WO2020057611A1

WO2020057611A1 - Novel bispecific ctla-4/pd-1 polypeptide complexes

Info

Publication number: WO2020057611A1
Application number: PCT/CN2019/106731
Authority: WO
Inventors: Zhuozhi Wang; 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, methods of producing the bispecific anti-CTLA-4 x PD-1 polypeptide 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 comprising the bispecific anti-CTLA-4 x PD-1 polypeptide complexes.

Description

Novel Bispecific CTLA-4/PD-1 Polypeptide Complexes

SEQUENCE LISTING

The instant application contains a sequence listing and is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present disclosure generally relates to bispecific anti-CTLA-4 x PD-1 polypeptide complexes and their uses for treating cancers.

BACKGROUND

Cancer immunotherapy has become a hot research area of 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. CD28 mediates one of 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 affinity binding of CTLA-4 to CD80 and CD86 than that of CD28, CTLA-4 can out-compete with CD28 binding on CD80 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 associated with Treg production of immunosuppressive cytokines such as transforming growth factor beta and iterleukin-10.

CTLA-4 blockade can induce tumor regression, demonstrating 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 of the interaction of CTLA-4, 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 5 mg/ml concentrate for solution for infusion. Ipilimumab is also under clinical investigation of other tumor types, including prostate and lung cancers. Another anti-CTLA-4 antibody 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 belong to 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 PD-1 pathway is to tune down inflammatory immune response in tissues and organs.

Immunotherapy with the combination of monoclonal antibodies (mAbs) that block PD-1 (nivolumab) and CTLA-4 (ipilimumab) has shown clinical benefit beyond that observed with either mAb alone.

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 effect superior to the effect of individual antibodies.

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 functional domain comprising a first antigen-binding moiety and a second functional domaincomprising a second antigen-binding moiety,

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

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

a) a heavy chain CDR1 comprising SEQ ID NO: 1, b) a heavy chain CDR2 comprising SEQ ID NO: 2, c) a heavy chain CDR3 comprising SEQ ID NO: 3, d) a light chain CDR1 comprising SEQ ID NO: 4, e) a light chain CDR2 comprising SEQ ID NO: 5, and f) a light chain CDR3 comprising SEQ ID NO: 6;

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

a) a heavy chain CDR1 comprising SEQ ID NO: 7, b) a heavy chain CDR2 comprising SEQ ID NO: 8, c) a heavy chain CDR3 comprising SEQ ID NO: 9, d) a light chain CDR1 comprising SEQ ID NO: 10, e) a light chain CDR2 comprising SEQ ID NO: 11, and f) a light chain CDR3 comprising SEQ ID NO: 12; or

a) a heavy chain CDR1 comprising SEQ ID NO: 13, b) a heavy chain CDR2 comprising SEQ ID NO: 14, c) a heavy chain CDR3 comprising SEQ ID NO: 15, d) a light chain CDR1 comprising SEQ ID NO: 16, e) a light chain CDR2 comprising SEQ ID NO: 17, and f) a light chain CDR3 comprising SEQ ID NO: 18.

In some embodiments, the anti-CTLA-4 binding moiety herein comprises:

a) a heavy chain CDR1 comprising SEQ ID NO: 1, b) a heavy chain CDR2 comprising SEQ ID NO: 2, c) a heavy chain CDR3 comprising SEQ ID NO: 3, d) a light chain CDR1 comprising SEQ ID NO: 4, e) a light chain CDR2 comprising SEQ ID NO: 5, and f) a light chain CDR3 comprising SEQ ID NO: 6; and the anti-PD-1 binding moiety herein comprises: a) a heavy chain CDR1 comprising SEQ ID NO: 7, b) a heavy chain CDR2 comprising SEQ ID NO: 8, c) a heavy chain CDR3 comprising SEQ ID NO: 9, d) a light chain CDR1 comprising SEQ ID NO: 10, e) a light chain CDR2 comprising SEQ ID NO: 11, and f) a light chain CDR3 comprising SEQ ID NO: 12; or

a) a heavy chain CDR1 comprising SEQ ID NO: 13, b) a heavy chain CDR2 comprising SEQ ID NO:14, c) a heavy chain CDR3 comprising SEQ ID NO: 15, d) a light chain CDR1 comprising SEQ ID NO: 16, e) a light chain CDR2 comprising SEQ ID NO: 17, and f) a light chain CDR3 comprising SEQ ID NO: 18.

In some embodiments, the anti-CTLA-4 binding moiety comprises:

a heavy chain variable region sequence comprising a) the amino acid sequence of SEQ ID NO: 19, b) an amino acid sequence at least 85%, 90%, or 95%identical to the amino acid sequence of SEQ ID NO: 19, or c) an amino acid sequence with addition, deletion and/or substitution of one or more amino acids as compared with the amino acid sequence of SEQ ID NO: 19; and

a light chain variable region sequence comprising a) the amino acid sequence of SEQ ID NO: 20, b) an amino acid sequence at least 85%, 90%, or 95%identical to the amino acid sequence of SEQ ID NO: 20, or c) an amino acid sequence with addition, deletion and/or substitution of one or more amino acids as compared with the amino acid sequence of SEQ ID NO: 20.

In some embodiments, the present disclosure provides a bispecific polypeptide complex, comprising:

a first functional domain comprising a CTLA-4 antigen-binding moiety and a second functional domain comprising a PD-1 antigen-binding moiety,

wherein the CTLA-4 antigen-binding moiety comprises a first heavy chain variable domain (VH) of an anti-CTLA-4 antibody operably linked to a first heavy chain constant region domain (CH1) , and a first light chain variable domain (VL) of the anti-CTLA-4 antibody operably linked to a first light chain constant region (CL) ,

wherein the PD-1 antigen-binding moiety comprises a second VH of an anti-PD-1 antibody operably linked to a second VL of the anti-PD-1 antibody, and

wherein: (A) the anti-CTLA-4 antibody comprises: a heavy chain complementarity determining region (CDRH) 1 consisting of SEQ ID NO: 1; a CDRH2 consisting of SEQ ID NO: 2; a CDRH3 consisting of SEQ ID NO: 3; a light chain complementarity determining region (CDRL) 1 consisting of SEQ ID NO: 4; a CDRL2 consisting of SEQ ID NO: 5; and a CDRL3 consisting of SEQ ID NO: 6; and (B) the anti-PD-1 antibody comprises: a CDRH1 selected from the group consisting of SEQ ID NOs: 7 and 13; a CDRH2 selected from the group consisting of SEQ ID NOs: 8 and 14; a CDRH3 selected from the group consisting of SEQ ID NOs: 9 and 15; a CDRL1 selected from the group consisting of SEQ ID NOs: 10 and 16; a CDRL2 selected from the group consisting of SEQ ID NOs: 11 and 17; and a CDRL3 selected from the group consisting of SEQ ID NOs: 12 and 18.

In some embodiments, the anti-PD-1 binding moiety herein comprisesa heavy chain variable region sequence comprising a) the amino acid sequence of SEQ ID NO: 21, b) an amino acid sequence at least 85%, 90%, or 95%identical to the amino acid sequence of SEQ ID NO: 21, or c) an amino acid sequence with addition, deletion and/or substitution of one or more amino acids as compared with the amino acid sequence of SEQ ID NO: 21; and

a light chain variable region sequence comprising a) the amino acid sequence of SEQ ID NO: 22, b) an amino acid sequence at least 85%, 90%, or 95%identical to the amino acid sequence of SEQ ID NO: 22, or c) an amino acid sequence with addition, deletion and/or substitution of one or more amino acids as compared with the amino acid sequence of SEQ ID NO: 22; or

the anti-PD-1 binding moiety comprisesa heavy chain variable region sequence comprising a) the amino acid sequence of SEQ ID NO: 23, b) an amino acid sequence at least 85%, 90%, or 95%identical to the amino acid sequence of SEQ ID NO: 23, or c) an amino acid sequence with addition, deletion and/or substitution of one or more amino acids as compared with the amino acid sequence of SEQ ID NO: 23; and

a light chain variable region sequence comprising a) the amino acid sequence of SEQ ID NO: 24, b) an amino acid sequence at least 85%, 90%, or 95%identical to the amino acid sequence of SEQ ID NO: 24, or c) an amino acid sequence with addition, deletion and/or substitution of one or more amino acids as compared with the amino acid sequence of SEQ ID NO: 24.

In some embodiments, the first functional domain and the second functional domain are linked, preferably via a linker.

In some embodiments, the first and/or second functional domain further comprises a Fc region, preferably a human Fc region, more preferably a human IgG Fc region, even more preferably a human IgG4 Fc region.

In some embodiments, the Fc region has a mutation of one or several amino acids.

In some embodiments, the human IgG4 Fc region has a mutation at amino acid 228, preferably a mutation of S to P at amino acid 228 (S228P) .

In some embodiments, the first functional domain and the second functional domain are independently selected from immunoglobin and the antigen-binding fragments thereof, such as Fab, F (ab) ’ 2 or scFv.

In some embodiments, the first functional domain is an immunoglobin and the second functional domain is scFv.

In some embodiments, the first antigen-binding moiety is an anti-CTLA-4 binding moiety and the second antigen-binding moiety is an anti-PD-1 binding moiety.

In some embodiments, the immunoglobin is selected from a group consisting of IgG, IgA, IgD, IgE and IgM, preferably IgG, such as IgG1, IgG2 or IgG4, more preferably IgG4, even more preferably IgG4 (S228P) .

In some embodiments, the second functional domain is linked to the C-terminus of the first functional domain.

In some embodiments, the bispecific polypeptide complex comprises a heavy chain sequence comprising a) the amino acid sequence of SEQ ID NO: 25 or 26, b) an amino acid sequence at least 85%, 90%, or 95%identical to the amino acid sequence of SEQ ID NO: 25 or 26, or c) an amino acid sequence with addition, deletion and/or substitution of one or more amino acids as compared with the amino acid sequence of SEQ ID NO: 25 or 26; and

a light chain sequence comprising a) the amino acid sequence of SEQ ID NO: 27, b) an amino acid sequence at least 85%, 90%, or 95%identical to the amino acid sequence of SEQ ID NO: 27, or c) an amino acid sequence with addition, deletion and/or substitution of one or more amino acids as compared with the amino acid sequence of SEQ ID NO: 27.

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 an isolated polynucleotide encoding the bispecific polypeptide complex provided herein.

In one aspect, the present disclosure provides an isolated vector comprising the polynucleotide provided herein.

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 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 drawings.

BRIEF DESCFRIPTION OF DRAWINGS

Figures 1A-1B present the result of SEC, which shows the purity of bispecific antibodies WBP3245A (W324-T1U6. G15-2. uIgG4. SP (dk) ) (Fig. 1A) and WBP3245B (W324-T1U8. G15-2. uIgG4. SP (dk) ) (Fig. 1B) .

Figure 2 shows bispecific antibodies bound to human CTLA-4, as tested by ELISA.

Figures 3A-3B show bispecific antibodies bound to human CTLA-4, as tested by FACS.

Figure 4 shows bispecific antibodies bound to cynomolgus CTLA-4, as tested by ELISA.

Figure 5 shows bispecific antibodies bound to cynomolgus CTLA-4, as tested by FACS.

Figure 6 shows bispecific antibodies bound to human PD-1, as tested by ELISA.

Figures 7A-7B show bispecific antibodies bound to human PD-1, as tested by FACS.

Figure 8 shows bispecific antibodies bound to cynomolgus PD-1, as tested by FACS.

Figures 9A-9B show bispecific antibodies binding simultaneously to human PD-1 and CTLA-4, as tested by ELISA.

Figure 10 show bispecific antibodies binding simultaneously to PD-1+ and CTLA-4+cells, as tested by FACS.

Figures 11A-11B show bispecific antibodies blocking CTLA4 ligand binding, as tested by competitive ELISA.

Figures 12A-12B show the blockage of human CTLA-4 protein binding to hCD86-expression cell by FACS.

Figure 13 shows the blockage of cyno CTLA-4 protein binding to hCD80-expression cell by FACS.

Figures 14A-14B show bispecific antibodies blocking human PD-1 Ligand binding, as tested by competitive ELISA

Figures 15A-15B show the blockage of human PD-1 binding to PD-1 ligand by FACS.

Figure 16 shows that the antibodies enhanced cytokine release inhuman allogenic MLR assay.

Figure 17 shows that the antibodies promoted cytokines production of PBMCs stimulated by SEB.

Figure 18 shows that the bispecific antibodies WBP3245A and WBP3245B are more potent than combo in inhibiting Tregs.

Figures 19A-19B show the serum stability of WBP3245A and WBP3245B over time.

Figure 20 shows the result of ADCC test.

Figure 21 shows the inhibition of WBP3245A on tumor growth in hCTLA-4 transgenic mice.

Figure 22 shows the inhibition of WBP3245A on tumor growth in hPD-1 transgenic mice.

Figure 23 shows the inhibition of WBP3245A on MC38 tumor in hCTLA+hPD1+ transgenic mice.

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, “a polypeptide 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, ” “a particular 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 “antigen-binding portion” or “antigen-binding fragment” of an antibody, which can be interchangeably used in the context of the application, refers to polypeptides comprising fragments of a full-length antibody, which retain the ability of specifically binding to an antigen that the full-length antibody specifically binds to, and/or compete with the full-length antibody for binding to the same antigen. Generally, see Fundamental Immunology, Ch. 7 (Paul, W., ed., the second edition, Raven Press, N.Y. (1989) , which is incorporated herein by reference for all purposes. Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and/or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries) , or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.

The term “variable domain” or “variable region” 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 or region 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.

Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F (ab’ ) 2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide) , or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc. ) , small modular immunopharmaceuticals (SMIPs) , and shark variable IgNAR domains, are also encompassed within the expression “antigen-binding fragment, ” as used herein. In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. The variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region. A hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule.

“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 region of a single light chain bound to the variable region 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 region 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) ) .

The term “human antibody” , as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies of the invention can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo) . However, the term “human antibody, ” as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

The term “humanized antibody” is intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences.

The term “chimeric antibody, ” as used herein, refers to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

The term “recombinant antibody, ” as used herein, refers to an antibody that is prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal that is transgenic for another species’ immunoglobulin genes, antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial antibody library, or antibodies prepared, expressed, created or isolated by any other means that involves splicing of immunoglobulin gene sequences to other DNA sequences.

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) , and may 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 that does 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.

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.

The term “identity, ” as used herein, refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. “Percent identity” means the percent of identical residues between the amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest of the molecules being compared. For these calculations, gaps in alignments (if any) are preferably addressed by a particular mathematical model or computer program (i.e., an “algorithm” ) . Methods that can be used to calculate the identity of the aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, A.M., ed. ) , 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, D.W., ed. ) , 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A.M., and Griffin, H.G., eds. ) , 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds. ) , 1991, New York: M. Stockton Press; and Carillo et al, 1988, SIAMJ. Applied Math. 48: 1073.

The term “EU numbering” refers to the EU numbering as in Kabat et al.. The Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) ) . The “EU numbering system” or “EU index” is generally used when referring to a residue in an immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra) . The “EU numbering as in Kabat” or “EU index as in Kabat” refers to the residue numbering of the human IgG1 EU antibody. Unless stated otherwise herein, references to residue numbers in the constant domain of antibodies means residue numbering by the EU numbering system. In certain embodiments, Fc modifications include, e.g., a mutation of serine ( “S” ) to proline ( “P” ) at position 228 of the amino acid sequence of human IgG4 Fc region.

The term “transfection, ” as used herein, refers to the process by which nucleic acids are introduced into eukaryoticcells, particularly mammalian cells. Protocols and techniques for transfection include but not limited to lipid transfection and chemical and physical methods such as electroporation. A number of transfection techniques are well known in the art and are disclosed herein. See, e.g., Graham et al., 1973, Virology 52: 456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al, 1981, Gene 13: 197. In a specific embodiment of the invention, human CD3/CD20 gene was transfected into 293F cells.

The term “fluorescence-activated cell sorting” or “FACS, ” as used herein, refers to a specialized type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell (FlowMetric. “Sorting Out Fluorescence Activated Cell Sorting” . Retrieved 2017-11-09. ) . Instruments for carrying out FACS are known to those of skill in the art and are commercially available to the public. Examples of such instruments include FACS Star Plus, FACScan and FACSort instruments from Becton Dickinson (Foster City, Calif. ) Epics C from Coulter Epics Division (Hialeah, Fla. ) and MoFlo from Cytomation (Colorado Springs, Colo. ) .

The term “antibody-dependent cell-mediated cytotoxicity” or “ADCC, ” as used herein, refers to a form of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g. Natural Killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. The antibodies “arm” the cytotoxic cells and are absolutely required for such killing. The primary cells for mediating ADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991) . To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in US Patent No. 5,500,362 or 5,821,337 may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. PNAS (USA) 95: 652-656 (1998) .

The term “complement dependent cytotoxicity” or “CDC” refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (Clq) to antibodies (of the appropriate subclass) which are bound to their cognate antigen. To assess complement activation, a CDC assay, e.g. as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996) , may be performed.

Antigen-binding moiety

The bispecific polypeptide complex provided herein comprises a first antigen-binding moiety associated with a second antigen-binding moiety, and one of them is an anti-CTLA-4 binding moiety that specifically binds to CTLA-4, while the other is an anti-PD-1 binding moiety that specifically binds to PD-1.

a) Anti-CTLA-4 binding moiety

In one embodiment of 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-4 binding moiety is derived from an anti-CTLA-4 antibody shown in Table A below. The CDR sequences of the antibody are provided below.

Table A: CDR sequences of anti-CTLA-4 antibody

Heavy and light chain variable region sequences of this anti-CTLA-4 antibody are provided below with CDR sequences annotated in bold and underline.

Anti-CTLA-4 #1-VH

Anti-CTLA-4 #1-VL

CDRs are known to be responsible for antigen binding.

In certain embodiments, the anti-CTLA-4 binding moiety comprises a 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: 3. 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) ) .

In certain embodiments, the anti-CTLA-4 binding 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 comprises a heavy chain variable region sequence comprising SEQ ID NO: 19 and a light chain variable region 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 1) .

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 ^-9 M, no more than 4x10 ^-9 M, no more than 3x10 ^-9 M, no more than 2x10 ^-9 M, no more than 10 ^-9 M, no more than 5x10 ^-10 M, no more than 4x10 ^-10 M, no more than 3x10 ^-10 M, no more than 2x10 ^-10 M, no more than 10 ^-10 M, 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-4 expressed 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 ₅₀ of 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 binding moiety

In one embodiment of the polypeptide complex provided herein, 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 one of the two anti-PD-1 antibodies shown in Table B below. The CDR sequences of the anti-PD-1 antibodies are provided below.

Table B: CDR sequences of anti-PD-1 antibodies

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

Anti-PD-1 #1-VH

Anti-PD-1 #1-VL

Anti-PD-1 #2-VH

Anti-PD-1 #2-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-1 antibody disclosed herein. In certain embodiments, the anti-PD-1 binding moiety provided herein comprises a heavy chain CDR3 sequence comprising SEQ ID NO: 9 or 15. 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) ) .

In certain embodiments, 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 is in a form of scFv.

In certain embodiments, the anti-PD-1 binding moiety provided herein comprises a heavy chain variable region sequence comprising SEQ ID NO: 21 and a light chain variable region sequence comprising SEQ ID NO: 22. In certain embodiments, the anti-PD-1 binding moiety provided herein comprises a heavy chain variable region sequence comprising SEQ ID NO: 23 and a light chain variable region sequence comprising SEQ ID NO: 24.

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 ^-9 M, no more than 1x10 ^-9 M, no more than 9x10 ¹⁰ M, no more than 8x10 ^-10 M, no more than 7x10 ^-10 M, no more than 6x10 ^-10 M, no more than 5x10 ^-10 M, no more than 4x10 ^-10 M, no more than 3x10 ^-10 M, no more than 2x10 ^-10 M, or no more than 1x10 ^-10 Mas 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 bispecific polypeptide complex disclosed herein is a bispecific antibody (alternatively, “BsAb” ) .

In certain embodiments, the present disclosure provides a bispecific polypeptide complex, comprising a first functional domain comprising a first antigen-binding moiety and a second functional domaincomprising a second antigen-binding moiety,

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

and

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

In certain embodiments, the anti-CTLA-4 binding moiety comprises: a heavy chain variable region sequence comprising a) the amino acid sequence of SEQ ID NO: 19, b) an amino acid sequence at least 85%, 90%, or 95%identical to the amino acid sequence of SEQ ID NO: 19, orc) an amino acid sequence with addition, deletion and/or substitution of one or more amino acids as compared with the amino acid sequence of SEQ ID NO: 19; and a light chain variable region sequence comprising a) the amino acid sequence of SEQ ID NO: 20, b) an amino acid sequence at least 85%, 90%, or 95%identical to the amino acid sequence of SEQ ID NO: 20, orc) an amino acid sequence with addition, deletion and/or substitution of one or more amino acids as compared with the amino acid sequence of SEQ ID NO: 20.

In certain embodiments, the anti-PD-1 binding moiety comprisesa heavy chain variable region sequence comprising a) the amino acid sequence of SEQ ID NO: 21, b) an amino acid sequence at least 85%, 90%, or 95%identical to the amino acid sequence of SEQ ID NO: 21, orc) an amino acid sequence with addition, deletion and/or substitution of one or more amino acids as compared with the amino acid sequence of SEQ ID NO: 21; anda light chain variable region sequence comprising a) the amino acid sequence of SEQ ID NO: 22, b) an amino acid sequence at least 85%, 90%, or 95%identical to the amino acid sequence of SEQ ID NO: 22, orc) an amino acid sequence with addition, deletion and/or substitution of one or more amino acids as compared with the amino acid sequence of SEQ ID NO: 22.

In certain embodiments, the anti-PD-1 binding moiety comprisesa heavy chain variable region sequence comprising a) the amino acid sequence of SEQ ID NO: 23, b) an amino acid sequence at least 85%, 90%, or 95%identical to the amino acid sequence of SEQ ID NO: 23, orc) an amino acid sequence with addition, deletion and/or substitution of one or more amino acids as compared with the amino acid sequence of SEQ ID NO: 23; anda light chain variable region sequence comprising a) the amino acid sequence of SEQ ID NO: 24, b) an amino acid sequence at least 85%, 90%, or 95%identical to the amino acid sequence of SEQ ID NO: 24, orc) an amino acid sequence with addition, deletion and/or substitution of one or more amino acids as compared with the amino acid sequence of SEQ ID NO: 24.

The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988) ) which has been incorporated into the ALIGN program (version 2.0) , using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percentage of identity between two amino acid sequences can be determined by the algorithm of Needleman and Wunsch (J. Mol. Biol. 48: 444-453 (1970) ) which has been incorporated into the GAP program in the GCG software package (available at http: //www. gcg. com) , using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

Additionally or alternatively, the protein sequences of the present invention can further be used as a “query sequence” to perform a search against public databases to, for example, identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. MoI. Biol. 215: 403-10. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to the antibody molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al, (1997) Nucleic Acids Res. 25 (17) : 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs {e.g., XBLAST and NBLAST) can be used. See www. ncbi. nlm. nih. gov.

In certain embodiments, the CDR amino acid sequences can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identical to the respective sequences set forth above. In other embodiments, the amino acid sequences of the variable regions can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identical to the respective sequences set forth above.

In certain embodiments, the CDRs of the antigen-binding moieties contain a conservative substitution of not more than 2 amino acids, or not more than 1 amino acid. The term “conservative substitution” , as used herein, refers to amino acid substitutions which would not disadvantageously affect or change the essential properties of a protein/polypeptide comprising the amino acid sequence. For example, a conservative substitution may be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions wherein an amino acid residue is substituted with another amino acid residue having a similar side chain, for example, a residue physically or functionally similar (such as, having similar size, shape, charge, chemical property including the capability of forming covalent bond or hydrogen bond, etc. ) to the corresponding amino acid residue. The families of amino acid residues having similar side chains have been defined in the art. These families include amino acids having alkaline side chains (for example, lysine, arginine and histidine) , amino acids having acidic side chains (for example, aspartic acid and glutamic acid) , amino acids having uncharged polar side chains (for example, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan) , amino acids having nonpolar side chains (for example, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine) , amino acids having β-branched side chains (such as threonine, valine, isoleucine) and amino acids having aromatic side chains (for example, tyrosine, phenylalanine, tryptophan, histidine) . Therefore, a corresponding amino acid residue is preferably substituted with another amino acid residue from the same side-chain family. Methods for identifying amino acid conservative substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32: 1180-1187 (1993) ; Kobayashi et al., Protein Eng. 12 (10) : 879-884 (1999) ; and Burks et al., Proc. Natl. Acad. Sci. USA 94: 412-417 (1997) , which are incorporated herein by reference) .

In certain embodiments, the bispecific polypeptide complex comprisesa heavy chain sequence comprising a) the amino acid sequence of SEQ ID NO: 25 or 26, b) an amino acid sequence at least 85%, 90%, or 95%identical to the amino acid sequence of SEQ ID NO: 25 or 26, orc) an amino acid sequence with addition, deletion and/or substitution of one or more amino acids as compared with the amino acid sequence of SEQ ID NO: 25 or 26; and a light chain sequence comprising a) the amino acid sequence of SEQ ID NO: 27, b) an amino acid sequence at least 85%, 90%, or 95%identical to the amino acid sequence of SEQ ID NO: 27, orc) an amino acid sequence with addition, deletion and/or substitution of one or more amino acids as compared with the amino acid sequence of SEQ ID NO: 27.

In certain embodiments, the first antigen-binding moiety and the second antigen-binding moiety of the bispecific polypeptide complex disclosed hereinmay be directly or indirectly linked to one another. In certain embodiments, the first antigen-binding moiety and the second antigen-binding moiety of the bispecific polypeptide complex disclosed hereinmay be linked to one another by a linker. In a specific embodiment, the linker is a peptide linker. In a more specific embodiment, the peptide linke is a peptide with the amino acid sequence SEQ ID NO: 31.

In certain embodiments, the first and/or second functional domain disclosed herein further comprises anFc region. The Fc regions of the bispecific polypeptide complexdisclosed herein may be human Fc regions. The Fc regions of the bispecific polypeptide complexdisclosed hereinmay be of e.g., IgG1, IgG2, IgG3 or IgG4. In one embodiment, the Fc regions are of the IgG4 isotype.

In one embodiment, the Fc regions may comprise one or more amino acid changes (e.g., insertions, deletions or substitutions) , without changing the desired functionality. For example, the bispecific polypeptide complex disclosed herein may comprise one or more modifications in the Fc region that results in a modified Fc region having a modified binding interaction (e.g., enhanced or diminished) between Fc and FcRn. Non-limiting examples of such Fc modifications include, e.g., a mutation of serine ( “S” ) to proline ( “P” ) at position 228 of the amino acid sequence of human IgG4 Fc region.

In certain embodiments of the bispecific polypeptide complex disclosed herein, the first functional domain and the second functional domain are independently selected from immunoglobin and the antigen-binding fragments thereof, such as Fab, F (ab) ’ ₂ or scFv. In one embodiment of the bispecific polypeptide complexdisclosed herein, the first functional domain is an immunoglobin and the second functional domain is scFv. In one embodiment, the first functional domain is an anti-CTLA-4 immunoglobin and the second functional domain is an anti-PD-1 scFv.

In certain embodiments, the immunoglobinmay be selected from a group consisting of IgG, IgA, IgD, IgE and IgM, preferably IgG, such as IgG1, IgG2 or IgG4, preferably IgG4, even more preferably IgG4 (S228P) (i.e., a human IgG4 including a mutation of serine ( “S” ) to proline ( “P” ) at position 228 of its amino acid sequence) .

The biospecific polypeptide complex disclosed herein may be in various formats, such as ScFab, TriFabs, Fab-Fab, Fab-Fv, MAb-Fv, IgG-Fv, ScFab-Fc-scFv ₂, ScFab-Fc-scFv, Appended IgG, DVD-Ig, and etc.

“ScFab” refers to a fusion polypeptide with a Fd linked to a light chain via a polypeptide linker, resulting in the formation of a single chain Fab fragment (scFab) .

“TriFabs” refers to a trivalent, bispecific fusion protein composed of three units with Fab-functionalities. TriFabs harbor two regular Fabs fused to an asymmetric Fab-like moiety.

“Fab-Fab” refers to a fusion protein formed by fusing the Fd chain of a first Fab arm to the N-terminus of the Fd chain of a second Fab arm.

“Fab-Fv” refers to a fusion protein formed by fusing a HCVR to the C-terminus of a Fd chain and a LCVR to the C-terminus of a light chain. A “Fab-dsFv” molecule can be formed by introducing an interdomain disulphide bond between the HCVR domain and the LCVR domain.

“MAb-Fv” or “IgG-Fv” refers to a fusion protein formed by fusion of HCVR domain to the C-terminus of one Fc chain and the LCVR domain either expressed separately or fused to the C-terminus of the other resulted in a bispecific, trivalent IgG-Fv (mAb-Fv) fusion protein, with the Fv stabilized by an interdomain disulphide bond.

“ScFab-Fc-scFv ₂” and “ScFab-Fc-scFv” refer to a fusion protein formed by fusion of a single-chain Fab with Fc and disulphide-stabilized Fv domains.

“Appended IgG” refers to a fusion protein with a Fab arm fused to an IgG to form the format of bispecific (Fab) ₂-Fc. It can form a “IgG-Fab” or a “Fab-IgG” , with a Fab fused to the C-terminus or N-terminus of an IgG molecule with or without a connector. In certain embodiments, the appended IgG can be further modified to a format of IgG-Fab ₄ (see, Brinkman et al., 2017, Supra) .

In one embodiment of the bispecific polypeptide complex disclosed herein, the second functional domain is linked to the C-terminus of the first functional domain, optionally by a linker. In one particular embodiment, the first functional domain is an anti-CTLA-4 immunoglobin and the second functional domain is an anti-PD-1 scFv.

In certain embodiments, the present disclosure provides two bispecific polypeptide complexes WBP3245A (W3245-T1U6-G15-2. uIgG4. SP) and WBP3245B (W3245-T1U8-G15-2. uIgG4. SP) .

The amino acid sequence of heavy chain of WBP3245A is as follows:

The amino acid sequence of heavy chain of WBP3245B is as follows:

The amino acid sequence of light chain of WBP3245A or WBP3245B is as follows:

The bispecific polypeptide complexes disclosed herein have longer in vivo half-life and showed stronger anti-tumor activities when compared to other bispecific polypeptide complexes.

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 expresses the bispecific polypeptide complex disclosed herein.

A vector in the context of the present invention may be any suitable vector, including chromosomal, non-chromosomal, and synthetic nucleic acid vectors (a nucleic acid sequence comprising a suitable set of expression control elements) . Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, a CD20 or a CD3 antibody-encoding nucleic acid is comprised in a naked DNA or RNA vector, including, for example, a linear expression element (as described in for instance Sykes and Johnston, Nat Biotech 17, 355-59 (1997) ) , a compacted nucleic acid vector (as described in for instance US 6,077,835 and/or WO 00/70087) , a plasmid vector such as pBR322, pUC 19/18, or pUC 118/119, a “midge” minimally-sized nucleic acid vector (as described in for instance Schakowski et al., Mol Ther 3, 793-800 (2001) ) , or as a precipitated nucleic acid vector construct, such as a CaP04-precipitated construct (as described in for instance WO200046147, Benvenisty and Reshef, PNAS USA 83, 9551-55 (1986) , Wigler et al., Cell 14, 725 (1978) , and Coraro and Pearson, Somatic Cell Genetics 7, 603 (1981) ) . Such nucleic acid vectors and the usage thereof are well known in the art (see for instance US 5,589,466 and US 5,973,972) .

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 encodingthe first functional domain and one or more polynucleotides encoding the second functional domain, 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 region 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 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 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.

The term “prevent, ” “prevention” or “preventing, ” as used herein, with reference to a certain disease condition in a mammal, refers to preventing or delaying the onset of the disease, or preventing the manifestation of clinical or subclinical symptoms 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-α and TNF-β. Agents that inactivate immunosuppressive targets can also be used, for example, TGF-beta inhibitors, IL-10 inhibitors, and Fas ligand inhibitors. Another group of agents include those that activate immune responsiveness to tumor or cancer cells, for example, those enhance T cell activation (e.g. agonist of T cell costimulatory molecules such as 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 hereinis associated 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.

Sequence Listing Summary

The following Table C provides a summary of the included sequences:

Table C: Sequences Summary

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

The present invention, thus generally described, will be understood more readily by reference to the following Examples, which are provided by way of illustration and are not intended to be limiting of the instant invention. The Examples are not intended to represent that the experiments below are all or the only experiments performed.

EXAMPLE 1: Materials and Methods

1.1 General Materials

General research materials and their sources are listed in Table 1 below.

Table 1: General research materials and their sources

1.2 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 C-terminal. Protein of human, cyno and mouse CTLA-4 and mouse and cyno 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 Ni-NTA column (GE Healthcare, 175248) . The obtained human PD-1 was QC’ed by SDS-PAGE and SEC, and then stored at -80 ℃.

1.3 Generation of BMK antibodies

DNA sequence encoding the variable region of anti-CTLA-4 antibody (WBP316-BMK1 (Ipilimumab) ) , anti-PD-1 antibody (WBP305-BMK1 (Nivolumab) ) was synthesized in Sangon Biothech (Shanghai, China) , and then subcloned into modified pcDNA3.4 expression vectors with 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 in house after immunizing rats with human PD-1 and mouse PD-1 and were converted to IgG4 (S228P) format. Anti-CTLA-4 antibody W3162_1.154.8-z35-IgG1k as disclosed in WO2018209701A was prepared in house. DNA sequences encoding a benchmark bispecific anti-CTLA-4 x PD-1 antibody, WBP324-BMK1. IgG1. KDL (i.e. “BiAb004” disclosed in CN106967172A) were synthesized.

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

1.4 Generation of target-expressing cell lines

Using Lipofectamine 2000, CHO-Sor 293F cells were transfected with the expression vector containing gene encoding full length human PD-1 or mouse PD-1. The cells were cultured in medium containing proper selection marker. 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.

1.5 Generation of Bispecific Anti-CTLA-4/PD-1antibodies

Construction of G15 format bispecific antibodies WBP3245A: DNA sequence encoding anti-CTLA-4 variable light chain on the N-terminal constant region of light chain was cloned into modified pcDNA3.3 expression vector. DNA sequence encoding scFv (VH- (G4S) ₃-VL) of anti-PD-1 antibody WBP3055-1.153.7. uIgG4k on the C-terminus of anti-CTLA-4 constant region of human IgG4 (S228P) heavy chain was cloned into modified pcDNA3.3 expression vector.

Construction of G15 format bispecific antibodies WBP3245B: DNA sequence encoding anti-CTLA-4 variable light chain on the N-terminal constant region of light chain was cloned into modified pcDNA3.3 expression vector. DNA sequence encoding scFv (VH- (G4S) ₃-VL) of anti-PD-1 antibody WBP3055-1.103.11. uIgG4k on the C-terminal of anti-CTLA-4 constant region of human IgG4 (S228P) heavy chain was cloned into modified pcDNA3.3 expression vector.

The amino acid sequence of heavy chain of WBP3245A is as follows:

The amino acid sequence of heavy chain of WBP3245B is as follows:

The amino acid sequence of light chain of WBP3245A or WBP3245B is as follows:

Heavy chain and light chain expression plasmids 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. As shown in Figs. 1A-1B, the purity of bispecific antibodies was above 99%as measured by SEC.

EXAMPLE 2: In vitro characterization

2.1Differential scanning fluorimetry (DSF)

A DSF assay was performed using 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 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.

Results: The measured Th1 of the two bispecific antibodies was 55-57 ℃.

2.2 Human CTLA-4-binding ELISA and FACS

For ELISA binding, non-tissue culture treated flat-bottom 96-well plates (Nunc MaxiSorp, ThermoFisher) were pre-coated with 1.0 μg/ml in house human CTLA-4 protein W316-hPro1. ECD. His overnight at 4℃. After 2%BSA blocking, 100 μL 3.16-fold titrated Abs from 25 nM to 0.00025 nM Abs were pipetted into each well and incubated for 1 hour at ambient temperature. Following removal of the unbound substances, 100 μL 1: 5000 diluted HRP-labeled goat anti-human IgG (Bethyl A80-304P) were added to the wells and incubated for 1 hour. The color was developed by dispensing 100 μL TMB substrate, and then stopped by 100 μL 2N HCl. The absorbance was read at 450 nm using a Microplate Spectrophotometer (

M5 ^e) .

Due to the differences of HRP-labeled goat anti-human IgG (Bethyl A80-304P) to Human IgG1 and IgG4 in affinity, we replace it with HRP-labeled mouse anti-Human IgG Fc (CH2) (Thermo MA5-16859 Monoclonal 1: 5000) when comparing to WBP324-BMK1. IgG1. KDL (inhouse) .

For FACS binding, 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 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.

Results: The binding of the bispecific antibodies WBP3245A and WBP3245B to human CTLA-4 were measured by both ELISA (Figure 2) and FACS (Figures 3A-3B) , which showed the two bispecific antibodies were both bound to human CTLA-4. The ELISA and FACS binding EC50 of the bispecific antibodies were provided in Tables 2 (ELISA) and 3 (FACS) below.

Table 2: ELISA binding EC50 of the bispecific antibodies to human CTLA-4

Table 3: FACS binding EC50 of the bispecific antibodies to human CTLA-4

BsAbs	EC50 (nM)	Ratio
WBP3245A (IgG4)	3.265	1.35
WBP3245B (IgG4)	2.603	1.07
WBP316BMK1 (IgG4)	2.426	/

2.3 Cynomolgus CTLA-4-binding (ELISA and FACS)

For ELISA binding, non-tissue culture treated flat-bottom 96-well plates (Nunc MaxiSorp, ThermoFisher) were pre-coated with 1.0 μg/ml in house human CTLA-4 protein W316-cPro1. ECD. His overnight at 4℃. After 2%BSA blocking, 100 μL 3.16-fold titrated Abs from 5.0 μg/ml to 0.0003 μg/ml Abs were pipetted into each well and incubated for 1 hour at ambient temperature. Following removal of the unbound substances, 100 μL 1: 10000 diluted HRP-labeled goat anti-human IgG (Bethyl A80-304P) were added to the wells and incubated for 1 hour. The color was developed by dispensing 100 μL TMB substrate, and then stopped by 100 μL 2N HCl. The absorbance was read at 450 nm using a Microplate Spectrophotometer (

M5 ^e) .

For FACS binding, 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 1: 150 dilutedPE-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.

Results: The binding of the bispecific antibodies to cynomolgus CTLA-4 were measured by both ELISA (Figure 4) and FACS (Figure 5) , which showed the two bispecific antibodies were both bound to cynomolgus CTLA-4. The ELISA and FACS biding EC50 of the bispecific antibodies were provided in Tables 4 (ELISA) and 5 (FACS) below.

Table 4: ELISA binding EC50 of the bispecific antibodies to cyno CTLA-4

BsAb	EC50 (nM)	Ratio
WBP3245A	0.1065	1.70
WBP3245B	0.1664	2.65
WBP316-BMK1 IgG4	0.0627	/

Table 5: FACS binding EC50 of the bispecific antibodies to cyno CTLA-4

BsAb	EC50 (nM)	Ratio
WBP3245A	0.8367	1.20
WBP3245B	1.2760	1.84
WBP316-BMK1 IgG4	0.6953	/

2.4 Human PD-1-binding ELISA and FACS

For ELISA binding, 96-well plates (Nunc MaxiSorp, ThermoFisher) were coated with 1.0 μg/ml human PD-1 protein W305-hPro1. ECD. mFc overnight at 4℃. After 2%BSA blocking, 100 μL 3.16-fold titrated antibodies from 25 nM to 0.00025 nM were pipetted into each well and incubated for 1 hour at ambient temperature. After removal of the unbound antibodies, 100 μL 1:5000 diluted HRP-labeled goat anti-human IgG (Bethyl A80-304P) were added to the wells and incubated for 1 hour. The color was developed by dispensing 100 μL TMB substrate, and then stopped by 100 μL 2N HCl. The absorbance was read at 450 nm using a Microplate Spectrophotometer (

M5 ^e) .

Due to the differences of HRP-labeled goat anti-human IgG (Bethyl A80-304P) to Human IgG1 and IgG4 in affinity, we later replaced it with HRP-labeled mouse anti-human IgG Fc (CH2) antibody (Thermo MA5-16859 Monoclonal 1: 5000) when comparing to WBP324-BMK1. IgG1. KDL (in house) .

For FACS binding, 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 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.

Results: The binding of the bispecific antibodies WBP3245A and WBP3245B to human PD-1 were measured by both ELISA (Figure 6) and FACS (Figures 7A-7B) , which showed the two bispecific antibodies both bound to human PD-1. The ELISA and FACS biding EC50 of the bispecific antibodies were provided in Tables 6 (ELISA) and 7 (FACS) below. The binding activity of WBP3245A was slightly better than WBP3245B in these ELISA or FACS test.

Table 6: ELISA binding EC50 of the bispecific antibodies to human PD-1

Table 7: FACS binding EC50 of the bispecific antibodies to human PD-1

BsAbs	EC50 (nM)
WBP3245A (IgG4)	5.939
WBP3245B (IgG4)	11.510
WBP305BMK1 (IgG4)	0.798

2.5 Cyno PD-1-binding (ELISA and FACS)

For ELISA binding, non-tissue culture treated flat-bottom 96-well plates (Nunc MaxiSorp, ThermoFisher) were pre-coated with 0.5 μg/ml mouse anti-His mAb (GenScript-A00186) overnight at 4 ℃. After 2%BSA blocking, 100 μL 0.5 μg/ml in house cyno protein W316-cPro1. ECD. His were added to each well. After wash, 100 μL 5.0-fold titrated Abs from 10.0 μg/ml to 0.00064μg/ml were pipetted into each well and incubated for 1 hour at ambient temperature. Following removal of the unbound substances, 100 μL 1: 5000 diluted HRP-labeled goat anti-human IgG (Bethyl A80-304P) were added to the wells and incubated for 1 hour. The color was developed by dispensing 100 μL TMB substrate, and then stopped by 100 μL 2N HCl. The absorbance was read at 450 nm using a Microplate Spectrophotometer (

M5e) .

For FACS binding, engineered cyno 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 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.

Results: The binding of the bispecific antibodies to cynomolgus PD-1 were measured by FACS (Figure 8) , which showed the two bispecific antibodies both bound to cynomolgus PD-1. The FACS biding EC50 of the bispecific antibodies were provided in Table 8 below. The binding activity of WBP3245A was slightly better than WBP3245B in the FACS test.

Table 8: FACS binding EC50 of the bispecific antibodies to cynomolgus PD-1

BsAbs	EC50 nM
WBP3245A	4.249
WBP3245B	7.117
WBP305-BMK1	1.496

2.6 hPD-1 and hCTLA-4 dual binding ELISA

In order to test whether the bispecific antibodies could bind to both hPD-1 and hCTLA-4, an ELISA assay was developed as 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 house) ) 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 are incubated on the plates for 1 hour at room temperature. Following the incubation, plates are 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 (in house) ) was added to plates and incubation 1 hour. After washing the plates three times, Streptavidin-HRP (Lifetechnologies, #SNN1004) (1: 20000 diluted) is 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 (in house) is added for the detection per well. The reaction is stopped after approximate 5 minutes through the addition of 100 μL per well of 2 M HCl. The absorbance of the wells is measured at 450 nm with a multiwall plate reader (

M5 ^e) .

Results: As shown in Figures 9A-9B and Table 9, WBP3245A and WBP3245B could bind to both PD-1 and CTLA-4 with EC50 at 0.0839 to 0.1200 nM, indicating that they are more potent than a bispecific reference antibody WBP324 BMK1 (EC50 =0.4455 nM) .

Table 9: ELISA Binding EC50 of the bispecific antibodies to hPD-1 and hCTLA-4 simultaneously

BsAbs	EC50 (nM)	Ratio
WBP3245A (IgG4)	0.0839	0.19
WBP3245B (IgG4)	0.1200	0.27
WBP324 BMK1 (IgG4)	0.4455	/
Anti-PD-1 WBP305 BMK1	No binding	NA
Anti-CTLA-4 WBP316 BMK1	No binding	NA

“NA” : not applicable.

2.7 hPD-1 and hCTLA-4 dual binding FACS

In order to test whether the bispecific antibodies could bind to both hPD-1 and hCTLA-4, a FACS assay was developed as 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) 50nM and Far red (Invitrogen-C34572) 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 0.83nM were added to the cells. The plates were incubated at 4 ℃ for 1.5 hour. The cells was tested by flow cytometry and the percentage of double positive cells was analyzed by FlowJo.

Results: As shown in Figure 10, both WBP3245A and WBP3245B could simultaneously bind to PD-1+ and CTLA-4+ cells.

2.8 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 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 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.

Results: As shown in Table 10, WBP3245A and WBP3245B have affinity to human CTLA-4 as 3.44 nM and 3.86 nM, respectively; and have affinity to human PD-1 as 11.1 nM and 32.2 nM, respectively.

Table 10: HumanCTLA-4-and PD-1-Binding (Biacore)

2.9 Non-specific binding

Both FACS and ELISA assays were used to test whether the antibodies binding to other targets. In the ELISA assay, the testing antibodies, isotype control antibodies were tested binding to different proteins including Factor VIII, FGFR-ECD, PD-1, CTLA-4. ECD, HER3. ECD, OX40. ECD, 4-1BB. ECD, CD22. ECD, CD3e. ECD. Several 96-well plates (Nunc-Immuno Plate, Thermo Scientific) was coated with the individual antigens (2 μg/mL) at 4 ℃ overnight. After 1 hour blocking with 2%BSA in PBS, wash plate 3 times with 300 μl PBST. Testing antibodies, as well as isotype control antibodies were diluted to 10 μg/ml in PBS containing 2%BSA, then were added to the plate and incubated at room temperature for 2 hours. After 3 times washing with 300 μl PBST, HRP-conjugated goat anti-human IgG antibody (1: 5000 diluted in 2%BSA) was added to the plate and incubated at room temperature for 1 hours. Finally, the plates were washed six times with 300 μl PBST. The color was developed by dispensing 100 μL of TMB substrate for 12 min, and then stopped by 100 μL of 2M HCl. The absorbance at 450 nM was measured using a microplate spectrophotometer.

In FACS assay, different cell lines (Ramos, Raji, MDA-MB-453, BT474, Jurkat, Hut78, A431, A204, CaLu-6, A375, HepG2, BxPC-3, HT29, FaDu, 293F, CHO-K1) were adjusted to 1 x 10 ⁵ cells per well. Testing antibodies and Isotype control antibodies were diluted to 10 μg/ml in PBS containing 1%BSA and incubated with cells at 4 ℃ for 1 hr. The cells were washed twice with 180 μL PBS containing 1%BSA. PE conjugated goat anti-human IgG Fc fragment (Jackson, Catalog number 109-115-098) was diluted to final concentration 5 μg/ml in PBS with 1%BSA, then added to re-suspend cells and incubated at 4 ℃ in the dark for 30 min. Additional washing steps were performed twice with 180 μL PBS containing 1%BSA followed by centrifugation at 1500 rpm for 4 minutes at 4℃. Finally, the cells were re-suspended in 100 μL PBS containing 1%BSA and fluorescence values were measured by flow cytometry (BD Canto II) and analyzed by FlowJo.

Results: No detectable binding of the antibodies WBP3245A and WBP3245B to other tested targets was observed (data not shown) .

2.10 Blockage of human/cynomolgus CTLA-4 binding to human CD80 or CD86

In order to test whether the bispecific antibodies could block hCTLA-4 binding to hCD80 protein, we used competitive ELISA.

In an ELISA, flat-bottom 96-well plates (Nunc MaxiSorp, ThermoFisher) were pre-coated with 0.5 μg/ml W316-hPro1. ECD. hFc (in house) overnight at 4 ℃. After 2%BSA blocking, 100 μL 3.16-fold titrated Abs from 400 nM to 0.04 nM Abs coupled with 0.5 μg/ml in house 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 per well and incubation 1 hour. After wash 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 (in house) substrate, and then stopped by 100 μL of 2N HCl. The absorbance was read at 450 nm using a Microplate Spectrophotometer (

M5 ^e) .

To test whether the antibodies could block human or cynomolgus CTLA-4 binding to hCD80 or hCD86 on cell surface, we used competitive FACS.

Human CD80-or CD86-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, positive and negative controls were mixed with biotinylated human CTLA-4. ECD. hFc (in house) . Due to different density of ligands on cell surface, 0.066&0.037 μg/mL of hCTLA-4. ECD. hFc-Biotin was used for human CD80&86 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.

Results: Both ELISA and FACS were used to test the bispecific antibodies’ blockage of CTLA-4 with its ligand CD80 and CD86. WBP3245A and WBP3245B blocked CTLA-4 binding to CD80 with IC50 of 0.4065 and 0.4273 nM (Figures 11A-11B and Tables 11A-11B) , and with similar IC50 of IC50 5.130 and 2.926 nM in FACS (Figure 12 and Table 12) . Similarly, the bispecific antibodies could also block cyno CTLA-4 binding to human CD80+ cells (Figure 13 and Table 13) . In conclusion, WBP3245A and WBP3245B can block CD80 binding to CTLA4 and have similar IC50 as anti-CTLA4 BMK (ELISA) , and can block human CTLA4 binding to its ligand and block CTLA-4 protein binding to hCD80 expressed cell line (FACS) .

Table 11A: IC50 of WBP3245A blocking CTLA-4 binding to CD80 tested by ELISA

BsAbs	IC50 (nM)	Ratio
WBP3245A (IgG4)	0.4065	1.44
WBP316BMK1 (IgG4)	0.2827	/

Table 11B: IC50 of WBP3245B blocking CTLA-4 binding to CD80 tested by ELISA

BsAbs	IC50 (nM)	Ratio
WBP3245B (IgG4)	0.4273	1.51
WBP316BMK1 (IgG4)	0.2827	/

Table 12: IC50 of the bispecific antibodies blocking human CTLA-4 binding to hCD86expression cell tested by FACS

BsAbs	IC50 (nM)	Ratio
WBP3245A (IgG4)	5.130	1.41
WBP3245B (IgG4)	2.926	0.81
WBP316BMK1 (IgG4)	3.624	/

Table 13: Blockage of the bispecific antibodies on cyno CTLA-4 binding to hCD80 expression cell tested by FACS

Antibody	IC ₅₀ (nM)	Inhibition %
WBP3245A	9.01	75.6
WBP3245B	8.023	92.8
WBP324-BMK1. IgG1. KDL	2.553	97.9
WBP316-BMK1. IgG4	19.82	90.9

2.11 Human PD-1-competitive ELISA and FACS

In order to test whether the bispecific antibodies could block hPD-L1 binding to hPD-1 protein, we used competitive ELISA and FACS.

In an ELISA, flat-bottom 96-well plates (Nunc MaxiSorp, ThermoFisher) were pre-coated with 1.0 μg/ml W305-hPro1. ECD. hFc (in house) overnight at 4 ℃. After 2%BSA blocking, 100 μL 3.16-fold titrated Abs from 400 nM to 0.01 nM Abs coupled with 5.0 μg/ml in house human PD-L1 protein W315-hPro1. ECD. mFc 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 1: 5000 HRP-labeled goat anti mouse IgG (Bethyl A90-231P) was added to plate per well and incubation 1 hour. After wash 6 times, the color was developed by dispensing 100 μL of TMB (in house) substrate, and then stopped by 100 μL of 2N HCl. The absorbance was read at 450 nm using a Microplate Spectrophotometer (

M5 ^e) .

For blocking human PD-L1 binding to human PD-1 by FACS, 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 coupled with 5ug/ml in house 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 cell expressive 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.

Results: Both ELISA and FACS were used to test the bispecific antibodies’ blockage of PD-1 with its ligand PD-L1. WBP3245A and WBP3245B blocked PD-1 binding to PD-L1 with IC50 of 4.85 nM and 5.92 nM in ELISA (Figures 14A-14B and Table 14) , and they blocked PD-1 and cell surface PD-L1 interaction with IC50 of 4.464 nM and 6.436 nM (Figure 15 and Table 15) . In conclusion, WBP3245A and WBP3245B can block human PD-1 binding to its ligand.

Table 14: IC50 of bispecfic antibodies blocking PD-1 binding to PD-L1 tested by ELISA

BsAbs	IC50 (nM)	Ratio
WBP3245A (IgG4)	4.85	3.28
WBP3245B (IgG4)	5.92	4
WBP305BMK1 (IgG4)	1.48	/

Table 15: IC50 of the bispecific antibodies blocking human PD-1 binding to PD-L1 tested by FACS

BsAbs	IC50 (nM)
WBP3245A (IgG4)	4.464
WBP3245B (IgG4)	6.436
WBP305BMK1 (IgG4)	0.4816

2.12 Cynomolgus PD-1 competitive ELISA

An ELISA assay was used to test whether the bispecific antibodies could block cPD-L1 binding to cPD-1 protein. Flat-bottom 96-well plates (Nunc MaxiSorp, ThermoFisher) were pre-coated with 1.0 μg/ml cynoPD-1. hFc protein (SinoBiological-90311-C02H) overnight at 4 ℃. After 2%BSA blocking, 100 μL 3-fold serially diluted antibodies from 40 μg/ml to 0.002 μg/ml was mixed with 2.5 μg/ml in house cynoPD-L1. hFc-Biotin. The mixture was pipetted into each well and incubated for 1 hour at ambient temperature. Following the incubation, the plates were washed 3 times with 300 μL per well of PBS containing 0.5% (v/v) Tween 20. A volume of 100 μL/well 1: 20000 HRP-conjugated streptavidin (Life-SNN1004) was added to the plate and incubated for 1 hour. After wash 6 times, the color was developed by dispensing 100 μL/well of TMB substrate, and then stopped by 100 μL/well of 2N HCl. The absorbance was read at 450 nm using a Microplate Spectrophotometer (

M5e) .

Results: Similarly, WBP3245A and WBP3245B can block cyno PD1 binding to its ligand (data not shown) .

2.13 Human mixed lymphocyte reaction (MLR)

Mixed lymphocyte reaction (MLR) was used to test the agonistic effect of antibodies on cytokine secretion of activated CD4 ⁺ T cells.

2.13.1 Cell isolation, cell culture and induction

Human peripheral blood mononuclear cells (PBMCs) were freshly isolated from healthy donors using Ficoll-Paque (STEMCELL-07861) PLUS gradient centrifugation. Isolated PBMCs were cultured in complete RPMI-1640 (containing 10%FBS and 1%PS) supplemented with 100 U recombinant human IL-2.

Human monocytes were isolated using Human Monocyte Enrichment Kit (Miltenyi Biotec-130-050-201) according to the manufacturer’s instructions. Cell concentration was adjusted in complete RPMI-1640 medium (Gibco-22400089) supplemented with 800 U/mL recombinant human GM-CSF and 50 ng/mL rhIL-4. Cell suspension was seeded at a concentration of 2×10 ⁶ cells/mL, 2.5 mL/well in 6-well plate. Cells were cultured for 5 to 7 days to differentiate into immature dendritic cells (iDCs) . Cytokines were replenished every 2-3 days by replacing half of the media with fresh media supplemented with cytokines.

Human CD4+ T cells were isolated using Human CD4+ T cell Enrichment kit (STEMCELL-19052) according to the manufacturer’s protocol.

2.13.2 Mixed lymphocyte reaction

For allogeneic MLR, isolated CD4+ T cells were co-cultured with immature DCs.

CD4+ T cells, DCs and various concentrations of antibodies (2-fold, 2.5-fold and 10-fold serially diluted from 335 nM to 0.067 nM) were added to 96-well round bottom plates in complete RPMI-1640 medium. The plates were incubated at 37℃ in a 5%CO ₂ incubator. IL-2 and IFN-γ in the supernatant were quantified on day 3 and day 5 respectively.

2.13.3 Primary PBMC Activation (stimulated with SEB)

PBMCs, various concentrations of antibodies (2-fold, 2.5-fold and 10-fold serially diluted from 335 nM to 0.067 nM) and SEB (Staphylococcal enterotoxin B) at the concentration of 10 ng/mL were added to 96-well round bottom plates in complete RPMI-1640 medium. The plates were incubated at 37℃, 5%CO ₂. IL-2 and IFN-γ quantitation were determined on day 3 and day 5 respectively.

2.13.4 Cytokine quantification

Human IFN-γ and IL-2 were measured by ELISA using matched antibody pairs. Recombinant human IFN-γ and recombinant human IL-2 were used as standards, respectively. The plates were pre-coated with capture antibody specific for human IFN-γ or IL-2, respectively. After blocking, standards or samples were pipetted into each well and incubated for 2 hours at ambient temperature. Following removal of the unbound substances, the biotin-conjugated detecting antibody specific for IFN-γ or IL-2 was added to the wells and incubated for 1 hour, respectively. The HRP-conjugated streptavidin was then added to the wells for 30 minutes at ambient temperature. 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) .

2.13.5 Treg Suppression

Human CD4+CD25+ Treg cells were separated from fresh hPBMC by isolation Kit (Miltenyi 130-093-631) and amplified for 2 weeks.

Human CD4+ T cells separated from another donor by Human CD4+ T cell Enrichment kit (STEMCELL-19052) were mixed with Treg, iDC and test antibodies (10-fold dilution, from 335 nM to 3.35 nM) . The amount of Treg, CD4+ T and iDC cells were 1×10 ⁵, 1×10 ⁵ and 1×10 ⁴per well and incubated in 96-well plates. The plates were kept at 37℃ in a 5%CO2 incubator for 5 days. IFN-γ in the supernatant was quantified by ELISA and T cell proliferation was measured by 3H-thymidine incorporation.

Results: In the allogenic MLR assay, the two bispecific antibodies WBP3245A and WBP3245B enhanced IFNgamma release in a dose-dependent manner, more potent than a reference bispecific antibody WBP324-BMK1 or the combo (combination of anti-CTAL-4 and anti-PD-1) (Figure 16) . In the SEB stimulated PBMC assay, the bispecific antibodies WBP3245A and WBP3245B enhanced IL-2 release, but less potent than anti-CTLA-4 antibody or the combo (Figure 17) . In the Treg assay, the bispecific antibodies stimulated INFgamma release, more potent than anti-PD-1, anti-CTLA-4 or the combo (Figure 18) .

2.14 Human Serum stability

Antibodies were incubated in freshly isolated human serum (serum content > 90%) at 37℃. On indicated time points, an aliquot of serum treated sample were removed from the incubator and snap frozen in liquid N2, and then stored at -80℃ until ready for test. The samples were quickly thawed immediately prior to the stability test. Briefly, plates were pre-coated with 0.5 μg/mL of CTLA4 (W316-hPro1. ECD. hFc) madein house at 4℃ overnight. After 1-hour blocking, testing antibodies were added to the plates at various concentrations (4-fold serially diluted from 5.0 nM to 0.0003 nM) . The plates were incubated at ambient temperature for 1 hour. Following the incubation, plates are washed three times with 300 μL per well of PBS containing 0.5% (v/v) Tween 20.0.1 μg/ml human PD-1 (W305-hPro1. ECD. hFc. Biotin) made in house was added to plates and incubation 1 hour. After washing the plates three times, Streptavidin-HRP (Lifetechnologies, #SNN1004) (1: 20000 diluted) is 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

M5e) .

Results: The two bispecific antibodies WBP3245A and WBP3245B were incubated at 37℃ human serum for 14 days, and their dual binding to human CTLA-4 and PD-1 was measured in an ELISA. As shown in Figures19A-19B, WBP3245A and WBP3245B 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.

Table 16: The EC50 of WBP3245A and WBP3245B dual binding to the targets over time

2.15 ADCC assay

The ADCC assay of WBP324 Antibodies bases on

EuTDA Cytotoxicity Reagents (PerkinElmer AD0116) .

In house engineered human CTLA-4 expressing cells W316-293F. hPro1. FL and W305-CHO. S. hPro1. C6 (loaded by BATDA reagent) 1×10 ⁵per well and various concentrations of antibodies (25-fold serially diluted from 10 μg/ml to 0.016 μg/ml) were pre-incubated in 96-well plate, and then PBMCs were added at the effector/target ratio of 50: 1. The plates were kept at 37℃ in a 5%CO ₂ incubator for 4 hours. Target cell lysis was determined by DELFIA Europium Solution. The europium and the ligand form a highly fluorescent and stable chelate (EuTDA) , then read by

M5e.

In house engineered human CTLA-4 expressing cells W316-293F. hPro1. F1 and various concentrations of anti-CTLA4 antibodies (10-fold serially diluted from 201 nM to 0.201 nM) were mixed in 96-well plates. Human complement was added at the dilution ratio of 1: 50. The plate was kept at 37℃ in a 5%CO ₂ incubator for 4 hours. Target cell lysis was determined by CellTiter-Glo. Rituxan-induced Raji cell lysis was used as positive control.

Results: As the isotype of WBP3245A and WBP3245B are human IgG4 (S228P) , these two antibodies did not induce significant ADCC effect on CTLA-4+ cells. In contrast, anti-CTLA-4 antibody in human IgG1 isotype induced up to 30%cell lysis in dose-dependent manner (Figure 20) . The lack of ADCC effect may reduce the toxicity of these two bispecific antibodies.

EXAMPLE 3: In vivo Characterization

3.1 In vivo efficacy study of WBP3245A in MC38 syngeneic human CTLA-4 KI transgenic mouse model

3.1.1 Study Objective and Regulatory Compliance

The objective of this study was to evaluate in vivo anti-tumor efficacy of WBP3245A in MC38 syngeneic model in hCTLA-4 KI transgenic mice.

3.1.2 Experimental Design

Table 17: Description of Experimental Design

Note:

a. N: number of animals per group;

b. Dose volume: adjust dosing volume based on body weight 10 l/g.

3.1.3 Animals and housing conditions

Species: Mus musculus

Strain: hCTLA-4 KI transgenic C57BL/6J

Age: 5-8 weeks

Sex: female

Body weight: 16-18 g

Number of animals: 50 mice including spare

Animal supplier: Beijing Biocytogen Co., LTD

The mice were kept in individual ventilation cages at constant temperature and humidity with 5 animals in each cage.

● Temperature: 20-26 ℃.

● Humidity 40-70 %.

Cages: Made of polycarbonate. The size is 300 mm x 200 mm x 180 mm. The bedding material is corn cob, which was changed twice per week.

Diet: Animals had free access to irradiation sterilized dry granule food during the entire study period.

Water: Animals had free access to sterile drinking water.

Cage identification: The identification labels for each cage contained the following information: number of animals, sex, strain, date received, treatment, study number, group number and the starting date of the treatment.

Animal identification: Animals were marked by ear coding.

3.1.4 Test articles

Product identification: W332-1.80.12. xAb. hIgG4

Manufacturer: WuXi Biologics

Package and storage condition: 9.38 mg/mL, stored at -80 ℃

Product identification: WBP3245A

Manufacturer: WuXi Biologics

Package and storage condition: 3.1 mg/mL, stored at -80 ℃

Product identification: Anti-CTLA4-IgG1

Manufacturer: WuXi Biologics

Package and storage condition: 4.6 mg/mL, stored at -80 ℃

Product identification: WBP305-BMK1-IgG4

Manufacturer: WuXi Biologics

Package and storage condition: 10.82 mg/mL, stored at -80 ℃

3.1.5 Experimental Methods and Procedures

The MC38 tumor cells (NTCC) were maintained in vitro as a monolayer culture in RPMI-1640 medium supplemented with 10%fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin at 37℃ in an atmosphere of 5%CO ₂ in air. The tumor cells were routinely sub-cultured twice weekly by trypsin-EDTA treatment. The cells growing in an exponential growth phase were harvested and counted for tumor inoculation.

Each mouse was inoculated subcutaneously at the right axillary (lateral) with MC38 tumor cells (3 x 10 ⁵) in 0.1 ml of PBS. The treatments were started on day 8 after tumor inoculation when the average tumor volume reached 61mm ³. The test articles administration and the animal numbers in each group were shown in the following experimental design table 1.

3.1.6 Observations

All the procedures related to animal handling, care and the treatment in the study were performed according to the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of WuXi AppTec following the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) . At the time of routine monitoring, the animals were daily checked for any effects of tumor growth and treatments on normal behavior such as mobility, food and water consumption (by looking only) , body weight gain/loss (body weights were measured twice weekly) , eye/hair matting and any other abnormal effect as stated in the protocol. Death and observed clinical signs were recorded on the basis of the numbers of animals within each subset.

3.1.7 Tumor Measurements and Endpoints

The major endpoint was to see if the tumor growth could be delayed or mice could be cured. Tumor size was measured twice weekly in two dimensions using a caliper, and the volume was expressed in mm ³ using the formula: V = 0.5 a x b ² where a and b are the long and short diameters of the tumor, respectively. The T/C value (in percent) is an indication of antitumor effectiveness.

TGI was calculated for each group using the formula: TGI (%) = [1- (T _i-T ₀) / (V _i-V ₀) ] ×100; Ti is the average tumor volume of a treatment group on a given day, T ₀ is the average tumor volume of the treatment group on the day of treatment start, V _i is the average tumor volume of the vehicle control group on the same day with T _i, and V ₀ is the average tumor volume of the vehicle group on the day of treatment start.

3.1.8 Statistical Analysis

Summary statistics, including mean and the standard error of the mean (SEM) , are provided for the tumor volume of each group at each time point. Statistical analysis of differences in tumor volumes and tumor weights among the groups were conducted on the data obtained at the best therapeutic time point (the 11 ^th day after treatment) . Aone-way ANOVA was performed to compare tumor volume and tumor weight among groups, and whena significant F -statistics (a ratio of treatment variance to the error variance) was obtained, comparisons between groupswere carried out with Games-Howell. All data were analyzed using SPSS 17.0. p< 0.05 was considered to be statistically significant.

3.1.9 Results: Efficacy in hCTLA-4 knock-in mice

Mean tumor volumes over time in female hCTLA-4 KI transgenic mice bearing MC38 syngeneic model dosed with test articles were shown in Table18.

Table 18: Tumor volumes over time

Note: a. Mean ± SEM;

b. days after the start of treatment

Tumor growth curves is shown in Figure 21. WBP3245A significantly inhibited tumor growth in a dose-dependent manner. At 10 mg/kg, WBP3245A showed to be more efficacious than hIgG control or anti-PD-1 antibody, but slightly less efficacious than anti-CTLA-4, which might be due to anti-CTLA4 antibody being a human IgG1 antibody.

Death of mice was found 0.5h post administration of anti-CTLA4-IgG1 at the doses of 3 mg/kg and 10 mg/kg, which might be due to drug toxicity. WBP3245A and WBP305-BMK1-IgG4 were well tolerated by the tumor-bearing animals at tested doses. No body weight loss was observed in all treatment groups.

3.2 In vivo efficacy study of WBP3245A in MC38 syngeneic hPD-1 KI transgenic mouse model

3.2.1 Study objective and regulatory compliance

The objective of the project is to evaluate the in vivo anti-tumor efficacy of antibody WBP3245A administrated by intravenous injection (I. V. ) in MC38 colon cancer model in human PD-1 transgenic mice.

3.2.2 Animals and housing condition

Species: Mus musculus

Strain: B-hPD-1 mice (PD-1 transgenic mice)

Age: 6-8 weeks

Sex: male

Body weight: 21-25 g

Number of animals: 89 mice plus spare

Animal supplier: Biocytogen Co., Ltd.

Certificate of conformity: 201711464

● Temperature: 20-26 ℃.

● Humidity 40-70 %.

Cages: Made of polycarbonate. The size is 300 mm x 180 mm x 150 mm. The bedding material is corn cob, which is changed twice per week.

Water: Animals had free access to sterile drinking water.

Animal identification: Animals were marked by ear coding.

3.2.3 Test articles

Product identification: W332-1.80.12. xAb. hlgG4

Supplier: WuXi Biologics

Physical description: Solution

Lot #: 2017-06-14

Molecular weight: NA, Formula weight: NA, Salt factor: NA, Purity: 98.90%

Package: 1.0 mL/vial × 1 vials, 0.025 mL/vial × 28 vials

Concentration: 10 /7.5 mg/kg

Store condition: -80 ℃

Product identification: WBP3245A

Supplier: WuXi Biologics

Physical description: Solution

Lot #: 2017-06-14

Molecular weight: NA, Formula weight: NA, Salt factor: NA, Purity: 87.10%

Package: 0.2 mL/vial × 22 vials

Concentration: 0.5 mg/kg, 3 mg/kg, 10 mg/kg

Store condition: -80 ℃

Product identification: Anti-PD1 antibody (WBP305-BMK1-IgG4)

Supplier: WuXi Biologics

Physical description: Solution

Lot #: 2017-07-14

Molecular weight: NA, Formula weight: NA, Salt factor: NA, Purity: 98.68%, 96.38%

Package: 0.17mL/vial × 2 vials, 0.1 mL/vial × 20 vials

Concentration: 0.5 /0.4 mg/kg, 3 /2.5 mg/kg, 10 /7.5 mg/kg

Store condition: -80 ℃

Product identification: Anti-CTLA4 antibody

Supplier: WuXi Biologics

Physical description: Solution

Lot #: 2017-01-20

Molecular weight: NA, Formula weight: NA, Salt factor: NA Purity: 99.62%

Package: 1mL/vial × 12 vials

Concentration: 10 /7.5 mg/mL

Store condition: -80 ℃

3.2.4 Experimental methods and procedures

The MC38-huPD-L1 cells will be maintained in vitro as a monolayer culture in RPMI-1640 medium supplemented with 10%fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin at 37℃ in an atmosphere of 5%CO ₂ in air. The tumor cells will be routinely subcultured twice weekly by trypsin-EDTA treatment. The cells growing in an exponential growth phase will be harvested and counted for tumor inoculation.

Each mouse was inoculated subcutaneously at the right axillary (lateral) with MC38-huPD-L1 tumor cell (3 x 10 ⁵) in 0.1 ml of PBS for tumor development. The animals were randomly grouped as 8 mice/group when the average tumor volume reached 59 mm ³, and then treatment started for the efficacy study. The tumor-bearing mice were intravenously dosed antibodies twice weekly for 2-3 weeks.

3.2.5Observations

All the procedures related to animal handling, care and the treatment in the study were performed according to the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of WuXi AppTec following the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) . At the time of routine monitoring, the animals were daily checked for any effects of tumor growth and treatments on normal behavior such as mobility, food and water consumption (by looking only) , body weight gain/loss (body weights were measured once every day) , eye/hair matting and any other abnormal effect as stated in the protocol. Death and observed clinical signs were recorded on the basis of the numbers of animals within each subset.

3.2.6 Tumor measurements and endpoints

The major endpoint is to see if the tumor growth can be delayed or mice can be cured. Tumor sizes will be measured three times weekly in two dimensions using a caliper, and the volume will be expressed in mm ³ using the formula: V = 0.5 a x b ² where a and b are the long and short diameters of the tumor, respectively. The tumor sizes are then used for the calculations of T/C (%) values. T/C (%) of relative tumor proliferation rate was calculated using the formula: T/C %= T _RTV/C _RTV x 100 % (T _RTV: treatment group RTV; C _RTV: negative control RTV) . The relative tumor volume was calculated based on the tumor measurements, the calculation formula was: RTV = V _t/V ₀, V ₀ is the average tumor volume on the day of treatment starts, V _t is the average tumor volume ofone time measure, T _RTV used data of day the same with C _RTV.

TGI is calculated for each group using the formula: TGI (%) = [1- (T _i-T ₀) / (V _i-V ₀) ] ×100; T _i is the average tumor volume of a treatment group on a given day, T ₀ is the average tumor volume of the treatment group on the first day of treatment, Vi is the average tumor volume of the vehicle control group on the same day with T _i, and V ₀ is the average tumor volume of the vehicle group on the first day of treatment.

3.2.7 Statistical analysis

Summary statistics, including mean and the standard error of the mean (SEM) , are provided for the tumor volume of each group at each time point (detailed in Section 5.3 Table 4) . Statistical analysis of difference in tumor volume among the groups and the analysis of drug interaction were conducted on the data obtained at the best therapeutic time point. It was on the 11 ^st day post treatment in this study.

One-way ANOVA was performed to compare tumor volume among groups, and whena significant F -statistics (a ratio of treatment variance to the error variance) was obtained, comparisons between groupswere carried out with Games-Howell test. All data were analyzed using SPSS 17.0. p< 0.05 was considered to be statistically significant.

3.2.8 Results: Efficacy in human PD-1 KI mice

Mean tumor volumes over time in MC38-huPD-L1 tumor-bearing mice dosed with test antibodies were shown in Table 19.

Table 19: Tumor volume over time

Tumor volume (mm ³) ^a

Note:

a. Mean ± SEM;

b. Days after the start of treatment

Tumor growth inhibition analysis

T/C and TGI values were calculated based on tumor size at day 11 post treatment. The results were shown in Table 20. The statistical differences between each treatment group and W332-1.80.12. xAb. hlgG4 were calculated by One-Way ANOVA.

Table 20: Tumor growth inhibition at day 11 (n=8)

Note:

a. Mean ± SEM.

b. T/C=T _RTV/V _RTV; TGI (%) = [1- (T ₁₁-T ₀) / (V ₁₁-V ₀) ] ×100.

c. p value was calculated based on tumor size by One-Way ANOVA, compared with W332-1.80.12. xAb. hlgG4.

Tumor growth curve and summary

In this study, the therapeutic efficacy of antibody WBP3245A was evaluated in MC38-huPD-L1 colon cancer model in human PD-1 transgenic mouse model. Tumor growth curve was shown in Figure 22.

The mean tumor size of W332-1.80.12. xAb. hlgG4 group reached 598 mm ³ at day 11 post treatment. WBP3245A 3 mg/kg and 10 mg/kg showed strong anti-tumor activities (TV _mean=143 mm ³ and 97 mm ³; T/C=23.96%and 15.92%; TGI=84.21%and 93.00%; p<0.001, respectively) . However, WBP3245A 0.5 mg/kg, WBP305-BMK1-IgG4 0.5/0.4 mg/kg, WBP305-BMK1-IgG4 3/2.5 mg/kg, WBP305-BMK1-IgG4 10/7.5mg/kg, and anti-CTLA4-IgG1 10/7.5mg/kg did not present anti-tumor efficacy (TV _mean=497 mm ³, 557 mm ³, 463 mm ³, 493 mm ³, and 612 mm ³, T/C=83.22%, 90.28%, 74.65%, 78.55%, and 101.49%, TGI=18.67%, 7.87%, 25.33%, 19.86%, and -2.60%, p=0.400, 0.709, 0.246, 0.319, and 0.919, respectively) .

In summary, antibody WBP3245A 3 mg/kg and 10 mg/kg demonstrated significant antitumor activity against the MC38-huPD-L1 colon cancer model in this study. In conclusion, WBP3245A can inhibit tumor growth in dose-dependent manner, and is more efficacious than anti-PD1 antibody.

3.3 In vivo efficacy study of WBP3245A in MC38 syngeneic hCTLA-4 and hPD-1 KI transgenic mouse model

3.3.1 Study design

Table 21: Description of Study design

Tumor volume (mm ³) = 0.5 × length × wideth ²

3.3.2 Results: Efficacy in hPD-1 and hCTLA-4 Knock in mice

In this study, the efficacy of antibody WBP3245A was evaluated in MC38-huPD-L1 colon cancer model in human PD-1 and CTLA-4 transgenic mouse model. Tumor growth curve was shown in Figure 23. Compared with IgG control, WBP3245A significantly inhibited tumor growth. The bispecific antibody WBP3245A had same efficacy as anti-PD-1 antibody, which might be due to the insensitivity of this model to anti-CTLA-4 arm, evidenced by no efficacy of anti-CTLA-4 antibody group.

Those skilled in the art will further appreciate that the present invention may be embodied in other specific forms without departing from the spirit or central attributes thereof. In that the foregoing description of the present disclosure discloses only exemplary embodiments thereof, it is to be understood that other variations are contemplated as being within the scope of the present invention. Accordingly, the present invention is not limited to the particular embodiments that have been described in detail herein. Rather, reference should be made to the appended claims as indicative of the scope and content of the invention.

Claims

A bispecific polypeptide complex, comprising:

a CTLA-4 antigen-binding moiety and a PD-1 antigen-binding moiety,

wherein the CTLA-4 antigen-binding moiety comprises a first heavy chain variable domain (VH) of an anti-CTLA-4 antibody operably linked to a first heavy chain constant region domain (CH1) , and a first light chain variable domain (VL) of the anti-CTLA-4 antibody operably linked to a first light chain constant region (CL) ,

wherein the PD-1 antigen-binding moiety comprises a second VH of an anti-PD-1 antibody operably linked to a second VL of the anti-PD-1 antibody, and

wherein:

(A) the anti-CTLA-4 antibody comprises:

a heavy chain complementarity determining region (CDRH) 1 consisting of SEQ ID NO: 1;

a CDRH2 consisting of SEQ ID NO: 2;

a CDRH3 consisting of SEQ ID NO: 3;

a light chain complementarity determining region (CDRL) 1 consisting of SEQ ID NO: 4;

a CDRL2 consisting of SEQ ID NO: 5; and

a CDRL3 consisting of SEQ ID NO: 6; and

(B) the anti-PD-1 antibody comprises:

a CDRH1 selected from the group consisting of SEQ ID NOs: 7 and 13;

a CDRH2 selected from the group consisting of SEQ ID NOs: 8 and 14;

a CDRH3 selected from the group consisting of SEQ ID NOs: 9 and 15;

a CDRL1 selected from the group consisting of SEQ ID NOs: 10 and 16;

a CDRL2 selected from the group consisting of SEQ ID NOs: 11 and 17; and

a CDRL3 selected from the group consisting of SEQ ID NOs: 12 and 18.
The bispecific polypeptide complex of claim 1, wherein the anti-PD-1 antibody comprises:

a CDRH1 consisting of SEQ ID NO: 7;

a CDRH2 consisting of SEQ ID NO: 8;

a CDRH3 consisting of SEQ ID NO: 9;

a CDRL1 consisting of SEQ ID NO: 10;

a CDRL2 consisting of SEQ ID NO: 11; and

a CDRL3 consisting of SEQ ID NO: 12.
The bispecific polypeptide complex of claim 1, wherein the anti-PD-1 antibody comprises:

a CDRH1 consisting of SEQ ID NO: 13;

a CDRH2 consisting of SEQ ID NO: 14;

a CDRH3 consisting of SEQ ID NO: 15;

a CDRL1 consisting of SEQ ID NO: 16;

a CDRL2 consisting of SEQ ID NO: 17; and

a CDRL3 consisting of SEQ ID NO: 18.
The bispecific polypeptide complex of any of the preceding claims, wherein the VH of the CTLA-4 antigen binding moiety comprises the amino acid sequence of SEQ ID NO: 19, and the VL of the CTLA-4 antigen binding moiety comprises the amino acid sequence of SEQ ID NO: 20.
The bispecific polypeptide complex of any of the preceding claims, whereinthe VH of the PD-1 antigen binding moiety comprisesthe amino acid sequence of SEQ ID NO: 21 or 23, and the VL of the PD-1 antigen binding moiety comprises the amino acid sequence of SEQ ID NO: 22 or 24.
The bispecific polypeptide complex of any of the preceding claims, wherein from N terminal to C terminal, the CTLA-4 antigen binding moiety is operably linked to an Fc region, and the Fc region is operably linked to the PD-1 antigen binding moiety.
The bispecific polypeptide complex ofclaim 6, wherein the Fc region is a human Fc region, preferably a human IgG Fc region, more preferably a human IgG4 Fc region.
The bispecific polypeptide complex ofclaim 7, wherein the human IgG4 Fc region has a mutation at amino acid 228, preferably a mutation of S to P at amino acid 228 (S228P) .
The bispecific polypeptide complex of any of the preceding claims, comprising a heavy chain and a light chain, wherein the heavy chain comprises, from N terminal to C terminal, domains operably linked as in VH-CH1-hinge-CH2-CH3-linker-scFv, and the light chain comprises domains operably linked as in VL-CL, wherein:

the VH, CH1, VL and CL are from the CTLA-4 antigen binding moiety, and the scFv is from the PD-1 antigen binding moiety.
The bispecific polypeptide complex of claim 9, wherein the VH and VL of the scFv is linked via a peptide sequence of (G4S) 3.
The bispecific polypeptide complex of any of the preceding claims, wherein the bispecific polypeptide complex comprises:

a heavy chain sequence comprising the amino acid sequence of SEQ ID NO: 25 or 26, and

a light chain sequence comprising the amino acid sequence of SEQ ID NO: 27.
A conjugate comprising the bispecific polypeptide complex of any of the preceding claims, conjugated to a moiety.
An isolated polynucleotide encoding the bispecific polypeptide complex of any of claims 1-11.
An isolated vector comprising the polynucleotide of claim 13.
A host cell comprising the isolated polynucleotide of claim 13 or the isolated vector of claim 14.
A method of expressing the bispecific polypeptide complex of any of claims 1-11, comprising culturing the host cell of claim 15 under the condition at which the bispecific polypeptide complex is expressed.
A method of producing the bispecific polypeptide complex of any of claims 1-11, comprising:

a) introducing to a host cell one or more polynucleotides encoding the first functional domain and one or more polynucleotides encoding the second functional domain; and

b) allowing the host cell to express the bispecific polypeptide complex.
The method of any of claims 16-17, further comprising isolating the bispecific polypeptide complex.
A composition comprising the bispecific polypeptide complex of any of claims 1-11.
A pharmaceutical composition comprising the bispecific polypeptide complex of any of claims 1-11 and a pharmaceutically acceptable carrier.
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 of any of claims 1-11, the composition of claim 19 or the pharmaceutical composition of claim 20.
A method of preventing or treating a cancer in a subject in need thereof, comprising administrating to the subject a therapeutically effective amount of the bispecific polypeptide complex of any of claims 1-11, the composition of claim 19 or the pharmaceutical composition of claim 20.
The method of claim 22, 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 1-11 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 1-11 in the manufacture of a medicament for preventing or treating a cancer in a subject in need thereof.
The bispecific polypeptide complex of any of claims 1-11 for use in modulating an immune response in a subject in need thereof.
The bispecific polypeptide complex of any of claims 1-11 for use in preventing or treating a cancer in a subject in need thereof.
A kit comprising the bispecific polypeptide complex of any of claims 1-11.