.

hPD-1 and hCTLA-4 dual binding by ELISA

In order to test whether the bispecific antibodies could bind to both hPD-1 and hCTLA-4, an ELISA assay was developed as described below. A 96-well ELISA plate (Nunc MaxiSorp, ThermoFisher) was coated overnight at 4 ℃ with 0.5 μg/ml antigen-1 (hPD-1-ECD, W305-hPro1. ECD. mFc) in carbonate-bicarbonate buffer. After a 1 hour blocking step with 2%(w/v) bovine serum albumin (Pierce) dissolved in PBS, serial dilutions of the different PD-1×CTLA-4 bispecific antibodies in PBS containing 2%BSA PBS were incubated on the plates for 1 hour at room temperature. Following the incubation, plates were washed three times with 300 μL per well of PBS containing 0.5% (v/v) Tween 20.0.5 μg/ml antigen-2 (hCTLA-4-ECD, W316-hPro1. ECD. hFc. Biotin) was added to plates and the mixture was incubated for 1 hour. After washing the plates three times, Streptavidin-HRP (Lifetechnologies, #SNN1004) (1: 20000 diluted) was added and incubated on the plates for 1 hour at room temperature. After washing six times with 300 μL per well of PBS containing 0.5% (v/v)

Tween

20, 100 μL tetramethylbenzidine (TMB) substrate was added for the detection per well. The reaction was stopped after approximately 5 minutes through the addition of 100 μL per well of 2 M HCl. The absorbance of the wells was measured at 450 nm with a multiwall plate reader (

M5e) .

hPD-1 and hCTLA-4 dual binding by FACS

In order to test whether the bispecific antibodies could bind to both hPD-1 and hCTLA-4, a FACS assay was developed as described below. Engineered human PD-1 and CTLA-4 expressing cells W305-CHO-S. hPro1. C6 and W316-293F. hPro1. F1 were stained with Calcein-AM (Corning-354216) at 50 nM and Far red (Invitrogen-C34572) at 20 nM, respectively, for 20mins at 37 ℃. After wash with 1% (w/v) bovine serum albumin (Pierce) dissolved in PBS twice, mixed hPD-1 (5E4) and hCTLA-4 (5E4) cells were seeded at 1×10 ⁵ cells/well in U-bottom 96-well plates (COSTAR 3799) . After removal of the supernatant, 3 x serially diluted antibodies with 1%BSA DPBS from 7.5 nM to 0.83 nM were added to the cells. The plates were incubated at 4 ℃ for 1.5 hour. The cells were tested by flow cytometry and the percentage of double positive cells was analyzed by FlowJo.

Human PD-1-competitive FACS

In order to test whether the bispecific antibodies could block hPD-L1 binding to hPD-1 protein, a competitive FACS was conducted. Briefly, engineered human PD-1 expressing cells W305-CHO-S. hPro1. C6 (in house) were seeded at 1×10 ⁵ cells/well in U-bottom 96-well plates (COSTAR 3799) , 200 nM to 0.002 nM human PD-L1 coupled with 5 ug/ml human PD-L1 protein W315-hPro1. ECD. mFc were added to the cells. Plates were incubated at 4 ℃ for 1 hour. After wash, the binding of W315-hPro1. ECD. mFc to cells expressing human PD-1 was detected by FITC-labeled goat anti-mouse antibody (abcam 98716 1: 125) . The competition binding of antibodies to the cells was tested by flow cytometry and the mean fluorescence intensity (MFI) was analyzed by FlowJo.

Blockage of human/cynomolgus CTLA-4 binding to human CD80

ELISA was used to test whether the bispecific antibodies could block hCTLA-4 binding to hCD80 protein. Briefly, flat-bottom 96-well plates (Nunc MaxiSorp, ThermoFisher) were pre-coated with 0.5 μg/ml W316-hPro1. ECD. hFc overnight at 4 ℃. After 2%BSA blocking, 100 μL of 3.16-fold titrated Abs from 400 nM to 0.04 nM Abs coupled with 0.5 μg/ml human CD80 protein W316-hPro1L1. ECD. His were pipetted into each well and incubated for 1 hour at ambient temperature. Following the incubation, plates are washed 3 times with 300 μL per well of PBS containing 0.5% (v/v) Tween 20.100 μL 0.5 μg/ml Biotin-labeled anti-His mAb (GenScript-A00613) was added to plate pre well and incubatedfor 1 hour. After washing for 6 times, the binding of W315-hPro1L1. ECD. His to WBP316-hPro1. ECD. hFc was detected by Streptavidin-HRP (Lifetechnologies, #SNN1004) (1: 20000 diluted) . The color was developed by dispensing 100 μL of TMB substrate, and then stopped by 100 μL of 2N HCl. The absorbance was read at 450 nm using a Microplate Spectrophotometer (

M5e) .

Competitive FACS was used to test whether the antibodies could block human or cynomolgus CTLA-4 binding to hCD80 on the cell surface. Briefly, human CD80-expressing CHO-K1 cells were added to each well of a 96-well plate (COSTAR 3799) at 1 x 10 ⁵ per well and centrifuged at 1500 rpm for 4 minutes at 4℃ before removing the supernatant. Serial dilutions of test antibodies, and positive and negative controls were mixed with biotinylated human CTLA-4. ECD. hFc. Due to different density of ligands on the cell surface, 0.066-0.037 μg/mL of hCTLA-4. ECD. hFc-Biotin was used for human CD80-expressing cells. Then the mixtures of antibody and CTLA-4 were added to the cells and incubated for 1 hour at 4 ℃. The cells were washed two times with 200 μl FACS washing buffer (DPBS containing 1%BSA) . Streptavidin PE (BD Pharmingen-554061) 1 to 600 diluted in FACS buffer was added to the cells and incubated at 4 ℃ for 1 hour. Additional washing steps were performed two times with 200 μL FACS washing buffer followed by centrifugation at 1500 rpm for 4 minutes at 4 ℃. Finally, the cells were resuspended in 100 μL FACS washing buffer and fluorescence values were measured by flow cytometry and analyzed by FlowJo.

Affinity to CTLA-4 and PD-1

SPR technology was used to measure the on-rate constant (ka) and off-rate constant (kd) of the antibodies to ECD of CTLA-4 or PD-1. The affinity constant (KD) was consequently determined.

Biacore T200, Series S Sensor Chip CM5, Amine Coupling Kit, and 10x HBS-EP were purchased from GE Healthcare. Goat anti-human IgG Fc antibody was purchased from Jackson ImmunoResearch Lab (catalog number 109-005-098) . In the immobilization step, the activation buffer was prepared by mixing 400 mM EDC and 100 mM NHS immediately prior to injection. The CM5 sensor chip was activated for 420 s with the activation buffer. 30 μg/mL of goat anti-human IgG Fcγ antibody in 10 mM NaAc (pH 4.5) was then injected to Fc1-Fc4 channels for 200s at a flow rate of 5 μL/min. The chip was deactivated by 1 M ethanolamine-HCl (GE) . Then the antibodies were captured on the chip. Briefly, 4 μg/mL antibodies in running buffer (HBS-EP+) was injected individually to the Fc3 channel for 30 s at a flow rate of 10 μL/min. Eight different concentrations (20, 10, 5, 2.5, 1.25, 0.625, 0.3125, and 0.15625 nM) of analyte ECD of CTLA-4 or PD-1 and blank running buffer were injected orderly to Fc1-Fc4 channels at a flow rate of 30 μL/min for an association phase of 120 s, followed by 2400 s dissociation phase. Regeneration buffer (10 mM Glycine pH 1.5) was injected at 10 μL/min for 30 s following every dissociation phase.

Human serum stability

The antibodies were incubated in freshly isolated human serum at 37℃. On indicated time points, an aliquot of serum treated sample was removed from the incubator and snap frozen in liquid nitrogen, and then stored at -80℃ until ready for a dual-binding ELISA test. The frozen samples were quickly thawed immediately prior to the stability test. Briefly, plates were pre-coated with 0.5 μg/mL of hCTLA4. ECD. hFc (in house) at 4℃ overnight. After 1-hour blocking, the testing antibodies were added to the plates at various concentrations. The plates were incubated at ambient temperature for 1 hour. Following the incubation, the plates were washed three times with 300 μL per well of PBS containing 0.5% (v/v) Tween 20. Then 0.1 μg/ml hPD-1-ECD. Biotin was added to the plates and the mixture was incubated for 1 hour. After washing the plates three times, Streptavidin-HRP (Lifetechnologies, #SNN1004) (1: 20000 diluted) was added and incubated on the plates for 1 hour at room temperature. After washing six times with 300 μL per well of PBS containing 0.5% (v/v)

Tween

20, 100 μL tetramethylbenzidine (TMB) substrate wasadded for the detection per well. The reaction was stopped after approximately 5 minutes by addition of 100 μL per well of 2 M HCl. The absorbance of the wells was measured at 450 nm with a multiwall plate reader (

M5e) .

Mouse pharmacokinetics study

Female C57BL/6 mice (Shanghai Lingchang Biotech Co., Ltd) of 10 weeks-old were used in the study. Ten animals were divided into two groups (5 animal/group) . The animals were administered with the antibodies at 10 mg/kg by tail vein injection respectively with a dose volume of 10 ml/kg. After anesthesiaby using Isoflurane inhalation, blood samples were collected with EDTA-K2 anticoagulation at 0.5h, 2h, 6h, 24h, Day2, Day4 and Day7 after injection. The plasma samples were then prepared by centrifuging the blood samples at approximately 4℃, 5000 g for 5 minutes. All plasma samples were then quickly frozen over dry ice and kept at -80℃ until ELISA analysis. The concentrations of W3248-U6T1. G25R-1. uIgG4. SP and W3248-U6T5. G25-1. uIgG4. SP in plasma samples were determined by ELISA. The linear/log trapezoidal rule was applied in obtaining the PK parameters. The values of 3 parameters were obtained (C ₀ -initial plasma concentration; AUC-area under the concentration-time curve after a single dose; and terminal T _1/2-half life of plasma clearance) and data was expressed asmean±SD.

Results

Expression and purification of bispecific antibodies

The purity of the bispecific antibodies was above 90%, analyzed by both SDS-PAGE (Figure 3A) and SEC-HPLC (Figure 3B) .

DSF of WuXiBody

DSF was used to measure Tm of WuXiBody. As shown in Figure 4, W3248-U6T1. G25R-1. uIgG4. SP and WBP3248-U6T5. G25-1-uIgG4. SP have Th1 at 60.8 and 63.4 ℃, respectively.

Binding to human and cynomolgus PD-1

The bispecific antibodies could bind to human PD-1 (Figure 5) and cynomolgus PD-1 (Figure 6) . The human PD-1-binding activity of W3248-U6T1. G25R-1. uIgG4. SP was slightly better than WBP3248-U6T5. G25-1-uIgG4. SP in FACS. W3248-U6T1. G25R-1. uIgG4. SP and WBP3248-U6T5. G25-1-uIgG4. SP have affinity to human PD-1 at 1.24 nM and 1.32 nM, respectively (Figure 9) .

Binding to human and cynomolgus CTLA-4

The purified bispecific antibodies bound to human CTLA-4, as tested in FACS (Figure 7) . The two bispecific antibodies also bound to cynomolgus CTLA-4 (Figure 8) . W3248-U6T1. G25R-1. uIgG4. SP and WBP3248-U6T5. G25-1-uIgG4. SP have affinity to human CTLA-4 at 0.0356 nM and 0.357 nM, respectively (Figure 9) .

Simultaneous binding to CTLA-4 and PD-1

In order to test whether the bispecific antibodies can bind to both targets, ELISA and FACS were used. In the ELISA, human PD-1 was coated on the plate. After adding bispecific antibodies, biotinylated CTLA-4 was used to detect bound bispecific antibodies. As shown in Figure 13, W3248-U6T1. G25R-1. uIgG4. SP and WBP3248-U6T5. G25-1-uIgG4. SP could bind to both PD-1 and CTLA-4 with EC50 at 0.1072 to 0.0710 nM, comparable with a bispecific reference antibody WBP324 BMK1 (EC50 =0.0599 nM) . In the FACS, both W3248-U6T1. G25R-1. uIgG4. SP and WBP3248-U6T5. G25-1-uIgG4. SP could simultaneously bind to PD-1+ and CTLA-4+ cells (Figure 14) .

Blocking human or cynomolgus CTLA-4 binding to CD80 binding

A competitive FACS was used to test the bispecific antibodies’ blockage of CTLA-4 with its ligand CD80. W3248-U6T1. G25R-1. uIgG4. SP and WBP3248-U6T5. G25-1-uIgG4. SP blocked CTLA-4 binding to CD80 with IC ₅₀ of 4.300 and 0.7581 nM (Figure 11) . Similarly, the bispecific antibodies could also block cynomolgus CTLA-4 binding to human CD80+ cells (Figure 12) .

Blocking PD-1 binding to its ligand

A competitive FACS was used to test the bispecific antibodies’ blockage of PD-1 with its ligand PD-L1. W3248-U6T1. G25R-1. uIgG4. SP and WBP3248-U6T5. G25-1-uIgG4. SP blocked PD-1 binding to PD-L1 with IC ₅₀ of 1.670 nM and 1.917 nM (Figure 10) .

Serum stability

The two bispecific antibodies were incubated at 37 ℃ human serum for 14 days, and their dual binding to human CTLA-4 and PD-1 was measured in ELISA. As shown in Figures 15A and 15B, W3248-U6T1. G25R-1. uIgG4. SP and WBP3248-U6T5. G25-1-uIgG4. SP dual binding to the targets did not change over time, indicating that these two bispecific antibodies were stable in 37℃ human serum for at least 14 days.

Mouse PK study

After asingle intravenous injection in C57BL/6 female mice, as shown in Figure 16, the C ₀, AUC _0-day7 and terminal T _1/2of W3248-U6T1. G25R-1. uIgG4. SP were 187±16.7μg/ml, 9450±670μg/ml*h and 113±16.3h, respectively; the C ₀, AUC _0-day7 and terminal T _1/2 of W3248-U6T5. G25-1. uIgG4. SP were 197±39μg/ml, 9014±443μg/ml*h and 138±56.5h, respectively. The two antibodies showed similar PK profilesat 10mg/kg in C57BL/6 mice.

Claims

A bispecific polypeptide complex, comprising a first antigen-binding moiety associated with a second antigen-binding moiety, wherein:

the first antigen-binding moiety comprises:

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 first light chain variable domain (VL) of the first antibody operably linked to a second TCR constant region (C2) , and

the second antigen-binding moiety comprises:

a second VH of a second antibody operably linked to an antibody heavy chain CH1 domain, and a second VL of the second antibody operably linked to an antibody light chain constant (CL) domain,

wherein: (a) one of the first and the second antigen-binding moiety is an anti-CTLA-4 binding moiety, and the other one is an anti-PD-1 binding moiety,

(b) the anti-CTLA-4 binding moiety is derived from an anti-CTLA-4 antibody comprising:

(i) a a heavy chain complementarity determining region (CDRH) 1 consisting of SEQ ID NO: 5, a CDRH2 consisting of SEQ ID NO: 6, a CDRH3 consisting of SEQ ID NO: 7, a light chain complementarity determining region (CDRL) 1 consisting of SEQ ID NO: 8, a CDRL2 consisting of SEQ ID NO: 9, and a CDRL3 consisting of SEQ ID NO: 10;

or (ii) a CDRH1 consisting of SEQ ID NO: 11, a CDRH2 consisting of SEQ ID NO: 12, a CDRH3 consisting of SEQ ID NO: 13, a CDRL1 consisting of SEQ ID NO: 14, a CDRL2 consisting of SEQ ID NO: 15, and a CDRL3 consisting of SEQ ID NO: 16,

(c) the anti-PD-1 binding moiety is derived from an anti-PD-1 antibody comprising:

a CDRH1 consisting of SEQ ID NO: 21, a CDRH2 consisting of SEQ ID NO: 22, a CDRH3 consisting of SEQ ID NO: 23, a CDRL1 consisting of SEQ ID NO: 24, a CDRL2 consisting of SEQ ID NO: 25, and a CDRL3 consisting of SEQ ID NO: 26.
The bispecific polypeptide complex of claim 1, wherein C1 comprises an engineered TCR beta constant region comprising one or more mutated residues selected from the group consisting of K9E, S56C, N69Q and C74A relative to a native human TCR beta constant region comprising the amino acid sequence of SEQ ID NO: 37; and/or

C2 comprises an engineered TCR alpha constant region comprising one or more mutated residues selected from the group consisting of N32Q, T47C, N66Q and N77Q relative to a native human TCR alpha constant region comprising the amino acid sequence of SEQ ID NO: 35.
The bispecific polypeptide complex of claim 2, wherein the engineered TCR beta constant region comprises a S56C mutation and the engineered TCR alpha constant region comprises a T47C mutation to form a non-native interchain disulphide bond.
The bispecific polypeptide complex of claim 3, wherein the engineered TCR beta constant region comprises SEQ ID NO: 1 and the engineered TCR alpha constant region comprises SEQ ID NO: 2.
The bispecific polypeptide complex of any of claims 1-4, comprising a combination of three polypeptide sequences, wherein:

the first polypeptide sequence comprises a VL of the anti-PD-1 binding moiety operably linked to a CL domain of the anti-PD-1 binding moiety,

the second polypeptide sequence comprisesa VL of the anti-CTLA-4 binding moiety operably linked to C2 at a second conjunction domain, and

the third polypeptide sequence comprises (i) a VH of the anti-PD-1 binding moiety operably linked to a CH1 domain of the anti-PD-1 binding moiety, and (ii) a VH of the anti-CTLA-4 binding moiety operably linked to C1 at a first conjunction domain, wherein the CH1 domain in (i) and the VH in (ii) are operably linked via a linker, and wherein:

the anti-CTLA-4 binding moiety is derived from an anti-CTLA-4 antibody comprising: a CDRH1 consisting of SEQ ID NO: 5, a CDRH2 consisting of SEQ ID NO: 6, a CDRH3 consisting of SEQ ID NO: 7, a CDRL1 consisting of SEQ ID NO: 8, a CDRL2 consisting of SEQ ID NO: 9, and a CDRL3 consisting of SEQ ID NO: 10.
The bispecific polypeptide complex of claim 5, further comprising an antibody CH2 domain, and/or an antibody CH3 domain, wherein C1 in the third polypeptide sequence is operably linked to the antibody CH2 domain at a third conjunction domain.
The bispecific polypeptide complex of claim 5, wherein the first conjunction domain comprises SEQ ID NO: 3, the second conjunction domain comprises SEQ ID NO: 4, the third conjunction domain comprises SEQ ID NO: 46, and the linker comprises SEQ ID NO: 45.
The bispecific polypeptide complex of any of the preceding claims, wherein the anti-CTLA-4 binding moiety comprises a heavy chain variable domain sequence comprising SEQ ID NO: 17 and a light chain variable domain sequence comprising SEQ ID NO: 18.
The bispecific polypeptide complex of any of the preceding claims, wherein the anti-PD-1 binding moiety comprises a heavy chain variable domain sequence comprising SEQ ID NO: 27 and a light chain variable domain sequence comprising SEQ ID NO: 28.
The bispecific polypeptide complex of any of the preceding claims, comprising a combination of three polypeptide sequences: SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31.
The bispecific polypeptide complex of any of claims 1-4, comprising a combination of three polypeptide sequences, wherein:

the first polypeptide sequence comprises a VL of the anti-PD-1 binding moiety operably linked to C2 at a second conjunction domain,

the second polypeptide sequence comprisesa VL of the anti-CTLA-4 binding moiety operably linked to a CL domain of the anti-CTLA-4 binding moiety, and

the third polypeptide sequence comprises (i) a VH of the anti-PD-1 binding moiety operably linked to C1 at a first conjunction domain, and (ii) a VH of the anti-CTLA-4 binding moiety operably linked to a CH1 domain of the anti-CTLA-4 binding moiety, wherein C1 in (i) and the VH in (ii) are operably linked via a linker, and wherein:

the anti-CTLA-4 binding moiety is derived from an anti-CTLA-4 antibody comprising a CDRH1 consisting of SEQ ID NO: 11, a CDRH2 consisting of SEQ ID NO: 12, a CDRH3 consisting of SEQ ID NO: 13, a CDRL1 consisting of SEQ ID NO: 14, a CDRL2 consisting of SEQ ID NO: 15, and a CDRL3 consisting of SEQ ID NO: 16.
The bispecific polypeptide complex of claim 11, further comprising an antibody CH2 domain, and/or an antibody CH3 domain.
The bispecific polypeptide complex of any of claims 11-12, wherein the first conjunction domain comprises SEQ ID NO: 3, the second conjunction domain comprises SEQ ID NO: 4, and the linker comprises SEQ ID NO: 45.
The bispecific polypeptide complex of any of claims 11-13, wherein the anti-CTLA-4 binding moiety comprises a heavy chain variable domain sequence comprising SEQ ID NO: 19 and a light chain variable domain sequence comprising SEQ ID NO: 20.
The bispecific polypeptide complex of any of claims 11-14, wherein the anti-PD-1 binding moiety comprises a heavy chain variable domain sequence comprising SEQ ID NO: 27 and a light chain variable domain sequence comprising SEQ ID NO: 28.
The bispecific polypeptide complex of any of claims 11-15, comprising at least a portion of an antibody hinge region, optionally derived from IgG1, IgG2, or IgG4.
The bispecific polypeptide complex of any of claims 11-16, comprising a combination of three polypeptide sequences: SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34.
A conjugate comprising the bispecific polypeptide complex of any of the preceding claims, conjugated to a moiety.
A host cell expressing the bispecific polypeptide complex of any of claims 1-17.
A method of expressing the bispecific polypeptide complex of any of claims 1-17, comprising culturing the host cell of claim 18 under a condition at which the bispecific polypeptide complex is expressed.
A method of producing a bispecific polypeptide complex comprising:

a) introducing to a host cell one or more polynucleotides encoding the bispecific polypeptide complex of any of claims 1-17; and

b) allowing the host cell to express the bispecific polypeptide complex.
The method of any of claims 20-21, further comprising isolating the bispecific polypeptide complex.
A composition comprising the bispecific polypeptide complex of any of claims 1-17.
A pharmaceutical composition comprising the bispecific polypeptide complex of any of claims 1-17 and a pharmaceutically acceptable carrier.
A method of treating a disease or condition in a subject in need thereof, comprising administrating to the subject a therapeutically effective amount of the bispecific polypeptide complex of any of claims 1-17.
The method of claim 25, wherein the disease or condition is cancer.
The method of claim 26, wherein the cancer islymphoma, lung cancer, liver cancer, cervical cancer, colon cancer, breast cancer, ovarian cancer, pancreatic cancer, melanoma, glioblastoma, prostate cancer, esophageal cancer, or gastric cancer.
Use of the bispecific polypeptide complex of any of claims 1-17 in the manufacture of a medicament for modulating an immune response in a subject in need thereof.
Use of the bispecific polypeptide complex of any of claims 1-17 in the manufacture of a medicament for preventing or teating a disease or condition in a subject in need thereof.
The use of claim 29, wherein the disease or condition is cancer.
The use of claim 30, wherein the cancer islymphoma, lung cancer, liver cancer, cervical cancer, colon cancer, breast cancer, ovarian cancer, pancreatic cancer, melanoma, glioblastoma, prostate cancer, esophageal cancer, or gastric cancer.
A kit comprising the bispecific polypeptide complex of any of claims 1-17.
The kit of claim 32, wherein the kit isused for detection, diagnosis, prognosis, or treatment of a disease or condition.