WO2019179391A1

WO2019179391A1 - Novel bispecific pd-1/ctla-4 antibody molecules

Info

Publication number: WO2019179391A1
Application number: PCT/CN2019/078484
Authority: WO
Inventors: Zhuozhi Wang; Yunying CHEN; Jing Li; Yong Zheng
Original assignee: Wuxi Biologics (Shanghai) Co., Ltd.; WuXi Biologics Ireland Limited
Priority date: 2018-03-19
Filing date: 2019-03-18
Publication date: 2019-09-26

Abstract

The present disclosure provides anti-CTLA-4/PD-1 bispecific antibody molecules, polynucleotides encoding the same, pharmaceutical compositions comprising the same, and the uses thereof.

Description

NOVEL BISPECIFIC PD-1/CTLA-4 ANTIBODY MOLECULES

PRIORITY CLAIM

The present application claims the priority to PCT Application Number PCT/CN2018/079501, filed on March 19, 2018.

FIELD OF THE INVENTION

The present disclosure generally relates to novel bispecific antibody molecules directed to human PD-1 and human CTLA-4.

BACKGROUND

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

Increasing evidences from preclinical and clinical results have shown that targeting immune checkpoints is becoming the most promising approach to treat patients with cancers. Programmed cell death 1 (PD-1) , one of immune-checkpoint proteins, play a major role in limiting the activity of T cells that provide a major immune resistance mechanism by which tumor cells escaped immune surveillance. The interaction of PD-1 expressed on activated T cells, and PD-L1 expressed on tumor cells negatively regulate immune response and damp anti-tumor immunity.

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. 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. Another anti-CTLA-4 antibody tremelimumab was evaluated as monotherapy in melanoma and malignant mesothelioma

Despite of the development of therapeutics targeting the targets respectively, there is a significant need for novel bispecific antibodies that can acts on dual targets.

BRIEF SUMMARY OF THE INVENTION

Throughout the present disclosure, 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, “an antibody” means one antibody or more than one antibody.

The present disclosure provides novel bispecific PD-1/CTLA-4 antibody molecules, amino acid and nucleotide sequences thereof, and uses thereof.

In one aspect, the present disclosure provides herein a bispecific antibody molecule comprising a CTLA-4-binding domain and a PD-1-binding domain, wherein:

the CTLA-4-binding domain comprises:

1, 2, or 3 heavy chain complementarity determining region (CDR) sequences selected from the group consisting of: SEQ ID NOs: 1-4; and/or

the PD-1-binding domain comprises:

1, 2, or 3 heavy chain complementarity determining region (CDR) sequences selected from the group consisting of: SEQ ID NOs: 11-13 and 21-23; and/or

1, 2, or 3 light chain CDR sequences selected from the group consisting of: SEQ ID NOs: 14-16 and 24-26,

the CTLA-4-binding domain comprises a VHH domain; and

the PD-1-binding domain comprises a Fab.

In certain embodiments, the CTLA-4-binding domain comprises a heavy chain variable region comprising SEQ ID NOs: 1, 4 and 3.

In certain embodiments, the CTLA-4-binding domain comprises a heavy chain variable region selected from the group consisting of SEQ ID NOs: 5, 7 and 9, and a homologous sequence thereof having at least 80%sequence identity yet retaining specific binding affinity to CTLA-4.

In certain embodiments, the CTLA-4-binding domain comprises a heavy chain variable region comprising SEQ ID NO: 9.

In certain embodiments, the PD-1-binding domain comprises a heavy chain variable region selected from the group consisting of:

a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 21-23; and

a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 11-13, and/or

a light chain variable region selected from the group consisting of:

a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 24-26; and

a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 14-16.

In certain embodiments, the PD-1-binding domain comprises:

a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 21-23; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 24-26; or

a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 11-13; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 14-16.

In certain embodiments, the PD-1-binding domain comprises a heavy chain variable region selected from the group consisting of SEQ ID NOs: 17 and 27, and a homologous sequence thereof having at least 80%sequence identity yet retaining specific binding affinity to PD-1.

In certain embodiments, the PD-1-binding domain comprises a light chain variable region selected from the group consisting of SEQ ID NOs: 18 and 28, and a homologous sequence thereof having at least 80%sequence identity yet retaining specific binding affinity to PD-1.

In certain embodiments, the PD-1-binding domain comprises:

a heavy chain variable region comprising SEQ ID NO: 27 and a light chain variable region comprising SEQ ID NO: 28; or

a heavy chain variable region comprising SEQ ID NO: 17 and a light chain variable region comprising SEQ ID NO: 18.

In certain embodiments, the CTLA-4-binding domain further comprises one or more amino acid residue substitutions or modifications yet retains specific binding affinity to CTLA-4, and/or the PD-1-binding domain further comprises one or more amino acid residue substitutions or modifications yet retains specific binding affinity to PD-1.

In certain embodiments, at least one of the substitutions or modifications is in one or more of the CDR sequences, and/or in one or more of the VH or the VL sequences but not in any of the CDR sequences.

In certain embodiments, the bispecific antibody molecule further comprises an immunoglobulin constant region, optionally a constant region of human Ig, or optionally a constant region of human IgG.

In certain embodiments, the CTLA-4-binding domain is operably linked to the N terminus or the C terminus of the PD-1-binding domain.

In certain embodiments, the CTLA-4-binding domain comprises the sequence of SEQ ID NO: 9, and the PD-1-binding domain comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 17 and a light chain variable region comprising the sequence of SEQ ID NO: 18.

In certain embodiments, the CTLA-4-binding domain comprises the sequence of SEQ ID NO: 9, and the PD-1-binding domain comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 27 and a light chain variable region comprising the sequence of SEQ ID NO: 28.

In certain embodiments, the CTLA-4-binding domain is operably linked to the C terminus of the heavy chain of the PD-1-binding domain.

In certain embodiments, the bispecific antibody molecule comprise a heavy chain in the format of: VH (anti-PD-1) -CH1-Hinge-CH2-CH3-spacer-VHH (anti-CTLA-4) , associated with a light chain in the format of VL (anti-PD-1) -CL.

In certain embodiments, the bispecific antibody molecule comprising a heavy chain comprising the sequence of SEQ ID NO: 34 and a light chain comprising the sequence of SEQ ID NO: 35.

In certain embodiments, the bispecific antibody molecule comprising a heavy chain comprising the sequence of SEQ ID NO: 36 and a light chain comprising the sequence of SEQ ID NO: 37.

In certain embodiments, the CTLA-4-binding domain and/or the PD-1-binding domain is fully human or humanized.

In certain embodiments, the bispecific antibody molecule as provided herein is linked to one or more conjugate moieties.

In certain embodiments, the conjugate moiety comprises a clearance-modifying agent, a chemotherapeutic agent, a toxin, a radioactive isotope, a lanthanide, a luminescent label, a fluorescent label, an enzyme-substrate label, a DNA-alkylators, a topoisomerase inhibitor, a tubulin-binders, or other anticancer drugs.

In another aspect, the present disclosure provides a pharmaceutical composition comprising the bispecific antibody molecule as provided herein, and a pharmaceutically acceptable carrier.

In another aspect, the present disclosure provides a polynucleotide encoding the bispecific antibody molecule as provided herein.

In certain embodiments, the polynucleotide comprising a nucleotide sequence selecting from a group consisting of SEQ ID NOs: 6, 8, 10, 19, 20, 29 and 30, and/or a homologous sequence thereof having at least 80% (e.g. at least 85%, 88%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity, and/or a variant thereof having only degenerate substitutions.

In another aspect, the present disclosure provides a vector comprising the polynucleotide as provided herein.

In another aspect, the present disclosure provides a host cell comprising the vector as provided herein.

In another aspect, the present disclosure provides a method of expressing the bispecific antibody molecule as provided herein, comprising culturing the host cell as provided herein under the condition at which the vector as provided herein is expressed.

In another aspect, the present disclosure provides a method of treating a disease or condition in a subject that would benefit from up-regulation of an immune response, comprising administering to the subject a therapeutically effective amount of the bispecific antibody molecule as provided herein or the pharmaceutical composition as provided herein.

In certain embodiments, the disease or condition that would benefit from up-regulaltion of an immune response is selected from the group consisting of cancer, a viral infection, a bacterial infection, a protozoan infection, a helminth infection, asthma associated with impaired airway tolerance, a neurological disease, multiple sclerosis, and an immunosuppressive disease.

In certain embodiments, the disease or condition is PD-1-related and/or CTLA-4-related.

In certain embodiments, the PD-1-related disease or condition is cancer, autoimmune disease, inflammatory disease, or infectious disease.

In certain embodiments, the CTLA-4-related disease or condition is cancer, autoimmune disease, inflammatory disease, or infectious disease.

In certain embodiments, the cancer is lymphoma, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, uterine or endometrial cancer, rectal cancer, esophageal cancer, head and neck cancer, anal cancer, gastrointestinal cancer, intra-epithelial neoplasm, kidney or renal cancer, leukemia, liver cancer, lung cancer, melanoma, myeloma, pancreatic cancer, prostate cancer, sarcoma, skin cancer, squamous cell cancer, stomach cancer, testicular cancer, vulval cancer, cancer of the endocrine system, cancer of the parathyroid gland, cancer of the adrenal gland, penile carcinoma, solid tumors of childhood, tumor angiogenesis, spinal axis tumor, pituitary adenoma, or epidermoid cancer.

In certain embodiments, the disease or condition is an environmentally induced cancer induced by asbestos or hematologic malignancies, wherein said cancer is selected from multiple myeloma, B-cell lymphoma, Hodgkin lymphoma, primary mediastinal B-cell lymphoma, non-Hodgkin's lymphoma, acute myeloid lymphoma, chronic myelogenous leukemia, chronic lymphoid leukemia (CLL) , follicular lymphoma, diffuse large B-cell lymphoma (DLBCL) , Burkitt's lymphoma, immunoblastic large cell lymphoma, precursor B- lymphoblastic lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia (ALL) , mycosis fungoides, anaplastic large cell lymphoma, T-cell lymphoma, and precursor T-lymphoblastic lymphoma, and any combinations of said cancers.

In certain embodiments, the subject is human.

In certain embodiments, the administration is via oral, nasal, intravenous, subcutaneous, sublingual, or intramuscular administration.

In another aspect, the present disclosure provides a method of modulating CTLA-4 activity in a CTLA-4-expressing cell, comprising exposing the CTLA-4-expressing cell to the bispecific antibody molecule as provided herein.

In another aspect, the present disclosure provides use of the bispecific antibody molecule as provided herein in the manufacture of a medicament for treating a disease or condition that would benefit from up-regulation of an immune response.

In another aspect, the present disclosure provides use of the bispecific antibody molecule as provided herein in the manufacture of a medicament for treating a disease or condition that is PD-1 and/or CTLA-4-related.

BRIEF DESCFRIPTION OF FIGURES

Figure 1 shows the result of antibody expression, purity and thermal stability. W3246-U3T9 and W3246-U6T9 have good expression level, thermal stability and purity.

Figure 2A shows the result of hCTLA-4-Binding ELISA. W3246-U3T9 and W3246-U6T9 can bind to hCTLA-4 protein as well as benchmark controls.

Figure 2B shows the result of hCTLA-4-Binding FACS. W3246-U3T9 and W3246-U6T9 can bind to hCTLA-4 cell.

Figure 3A shows the result of hPD-1-Binding ELISA. W3246-U3T9 and W3246-U6T9 can bind to hPD-1 protein as well as benchmark controls.

Figure 3B shows the result of hPD-1-Binding FACS. W3246-U3T9 and W3246-U6T9 can bind to hPD-1 by FACS as well as parental and benchmark antibodies.

Figure 4A shows the result of cyno CTLA-4-Binding ELISA. W3246-U3T9 and W3246-U6T9 can bind to cCTLA-4 protein as well as benchmark controls.

Figure 4B shows the result of cyno CTLA-4-Binding FACS. W3246-U3T9 and W3246-U6T9 can bind to cCTLA-4 cell.

Figure 5A shows the result of cynoPD-1-binding FACS. W3246-U3T9 and W3246-U6T9 can bind to cPD-1 cell.

Figure 5B shows the result of cynoPD-1-binding ELISA. W3246-U3T9 and W3246-U6T9 can bind to cPD-1 protein as well as benchmark controls.

Figure 6 shows the result of Dual-Binding ELISA. W3246-U3T9 and W3246-U6T9 can bind to hPD-1 and hCTLA-4 simultaneously.

Figure 7 shows the results of dual binding FACS. W3246-U3T9 and W3246-U6T9 have higher double positive percentage than the benchmark control.

Figure 8A shows the result of blocking hCTLA-4 binding to hCD80 on cell surface (FACS) . W3246-U3T9 and W3246-U6T9 can block hCD80 binding to hCTLA-4+ cells.

Figure 8B shows the result of blocking hCTLA-4 binding to hCD86 on cell surface (FACS) . W3246-U3T9 and W3246-U6T9 can block hCD86 binding to hCTLA-4+ cells.

Figure 9A shows the result of W3246-U3T9 and W3246-U6T9 blocking cyno CTLA-4 binding to hCD80+ cell (FACS) .

Figure 9B shows the result of W3246-U3T9 and W3246-U6T9 blocking cyno CTLA-4 binding to hCD86+ cells (FACS) .

Figure 10A shows the result of hPD-1-Competition FACS. W3246-U3T9 and W3246-U6T9 can block hPDL1 binding to hPD-1.

Figure 10B shows the result of mPD-1-Competition FACS. W3246-U3T9 and W3246-U6T9 can block mPD-L1 binding to hPD-1.

Figure 11 shows that W3246-U3T9 and W3246-U6T9 enhance cytokine release in Human Allogeneic MLR Assay

Figures 12A and 12B show that W3246-U3T9 and W3246-U6T9 enhance cytokine release of Human PBMC stimulated by SEB

Figure 13A shows the result of Human Treg MLR Assay (Proliferation) . W3246-U3T9 and W3246-U6T9 significantly enhance T cell proliferation: W3246-U3T9 or W3246-U6T9 > combination of W316-BMK1 and W305-BMK1 > anti-PD-1 (W305-BMK1) > anti-CTLA-4 (W316-BMK1) > bispecific benchmark control.

Figure 13B shows the result of Human Treg MLR Assay (INFgamma) . W3246-U3T9 and W3246-U6T9 significantly enhance IFN-γ secretion: W3246-U3T9 or W3246- U6T9 >Bispecific benchmark control = combination of W316-BMK1 and W305-BMK1 >anti-PD-1 (W305-BMK1) > anti-CTLA-4 (W316-BMK1) .

Figure 14 shows that W3246-U3T9 and W3246-U6T9 enhance cytokine release in Human Treg Suppression Assay

Figure 15 shows the result of Human Serum Stability ELISA test. W3246-U3T9 and W3246-U6T9 remain stable in human serum at 37 ℃ for two weeks.

Figure 16 shows the result of Cross-Family ELISA test. W3246-U3T9 and W3246-U6T9 have no-cross reaction with hCD28, hICOS and hBTLA.

Figure 17 W3246-U3T9 can bind to mPD-1 protein with EC ₅₀ of 0.0785 nM by ELISA.

Figure 18 W3246-U3T9 can bind to mPD-1 protein with EC ₅₀ of 4.579 nM by FACS.

DETAILED DESCRIPTION OF THE INVENTION

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

Definitions

The term “antibody” as used herein includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multispecific antibody, or bispecific antibody that binds to a specific antigen. A native intact antibody comprises two heavy (H) chains and two light (L) chains. Mammalian heavy chains are classified as alpha, delta, epsilon, gamma, and mu, each heavy chain consists of a variable region (V _H) and a first, second, and third constant region (C _H1, C _H2, C _H3, respectively) ; mammalian light chains are classified as λ or κ, while each light chain consists of a variable region (V _L) and a constant region. 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 disulfide 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 chain CDRs including LCDR1, LCDR2, and LCDR3, heavy chain CDRs including HCDR1, HCDR2, HCDR3) . CDR boundaries for the antibodies and antigen-binding domains disclosed herein may be defined or identified by the conventions of Kabat, IMGT, AbM, 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) ; Whitelegg et al, Protein Engineering, v13 (12) , 819-824 (2000) ; 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) ; Marie-Paule Lefranc et al, Developmental and Comparative Immunology, 27: 55-77 (2003) ; Marie-Paule Lefranc et al, Immunome Research, 1 (3) , (2005) ; Marie-Paule Lefranc, Molecular Biology of B cells (second edition) , chapter 26, 481-514, (2015) ) . 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. 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 alpha, delta, epsilon, gamma, and mu heavy chains, respectively. Several of the major antibody classes are divided into subclasses such as IgG1 (gamma1 heavy chain) , IgG2 (gamma2 heavy chain) , IgG3 (gamma3 heavy chain) , IgG4 (gamma4 heavy chain) , IgA1 (alpha1 heavy chain) , or IgA2 (alpha2 heavy chain) .

The term “antibody molecule” as used herein refers to an antigen-binding protein or polypeptide comprising at least one antibody fragment (such as CDR, and/or variable region sequence) . An antibody molecule includes, for example, a monoclonal antibody, an antibody fragment or domain, a fusion protein comprising an antibody fragment or domain, a polypeptide complex comprising an antibody fragment or domain, and so on.

The term “bivalent” as used herein refers to an antibody or an antigen-binding domain having two antigen-binding sites; the term “monovalent” refers to an antibody or an antigen-binding domain having only one single antigen-binding site; and the term “multivalent” refers to an antibody or an antigen-binding domain having multiple antigen- binding sites. In some embodiments, the antibody or antigen-binding domain thereof is bivalent.

The term “antigen-binding domain” (e.g. CTLA-4-binding domain or PD-1-binding domain) as used herein refers to an antibody fragment formed 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 domain include, without limitation, a diabody, a Fab, a Fab', a F (ab') ₂, an Fv fragment, a disulfide stabilized Fv fragment (dsFv) , a (dsFv) ₂, a bispecific dsFv (dsFv-dsFv') , a disulfide stabilized diabody (ds diabody) , a single-chain antibody molecule (scFv) , an scFv dimer (bivalent diabody) , a bispecific antibody, a multispecific antibody, a camelized single domain antibody, a nanobody, a domain antibody, and a bivalent domain antibody. An antigen-binding domain is capable of binding to the same antigen to which the parent antibody binds. In certain embodiments, an antigen-binding domain 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 domain are described in Spiess et al, 2015 (Supra) , and Brinkman et al., mAbs, 9 (2) , pp. 182–212 (2017) , which are incorporated herein by entirety reference.

“Fab” with regard to an antibody refers to that portion of the antibody consisting of a single light chain (both variable and constant regions) bound to the variable region and first constant region of a single heavy chain by a disulfide bond.

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

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

A “fragment difficult (Fd) ” with regard to an antibody refers to the amino-terminal half of the heavy chain fragment that can be combined with the light chain to form a Fab. For example, Fd fragment may consists of the VH and CH1 domains

“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. Fvs constructs containing a variable domain of a heavy or light immunoglobulin chain associated to the variable and constant domain of the corresponding immunoglobulin heavy or light chain have also been produced. Fvs have also been multimerised to form diabodies and triabodies (Maynard et al., Annu Rev Biomed Eng 2 339-376 (2000) ) .

“Single-chain Fv antibody” or “scFv” refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region connected to one another directly or via a peptide linker sequence (Huston JS et al. Proc Natl Acad Sci USA, 85: 5879 (1988) ) .

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

A “dsFv” refers to a disulfide-stabilized Fv fragment that the linkage between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond. In some embodiments, a “ (dsFv) ₂” or “ (dsFv-dsFv') ” comprises three peptide chains: two V _H moieties linked by a peptide linker (e.g., a long flexible linker) and bound to two V _L moieties, respectively, via disulfide bridges. In some embodiments, dsFv-dsFv' is bispecific in which each disulfide paired heavy and light chain has a different antigen specificity.

“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) .

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

“Camelized single domain antibody, ” “heavy chain antibody, ” or “HCAb” refers to an antibody that contains two VH domains and no light chains (Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10; 231 (1-2) : 25-38 (1999) ; Muyldermans S., J Biotechnol. Jun; 74 (4) : 277-302 (2001) ; WO94/04678; WO94/25591; U.S. Patent No. 6,005,079) . Heavy chain antibodies were originally derived from Camelidae (camels, dromedaries, and llamas) . Although devoid of light chains, camelized antibodies have an authentic antigen-binding repertoire (Hamers-Casterman C. et al., Nature. Jun 3; 363 (6428) : 446-8 (1993) ; Nguyen VK. et al. “Heavy-chain antibodies in Camelidae; a case of evolutionary innovation, ” Immunogenetics. Apr; 54 (1) : 39-47 (2002) ; Nguyen VK. et al.Immunology. May; 109 (1) : 93-101 (2003) ) . The variable domain of a heavy chain antibody (VHH domain) represents the smallest known antigen-binding unit generated by adaptive immune responses (Koch-Nolte F. et al., FASEB J. Nov; 21 (13) : 3490-8. Epub 2007 Jun 15 (2007) ) .

A “nanobody” refers to an antibody fragment that consists of a VHH domain from a heavy chain antibody and two constant domains, CH2 and CH3.

A “domain antibody” or a “single domain antibody” refers to an antibody fragment containing only the variable region of a heavy chain or the variable region of a light chain. In certain instances, two or more V _H domains are covalently joined with a peptide linker to create a bivalent or multivalent domain antibody. The two V _H domains of a bivalent domain antibody may target the same or different antigens.

The term “chimeric” as used herein, means an antibody or antigen-binding domain, having a portion of heavy and/or light chain derived from one species, and the rest of the heavy and/or light chain derived from a different species. In an illustrative example, a chimeric antibody may comprise a constant region derived from human and a variable region from a non-human animal, such as from mouse. In some embodiments, the non-human animal is a mammal, for example, a mouse, a rat, a rabbit, a goat, a sheep, a guinea pig, or a hamster.

The term “humanized” as used herein means that the antibody or antigen-binding domain comprises CDRs derived from non-human animals, FR regions derived from human, and when applicable, the constant regions derived from human.

The term “fully human” as used herein, with reference to antibody or antigen-binding domain, means that the antibody or the antigen-binding domain has or consists of amino acid sequence (s) corresponding to that of an antibody produced by a human or a human immune cell, or derived from a non-human source such as a transgenic non-human animal that utilizes human antibody repertoires or other human antibody-encoding sequences. In certain embodiments, a fully human antibody does not comprise amino acid residues (in particular antigen-binding residues) derived from a non-human antibody.

The term “operably link” or “operably linked” refers to a juxtaposition, with or without a spacer or a linker or an intervening sequence, 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. For another example, an antigen-binding domain can be operably linked to another antigen-binding domain with an intervening sequence there between, and such intervening sequence can be a spacer or can comprise a much longer sequence such as a constant region of an antibody. 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 which 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.

An “antigen” 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.

“CTLA-4” as used herein, refers to the Cytotoxic T-lymphocyte-associated protein 4 derived from any vertebrate source, including mammals such as primates (e.g. humans, monkeys) and rodents (e.g., mice and rats) . Exemplary sequence of human CTLA-4 includes Homo sapiens (human) CTLA-4 protein (NCBI Ref Seq No. AAL07473.1) . Exemplary sequence of CTLA-4 includes Macaca fascicularis (monkey) CTLA-4 protein (NCBI Ref Seq No XP_005574071.1) .

The term “CTLA-4” as used herein is intended to encompass any form of CTLA-4, for example, 1) native unprocessed CTLA-4 molecule, “full-length” CTLA-4 chain or naturally occurring variants of CTLA-4, including, for example, splice variants or allelic variants; 2) any form of CTLA-4 that results from processing in the cell; or 3) full length, a fragment (e.g., a truncated form, an extracellular/transmembrane domain) or a modified form (e.g. a mutated form, a glycosylated/PEGylated, a His-tag/immunofluorescence fused form) of CTLA-4 subunit generated through recombinant method.

The term “anti-CTLA-4 antibody” , “anti-CTLA-4 binding domain” or “CTLA-4-binding domain” refers to an antibody or antigen-binding domain that is capable of specific binding CTLA-4 (e.g. human or monkey CTLA-4) .

“PD-1” as used herein refers programmed cell death protein, which belongs to the superfamily of immunoglobulin and functions as co-inhibitory receptor to negatively regulate the immune system. PD-1 is a member of the CD28/CTLA-4 family, and has two known ligands including PD-L1 and PD-L2. Representative amino acid sequence of human PD-1 is disclosed under the NCBI accession number: NP_005009.2, and the representative nucleic acid sequence encoding the human PD-1 is shown under the NCBI accession number: NM_005018.2.

“PD-L1” as used herein refers to programmed cell death ligand 1 (PD-L1, see, for example, Freeman et al. (2000) J. Exp. Med. 192: 1027) . Representative amino acid sequence of human PD-L1 is disclosed under the NCBI accession number: NP_054862.1, and the representative nucleic acid sequence encoding the human PD-L1 is shown under the NCBI accession number: NM_014143.3. PD-L1 is expressed in placenta, spleen, lymph nodes, thymus, heart, fetal liver, and is also found on many tumor or cancer cells. PD-L1 binds to its receptor PD-1 or B7-1, which is expressed on activated T cells, B cells and myeloid cells. The binding of PD-L1 and its receptor induces signal transduction to suppress TCR-mediated activation of cytokine production and T cell proliferation. Accordingly, PD-L1 plays a major role in suppressing immune system during particular events such as pregnancy, autoimmune diseases, tissue allografts, and is believed to allow tumor or cancer cells to circumvent the immunological checkpoint and evade the immune response.

“Anti-PD-1 antibody” , “anti-PD-1 binding domain” or “PD-1 binding domain” as used herein refers to an antibody or antigen-binding domain that is capable of specific binding to PD-1 (e.g. human or monkey PD-1) with an affinity which is sufficient to provide for diagnostic and/or therapeutic use.

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 antibody molecules or antigen-binding domains provided herein specifically bind to human PD-1 and/or human CTLA-4 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, ≤4x10 ^-9M, ≤3x10 ^-9M, ≤2x10 ^-9 M, or ≤10 ^-9 M) . K _D used herein refers to the ratio of the dissociation rate to the association rate (k _off/k _on) , which may be determined by using any conventional method known in the art, including but are not limited to surface plasmon resonance method, microscale thermophoresis method, HPLC-MS method and flow cytometry (such as FACS) method. In certain embodiments, the K _D value can be appropriately determined by using flow cytometry.

The ability to “block binding” or “compete for the same epitope” as used herein refers to the ability of an antibody or antigen-binding domain to inhibit the binding interaction between two molecules (e.g. human CTLA-4 and an anti-CTLA-4 antibody, human PD-1 and an anti-PD-1 antibody) to any detectable degree. In certain embodiments, an antibody or antigen-binding domain that blocks binding between two molecules inhibits the binding interaction between the two molecules by at least 85%, or at least 90%. In certain embodiments, this inhibition may be greater than 85%, or greater than 90%.

The term “epitope” as used herein refers to the specific group of atoms or amino acids on an antigen to which an antibody binds. Epitopes can be formed both from contiguous amino acids (also called linear or sequential epitope) or noncontiguous amino acids juxtaposed by tertiary folding of a protein (also called configurational or conformational epitope) . 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. Two antibodies may bind the same or a closely related epitope within an antigen if they exhibit competitive binding for the antigen. For example, if an antibody or antigen-binding domain blocks binding of a reference antibody to the antigen by at least 85%, or at least 90%, or at least 95%, then the antibody or antigen-binding domain may be considered to bind the same/closely related epitope as the reference antibody.

The antibody names as used herein may include one or more suffix symbols which usually indicates the type of the antibody or particular modifications made to the antibody. For example, “uIgG1” or “uIgG4” means an antibody with human constant region of IgG1 isotype or a IgG4 isotype, “hAb” or “uAb” means human antibody, “z” means humanized antibody, “SP” refers to mutation of S228P in the constant region of human IgG4 (i.e. S228P) .

A “conservative substitution” with reference to amino acid sequence refers to replacing an amino acid residue with a different amino acid residue having a side chain with similar physiochemical properties. For example, conservative substitutions can be made among amino acid residues with hydrophobic side chains (e.g. Met, Ala, Val, Leu, and Ile) , among residues with neutral hydrophilic side chains (e.g. Cys, Ser, Thr, Asn and Gln) , among residues with acidic side chains (e.g. Asp, Glu) , among amino acids with basic side chains (e.g. His, Lys, and Arg) , or among residues with aromatic side chains (e.g. Trp, Tyr, and Phe) . As known in the art, conservative substitution usually does not cause significant change in the protein conformational structure, and therefore could retain the biological activity of a protein.

The term “homolog” and “homologous” as used herein are interchangeable and refer to nucleic acid sequences (or its complementary strand) or amino acid sequences that have sequence identity of at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) to another sequences when optimally aligned.

“Percent (%) sequence identity” with respect to amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues in a reference sequence, after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum number of identical amino acids (or nucleic acids) . Conservative substitution of the amino acid residues may or may not be considered as identical residues. Alignment for purposes of determining percent amino acid (or nucleic acid) sequence identity can be achieved, for example, using publicly available tools such as BLASTN, BLASTp (available on the website of U.S. National Center for Biotechnology Information (NCBI) , see also, Altschul S.F. et al, J. Mol. Biol., 215: 403–410 (1990) ; Stephen F. et al, Nucleic Acids Res., 25: 3389–3402 (1997) ) , ClustalW2 (available on the website of European Bioinformatics Institute, see also, Higgins D. G. et al, Methods in Enzymology, 266: 383-402 (1996) ; Larkin M.A. et al, Bioinformatics (Oxford, England) , 23 (21) : 2947-8 (2007) ) , and ALIGN or Megalign (DNASTAR) software. Those skilled in the art may use the default parameters provided by the tool, or may customize the parameters as appropriate for the alignment, such as for example, by selecting a suitable algorithm.

“Effector functions” as used herein refer to biological activities attributable to the binding of Fc region of an antibody to its effectors such as C1 complex and Fc receptor. Exemplary effector functions include: complement dependent cytotoxicity (CDC) induced by interaction of antibodies and C1q on the C1 complex; antibody-dependent cell-mediated cytotoxicity (ADCC) induced by binding of Fc region of an antibody to Fc receptor on an effector cell; and phagocytosis.

“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 term “subject” or “individual” or “animal” or “patient” as used herein refers to 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 disorder. 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 “vector” as used herein refers to a vehicle into which a polynucleotide encoding a protein may be operably inserted so as to bring about the expression of that protein. A vector may be used to transform, transduce, or transfect a host cell so as to bring about expression of the genetic element it carries within the host cell. Examples of vectors include plasmids, phagemids, cosmids, and artificial chromosomes such as yeast artificial chromosome (YAC) , bacterial artificial chromosome (BAC) , or P1-derived artificial chromosome (PAC) , bacteriophages such as lambda phage or M13 phage, and animal viruses. Categories of animal viruses used as vectors include retrovirus (including lentivirus) , adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus) , poxvirus, baculovirus, papillomavirus, and papovavirus (e.g., SV40) . A vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. In addition, the vector may contain an origin of replication. A vector may also include materials to aid in its entry into the cell, including but not limited to a viral particle, a liposome, or a protein coating. A vector can be an expression vector or a cloning vector.

The phrase “host cell” as used herein refers to a cell into which an exogenous polynucleotide and/or a vector has been introduced.

A “CTLA-4-related” disease or condition as used herein refers to any disease or condition caused by, exacerbated by, or otherwise linked to increased or decreased expression or activities of CTLA-4. In some embodiments, the CTLA-4 related condition is immune-related disorder, such as, for example, cancer, autoimmune disease, inflammatory disease or infectious disease, graft versus host disease (GVHD) , or transplant rejection.

A “PD-1-related” disease or condition as used herein refers to any condition that is caused by, exacerbated by, or otherwise linked to increased or decreased expression or activities of PD-1 (e.g. a human PD-1) .

“Cancer” as used herein refers to any medical condition characterized by malignant cell growth or neoplasm, abnormal proliferation, infiltration or metastasis, and includes both solid tumors and non-solid cancers (hematologic malignancies) such as leukemia. As used herein “solid tumor” refers to a solid mass of neoplastic and/or malignant cells. Examples of cancer or tumors include hematological malignancies, oral carcinomas (for example of the lip, tongue or pharynx) , digestive organs (for example esophagus, stomach, small intestine, colon, large intestine, or rectum) , peritoneum, liver and biliary passages, pancreas, respiratory system such as larynx or lung (small cell and non-small cell) , bone, connective tissue, skin (e.g., melanoma) , breast, reproductive organs (fallopian tube, uterus, cervix, testicles, ovary, or prostate) , urinary tract (e.g., bladder or kidney) , brain and endocrine glands such as the thyroid. In certain embodiments, the cancer is selected from ovarian cancer, breast cancer, head and neck cancer, renal cancer, bladder cancer, hepatocellular cancer, and colorectal cancer. In certain embodiments, the cancer is selected from a lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma and B-cell lymphoma.

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. Bispecific Antibody Molecule

In one aspect, the present disclosure provides herein a bispecific antibody molecule. The term “bispecific” as used herein means that, there are at least two antigen-binding domains (i.e. could be dual specific or multispecific) , each of which is capable of specifically binding to a different epitope. The bispecific antibody molecule provided herein comprises a CTLA-4-binding domain and a PD-1-binding domain capable of specifically binding to PD-1, and the CTLA-4-binding domain comprises a VHH domain; and the PD-1-binding domain comprises a Fab.

i. CTLA-4-binding domain

In certain embodiments, the CTLA-4-binding domain comprises one or more (e.g. 1, 2, or 3) CDR sequences of an anti-CTLA-4 single domain antibody W3166.

“W3166” as used herein refers to a VHH antibody having a heavy chain variable region of SEQ ID NO: 5.

“W3166-z13” as used herein refers to a humanized VHH antibody based on W3166 that comprises a heavy chain variable region of SEQ ID NO: 7. W3166-z13 has comparable affinity to the antigen as compared with its parent antibody W3166.

“W3166-z17” as used herein refers to a humanized VHH antibody based on W3166 that comprises a heavy chain variable region of SEQ ID NO: 9. W3166-z17 has comparable affinity to the antigen as compared with its parent antibody W3166.

In certain embodiments, the CTLA-4-binding domain comprises a heavy chain CDR1 comprising the sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the sequence selected from SEQ ID NOs: 2 and 4, and a heavy chain CDR3 comprising the sequence of SEQ ID NO: 3.

Table 1 shows the CDR sequences of the anti-CTLA-4 single domain antibodies. The heavy chain variable region sequences are also provided below in Table 2 and Table 3.

Table 1 CDR amino acid sequences

Table 2 Variable region amino acid sequences

Table 3 Variable region nucleotide sequences

In certain embodiments, the CTLA-4-binding domains provided herein are derived from single domain antibodies. Examples of single domain antibodies include but not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies.

In certain embodiments, the heavy chain variable domain of the antibody polypeptides provided herein is derived from a VHH domain. VHH domains are heavy chain variable domains derived from antibodies naturally devoid of light chains, for example, antibodies derived from Camelidae species (see, e.g. WO9404678) , for example in camel, llama, dromedary, alpaca and guanaco. VHH domains are single polypeptides, and are stable.

In certain embodiments, the heavy chain variable domain of the antibody polypeptides provided herein is of camelid origin.

CDRs are known to be responsible for antigen binding, however, it has been found that not all of the 6 CDRs are indispensable or unchangeable. In other words, it is possible to replace or change or modify one or more CDRs provided herein for CTLA-4-binding domains, yet substantially retain the specific binding affinity to CTLA-4.

In certain embodiments, the CTLA-4-binding domains provided herein comprise a heavy chain CDR3 sequence of SEQ ID NO: 3 (i.e. the anti-CTLA-4 VHH antibody W3166 or W3166-z13, W3166-z17) .

Heavy chain CDR3 regions are located at the center of the antigen-binding site, and therefore are believed to make the most contact with antigen and provide the most free energy to the affinity of antibody to antigen. It is also believed that the heavy chain CDR3 is by far the most diverse CDR of the antigen-binding site in terms of length, amino acid composition and conformation by multiple diversification mechanisms (Tonegawa S. Nature. 302: 575-81) . The diversity in the heavy chain CDR3 is sufficient to produce most antibody specificities (Xu JL, Davis MM. Immunity. 13: 37-45) as well as desirable antigen-binding affinity (Schier R, etc. J Mol Biol. 263: 551-67) .

In certain embodiments, the CTLA-4-binding domains provided herein comprise any suitable framework region (FR) sequences, as long as the antigen-binding domains can specifically bind to CTLA-4. In certain embodiments, the CDR sequences provided in Table 1 are obtained from camelid antibodies, but they can be grafted to any suitable FR sequences of any suitable species such as mouse, human, rat, rabbit, among others, using suitable methods known in the art such as recombinant techniques.

In certain embodiments, the CTLA-4-binding domains provided herein are humanized. A humanized antigen-binding domain is desirable in its reduced immunogenicity in human. A humanized antigen-binding domain is chimeric in its variable regions, as non-human CDR sequences are grafted to human or substantially human FR sequences. Humanization of an antigen-binding domain can be essentially performed by substituting the non-human (such as murine) CDR genes for the corresponding human CDR genes in a human immunoglobulin gene (see, for example, Jones et al. (1986) Nature 321: 522-525; Riechmann et al. (1988) Nature 332: 323-327; Verhoeyen et al. (1988) Science 239: 1534-1536) .

Suitable human heavy chain and light chain variable domains can be selected to achieve this purpose using methods known in the art. In an illustrative example, “best-fit” approach can be used, where a non-human (e.g. rodent) antibody variable domain sequence is screened or BLASTed against a database of known human variable domain sequences, and the human sequence closest to the non-human query sequence is identified and used as the human scaffold for grafting the non-human CDR sequences (see, for example, Sims et al, (1993) J. Immunol. 151: 2296; Chothia et al. (1987) J. Mot. Biol. 196: 901) . Alternatively, a framework derived from the consensus sequence of all human antibodies may be used for the grafting of the non-human CDRs (see, for example, Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89: 4285; Presta et al. (1993) J. Immunol., 151: 2623) .

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

In certain embodiments, the humanized CTLA-4-binding domains provided herein comprise one or more FR sequences of W3166-z13, or W3166-z17.

The two exemplary humanized anti-CTLA-4 single domain antibodies W3166-z13 and W3166-z17 both retained the specific binding affinity to CTLA-4, and are at least comparable to, or even better than, the parent camelid antibodies in that aspect.

In some embodiments, the FR regions derived from human may comprise the same amino acid sequence as the human immunoglobulin from which it is derived. In some embodiments, one or more amino acid residues of the human FR are substituted with the corresponding residues from the parent non-human antibody. This may be desirable in certain embodiments to make the humanized antibody or its fragment closely approximate the non-human parent antibody structure. In certain embodiments, the humanized CTLA-4-binding domain provided herein comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue substitutions in each of the human FR sequences, or no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue substitutions in all the FRs of a heavy or a light chain variable domain. In some embodiments, such change in amino acid residue could be present in heavy chain FR regions only, in light chain FR regions only, or in both chains.

In certain embodiments, the CTLA-4-binding domains provided herein comprise a heavy chain variable domain sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9.

In some embodiments, the CTLA-4-binding domains provided herein comprise all or a portion of the heavy chain variable domain. In one embodiment, the CTLA-4-binding domains provided herein are a single domain antibody which consists of all or a portion of the heavy chain variable domain provided herein. More information of such a single domain antibody is available in the art (see, e.g., U.S. Pat. No. 6,248,516) .

ii. PD-1-binding domain

In certain embodiments, the PD-1 binding domain of the bispecific antibody molecule is capable of specifically binding to PD-1 (such as human PD-1) . In certain embodiments, the PD-1 binding domain or the bispecific antibody molecule is capable of specifically binding to PD-1 (such as human PD-1) and comprises one independently selected from the group consisting of: a Fab and a VHH domain.

In certain embodiments, the PD-1-binding domain comprises one or more (e.g. 1, 2, 3, 4, 5, or 6) CDR sequences of an anti-PD-1 antibody selected from the group consisting of: W3052-2E5 and W3055-1.153.7 hAb.

“W3052-2E5” as used herein refers to a humanized monoclonal antibody having a heavy chain variable region of SEQ ID NO: 17, and a kappa light chain variable region of SEQ ID NO: 18.

“W3055-1.153.7 hAb” as used herein refers to a fully human monoclonal antibody having a heavy chain variable region of SEQ ID NO: 27, and a kappa light chain variable region of SEQ ID NO: 28.

In certain embodiments, the PD-1-binding domain comprises heavy chain CDR1 comprising the sequence of SEQ ID NO: 11, heavy chain CDR2 comprising the sequence of SEQ ID NO: 12, and heavy chain CDR3 comprising the sequence of SEQ ID NO: 13, and/or light chain CDR1 comprising the sequence of SEQ ID NO: 14, light chain CDR2 comprising the sequence of SEQ ID NO: 15, and light chain CDR3 comprising the sequence of SEQ ID NO: 16.

In certain embodiments, the PD-1-binding domain comprises heavy chain CDR1 comprising the sequence of SEQ ID NO: 21, heavy chain CDR2 comprising the sequence of SEQ ID NO: 22, and heavy chain CDR3 comprising the sequence of SEQ ID NO: 23, and/or light chain CDR1 comprising the sequence of SEQ ID NO: 24, light chain CDR2 comprising the sequence of SEQ ID NO: 25, and light chain CDR3 comprising the sequence selected from SEQ ID NO: 26.

Table 4 shows the CDR sequences of the 2 anti-PD-1 antibodies. The heavy chain and light chain variable region sequences are also provided below in Table 5 and Table 6.

Table 4 CDR amino acid sequences

Table 5. Variable region amino acid sequences

Table 6. Variable region nucleotide sequences

CDRs are known to be responsible for antigen binding, however, it has been found that not all of the 6 CDRs are indispensable or unchangeable. In other words, it is possible to replace or change or modify one or more CDRs provided herein for PD-1-binding domains, yet substantially retain the specific binding affinity to PD-1 (e.g. human PD-1) .

In certain embodiments, the PD-1-binding domains provided herein comprise a heavy chain CDR3 sequence of one of the anti-PD-1 antibodies W3052-2E5 and W3055-1.153.7 hAb.

In certain embodiments, the PD-1-binding domains provided herein are fully human. For example, the PD-1-binding domains derived from W3055-1.153.7 hAb is fully human.

In certain embodiments, the PD-1-binding domains provided herein are not fully human. In certain embodiments, the PD-1-binding domains provided herein comprise suitable framework region (FR) sequences, as long as the antigen-binding domains can specifically bind to PD-1, respectively. In certain embodiments, the CDR sequences of W3052-2E5 are obtained from rat antibodies, but they can be grafted to any suitable FR sequences of any suitable species such as mouse, human, rat, rabbit, among others, using suitable methods known in the art such as recombinant techniques.

In certain embodiments, the PD-1-binding domains provided herein are humanized. The exemplary humanized anti-PD-1 antibodies W3052-2E5, retained the specific binding affinity to PD-1, and are at least comparable to, or even better than, the parent rat antibodies in that aspect.

In some embodiments, the FR regions derived from human may comprise the same amino acid sequence as the human immunoglobulin from which it is derived. In some embodiments, one or more amino acid residues of the human FR are substituted with the corresponding residues from the parent non-human antibody. This may be desirable in certain embodiments to make the humanized antibody or its fragment closely approximate the non- human parent antibody structure. In certain embodiments, the humanized PD-1 binding domain provided herein comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue substitutions in each of the human FR sequences, or no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue substitutions in all the FRs of a heavy or a light chain variable domain. In some embodiments, such change in amino acid residue could be present in heavy chain FR regions only, in light chain FR regions only, or in both chains.

In certain embodiments, the PD-1-binding domains provided herein comprise a heavy chain variable domain sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 27, and a homologous sequence thereof having at least 80%sequence identity yet retaining specific binding affinity to PD-1. In certain embodiments, PD-1-binding domains provided herein comprise a light chain variable domain sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 28, and a homologous sequence thereof having at least 80%sequence identity yet retaining specific binding affinity to PD-1.

In some embodiments, the PD-1-binding domains provided herein comprise all or a portion of the heavy chain variable domain and/or all or a portion of the light chain variable domain. In one embodiment, the PD-1-binding domains provided herein are a single domain antibody which consists of all or a portion of the heavy chain variable domain provided herein. More information of such a single domain antibody is available in the art (see, e.g., U.S. Pat. No. 6,248,516) .

iii. Bispecific Antibody Molecule

In certain embodiments, the bispecific antibody molecule provided herein comprises an CTLA-4-binding domain comprising one or more (e.g. 1, 2, or 3) CDR sequences of SEQ ID NOs: 1-4 (i.e. derived from W3166, W3166-z13 and W3166-z17) and a PD-1 binding domain comprising one or more (e.g. 1, 2, 3, 4, 5, or 6) CDR sequences of SEQ ID NOs: 11-16 and 21-26 (i.e. derived from W3052-2E5 and W3055_1.153.7 hAb) , and the CTLA-4-binding domain comprises a VHH domain, the PD-1-binding domain comprises a Fab.

In certain embodiments, the bispecific antibody molecule provided herein comprises an CTLA-4-binding domain comprising:

a) heavy chain CDR1 comprising the sequence of SEQ ID NO: 1, heavy chain CDR2 comprising the sequence of SEQ ID NO: 2, and heavy chain CDR3 comprising the sequence of SEQ ID NO: 3; or

b) heavy chain CDR1 comprising the sequence of SEQ ID NO: 1, heavy chain CDR2 comprising the sequence of SEQ ID NO: 4, and heavy chain CDR3 comprising the sequence of SEQ ID NO: 3; and

a PD-1-binding domain comprising:

c) heavy chain CDR1 comprising the sequence of SEQ ID NO: 11, heavy chain CDR2 comprising the sequence of SEQ ID NO: 12, and heavy chain CDR3 comprising the sequence of SEQ ID NO: 13, and/or light chain CDR1 comprising the sequence of SEQ ID NO: 14, light chain CDR2 comprising the sequence of SEQ ID NO: 15, and light chain CDR3 comprising the sequence of SEQ ID NO: 16; or

d) heavy chain CDR1 comprising the sequence of SEQ ID NO: 21, heavy chain CDR2 comprising the sequence of SEQ ID NO: 22, and heavy chain CDR3 comprising the sequence of SEQ ID NO: 23, and/or light chain CDR1 comprising the sequence of SEQ ID NO: 24, light chain CDR2 comprising the sequence of SEQ ID NO: 25, and light chain CDR3 comprising the sequence selected from SEQ ID NO: 26, and

the CTLA-4-binding domain comprises a VHH domain, and the PD-1-binding domain comprises a Fab.

In certain embodiments, the CTLA-4-binding domain comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 5, 7, 9, or a homologous sequence thereof having at least 80%sequence identity yet retaining specific binding affinity to CTLA-4 (e.g. human CTLA-4) .

In certain embodiments, the PD-1 binding domain comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 17, 27, or a homologous sequence thereof having at least 80%sequence identity yet retaining specific binding affinity to PD-1 (e.g. human PD-1) , and/or a light chain variable region comprising the sequence of SEQ ID NO: 18, 28, or a homologous sequence thereof having at least 80%sequence identity yet retaining specific binding affinity to PD-1 (e.g. human PD-1) .

In certain embodiments, the CTLA-4-binding domain comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 9 (i.e. derived from W3166-z17) , and the PD-1 binding domain comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 17 and a light chain variable region comprising the sequence of SEQ ID NO: 18 (i.e. derived from W3052-2E5) (such bispecific antibody molecules are also referred to as “W3246-U3T9” herein) .

In certain embodiments, the CTLA-4-binding domain comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 9 (i.e. derived from W3166-z17) , and the PD-1 binding domain comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 27 and a light chain variable region comprising the sequence of SEQ ID NO: 28 (i.e. derived from W3055-1.153.7) (such bispecific antibody molecules are also referred to as “W3246-U6T9” herein) .

The CTLA-4-binding domains and/or the PD-1-binding domains provided herein comprise one independently selected from the group consisting: a Fab and a VHH domain.

Various techniques can be used for the production of such antigen-binding domains. Illustrative methods include, enzymatic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992) ; and Brennan et al., Science, 229: 81 (1985) ) , recombinant expression by host cells such as E. Coli (e.g. for Fab, Fv and ScFv antibody fragments) , screening from a phage display library as discussed above (e.g. for ScFv) , and chemical coupling of two Fab'-SH fragments to form F (ab') ₂ fragments (Carter et al., Bio/Technology 10: 163-167 (1992) ) . Other techniques for the production of antibody fragments will be apparent to a skilled practitioner.

In certain embodiments, the CTLA-4-binding domain is a VHH domain. In certain embodiments, the CTLA-4-binding VHH domain comprises the sequence of SEQ ID NO: 5, 7, or 9. Various techniques can be used for the production of VHH or single domain antibodies. For example, VHHs may be obtained using methods known in the art such as by immunising a camel and obtaining hybridomas therefrom, or by cloning a library of single domain antibodies using molecular biology techniques known in the art and subsequent selection by using phage display.

In certain embodiments, the PD-1-binding domain is a Fab. In certain embodiments, the PD-1-binding Fab comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 17 and a light chain variable region comprising the sequence of SEQ ID NO: 18; or a heavy chain variable region comprising the sequence of SEQ ID NO: 27 and a light chain variable region comprising the sequence of SEQ ID NO: 28, respectively. The heavy chain variable region and the light chain variable region can be disulfidely bonded. The term “disulfidely bonded” refers to linkage via one or more disulfide bond (optionally in addition to another bond) . A disulfide bond can be formed between, for example, one cysteine residue of an antibody heavy chain and another cysteine residue of the light chain.

In certain embodiments, the CTLA-4-binding and/or the PD-1-binding domains are multivalent, such as bivalent, trivalent, tetravalent. The term “valent” as used herein refers to the presence of a specified number of antigen binding sites in a given molecule. As such, the terms “bivalent” , “tetravalent” , and “hexavalent” denote the presence of two binding site, four binding sites, and six binding sites, respectively, in an antigen-binding molecule. A bivalent molecule can be monospecific if the two binding sites are both for specific binding of the same antigen or the same epitope. Similarly, a trivalent molecule can be bispecific, for example, when two binding sites are monospecific for a first antigen (or epitope) and the third binding site is specific for a second antigen (or epitope) . In certain embodiments, the CTLA-4-binding and/or the PD-1-binding domains in the bispecific antibody molecule provided herein can be bivalent, trivalent, or tetravalent, with at least two binding sites specific for the same antigen or epitope. This, in certain embodiments, provides for stronger binding to the antigen or the epitope than a monovalent counterpart. In certain embodiments, in a bivalent antigen-binding moiety, the first valent of binding site and the second valent of binding site are structurally identical (i.e. having the same sequences) , or structurally different (i.e. having different sequences albeit with the same specificity) . In certain embodiments, CTLA-4-binding and/or the PD-1-binding domains comprises two or more antigen binding sites (e.g. VHH, or scFv or Fab) operably linked together, with or without a spacer. In another aspect of the present disclosure, the CTLA-4-binding domain provided herein may comprise two or more single domain antibodies which have been joined. The single domain antibodies may be identical in sequence and directed against the same target or antigen. Depending on the number of VHHs linked, the CTLA-4-binding domain may be bivalent (2 VHHs) , trivalent (3 VHHs) , tetravalent (4 VHHs) or have a higher valency molecules.

In certain embodiments, the CTLA-4-binding domain is operably linked to the N terminus or the C terminus of the PD-1-binding domain. In certain embodiments, the PD-1-binding domain is operably linked to the N terminus or the C terminus of the CTLA-4-binding domain.

The operable linkage can be a direct chemical bond linkage or linkage via a spacer or via an intervening sequence. The term “spacer” 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 binding domains, such as between a VHH domain and a Fab. In certain embodiment, the spacer comprises GTDTTADTGRASGDNTT (SEQ ID NO: 42) . In certain embodiment, the spacer comprises 1, 2, 3, 4 or more sequential or tandem repeats of SEQ ID NOs: 38 and 39. In certain embodiments, the spacer comprises GGGGS (SEQ ID NO: 38) . In certain embodiments, the spacer comprises GGGGSGGGGS (SEQ ID NO: 39) , GGGGSGGGGSGGGGS (SEQ ID NO: 40) , GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 41) . The intervening sequence as used herein can be any amino acid sequence located between the CTLA-4-binding domain and the PD-1-binding domain, as long as both the CTLA-4-binding domain and the PD-1-binding domain are capable of binding to its respective antigen. In an illustrative example, the intervening sequence can comprise a heavy chain constant region, or a light chain constant region.

In certain embodiments, the CTLA-4-binding domain comprises a VHH domain and the PD-1 binding domain comprises a Fab or an IgG. In certain embodiments, the CTLA-4-binding VHH can be operably linked to the N terminus or C terminus of heavy chain of the PD-1-binding Fab or IgG (e.g. the C-terminus of the heavy chain constant region following the PD-1-binding Fab) , or to the N terminus or the C-terminus of the light chain of the anti-PD-1 binding Fab or IgG, or any combination thereof, and vice versa.

In an illustrative example, the bispecific antibody molecule can comprise a heavy chain in the format of: VH (anti-PD-1) -CH1-Hinge-CH2-CH3-VHH (anti-CTLA-4) or VHH (anti-CTLA-4) -VH (anti-PD-1) -CH1-Hinge-CH2-CH3, and a light chain VL (anti-PD-1) -CL. As used herein, VH (anti-PD-1) and VL (anti-PD-1) refer respectively to the heavy and light chain variable domain of the anti-PD-1 antibody provided herein; VHH (anti-CTLA-4) refers to a VHH derived from the anti-CTLA-4 VHH antibody provided herein, CL refers to the light chain constant region; and CH1-Hinge-CH2-CH3 are collectively heavy chain constant region.

In another illustrative example, the bispecific antibody molecule can comprise a light chain in the format of: VHH (anti-CTLA-4) -VL (anti-PD-1) -CL or VL (anti-PD-1) -CL-VHH (anti-CTLA-4) , and a heavy chain VH (anti-PD-1) -CH1-Hinge-CH2-CH3, by the same token.

The CTLA-4-binding domain may be monovalent (i.e. one VHH) or multivalent (e.g. more than one VHH) . The PD-1-binding domain may be monovalent or multivalent.

In certain embodiments, the bispecific antibody molecule can comprise a heavy chain in the format of: VH (anti-PD-1) -CH1-Hinge-CH2-CH3-VHH (anti-CTLA-4) , and a light chain VL (anti-PD-1) -CL, wherein the VHH (anti-CTLA-4) comprises a sequence of SEQ ID NO: 9, the VH (anti-PD-1) comprises the sequence of SEQ ID NO: 27, and the VL (anti-PD-1) comprises an amino acid sequence of SEQ ID NO: 28. In certain embodiment, the spacer comprises the sequence of SEQ ID NO: 42. In certain embodiments the heavy chain constant region is of human IgG4 isotype, and optionally contains mutations of S228P and/or L235E. In certain embodiments, the heavy chain constant region comprises the sequence of SEQ ID NO: 31. In certain embodiments, the light chain constant region comprises the sequence of SEQ ID NO: 32.

In certain embodiments, the bispecific antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 34 and a light chain comprising the amino acid sequence of SEQ ID NO: 35. This antibody is also called W3246-U6T9 in the present disclosure.

In certain embodiments, the bispecific antibody molecule can comprise a heavy chain in the format of: VH (anti-PD-1) -CH1-Hinge-CH2-CH3-VHH (anti-CTLA-4) , and a light chain VL (PD-1) -CL, wherein the VHH (anti-CTLA-4) comprises a sequence of SEQ ID NO: 9, the VH (anti-PD-1) comprises the sequence of SEQ ID NO: 17, and the VL (anti-PD-1) comprises an amino acid sequence of SEQ ID NO: 18. In certain embodimens, the spacer comprises the sequence of SEQ ID NO: 42. In certain embodiments the heavy chain constant region is of human IgG4 isotype, and optionally contains mutations of S228P and/or L235E. In certain embodiments, the heavy chain constant region comprises the sequence of SEQ ID NO: 31. In certain embodiments, the light chain constant region comprises the sequence of SEQ ID NO: 33.

In certain embodiments, the bispecific antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 36 and a light chain comprising the amino acid sequence of SEQ ID NO: 37. This antibody is also called W3246-U3T9 in the present disclosure.

Tables 7 and 8 show the combination of heavy chain and light chain sequences of the bispecific antibody molecules of W3246-U6T9 and W3246-U3T9.

Table 7

“CL” refers to light chain constant region; “CH” refers to heavy chain constant region; “VL” refers to light chain variable region; “VH” refers to heavy chain variable region;

“Anti-PD-1” refers to anti-PD-1 antibody, in particular, the sequence provided in the table is the sequence derived from anti-PD-1 antibody W3052-2E5 or W3055-1.153.7.

“Anti-CTLA-4” refers to anti-CTLA-4 antibody, in particular, the sequence provided in the table is the sequence derived from anti-CTLA-4 antibody W3166-z17.

Table 8

In certain embodiments, the bispecific antibody molecules provided herein may further comprise an immunoglobulin constant region. In some embodiments, an immunoglobulin constant region comprises a heavy chain and/or a light chain constant region. The heavy chain constant region comprises CH1, hinge, and/or CH2-CH3 regions. In certain embodiments, the heavy chain constant region comprises an Fc region. In certain embodiments, the light chain constant region comprises Cκ or Cλ.

The bispecific antibody molecules provided herein can have a constant region, for example a human IgG constant region. The constant region can be in any suitable isotype. In certain embodiments, the bispecific antibody molecules provided herein comprises a constant region of IgG1 isotype, which could induce ADCC or CDC. In some embodiments, the bispecific antibody molecules have a constant region of IgG2 or IgG4 isotype, which has reduced or depleted effector function.

In some embodiments, the bispecific antibody molecules provided herein have reduced or depleted effector function. In some embodiments, the bispecific antibody molecules provided herein have a constant region of IgG4 isotype, which has reduced or depleted effector function. Effector functions such as ADCC and CDC can lead to cytotoxicity to cells expressing PD-1. Many cells such as T cells normally express PD-1. In order to avoid potential unwanted toxicity to those normal cells, certain embodiments of the antibodies and antigen-binding fragments provided herein can possess reduced or even depleted effector functions. Various assays are known to evaluate ADCC or CDC activities, for example, Fc receptor binding assay, C1q binding assay, and cell lysis assay, and can be readily selected by people in the art. Without wishing to be bound to theory, but it is believed that antibodies with reduced or depleted effector functions such as ADCC or CDC would cause no or minimal cytotoxicity to PD-1-expressing cells, for example those T cells, and therefore spare them from unwanted side effects, whereas in the meantime, blocking of PD-1 would boost immune system for the treatment of conditions such as cancer or chronic infection.

In certain embodiments, the bispecific antibody molecules provided herein have reduced side effects. For example, the bispecific antibody molecules provided herein can comprise at least one fully human antigen-binding domain and Fc region and therefore reduced immunogenicity than a humanized antibody counterpart.

B. Characterization of the Bispecific Antibody Molecule

In some embodiments, the bispecific antibody molecules provided herein are capable of specifically binding to both human PD-1 and human CTLA-4. The bispecific antibody molecules provided herein retain the specific binding affinity to both PD-1 and CTLA-4, in certain embodiments are at least comparable to, or even better than, the parent antibodies in that aspect.

In certain embodiments, the bispecific antibody molecules provided herein have a specific binding affinity to CTLA-4 which is sufficient to provide for diagnostic and/or therapeutic use.

Binding of bispecific antibody molecules can be represented by “half maximal effective concentration” (EC ₅₀) value, which refers to the concentration of an antibody where 50%of its maximal effect (e.g., binding or inhibition etc. ) is observed. The EC ₅₀ value can be measured by methods known in the art, for example, sandwich assay such as ELISA, Western Blot, FACS assay, and other binding assay.

Binding affinity of the antigen-binding domains provided herein can also 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, FACS assay. In some embodiments, binding of the antibody to the antigen at different concentrations can be determined by FACS, 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 bispecific antibody molecules provided herein specifically bind to human PD-1 at an EC ₅₀ (i.e. 50%binding concentration) of no more than: 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM, 0.08 nM, 0.07 nM, or 0.06 nM by ELISA; or specifically bind to cell surface human PD-1 at an EC ₅₀ of no more than: 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1.5 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, or 0.5 nM by FACS.

In certain embodiments, the bispecific antibody molecules provided herein specifically bind to human CTLA-4 at an EC ₅₀ (i.e. 50%binding concentration) of no more than: 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, or 0.05 nM by ELISA; or specifically bind to cell surface human CTLA-4 at an EC ₅₀ of no more than: 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, or 0.2 nM by FACS.

In certain embodiments, the bispecific antibody molecules provided herein cross-react with Cynomolgus monkey PD-1, for example, Cynomolgus monkey PD-1 expressed on a cell surface, or a soluble recombinant Cynomolgus monkey PD-1. In certain embodiments, the bispecific antibody molecules provided herein cross-react with Cynomolgus monkey CTLA-4, for example, Cynomolgus monkey CTLA-4 expressed on a cell surface, or a soluble recombinant Cynomolgus monkey CTLA-4.

In certain embodiments, the bispecific antibody molecules provided herein specifically bind to Cynomolgus monkey PD-1 protein at an EC ₅₀ of no more than: 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM by ELISA; or specifically bind to cell surface Cynomolgus monkey PD-1 at an EC ₅₀ of no more than: 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM by FACS.

In certain embodiments, the bispecific antibody molecules provided herein specifically bind to mouse PD-1 protein at an EC ₅₀ of no more than: 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM, 0.08 nM, or 0.07 nM by ELISA; or specifically bind to cell surface mouse PD-1 at an EC ₅₀ of no more than: 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, or 4 nM by FACS.

In certain embodiments, the bispecific antibody molecules provided herein specifically bind to Cynomolgus monkey CTLA-4 protein with an EC ₅₀ of no more than: 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM, 0.08 nM, or 0.07 nM by ELISA; or specifically bind to cell surface Cynomolgus monkey CTLA-4 with an EC ₅₀ of no more than: 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1.5 nM, 1 nM, or 0.5 nM by FACS.

In some embodiments, the bispecific antibody molecules provided herein are capable of dual binding to human PD-1 and human CTLA-4 with an EC ₅₀ of no more than: 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, 0.02 nM, or 0.01 nM by ELISA.

In some embodiments, the bispecific antibody molecules provided herein are capable of blocking human CTLA-4 binding to human CD80 on cell surface with at an IC ₅₀ (i.e. 50%inhibiting concentration) of no more than: 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, or 2 nM by FACS.

In some embodiments, the bispecific antibody molecules provided herein are capable of blocking human CTLA-4 binding to human CD86 on cell surface with at an IC ₅₀ of no more than: 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM by FACS.

In some embodiments, the bispecific antibody molecules provided herein are capable of blocking Cynomolgus monkey CTLA-4 binding to human CD80 on cell surface with at an IC ₅₀ of no more than: 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, or 0.3 nM by FACS.

In some embodiments, the bispecific antibody molecules provided herein are capable of blocking Cynomolgus monkey CTLA-4 binding to human CD86 on cell surface with at an IC ₅₀ of no more than: 300 nM, 200 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 28 nM, 25 nM, 23 nM, or 22 nM by FACS.

In some embodiments, the bispecific antibody molecules provided herein are capable of blocking human PD-L1 binding to human PD-1 on cell surface with at an IC ₅₀ of no more than: 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1.5 nM, or 1 nM by FACS.

In some embodiments, the bispecific antibody molecules provided herein are capable of blocking mouse PD-L1 binding to human PD-1 on cell surface with at an IC ₅₀ of no more than: 500 nM, 400 nM, 200 nM, 150 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, or 20 nM by FACS.

In some embodiments, the bispecific antibody molecules provided herein are capable of specifically binding to human PD-1 with a binding affinity (K _D) of no more than: 80x10 ^-9 M, 70x10 ^-9 M, 60x10 ^-9 M, 50x10 ^-9 M, 40x10 ^-9 M, 30x10 ^-9 M, 20x10 ^-9 M, 10x10 ^-9 M, 9x10 ^-9 M, 8x10 ^-9 M, 7x10 ^-9 M, 6x10 ^-9 M, 5x10 ^-9 M, 4x10 ^-9 M, 3x10 ^-9 M, or 2x10 ^-9 M as measured by surface plasmon resonance (SPR) .

In some embodiments, the bispecific antibody molecules provided herein are capable of specifically binding to human CTLA-4 with a binding affinity (K _D) of no more than: 100x10 ^-9 M, 90x10 ^-9 M, 80x10 ^-9 M, 70x10 ^-9 M, 60x10 ^-9 M, 50x10 ^-9 M, 40x10 ^-9 M, 30x10 ^-9 M, 20x10 ^-9 M, 10x10 ^-9 M, 9x10 ^-9 M, 8x10 ^-9 M, 7x10 ^-9 M, 6x10 ^-9 M, 5x10 ^-9 M, or 4x10 ^-9 M as measured by SPR.

In some embodiments, the bispecific antibody molecules provided herein are capable of specifically binding to Cynomolgus monkey PD-1 with a binding affinity (K _D) of no more than: 50x10 ^-8 M, 40x10 ^-8 M, 30x10 ^-8 M, 20x10 ^-8 M, 10x10 ^-8 M, 9x10 ^-8 M, 8x10 ^-8 M, 7x10 ^-8 M, 6x10 ^-8 M, 5x10 ^-8 M, 4x10 ^-8 M, 3x10 ^-8 M, 2x10 ^-8 M, or 1x10 ^-8 M as measured by surface plasmon resonance (SPR) .

In some embodiments, the bispecific antibody molecules provided herein are capable of specifically binding to Cynomolgus monkey CTLA-4 with a binding affinity (K _D) of no more than: 50x10 ^-8 M, 40x10 ^-8 M, 30x10 ^-8 M, 20x10 ^-8 M, 10x10 ^-8 M, 9x10 ^-8 M, 8x10 ^-8 M, 7x10 ^-8 M, 6x10 ^-8 M, 5x10 ^-8 M, 4x10 ^-8 M, 3x10 ^-8 M, or 2x10 ^-8 M as measured by SPR.

In certain embodiments, the bispecific antibody molecules provided herein block binding of human PD-1 to its ligand and thereby providing biological activity including, for example, inducing cytokine production from the activated T cells (such as CD4+ T cells and CD8+ T cells) , inducing proliferation of activated T cells (such as CD4+ T cells and CD8+ T cells) , and reversing T reg’s suppressive function. Exemplary cytokines include IL-2 and IFNγ. The term “IL-2” refers to interleukin 2, a type of cytokine signaling molecule in the immune system that regulates the activities of white blood cells (e.g. leukocytes) . The term “Interferon gamma (IFNγ) ” is a cytokine that is produced by natural killer (NK) , NK T cells, CD4+ and CD8+T cells, which is a critical activator of macrophages and inducer of major histocompatibility complex (MHC) molecule expression. The cytokine production can be determined using methods known in the art, for example, by ELISA. Methods can also be used to detect proliferation of T cells, including [ ³H] thymidine incorporation assay.

In certain embodiments, the bispecific antibody molecules provided herein are capable of specifically enhancing IL-2 and/or IFN-γ production in CD4+ T cells stimulated with iDC (hMLR) , as measured by human Treg mixed lymphocyte reaction (MLR) .

In certain embodiments, the bispecific antibody molecules provided herein are capable of specifically enhancing T cell proliferation, as measured by human Treg mixed lymphocyte reaction (MLR) .

In certain embodiments, the bispecific antibody molecules provided herein do not cross-react with human CD28, human ICOS or human BTLA.

In certain embodiments, the bispecific antibody molecules provided herein are capable of simultaneous stimulating cells from both the innate and the adaptive immune system.

C. Format of the Bispecific Antibody Molecule

Bispecific antibody fragments are antigen-binding fragments that are derived from an antibody but lack some or all of the antibody constant domains. Examples of such a bispecific antibody fragment include, for example, such as single domain antibody, Fv, Fab and diabody etc.

In certain embodiments, the bispecific antibody molecules as provided herein are based on the format of a “whole” antibody, such as whole IgG or IgG-like molecules, and small recombinant formats.

The bispecific antibody molecules provided herein can be made with any suitable methods known in the art. In a conventional approach, two immunoglobulin heavy chain-light chain pairs having different antigen-binding specificities can be co-expressed in a host cell to produce bispecific antibodies in a recombinant way (see, for example, Milstein and Cuello, Nature, 305: 537 (1983) ) , followed by purification by affinity chromatography.

Recombinant approach may also be used, where sequences encoding the antibody heavy chain variable domains for the two specificities are respectively fused to immunoglobulin constant domain sequences, followed by insertion to an expression vector which is co-transfected with an expression vector for the light chain sequences to a suitable host cell for recombinant expression of the bispecific antibody (see, for example, WO 94/04690; Suresh et al., Methods in Enzymology, 121: 210 (1986) ) . Similarly, scFv dimers can also be recombinantly constructed and expressed from a host cell (see, e.g. Gruber et al., J. Immunol., 152: 5368 (1994) . )

D. Variants

The antigen-binding domains and bispecific antibody molecules provided herein also encompass various variants thereof. In certain embodiments, the variants comprise one or more modifications or substitutions in one or more CDR sequences as provided in Table 1, or Table 4, one or more variable region sequences (but not in any of the CDR sequences) provided in Table 2, or Table 5, and/or the constant region (e.g. Fc region) . Such variants retain specific binding affinity to CTLA-4 and/or PD-1 of their parent antibodies, but have one or more desirable properties conferred by the modification (s) or substitution (s) . For example, the variants may have improved antigen-binding affinity, improved productivity, improved stability, improved glycosylation pattern, reduced risk of glycosylation, reduced deamination, reduced or depleted effector function (s) , improved FcRn receptor binding, increased pharmacokinetic half-life, pH sensitivity, and/or compatibility to conjugation (e.g. one or more introduced cysteine residues) .

The parent antibody sequence may be screened to identify suitable or preferred residues to be modified or substituted, using methods known in the art, for example “alanine scanning mutagenesis” (see, for example, Cunningham and Wells (1989) Science, 244: 1081-1085) . Briefly, target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) can be identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) , and the modified antibodies are produced and screened for the interested property. If substitution at a particular amino acid location demonstrates an interested functional change, then the position can be identified as a potential residue for modification or substitution. The potential residues may be further assessed by substituting with a different type of residue (e.g. cysteine residue, positively charged residue, etc. ) .

In certain embodiments, the CTLA-4-binding domains and/or the PD-1 binding domains provided herein comprise one or more amino acid residue substitutions in one or more CDR sequences, and/or one or more FR sequences, and/or one or more variable region sequences. In certain embodiments, a variant comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitutions in the CDR sequences and/or FR sequences and/or one or more variable region sequences in total.

In certain embodiments, the CTLA-4-binding domains comprise 1, 2, or 3 CDR sequences having at least 80% (e.g. at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to SEQ ID NO: 1-3 or 4, and in the meantime retain the binding affinity to CTLA-4 at a level similar to or even higher than its parent antibody.

In certain embodiments, the anti-CTLA-4-binding domains comprise one or more variable region sequences having at least 80% (e.g. at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to SEQ ID NO: 5, 7, or 9, and in the meantime retain the binding affinity to CTLA-4 at a level similar to or even higher than its parent antibody. In some embodiments, a total of 1 to 10 amino acids have been substituted, inserted, or deleted in a variable region sequence comprising SEQ ID NOs: 5, 7, or 9. In some embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (e.g., in the FRs) .

In certain embodiments, the PD-1-binding domains comprise 1, 2, or 3 CDR sequences having at least 80% (e.g. at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to SEQ ID NOs: 11-16 or 21-26, and in the meantime retain the binding affinity to PD-1 at a level similar to or even higher than its parent antibody.

In certain embodiments, the PD-1-binding domains comprise one or more variable region sequences having at least 80% (e.g. at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to SEQ ID NO: 17, 18, 27, or 28, and in the meantime retain the binding affinity to PD-1 at a level similar to or even higher than its parent antibody. In some embodiments, a total of 1 to 10 amino acids have been substituted, inserted, or deleted in a variable region sequence comprising SEQ ID NO: 17, 18, 27, or 28. In some embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (e.g., in the FRs) .

i. Glycosylation variant

The antigen-binding domains and bispecific antibody molecules provided herein also encompass a glycosylation variant, which can be obtained to either increase or decrease the extent of glycosylation of the bispecific antibody molecules.

The antigen-binding domains and bispecific antibody molecules provided herein may comprise one or more amino acid residues with a side chain to which a carbohydrate moiety (e.g. an oligosaccharide structure) can be attached. Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue, for example, an asparagine residue in a tripeptide sequence such as asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline. O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly to serine or threonine. Removal of a native glycosylation site can be conveniently accomplished, for example, by altering the amino acid sequence such that one of the above-described tripeptide sequences (for N-linked glycosylation sites) or serine or threonine residues (for O-linked glycosylation sites) present in the sequence in the is substituted. A new glycosylation site can be created in a similar way by introducing such a tripeptide sequence or serine or threonine residue.

ii. Cysteine-engineered variant

The antigen-binding domains and bispecific antibody molecules also encompass a cysteine-engineered variant, which comprises one or more introduced free cysteine amino acid residues.

A free cysteine residue is one which is not part of a disulfide bridge. A cysteine-engineered variant is useful for conjugation with for example, a cytotoxic and/or imaging compound, a label, or a radioisoptype among others, at the site of the engineered cysteine, through for example a maleimide or haloacetyl. Methods for engineering antibody polypeptides to introduce free cysteine residues are known in the art, see, for example, WO2006/034488.

iii. Fc Variant

The antigen-binding domains and bispecific antibody molecules provided herein also encompass an Fc variant, which comprises one or more amino acid residue modifications or substitutions at its Fc region and/or hinge region.

In certain embodiments, the antigen-binding domains and bispecific antibody molecules comprise one or more amino acid substitution (s) that improves pH-dependent binding to neonatal Fc receptor (FcRn) . Such a variant can have an extended pharmacokinetic half-life, as it binds to FcRn at acidic pH which allows it to escape from degradation in the lysosome and then be translocated and released out of the cell. Methods of engineering an antibody molecule to improve binding affinity with FcRn are well-known in the art, see, for example, Vaughn, D. et al, Structure, 6 (1) : 63-73, 1998; Kontermann, R. et al, Antibody Engineering, Volume 1, Chapter 27: Engineering of the Fc region for improved PK, published by Springer, 2010; Yeung, Y. et al, Cancer Research, 70: 3269-3277 (2010) ; and Hinton, P. et al, J. Immunology, 176: 346-356 (2006) .

In certain embodiments, the antigen-binding domains and bispecific antibody molecules comprise one or more amino acid substitution (s) that alters the antibody-dependent cellular cytotoxicity (ADCC) . Certain amino acid residues at the Fc region (e.g. at the CH2 domain) can be substituted to provide for altered (e.g. enhanced, decreased, or depleted) ADCC activity. Alternatively or additionally, carbohydrate structures on the antibody can be changed to alter (e.g. enhance, decrease or deplete) ADCC activity. Methods of altering ADCC activity by antibody engineering have been described in the art, see for example, Shields RL. et al., J Biol Chem. 2001.276 (9) : 6591-604; Idusogie EE. et al., J Immunol. 2000.164 (8) : 4178-84; Steurer W. et al., J Immunol. 1995, 155 (3) : 1165-74; Idusogie EE. et al., J Immunol. 2001, 166 (4) : 2571-5; Lazar GA. et al., PNAS, 2006, 103 (11) : 4005-4010; Ryan MC. et al., Mol. Cancer Ther., 2007, 6: 3009-3018; Richards JO, . et al., Mol Cancer Ther. 2008, 7 (8) : 2517-27; Shields R.L. et al, J. Biol. Chem, 2002, 277: 26733-26740; Shinkawa T. et al, J. Biol. Chem, 2003, 278: 3466-3473.

In certain embodiments, the antigen-binding domains and bispecific antibody molecules comprise a human IgG4 constant region in which the 228 ^th amino acid residue is altered, for example from Ser228Pro (S228P, which may prevent or reduce strand exchange) , and/or the 235 ^th amino acid residue is altered, for example from Leu235Glu (L235E, which may alter Fc receptor interactions.

In certain embodiments, the antigen-binding domains and bispecific antibody molecules comprise one or more amino acid substitution (s) that alters Complement Dependent Cytotoxicity (CDC) , for example, by improving or diminishing C1q binding and/or CDC (see, for example, WO99/51642; Duncan &Winter Nature 322: 738-40 (1988) ; U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821) ; and WO94/29351 concerning other examples of Fc region variants.

In certain embodiments, the antigen-binding domains and bispecific antibody molecules comprise one or more amino acid substitution (s) in the interface of the Fc region to facilitate and/or promote heterodimerization. These modifications comprise introduction of a protuberance into a first Fc polypeptide and a cavity into a second Fc polypeptide, wherein the protuberance can be positioned in the cavity so as to promote interaction of the first and second Fc polypeptides to form a heterodimer or a complex. Methods of generating antibodies with these modifications are known in the art, e.g., as described in U.S. Pat. No. 5,731,168.

E. Conjugates

In some embodiments, the bispecific antibody molecules further comprise a conjugate moiety. The conjugate moiety can be linked to the bispecific antibody molecules. A conjugate moiety is a non-proteinaceous moiety that can be attached to the bispecific antibody molecules. It is contemplated that a variety of conjugate moieties may be linked to the bispecific antibody molecules provided herein (see, for example, “Conjugate Vaccines” , Contributions to Microbiology and Immunology, J.M. Cruse and R.E. Lewis, Jr. (eds. ) , Carger Press, New York, (1989) ) . These conjugate moieties may be linked to the bispecific antibody molecules by covalent binding, affinity binding, intercalation, coordinate binding, complexation, association, blending, or addition, among other methods.

In certain embodiments, the bispecific antibody molecules disclosed 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 antibody molecules may be linked to a conjugate moiety indirectly, or through another conjugate moieties. For example, the bispecific antibody molecules may be conjugated to biotin, then indirectly conjugated to a second conjugate moiety that is conjugated to avidin. The conjugate moieties can be a clearance-modifying agent, a toxin (e.g., a chemotherapeutic agent) , a detectable label (e.g., a radioactive isotope, a lanthanide, a luminescent label, a fluorescent label, or an enzyme-substrate label) , or purification moiety.

A “toxin” can be any agent that is detrimental to cells or that can damage or kill cells. Examples of toxin include, without limitation, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, MMAE, MMAF, DM1, 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) ) , anti-mitotic agents (e.g., vincristine and vinblastine) , a topoisomerase inhibitor, and a tubulin-binders.

Examples of detectable label may include a fluorescent labels (e.g. fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red) , enzyme-substrate labels (e.g. horseradish peroxidase, alkaline phosphatase, luceriferases, glucoamylase, lysozyme, saccharide oxidases or β-D-galactosidase) , radioisotopes (e.g. ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ³⁵S, ³H, ¹¹¹In, ¹¹²In, ¹⁴C, ⁶⁴Cu, ⁶⁷Cu, ⁸⁶Y, ⁸⁸Y, ⁹⁰Y, ¹⁷⁷Lu, ²¹¹At, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ²¹²Bi, and ³²P, other lanthanides) , luminescent labels, chromophoric moiety, digoxigenin, biotin/avidin, a DNA molecule or gold for detection.

In certain embodiments, the conjugate moiety can be a clearance-modifying agent which helps increase half-life of the antibody. Illustrative example include water-soluble polymers, such as PEG, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, copolymers of ethylene glycol/propylene glycol, and the like. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer are attached, they can be the same or different molecules.

In certain embodiments, the conjugate moiety can be a purification moiety such as a magnetic bead.

In certain embodiments, the bispecific antibody molecule provided herein is used for a base for a conjugate.

F. Polynucleotides and Recombinant Methods

The present disclosure provides polynucleotides that encode the bispecific antibody molecules provided herein.

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

In certain embodiments, the polynucleotides comprise one or more nucleotide sequences as shown in SEQ ID NOs: 6, 8, 10, 19, 20, 29 and 30, and/or a homologous sequence thereof having at least 80% (e.g. at least 85%, 88%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity, and/or a variant thereof having only degenerate substitutions, and encodes the variable region of the exemplary antibodies provided herein. DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody) . The encoding DNA may also be obtained by synthetic methods.

The isolated polynucleotide that encodes the bispecific antibody molecule (e.g. including the sequences as shown in Table 3 and Table 6) can be inserted into a vector for further cloning (amplification of the DNA) or for expression, using recombinant techniques known in the art. Many vectors are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g. SV40, CMV, EF-1α) , and a transcription termination sequence.

The present disclosure provides vectors (e.g., expression vectors) containing the nucleic acid sequence provided herein encoding the bispecific antibody molecules, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selection marker. Examples of vectors include, but are not limited to, retrovirus (including lentivirus) , adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus) , poxvirus, baculovirus, papillomavirus, papovavirus (e.g., SV40) , lambda phage, and M13 phage, plasmid pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT. RTM., pCDM8, pCDNA1.1/amp, pcDNA3.1, pRc/RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos etc.

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

Suitable host cells for the expression of glycosylated bispecific antibody molecules 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) ) ; 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) . In some preferable embodiments, the host cell is 293F cell.

Host cells are transformed with the above-described expression or cloning vectors for production of the bispecific antibody molecules and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. In another embodiment, the bispecific antibody molecules may be produced by homologous recombination known in the art.

The host cells used to produce the bispecific antibody molecule provided herein 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.

When using recombinant techniques, the bispecific antibody molecules can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody 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 bispecific antibody molecules are 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 antibody molecules thereof 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.

In certain embodiments, Protein A immobilized on a solid phase is used for immunoaffinity purification of the bispecific antibody molecules. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the bispecific antibody molecules. Protein A can be used to purify antibodies that are based on human gamma1, gamma2, or gamma4 heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983) ) . Protein G is recommended for all mouse isotypes and for human gamma3 (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 antibody molecule comprises a CH3 domain, the Bakerbond ABX ^TM 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 antibody molecule 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) .

G. Pharmaceutical Composition

The present disclosure further provides pharmaceutical compositions comprising the bispecific antibody molecule and one or more pharmaceutically acceptable carriers.

Pharmaceutical acceptable carriers for use in the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending/dispending agents, sequestering or chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.

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

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

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

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

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

In certain embodiments, a sterile, lyophilized powder is prepared by dissolving a bispecific antibody molecule 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 bispecific antibody molecule 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.

H. Methods of Use

In another aspect, methods are provided to treat a condition in a subject that would benefit from up-regulation of immune response, comprising administering a therapeutically effective amount of the bispecific antibody molecule as provided herein to a subject in need thereof. The disease or condition that would benefit from up-regulation of an immune response is selected from the group consisting of cancer, a viral infection, a bacterial infection, a protozoan infection, a helminth infection, asthma associated with impaired airway tolerance, a neurological disease, multiple sclerosis, and an immunosuppressive disease.

Therapeutic methods are also provided, comprising: administering a therapeutically effective amount of the bispecific antibody molecule as provided herein to a subject in need thereof, thereby treating or preventing a PD-1 related and/or a CTLA-4-related condition or a disorder.

PD-1-related conditions and disorders can be immune related disease or disorder, tumors and cancers, autoimmune diseases, or infectious disease. In certain embodiments, the PD-1-related conditions and disorders include tumors and cancers, for example, non-small cell lung cancer, small cell lung cancer, renal cell cancer, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric carcinoma, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, leukemia, lymphomas, myelomas, mycoses fungoids, merkel cell cancer, and other hematologic malignancies, such as classical Hodgkin lymphoma (CHL) , primary mediastinal large B-cell lymphoma, T-cell/histiocyte-rich B-cell lymphoma, EBV-positive and -negative PTLD, and EBV-associated diffuse large B-cell lymphoma (DLBCL) , plasmablastic lymphoma, extranodal NK/T-cell lymphoma, nasopharyngeal carcinoma, and HHV8-associated primary effusion lymphoma, Hodgkin's lymphoma, neoplasm of the central nervous system (CNS) , such as primary CNS lymphoma, spinal axis tumor, brain stem glioma. In certain embodiments, the tumors and cancers are metastatic, especially metastatic tumors expressing PD-L1.

In certain embodiments, the PD-1-related conditions and disorders include autoimmune diseases. Autoimmune diseases include, but are not limited to, Acquired Immunodeficiency Syndrome (AIDS, which is a viral disease with an autoimmune component) , alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune diabetes, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED) , autoimmune lymphoproliferative syndrome (ALPS) , autoimmune thrombocytopenic purpura (ATP) , Behcet's disease, cardiomyopathy, celiac sprue-dermatitis hepetiformis; chronic fatigue immune dysfunction syndrome (CFIDS) , chronic inflammatory demyelinating polyneuropathy (CIPD) , cicatricial pemphigold, cold agglutinin disease, crest syndrome, Crohn's disease, Degos' disease, dermatomyositis-juvenile, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP) , IgA nephropathy, insulin-dependent diabetes mellitus, juvenile chronic arthritis (Still's disease) , juvenile rheumatoid arthritis, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemacious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomena, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma (progressive systemic sclerosis (PSS) , also known as systemic sclerosis (SS) ) , Sjogren's syndrome, stiff-man syndrome, systemic lupus erythematosus, Takayasu arteritis, temporal arteritis/giant cell arteritis, ulcerative colitis, uveitis, vitiligo and Wegener's granulomatosis.

In certain embodiments, the PD-1-related conditions and disorders include infectious disease. Infectious disease include, for example, chronic viral infection, for example, fungus infection, parasite/protozoan infection or chronic viral infection, for example, malaria, coccidioiodmycosis immitis, histoplasmosis, onychomycosis, aspergilosis, blastomycosis, candidiasis albicans, paracoccidioiomycosis, microsporidiosis, Acanthamoeba keratitis, Amoebiasis, Ascariasis, Babesiosis, Balantidiasis, Baylisascariasis, Chagas disease, Clonorchiasis, Cochliomyia, Cryptosporidiosis, Diphyllobothriasis, Dracunculiasis, Echinococcosis, Elephantiasis, Enterobiasis, Fascioliasis, Fasciolopsiasis, Filariasis, Giardiasis, Gnathostomiasis, Hymenolepiasis, Isosporiasis, Katayama fever, Leishmaniasis, Lyme disease, Metagonimiasis, Myiasis, Onchocerciasis, Pediculosis, Scabies, Schistosomiasis, Sleeping sickness, Strongyloidiasis, Taeniasis, Toxocariasis, Toxoplasmosis, Trichinosis, Trichuriasis, Trypanosomiasis, helminth infection, infection of hepatitis B (HBV) , hepatitis C (HCV) , herpes virus, Epstein-Barr virus, HIV-1, HIV-2, cytomegalovirus, herpes simplex virus type I, herpes simplex virus type II, human papilloma virus, adenovirus, Kaposi West sarcoma associated herpes virus epidemics, thin ring virus (Torquetenovirus) , human T lymphotrophic viruse I, human T lymphotrophic viruse II, varicella zoster, JC virus or BK virus.

In some embodiments, the subject has been identified as being likely to respond to a PD-1 antagonist. The presence or level of PD-L1 on an interested biological sample can be indicative of whether the subject from whom the biological sample is derived could likely respond to a PD-1 antagonist. Various methods can be used to determine the presence or level of PD-L1 in a test biological sample from the subject. For example, the test biological sample can be exposed to anti-PD-L1 antibody or antigen-binding fragment thereof, which binds to and detects the expressed PD-L1 protein. Alternatively, PD-L1 can also be detected at nucleic acid expression level, using methods such as quantitative Polymerase Chain Reaction (qPCR) , reverse transcriptase PCR, microarray, Serial analysis of gene expression (SAGE) , Fluorescence in situ hybridization (FISH) , and the like. In some embodiments, the test sample is derived from a cancer cell or tissue, or tumor infiltrating immune cells. In certain embodiments, presence or up-regulated level of the PD-L1 in the test biological sample indicates likelihood of responsiveness. The term “up-regulated” as used herein, refers to an overall increase of no less than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%or greater, in the protein level of PD-L1 in the test sample, as compared to the PD-L1 protein level in a reference sample as detected using the same antibody. The reference sample can be a control sample obtained from a healthy or non-diseased individual, or a healthy or non-diseased sample obtained from the same individual from whom the test sample is obtained. For example, the reference sample can be a non-diseased sample adjacent to or in the neighborhood of the test sample (e.g. tumor) .

In some embodiments, the subject is resistant or has developed resistance to PD-1 antagonist therapy or PD-L1 inhibitor therapy. For example, the subject can be one who progressed (e.g., experienced tumor growth) during therapy with a PD-1 inhibitor (e.g., an antibody molecule as described herein) and/or a PD-L1 inhibitor (e.g., antibody molecule) .

The present disclosure also provides therapeutic methods comprising: administering a therapeutically effective amount of the bispecific antibody molecule as provided herein to a subject in need thereof, thereby treating or preventing a CTLA-4-related condition or a disorder. In some embodiment, the CTLA-4-related condition or a disorder is cancer, autoimmune disease, inflammatory disease, infectious disease, graft versus host disease (GVHD) , or transplant rejection.

Examples of cancer include but are not limited to, lymphoma, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, uterine or endometrial cancer, rectal cancer, esophageal cancer, head and neck cancer, anal cancer, gastrointestinal cancer, intra-epithelial neoplasm, kidney or renal cancer, leukemia, liver cancer, lung cancer (e.g. non-small cell lung cancer and small cell lung cancer) , melanoma, myeloma, pancreatic cancer, prostate cancer, sarcoma, skin cancer, squamous cell cancer, stomach cancer, testicular cancer, vulval cancer, cancer of the endocrine system, cancer of the parathyroid gland, cancer of the adrenal gland, penile carcinoma, solid tumors of childhood, tumor angiogenesis, spinal axis tumor, pituitary adenoma, or epidermoid cancer.

Examples of autoimmune diseases include, but are not limited to, Acquired Immunodeficiency Syndrome (AIDS, which is a viral disease with an autoimmune component) , alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED) , autoimmune lymphoproliferative syndrome (ALPS) , autoimmune thrombocytopenic purpura (ATP) , Behcet's disease, cardiomyopathy, celiac sprue-dermatitis hepetiformis; chronic fatigue immune dysfunction syndrome (CFIDS) , chronic inflammatory demyelinating polyneuropathy (CIPD) , cicatricial pemphigold, cold agglutinin disease, crest syndrome, Crohn's disease, Degos' disease, dermatomyositis-juvenile, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP) , IgA nephropathy, insulin-dependent diabetes mellitus, juvenile chronic arthritis (Still's disease) , juvenile rheumatoid arthritis, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemacious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomena, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma (progressive systemic sclerosis (PSS) , also known as systemic sclerosis (SS) ) , Sjogren's syndrome, stiff-man syndrome, systemic lupus erythematosus, Takayasu arteritis, temporal arteritis/giant cell arteritis, ulcerative colitis, uveitis, vitiligo and Wegener's granulomatosis.

Inflammatory disorders, include, for example, chronic and acute inflammatory disorders. Examples of inflammatory disorders include Alzheimer's disease, asthma, atopic allergy, allergy, atherosclerosis, bronchial asthma, eczema, glomerulonephritis, graft vs. host disease, hemolytic anemias, osteoarthritis, sepsis, stroke, transplantation of tissue and organs, vasculitis, diabetic retinopathy and ventilator induced lung injury.

Examples of infectious disease include, but are not limited to, fungus infection, parasite/protozoan infection or chronic viral infection, for example, malaria, coccidioiodmycosis immitis, histoplasmosis, onychomycosis, aspergilosis, blastomycosis, candidiasis albicans, paracoccidioiomycosis, microsporidiosis, Acanthamoeba keratitis, Amoebiasis, Ascariasis, Babesiosis, Balantidiasis, Baylisascariasis, Chagas disease, Clonorchiasis, Cochliomyia, Cryptosporidiosis, Diphyllobothriasis, Dracunculiasis, Echinococcosis, Elephantiasis, Enterobiasis, Fascioliasis, Fasciolopsiasis, Filariasis, Giardiasis, Gnathostomiasis, Hymenolepiasis, Isosporiasis, Katayama fever, Leishmaniasis, Lyme disease, Metagonimiasis, Myiasis, Onchocerciasis, Pediculosis, Scabies, Schistosomiasis, Sleeping sickness, Strongyloidiasis, Taeniasis, Toxocariasis, Toxoplasmosis, Trichinosis, Trichuriasis, Trypanosomiasis, helminth infection, infection of hepatitis B (HBV) , hepatitis C (HCV) , herpes virus, Epstein-Barr virus, HIV, cytomegalovirus, herpes simplex virus type I, herpes simplex virus type II, human papilloma virus, adenovirus, human immunodeficiency virus I, human immunodeficiency virus II, Kaposi West sarcoma associated herpes virus epidemics, thin ring virus (Torquetenovirus) , human T lymphotrophic viruse I, human T lymphotrophic viruse II, varicella zoster, JC virus or BK virus.

The therapeutically effective amount of an bispecific antibody molecule as 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 antibody molecule as provided herein may be administered at a therapeutically effective dosage of about 0.01 mg/kg to about 100 mg/kg. In certain of these embodiments, the bispecific antibody molecule 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, 3 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 antibody molecule disclosed 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 some embodiments, the bispecific antibody molecules disclosed herein may be administered alone or in combination with one or more additional therapeutic means or agents. For example, the bispecific antibody molecules disclosed herein may be administered in combination with another therapeutic agent, for example, a chemotherapeutic agent or an anti-cancer drug.

In certain of these embodiments, an bispecific antibody molecule as disclosed herein that is administered in combination with one or more additional therapeutic agents may be administered simultaneously with the one or more additional therapeutic agents, and in certain of these embodiments the bispecific antibody molecule and the additional therapeutic agent (s) may be administered as part of the same pharmaceutical composition. However, a bispecific antibody molecule administered “in combination” with another therapeutic agent does not have to be administered simultaneously with or in the same composition as the agent. A bispecific antibody molecule 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 bispecific antibody molecule and second agent are administered via different routes. Where possible, additional therapeutic agents administered in combination with the bispecific antibody molecule disclosed 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 2003 (Physicians' Desk Reference, 57th Ed; Medical Economics Company; ISBN: 1563634457; 57th edition (November 2002) ) or protocols well known in the art.

The present disclosure further provides methods of using the bispecific antibody molecule thereof.

In some embodiments, the present disclosure also provides use of the bispecific antibody molecule provided herein in the manufacture of a medicament for treating a PD-1 and/or CTLA-4 related disease or condition in a subject.

I. Advantages

The bispecific antibodies provided herein are advantageous over existing therapies in many aspects. For example, the bispecific antibodies provided herein can block both PD-1 and CTLA-4 pathways, and they particularly inhibit Treg function. The bispecific antibodies provided herein are superior to monospecific anti-PD-1 antibodies, or monospecific anti-CTLA-4 antibodies, or combination of monospecific anti-PD-1 antibodies and monospecific anti-CTLA-4 antibodies. The bispecific antibodies provided herein are also advantageous in that they are cross-reactive to human, monkey PD-1 and CTLA-4, and in some embodiments, cross-reactive to murine PD-1. The bispecific antibodies may be used to treat the patients who are resistant to or relapse from anti-PD-1 or anti-CTLA-4 monotherapy. The bispecific antibodies may also increase the response rate comparing with anti-PD-1 or anti-CTLA-4 alone. The bispecific antibodies may also reduce the toxicity of anti-CTLA-4 or anti-PD-1 by lowering therapeutic dose.

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 falling within the scope of the invention. One skilled in the art may develop equivalent compositions, materials, and methods without the exercise of inventive capacity and without departing from the scope of the invention. It will be understood that many variations can be made in the procedures herein described while still remaining within the bounds of the present invention. It is the intention of the inventors that such variations are included within the scope of the invention.

EXAMPLE

EXAMPLE 1: Generation and characterization of PD-1 humanized monoclonal antibody of W3052-2E5

The humanized monoclonal PD-1 antibody W3052-2E5 was generated as described in WO2018053709A1. Generally, SD rats were immunized with human PD-1 extracellular domain (ECD) protein and the B lymphocytes isolated from lymph node of the immunized SD rats were combined with myeloma cells to obtain a hybridoma, which was isolated, selected and sub-cloned. The total RNA of the hybridoma was extracted and the cDNA was synthesized and amplified. The framework region of the rat VH and VL genes were replaced with human frameworks by CDR-grafting technique and were cloned into expression vectors to create corresponding clones of humanized antibodies. The monoclonal antibody W3052-2E5 was obtained after affinity maturation by point mutation (s) in the CDR and/or framework regions.

The humanized W3052-2E5 antibody has a heavy chain variable region of SEQ ID NO: 17, a kappa light chain variable region of SEQ ID NO: 18, and a human IgG4 constant region. As described in PCT application No.: WO2018053709A1, W3052-2E5 binds to human PD-1 transfected CHO-Scells with EC ₅₀ of 2.20 nM, to mouse PD-1 transfected 293F cells with EC ₅₀ of 12.9 nM and to activated cynomolgus PBMC in a dose dependent way, as determined by flow cytometry. W3052-2E5 binds to human PD-1 with EC ₅₀ of 0.18 nM, to mouse PD-1 with EC ₅₀ of 0.37 nM and to cynomolgus PD-1 with EC ₅₀ of 0.25 nM by ELISA. W3052-2E5 binds specifically to human PD-1, but not to CD28 and CD47, as measured by flow cytometry. W3052-2E5 blocks human PD-L1 binding to PD-1 transfected CHO-S cells with IC ₅₀ of 2.14 nM, blocks mouse PD-L1 binding to PD-1 transfected 293F cells with IC ₅₀ of 13 nM, and blocks human PD-L2 binding to PD-1 in a dose-dependent manner, as determined by ELISA. The binding affinity of W3052-2E5 to human PD-1 by SPR assay was 6.13 nM (KD value) . The binding affinity of W3052-2E5 to mouse PD-1 by SPR assay was 3.99 nM (K _D value) . The binding affinity of W3052-2E5 to human PD-1 by FACS assay was 0.23 nM (K _D value) . The binding affinity of W3052-2E5 to mouse PD-1 by FACS assay was 29 nM (K _D value) .

Result of epitope binning test showed that the parent antibody of W3052-2E5 (i.e. W3052_r16.88.9) , is in the same or close epitope bin as benchmark antibodies nivolumab (clone of 5C4 from U.S. patent US9084776B2) and pembrolizumab (disclosed as clone hPD-1.09A in US8354509B2 and WO2008156712A1) . However, after setting an additional cutoff to the binding fold change (<0.55) , the final determined epitope residues revealed that W3052_r16.88.9 binds to both human and murine PD-1 while pembrolizumab only bound to the human PD-1. Result of epitope mapping showed that although both are functional in binding human PD-1 and blocking human PD-L1, they have obviously different epitopes.

W3052-2E5 increased IL-2 secretion, IFN-γ secretion in a dose-dependent manner, as measured by ELISA in both human and mouse T cell function assays. W3052-2E5 increased and CD4 ⁺ T cells proliferation in a dose-dependent manner, as measured by 3H-thymidine incorporation assay in both human and mouse T cell function assays. Tests in W3052-2E5 on cell proliferation and cytokine production by autologous antigen specific immune response showed that W3052-2E5 can enhance the function of human CD4 ⁺ T cell by increase IFN-γ secretion and CD4 ⁺ T cells proliferation in a dose-dependent manner. W3052-2E5 also can reverse the suppressive function of Tregs by restoring the IFN-γ secretion and the T-cell proliferation.

W3052-2E5 does not mediated ADCC or CDC activity on activated CD4 ⁺ T cells.

In vivo efficacy of W3052-2E5 were studied in CloudmanS91 syngeneic tumor model. A weak inhibitory effect was observed in 1 mg/kg W3052-2E5 group compared with the control group, and the tumor volume was 1, 089 mm ³ (T/C=68.1%, TGI=34.4%, p=0.367) , tumor growth delay was 0 days. A significant anti-tumor effect was observed in 3 mg/kg W3052-2E5 group compared with the solvent control group, and the tumor volume was 361 mm ³ (T/C=22.9%, TGI=81.0%, p=0.008) , tumor growth delay was 5 days. A significant anti-tumor effect was also observed in 10 mg/kg W3052-2E5 group compared with the solvent control group, the tumor volume was 614 mm ³ (T/C=39.4%, TGI=64.7%, p=0.036) , tumor growth delay was 5 days.

Compared to the solvent control group, W3052-2E5 prolonged the median survival time of tumor-bearing mice by 25% (p=0.077) at 1 mg/kg, and by 66.7% (p=0.001) at 3 mg/kg, and by 100% (p=0.022) at 10 mg/kg. W3052-2E5 was also shown to have good tolerability in all tumor-bearing mice.

EXAMPLE 2: Generation and characterization of monoclonal antibody of W3055-1.153.7 hAb

Fully human W3055-1.153.7 hAb was obtained as described in PCT application No.: PCT/CN2016/094624, having a heavy chain variable region of SEQ ID NO: 27, a kappa light chain variable region of SEQ ID NO: 28, and a human IgG4 constant region. As disclosed in PCT application No.: PCT/CN2016/094624, the affinities of W3055-1.153.7 hAb for recombinant human PD-1 was 2.79 nM by SPR. W3055-1.153.7 hAb bound to cynomolgus monkey PD-1 but did not bind to murine PD-1 as measured by FACS. W3055-1.153.7 hAb bound specifically to PD-1, but not to CD28 and CTLA-4 of PD-1 family. The results of SPR assay and FACS for the binning test showed that the epitope on human PD-1 bound by W3055-1.153.7 hAb was different from the existing PD-1 antibodies (i.e. benchmark antibodies nivolumab (disclosed as clone of 5C4 from U.S. patent US9084776B2) and pembrolizumab (disclosed as clone hPD-1.09A in US8354509B2 and WO2008156712A1) ) . [ ³H] thymidine incorporation assay showed that W3055-1.153.7 hAb enhanced concentration dependent T cell proliferation.

Human CD4 ⁺ T Cells were stimulated with allogeneic dendritic cells (DCs) in the presence of W3055-1.153.7 hAb, which increased IL-2 secretion, IFNγ secretion in a dose manner by ELISA. W3055-1.153.7 hAb enhanced concentration dependent CMV ⁺-CD4 ⁺ T cell proliferation stimulated with CMV pp65 peptide-loaded autologous DC, as assessed by [ ³H] thymidine incorporation. W3055-1.153.7 hAb abrogated Treg’s suppressive function and restored responding T cell proliferation and IFNγ secretion, as assessed by [ ³H] thymidine incorporation.

W3055-1.153.7 hAb has no ADCC and CDC function.

EXAMPLE 3: Generation and characterization of monoclonal antibody of humanized W3166-z17

The monoclonal human anti-CTLA-4 antibody W3166-z17 was generated as described in PCT/CN2018/079495. Generally, a Llama glama was immunized with human and Cynomolgus monkey (cyno) CTLA-4 extracellular domain (ECD) as antigen to obtain VHH antibodies. Peripheral blood mononuclear cells (PBMCs) from the immunized animal were collected for total RNA extraction and cDNA synthesis. The repertoire of PCR-amplified VHH from the cDNA was purified and ligated in phagemid vector pFL249 and electrotransformed into E. Coli TG1 for expression. After cell panning and screening, the production of selected VHH was confirmed by BL21 E. coli expression. Chimeric VHH-Fc (hIgG1) fusion antibodies, abbreviated as VHH-IgG, were prepared by fusing the VHH genes into a modified human IgG1 expression pcDNA3.3 vector to create corresponding clones of VHH-Fc (hIgG1) chimeric antibody. The vector was transiently transfected into Expi-293 cells.

Humanized VHH sequences were generated by replacing human CDR sequences in the top hit with VHH CDR sequences using Kabat CDR definition. Several residues in the framework region were back-mutated to VHH in order to maintain the affinity. Humanized genes, which were back-translated and codon optimized for mammalian expression, were synthesized by GENEWIZ. The re-amplified genes were cloned into a modified pcDNA3.3 vector to express bivalent humanized VHHs linked with human IgG1 Fc region.

Humanized W3166-z17 was obtained as described in PCT application No.: PCT/CN2018/079495, having a heavy chain variable region of SEQ ID NO: 9, and a human IgG1 constant region. Reference benchmark antibody W316-BMK1 was generated based on the variable region sequence of Ipilimumab (Yervoy) as disclosed in patent document US20150283234. Breifely the DNA sequences encoding the variable region were synthesized in Sangon Biotech (Shanghai, China) , and then cloned into modified pcDNA3.4 expression vectors with constant region of human IgG1, or human IgG4.

Human CTLA-4-binding (ELISA and FACS) . As disclosed in PCT application No.: PCT/CN2018/079495, W3166-z17 bound to cell surface human CTLA-4 and immobilized human CTLA-4 ECD protein in a dose-dependent manner. W3166-z17 bind to cell surface human CTLA-4 with EC ₅₀ values of 0.2975 nM; In comparison, W316-BMK1 bound to cell surface human CTLA-4 with an EC ₅₀ of 0.5898 nM (FACS) . In comparison, W316-BMK1 bound to immobilized human CTLA-4 ECD protein with an EC ₅₀ of 0.0800 nM. The binding EC ₅₀ of W3166-z17 is similar with that of W316-BMK1 (ELISA) .

Cynomolgus CTLA-4-binding (ELISA and FACS) . W3166-z17 bound to cell surface cyno CTLA-4 and immobilized cyno CTLA-4 ECD protein in a dose-dependent manner. W3166-z17 bound to cell surface cyno CTLA-4 with EC ₅₀ values of 1.162 nM; in comparison, W316-BMK1 binds to cell surface cyno CTLA-4 with an EC ₅₀ of 1.737 nM (FACS) . W3166-z17 bound to immobilized cyno CTLA-4 ECD protein with EC ₅₀ values of 0.0401 nM; in comparison, W316-BMK1 bound to immobilized cyno CTLA-4 ECD protein with an EC ₅₀ of 0.0348 nM (ELISA) . The binding EC ₅₀ of W3166-z17 is comparable to that of W316-BMK1.

Competition binding assay (ELISA) . W3166-z17 block the binding of hCD80 to hCTLA-4 with IC ₅₀ values of 1.1000 nM and 0.9076 nM, respectively, in comparison, W316-BMK1 blocks the binding of hCD80 to hCTLA-4 with an IC ₅₀ of 0.8379 nM, and that W3166-z13 and W3166-z17 block the binding of hCD86 to hCTLA-4 with IC ₅₀ values of 2.0610 nM and 1.6670 nM, respectively, in comparison W316-BMK1 blocks the binding of hCD86 to hCTLA-4 with an IC ₅₀ of 0.7546 nM, examined by competition ELISA. As can be seen, W3166-z13 and W3166-z17 can block the binding of hCTLA-4 to hCD80 or hCD86 protein as effectively as W316-BMK1.

Competition binding assay (FACS) . W3166-z17 blocked the binding of hCD80 to hCTLA-4 with IC ₅₀ values of 0.0786 nM, in comparison, W316-BMK1 blocks the binding of hCD80 to hCTLA-4 with an IC ₅₀ of 0.4281 nM, and that W3166-z17 blocked the binding hCD86 of hCTLA-4 with IC ₅₀ values of 0.1632 nM, in comparison W316-BMK1 blocked the binding of hCD86 to hCTLA-4 with an IC ₅₀ of 1.1140 nM, determined by competition FACS. This indicated that W3166-z17 blocked the binding of hCTLA-4 to cell surface hCD80 and hCD86 more effectively as compared with W316-BMK1.

W3166-z17 had better affinity K _D to cell surface human CTLA-4 than that of W316-BMK1 by FACS (4.9E-11M and 2.8E-10M, respectively ) , and bound to cell surface monkey CTLA-4 at KD of 1.4E-10M, which is lower than or comparable to that of the W316-BMK1 (2.8E-10M) .

In a kinetic binding affinity measured by surface plasmon resonance (SPR) , affinity of W3166-z17 to human CTLA-4 protein was similar to that of W316-BMK1 (3.13E-09M and 3.32E-09M, respectively) . W3166-z17 specifically bound to human CTLA-4 but did not cross-react with hICOS, BTLA, hCD28 and hPD-1.

W3166-z17 enhance IFN-γ and IL-2 production of primary PBMC in SEB stimulation assay in a dose-dependent manner. In an epitope binning assay, W3166-z17 had similar epitope binning with W316-BMK1.

W3166-z17 induced ADCC effect on hCTLA-4 transfected cells at an EC ₅₀ of 0.2474 nM, while W316-BMK1 showed an EC ₅₀ of 1.279 nM in inducing ADCC effect by ELISA. W3166-z17 did not induce CDC effect on hCTLA-4 transfected cells by ELISA.

W3166-z17 showed consistent EC ₅₀ values ranging from 0.1900-0.2212 nM by FACS, respectively, throughout the tested period (0-14 days) , demonstrating that it is stable in human serum stability test.

W3166-z17 had no non-specific binding. W3166-z17 was stable in DSF test and has a T _h1 of 54.1 ℃.

EXAMPLE 4: Generation and characterization of the bispecific antibodies

1. Materials and Methods

1.1 Antigen and Other Proteins Generation

1.1.1 Generate soluble antigens

DNA sequences encoding the extracellular domain sequence of human PD-1 (Uniport No.: Q15116) was 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 ℃.

1.1.2 Generate benchmark (BMK) antibodies

DNA sequences encoding the variable regions of anti-CTLA-4 antibody Ipilimumab, anti-PD-1 antibody Nivolumab, and anti anti-CTLA-4/anti-PD-1 bispecific antibody BIAB003 (sequences disclosed as clone “BIAB003” in Chinese Patent application publication No. CN106967172A) were synthesized in Sangon Biothech (Shanghai, China) , and then subcloned into modified pcDNA3.3 expression vectors with constant region of human IgG1 or human IgG4 (S228P) to generate the benchmark antibodies. The obtained benchmark anti-CTLA-4 antibody was named W316-BMK1 (with human IgG1 constant region) , the benchmark anti-PD-1 antibody was named W305-BMK1 (with human IgG4 (S228P) constant region) , and the benchmark anti-CTLA-4/anti-PD-1 bispecific antibody was named W324-BMK1 (with human IgG1 constant region) .

The plasmid containing VH and VL gene 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 ℃.

1.2 Cell Pool/Line Generation

1.2.1 Generate target-expressing cell lines

Lipofectamine 2000 was used to transfect CHO-Sor 293F cells with the expression vector containing gene encoding full length human PD-1, or mouse PD-1. Cells were cultured in medium containing proper selection pressure. Human PD-1 high expression stable cell line (W305. CHO-S. hPro1. C6) , mouse PD-1 high expression stable cell line (W305.293F. mPro1. B4) were obtained after limited dilution.

1.3 BsAb Generation

1.3.1 Construct expression vectors

Construction of G9 format bispecific antibodies: DNA sequence encoding anti-PD-1 IgG heavy chain of W3055-1.153.7 or W3052-2E5 at the N terminus, followed by a spacer (SEQ ID NO: 42) , and anti-CTLA-4 VHH antibody W3166-z17 at the C-terminus, was cloned into modified pcDNA3.3 expression vector, respectively. DNA sequence encoding light chain of anti-PD-1 antibody W3055-1.153.7 or W3052-2E5 was cloned into modified pcDNA3.3 expression vector. Two bispecific antibodies were obtained: W3246-U6T9 which was based on W3055-1.153.7; and W3246-U3T9 which was based on W3052-2E5. Each of the bispecific antibodies was operably linked to human antibody constant region of an IgG4 isotype with S228P mutation.

1.4.2 Small scale transfection, expression and purification

Heavy chain and light chain expression plasmids were co-transfected into ExpiCHO cells using ExpiCHO expression system kit (ThermoFisher-A29133) according to the manufacturer’s instructions. 10 days after transfection, the supernatants were collected and used for protein purification using Protein A column (GE Healthcare-17543802) and further size exclusion chromatography (GE Healthcare-17104301) . Antibody concentration was measured by Nano Drop. The purity of proteins was evaluated by SDS-PAGE and HPLC-SEC. Two bispecific antibodies, W3246-U6T9-3. uIgG4. SP (hereafter “W3246-U6T9” ) and W3246-U3T9-3. uIgG4. SP (hereafter “W3246-U3T9” ) , were obtained after expression and purification.

The antibody expression, purity and thermal stability is shown in Figure 1. W3246-U6T9 and W3246-U3T9 have good expression level, thermal stability and purity.

1.4 In vitro Characterization

1.4.1 Differential 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.

The thermal stability of the W3246-U3T9 and W3246-U6T9 are shown in Figure 1. Both antibodies are stable in DSF test and have a T _h1 of 57.1 ℃ and 55.1℃, respectively.

1.4.2 Human CTLA-4-binding (ELISA/FACS)

For ELISA binding, non-tissue culture treated flat-bottom 96-well plates (Nunc MaxiSorp, ThermoFisher) were pre-coated with 1.0 μg/ml human CTLA-4 protein W316-hPro1. ECD. His overnight at 4℃. Aft er 2%BSA blocking, 100 μL 3.16-fold titrated Abs from 5.0 μg/ml to 0.00005 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: 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 W324-BMK1 (IgG1 isotype) .

The result of hCTLA-4-Binding ELISA is shown in Figure 2A. Both W3246-U3T9 and W3246-U6T9 can bind to hCTLA-4 protein, with EC ₅₀ of 0.0533 nM and 0.0664 nM, respectively, wherein W316-BMK1 has an EC ₅₀ of 0.0631 nM and W324-BMK1 has an EC ₅₀ of 0.0578 nM.

In FACS assay, engineered human CTLA-4 expressing cells were W316-293F. hPro1. FL 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 20 μg/ml to 0.000076 μg/ml were added to the cells. The plates were incubated at 4 ℃ for 1 hour. After wash, 100 μL of 1: 150 diluted PE-labeled goat anti-human antibody (Jackson 109-115-098) was added to each well and the plates were incubated at 4 ℃ for 1 hour. The binding of the antibodies onto the cells was tested by flow cytometry and the mean fluorescence intensity (MFI) was analyzed by FlowJo.

The result of hCTLA-4-Binding FACS is shown in Figure 2B. Both W3246-U3T9 and W3246-U6T9 can bind to hCTLA-4 cell. W3246-U3T9 and W3246-U6T9 bound to hCTLA-4 expressing cells with EC ₅₀ of 0.2621 nM and 0.2849 nM, respectively, wherein W316-BMK1 has an EC ₅₀ of 0.4822 nM and W324-BMK1 has an EC ₅₀ of 4.36 nM, indicating that W3246-U3T9 and W3246-U6T9 are better than the benchmark Abs in binding to hCTLA-4 expressing cells.

1.4.3 Human 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 1.0 μg/ml in house human PD-1 protein W305-hPro1. ECD. mFc overnight at 4℃. After 2%BSA blocking, 100 μL 3.16-fold titrated Abs from 5.0 μg/ml to 0.00005 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: 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) .

The result of hPD-1-Binding ELISA is shown in Figure 3A. Both W3246-U3T9 and W3246-U6T9 can bind to hPD-1 protein as well as benchmark controls. W3246-U3T9 and W3246-U6T9 bound to hPD-1 protein with EC ₅₀ of 0.0677 nM and 0.0896 nM, respectively, wherein W305-BMK1 has an EC ₅₀ of 0.0738 nM and W324-BMK1 has an EC ₅₀ of 0.0613 nM.

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 W324-BMK1.

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) . 3.16-Fold titrated Abs with 1%BSA DPBS from 20 μg/ml to 0.0002 μg/ml were added to the cells. Plates were incubated at 4 ℃ for 1 hour. After wash, 100 μL 1: 125 diluted PE-labeled goat anti-human antibody (Jackson 109-095-008) 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.

The result of hPD-1-Binding FACS is shown in Figure 3B. Both W3246-U3T9 and W3246-U6T9 can bind to hPD-1 by FACS as well as parental and benchmark antibodies. W3246-U3T9 and W3246-U6T9 bound to hPD-1 expressing cells with EC ₅₀ of 0.5620 nM and 1.4110 nM, respectively, wherein W316-BMK1 has an EC ₅₀ of 0.7013 nM and W324-BMK1 has an EC ₅₀ of 1.0980 nM.

1.4.4 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 4.0-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) .

The result of cyno CTLA-4-Binding ELISA is shown in Figure 4A. Both W3246-U3T9 and W3246-U6T9 can bind to cyno CTLA-4 protein as well as BMK. W3246-U3T9 and W3246-U6T9 bind to cyno CTLA-4 protein with EC ₅₀ of 0.0754 nM and 0.0726 nM, respectively, wherein W316-BMK1 has an EC ₅₀ of 0.0931 nM and W324-BMK1 has an EC ₅₀ of 0.1378 nM.

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 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.

The result of cyno CTLA-4-Binding FACS is shown in Figure 4B. Both W3246-U3T9 and W3246-U6T9 can bind to cyno CTLA-4 cell. W3246-U3T9 and W3246-U6T9 bound to cyno CTLA-4 expressing cells with EC ₅₀ of 0.5384 nM and 0.6663 nM, respectively, wherein W316-BMK1 has an EC ₅₀ of 0.6709 nM and W324-BMK1 has an EC ₅₀ of 3.1330 nM.

1.4.5.1 Cynomolgus PD-1-binding ELISA and FACS

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.

The result of cyno PD-1-binding FACS is shown in Figure 5A. Both W3246-U3T9 and W3246-U6T9 can bind to cyno PD-1 cell. W3246-U3T9 and W3246-U6T9 bind to cyno PD-1 expressing cells with EC ₅₀ of 1.4790 nM and 1.9250 nM, respectively, wherein W305-BMK1 has an EC ₅₀ of 1.1430 nM and W324-BMK1 has an EC ₅₀ of 2.6330 nM.

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) .

The result of cyno PD-1-binding ELISA is shown in Figure 5B. Both W3246-U3T9 and W3246-U6T9 can bind to cyno PD-1 protein as well as BMK. Both W3246-U3T9 and W3246-U6T9 can bind to cyno PD-1 protein. W3246-U3T9 and W3246-U6T9 bound to cyno CTLA-4 protein with EC ₅₀ of 0.1002 nM and 0.1783 nM, respectively, wherein W305-BMK1 has an EC ₅₀ of 0.0583 nM and W324-BMK1 has an EC ₅₀ of 0.1150 nM.

1.4.5.2Mouse 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 1.0 μg/ml in house human PD-1 protein W305-mPro1. ECD. mFc overnight at 4℃. After 2%BSA blocking, 100 μL 3.16-fold titrated antibodies from 5.0 μg/ml to 0.00005μg/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 (

M5 ^e) .

The result of mPD-1-Binding ELISA is shown in Figure 17. W3246-U3T9 can bind to mPD-1 protein with EC ₅₀ of 0.0785 nM.

For FACS binding, engineered mouse PD-1 expressing cells W305-293F. mPro1. B4 were seeded at 1×10 ⁵ cells/well in U-bottom 96-well plates (COSTAR 3799) . 4.0-Fold titrated antibodies with 1%BSA DPBS from 80 μg/ml to 0.000076 μg/ml were added to the cells. Plates were incubated at 4 ℃ for 1 hour. After wash, 100 μL 1: 150 diluted FITC-labeled goat anti-human antibody (Jackson 109-095-008) 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.

The result of mPD-1-Binding ELISA is shown in Figure 18. W3246-U3T9 can bind to mPD-1 protein with EC ₅₀ of 4.579 nM.

1.4.6 PD-1 and CTLA-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 pre 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) .

The result of Dual-Binding ELISA is shown in Figure 6. Both W3246-U3T9 and W3246-U6T9 can bind to hPD-1 and hCTLA-4 proteins simultaneously, with EC ₅₀ at 0.0132 nM and 0.0133 nM, respectively.

1.4.7 PD-1 and CTLA-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. cynoPro1. F1. Pool were stained with Calcein-AM (Corning-354216) 50nM and Far red (Invitrogen-C34572) 20nM 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. After wash twice, the cells was tested by flow cytometry and the percentage of double positive cells was analyzed by FlowJo.

The bispecific antibodies could bind to both CTLA-4+ cells and PD-1+ cells. As shown in Figure 7, at different concentration, the percentage of cross-linked two kinds of cells by bispecific antibodies were much higher than the isotype control. The W3246 antibodies could cross-link more cells than W324-BMK1.

1.4.8 Affinity to CTLA-4 and PD-1 (SPR)

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. The results are shown in Table 9 and Table 10.

Table 9 is the summary of Human PD-1-and CTLA-4-binding (SPR) .

Table 10 is the summary of Cynomolgus PD-1-and CTLA-4-binding (SPR) .

1.4.9 Human CTLA-4 ligand-hCD80 or CD86 Competitive FACS

To test whether the antibodies could block hCTLA-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.

The result of blocking CTLA-4 binding to CD80 on cell surface (FACS) is shown in Figure 8A. Both W3246-U3T9 (IC ₅₀ of 2.298 nM) and W3246-U6T9 (IC ₅₀ of 2.869 nM) can block hCD80 binding to hCTLA-4+ cells better than benchmarks, where W316-BMK1 has an IC ₅₀ of 4.411 nM and W324-BMK1 has an IC ₅₀ of 3.043 nM. The result of blocking CTLA-4 binding to CD86 on cell surface (FACS) is shown in Figure 8B. Both W3246-U3T9 (IC ₅₀ of 1.156 nM) and W3246-U6T9 (IC ₅₀ of 1.295 nM) can block hCD86 binding to hCTLA-4 by FACS, where W316-BMK1 has an IC ₅₀ of 1.945 nM and W324-BMK1 has an IC ₅₀ of 1.058 nM.

Competitive FACS was also used to test whether the bispecific antibodies could block cyno CTLA-4 binding to CD80 or CD86 on cell surface, following a protocol simiar to the method used for human CTLA-4 binding, expect that cyno CTLA-4 and cells expressing CD80 or cyno CD86 were used. The bispecific antibodies can block cyno CTLA-4 binding to CD80 and CD86+ cells, as shown in Figure 9A and 9B. In the competitive FACS, both W3246-U3T9 and W3246-U6T9 block cyno CTLA-4 binding to CD80+ cells, with IC ₅₀ of 0.2897 and 0.3367 nM, respectively, much more potent than the monospecific (W316-BMK1, IC ₅₀ at 19.82 nM) or bispecific (W324-BMK1, IC ₅₀ at 2.553 nM) benchmark antibodies. In a similar FACS, both W3246-U3T9 and W3246-U6T9 can potently block cyno CTLA-4 binding to CD86+ cells, with IC ₅₀ of 20.53 and 28.81 nM, respectively, better than the W316-BMK1 (IC ₅₀ at 156 nM) .

1.4.10 Human PD-1-competitive ELISA and FACS

In order to test whether the bispecific antibodies could block hPD-L1 binding to hPD-1 protein.

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 4.0-fold titrated Abs from 20 μg/ml to 0.0012 μg/ml Abs coupled with 5.0 μg/ml in house hPD-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 pre 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) . 3.16-Fold titrated Abs from 80 μg/ml to 0.0008 μg/ml 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 expressing human PD-1 was detected by FITC-labeled goat anti-mouse antibody (abcam 98716 1: 125) . The competition binding of antibodies to the cells was tested by flow cytometry and the mean fluorescence intensity (MFI) was analyzed by FlowJo.

The result of hPD-1-Competition FACS is shown in Figure 10A. Both W3246-U3T9 (IC ₅₀ at 1.0390 nM) and W3246-U6T9 (IC ₅₀ at 2.6490 nM) can block hPD-L1 binding to hPD-1 expressing cell. The result of mPD-1-Competition FACS is shown in Figure 10B. W3246-U3T9 (IC ₅₀ at 19.320 nM) can block mPD-L1 binding to hPD-1 expressing cell by FACS.

1.4.11 Mixed lymphocyte reaction (MLR)

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

1.4.11.1 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.

1.4.11.2 Mix 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.

These two W3246 antibodies also enhanced cytokine release (IFN-γ) in allogeneic MLR assay (Figure 11) . W3246-U3T9 at high concentration was more potent than a bispecific reference antibody (W324-BMK1) , or monospecific antibodies against PD-1 (W305-BMK1) or CTLA-4 (W316-BMK1) . Another antibody W3246-U6T9 was comparable with the reference antibodies.

1.4.11.3 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%CO2. IL-2 and IFN-γ quantitation were determined on day 3 and day 5 respectively.

The W3246 bispecific antibodies can improve cytokine release in SEB stimulated PBMCs. As shown in Figures 12A and 12B, both W3246-U3T9 and W3246-U6T9 were more potent than a bispecific reference antibody (W324-BMK1) , or monospecific antibodies against PD-1 (W305-BMK1) or CTLA-4 (W316-BMK1) .

1.4.11.4 Cytokine quantification

Human IFN-γ was measured by ELISA using matched antibody pairs. Recombinant human IFN-γ was used as standards. The plates were pre-coated with capture antibody specific for human IFN-γ. 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 as 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 (

M5 ^e) .

1.4.11.5 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 number of Treg, CD4+ T and iDC cells were 1E5, 1E5 and 1E4 pre 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.

The result of Human Treg MLR Assay (INF-γ) is shown in Figure 13B. W3246 BsAbs significantly enhance IFN-γ secretion: W3246-U3T9 and/or W3246-U6T9 >W324-BMK1 (Bispecific benchmark control) = Combination of W305-BMK1 and W316-BMK1 >W305-BMK1 (anti-PD-1) > W316-BMK1 (anti-CTLA-4) . The result of Human Treg MLR Assay (Proliferation) is shown in Figure 13A. Both W3246-U3T9 and W3246-U6T9 significantly enhanced T cell proliferation. W3246-U3T9 and/or W3246-U6T9 showed the highest T cell proliferation, which was higher than the in combination of W305-BMK1 and W316-BMK1, and was much higher than the benchmark control anti-PD-1 monoclonal antibody W305-BMK1 or anti-CTLA-4 monoclonal antibody W316-BMK1. The benchmark control bispecific antibody W324-BMK1 showed the lowest T cell proliferation.

In another human Treg suppression assay, W3246-U3T9 and/or W3246-U6T9 also enhanced cytokine release from Treg suppressed CD4 effect cells (Figure 14) . W3246-U3T9 at high concentration was more potent than a bispecific reference antibody (W324-BMK1) , or monospecific antibodies against PD-1 (W305-BMK1) or CTLA-4 (W316-BMK1) . Another antibody W3246-U6T9, at low concentration, was more potent than the reference antibodies, and at high concentration, comparable with the reference antibodies.

1.4.12 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 W316-hPro1. ECD. hFc (in 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 antigen-2 (hPD-1-ECD, W305-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

M5 ^e) .

The result of Human Serum Stability ELISA test is shown in Figure 15. The BsAbs remain stable in human serum at 37 ℃ for two weeks

1.4.13 CTLA-4/PD-1 paralog-Binding ELISA

Human CTLA-4, ICOS, BTLA, CD28 and PD-1 were purchased from Sino Biological. HRP-conjugated goat anti-human IgG Fc was purchased from Bethyl (Cat: A80-304P) . A 96-well plate was coated with these five antigens (1.0 μg/mL) at 4 ℃ for 16-20 hours. After 1 hour blocking with 2%BSA in DBPS, testing antibodies, as well as positive and negative control antibodies were added to the plates and incubated at room temperature for 1 hour. The binding of the antibodies to the plates were detected by HRP-conjugated goat anti-human IgG antibody (1: 5000 dilution) with 1 hr incubation. The color was developed by dispensing 100 μL of TMB substrate for 8 mins, and then stopped by 100 μL of 2N HCl. The absorbance at 450 nM was measured using a microplate spectrophotometer.

The result of Cross-Family ELISA test is shown in Figure 16. Both Abs of W3246 have no-cross reaction with hCD28, hICOS and hBTLA.

2. References

1: Haratani K, Hayashi H, Tanaka T et al. Tumor Immune Microenvironment and Nivolumab Efficacy in CTLA4 Mutation-Positive Non-Small Cell Lung Cancer Based on T790M Status after Disease Progression During CTLA4-TKI Treatment. Ann Oncol. 2017 Apr 12. doi: 10.1093/annonc/mdx183. [Epub ahead of print] PubMed PMID: 28407039.

2: Okita R, Maeda A, Shimizu K, Nojima Y, Saisho S, Nakata M. PD-L1 overexpression is partially regulated by CTLA4/HER2 signaling and associated with poor prognosis in patients with non-small-cell lung cancer. Cancer Immunol Immunother. 2017 Mar 25. doi: 10.1007/s00262-017-1986-y. PubMed PMID: 28341875.

3: Soo RA, Kim HR, Asuncion BR, Fazreen Z, Omar MF, Herrera MC, Yun Lim JS, Sia G, Soong R, Cho BC. Significance of immune checkpoint proteins in CTLA4- mutantnon-small cell lung cancer. Lung Cancer. 2017 Mar; 105: 17-22. doi: 10.1016/j. lungcan. 2017.01.008. Epub 2017 Jan 15. PubMed PMID: 28236980.

4: Heigener DF, Reck M. PD-1 Axis Inhibition in CTLA4 Positives: A Blunt Sword? J Thorac Oncol. 2017 Feb; 12 (2) : 171-172. doi: 10.1016/j. jtho. 2016.12.013. PubMedPMID: 28115107.

5: Gettinger S, Politi K. PD-1 Axis Inhibitors in CTLA4-and ALK-Driven Lung Cancer: Lost Cause? Clin Cancer Res. 2016 Sep 15; 22 (18) : 4539-41. doi: 10.1158/1078-0432. CCR-16-1401. Epub 2016 Jul 28. PubMed PMID: 27470969.

6: Gainor JF, Shaw AT, Sequist LV et al. CTLA4 Mutations and ALK Rearrangements Are Associated with Low Response Rates to PD-1 Pathway Blockade in Non-Small Cell Lung Cancer: A Retrospective Analysis. Clin Cancer Res. 2016 Sep 15; 22 (18) : 4585-93. doi: 10.1158/1078-0432. CCR-15-3101. Epub 2016 May 25. PubMed PMID: 27225694; PubMed Central PMCID: PMC5026567.

7: Boussiotis VA. Molecular and Biochemical Aspects of the PD-1 Checkpoint Pathway. N Engl J Med. 2016 Nov 3; 375 (18) : 1767-1778. Review. PubMed PMID: 27806234.

8: Azuma K, Ota K, Kawahara A, Hattori S, Iwama E, Harada T, Matsumoto K, Takayama K, Takamori S, Kage M, Hoshino T, Nakanishi Y, Okamoto I. Association of PD-L1 overexpression with activating CTLA4 mutations in surgically resected nonsmall-cell lung cancer. Ann Oncol. 2014 Oct; 25 (10) : 1935-40. doi: 10.1093/annonc/mdu242. Epub 2014 Jul 9. PubMed PMID: 25009014.

9: Akbay EA, Koyama S, Carretero J, et al. Activation of the PD-1 pathway contributes to immune escape in CTLA4-driven lung tumors. Cancer Discov. 2013 Dec; 3 (12) : 1355-63. doi: 10.1158/2159-8290. CD-13-0310. Epub 2013 Sep 27. PubMed PMID: 24078774; PubMed Central PMCID: PMC3864135.

10: Ramalingam S, Hui R, Gandhi L, Carcereny E, Felip E, Ahn MJ, Eder JP, Balmanoukian AS, Leighl N, Aggarwal C, Horn L, Patnaik A, Middleton GW, Gubens M, Hellmann M, Soria JC, Lubiniecki GM, Zhang J, Piperdi B, Garon EB. P2.39: Long-Term OS for Patients With Advanced NSCLC Enrolled in the KEYNOTE-001 Study of Pembrolizumab: Track: Immunotherapy. J Thorac Oncol. 2016 Oct; 11 (10S) : S241-S242. doi: 10.1016/j. jtho. 2016.08.110. Epub 2016 Sep 22. PubMed PMID: 27676573.

Claims

A bispecific antibody molecule comprising a CTLA-4-binding domain and a PD-1-binding domain, wherein:

the CTLA-4-binding domain comprises:

1, 2, or 3 heavy chain complementarity determining region (CDR) sequences selected from the group consisting of: SEQ ID NOs: 1-4; and/or

the PD-1-binding domain comprises:

1, 2, or 3 heavy chain complementarity determining region (CDR) sequences selected from the group consisting of: SEQ ID NOs: 11-13 and 21-23; and/or

1, 2, or 3 light chain CDR sequences selected from the group consisting of: SEQ ID NOs: 14-16 and 24-26,

the CTLA-4-binding domain comprises a VHH domain; and

the PD-1-binding domain comprises a Fab.
The bispecific antibody molecule of claim 1, wherein the CTLA-4-binding domain comprises a heavy chain variable region comprising SEQ ID NOs: 1, 4 and 3.
The bispecific antibody molecule of any of the preceding claims, wherein the CTLA-4-binding domain comprises a heavy chain variable region selected from the group consisting of SEQ ID NOs: 5, 7 and 9, and a homologous sequence thereof having at least 80%sequence identity yet retaining specific binding affinity to CTLA-4.
The bispecific antibody molecule of any of the preceding claims, wherein the CTLA-4-binding domain comprises a heavy chain variable region comprising SEQ ID NO: 9.
The bispecific antibody molecule of any of the preceding claims, wherein the PD-1-binding domain comprises a heavy chain variable region selected from the group consisting of:

a) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 21-23; and

b) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 11-13, and/or

a light chain variable region selected from the group consisting of:

c) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 24-26; and

d) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 14-16.
The bispecific antibody molecule of any of the preceding claims, wherein the PD-1-binding domain comprises:

a) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 21-23; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 24-26; or

b) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 11-13; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 14-16.
The bispecific antibody molecule of any of the preceding claims, wherein the PD-1-binding domain comprises a heavy chain variable region selected from the group consisting of SEQ ID NOs: 17 and 27, and a homologous sequence thereof having at least 80%sequence identity yet retaining specific binding affinity to PD-1.
The bispecific antibody molecule of any of the preceding claims, wherein the PD-1-binding domain comprises a light chain variable region selected from the group consisting of SEQ ID NOs: 18 and 28, and a homologous sequence thereof having at least 80%sequence identity yet retaining specific binding affinity to PD-1.
The bispecific antibody molecule of any of the preceding claims, wherein the PD-1-binding domain comprises:

a) a heavy chain variable region comprising SEQ ID NO: 27 and a light chain variable region comprising SEQ ID NO: 28; or

b) a heavy chain variable region comprising SEQ ID NO: 17 and a light chain variable region comprising SEQ ID NO: 18.
The bispecific antibody molecule of any of the preceding claims, wherein the CTLA-4-binding domain further comprises one or more amino acid residue substitutions or modifications yet retains specific binding affinity to CTLA-4, and/or the PD-1-binding domain further comprises one or more amino acid residue substitutions or modifications yet retains specific binding affinity to PD-1.
The bispecific antibody molecule of any of the preceding claims, wherein at least one of the substitutions or modifications is in one or more of the CDR sequences, and/or in one or more of the VH or the VL sequences but not in any of the CDR sequences.
The bispecific antibody molecule of any of the preceding claims, wherein the bispecific antibody molecule further comprises an immunoglobulin constant region, optionally a constant region of human Ig, or optionally a constant region of human IgG.
The bispecific antibody molecule of any of the preceding claims, wherein the CTLA-4-binding domain is operably linked to the N terminus or the C terminus of the PD-1-binding domain.
The bispecific antibody molecule of any of the preceding claims, wherein the CTLA-4-binding VHH comprises the sequence of SEQ ID NO: 9, and the PD-1-binding Fab comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 17 and a light chain variable region comprising the sequence of SEQ ID NO: 18.
The bispecific antibody molecule of any of the preceding claims, wherein the CTLA-4-binding VHH comprises the sequence of SEQ ID NO: 9, and the PD-1-binding Fab comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 27 and a light chain variable region comprising the sequence of SEQ ID NO: 28.
The bispecific antibody molecule of any of the preceding claims, wherein the CTLA-4-binding domain is operably linked to the C terminus of the heavy chain of the PD-1-binding domain.
The bispecific antibody molecule of claim 16, wherein the bispecific antibody molecule comprise a heavy chain in the format of: VH (anti-PD-1) -CH1-Hinge-CH2-CH3-spacer-VHH(anti-CTLA-4) , associated with a light chain in the format of VL (anti-PD-1) -CL.
The bispecific antibody molecule of claim 17, comprising a heavy chain comprising the sequence of SEQ ID NO: 34 and a light chain comprising the sequence of SEQ ID NO: 35.
The bispecific antibody molecule of claim 17, comprising a heavy chain comprising the sequence of SEQ ID NO: 36 and a light chain comprising the sequence of SEQ ID NO: 37.
The bispecific antibody molecule of any of the preceding claims, wherein the CTLA-4-binding domain and/or the PD-1-binding domain is fully human or humanized.
The bispecific antibody molecule of any of the preceding claims linked to one or more conjugate moieties.
The bispecific antibody molecule of claim 21, wherein the conjugate moiety comprises a clearance-modifying agent, a chemotherapeutic agent, a toxin, a radioactive isotope, a lanthanide, a luminescent label, a fluorescent label, an enzyme-substrate label, a DNA-alkylators, a topoisomerase inhibitor, a tubulin-binders, or other anticancer drugs.
A pharmaceutical composition comprising the bispecific antibody molecule of any of the preceding claims, and a pharmaceutically acceptable carrier.
A polynucleotide encoding the bispecific antibody molecule of claims 1-20.
The polynucleotide of claim 24, comprising a nucleotide sequence selecting from a group consisting of SEQ ID NOs: 6, 8, 10, 19, 20, 29 and 30, and/or a homologous sequence thereof having at least 80% (e.g. at least 85%, 88%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity, and/or a variant thereof having only degenerate substitutions.
A vector comprising the polynucleotide of claim 24 or 25.
A host cell comprising the vector of claim 26.
A method of expressing the bispecific antibody molecule of any of claims 1-20, comprising culturing the host cell of claim 27 under the condition at which the vector of claim 26 is expressed.
A method of treating a disease or condition in a subject that would benefit from up-regulation of an immune response, comprising administering to the subject a therapeutically effective amount of the bispecific antibody molecule of any of claims 1-22 or the pharmaceutical composition of claim 23.
The method of claim 29, wherein the disease or condition that would benefit from up-regulaltion of an immune response is selected from the group consisting of cancer, a viral infection, a bacterial infection, a protozoan infection, a helminth infection, asthma associated with impaired airway tolerance, a neurological disease, multiple sclerosis, and an immunosuppressive disease.
The method of claim 29 or 30, wherein the disease or condition is PD-1-related and/or CTLA-4-related.
The method of any of claims 29-31, wherein the PD-1-related disease or condition is cancer, autoimmune disease, inflammatory disease, or infectious disease.
The method of any of claims 29-31, wherein the CTLA-4-related disease or condition is cancer, autoimmune disease, inflammatory disease, or infectious disease.
The method of claim 32 or 33, wherein the cancer is lymphoma, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, uterine or endometrial cancer, rectal cancer, esophageal cancer, head and neck cancer, anal cancer, gastrointestinal cancer, intra-epithelial neoplasm, kidney or renal cancer, leukemia, liver cancer, lung cancer, melanoma, myeloma, pancreatic cancer, prostate cancer, sarcoma, skin cancer, squamous cell cancer, stomach cancer, testicular cancer, vulval cancer, cancer of the endocrine system, cancer of the parathyroid gland, cancer of the adrenal gland, penile carcinoma, solid tumors of childhood, tumor angiogenesis, spinal axis tumor, pituitary adenoma, or epidermoid cancer.
The method of any of claims 29-34, wherein the disease or condition is an environmentally induced cancer induced by asbestos or hematologic malignancies, wherein said cancer is selected from multiple myeloma, B-cell lymphoma, Hodgkin lymphoma, primary mediastinal B-cell lymphoma, non-Hodgkin's lymphoma, acute myeloid lymphoma, chronic myelogenous leukemia, chronic lymphoid leukemia (CLL) , follicular lymphoma, diffuse large B-cell lymphoma (DLBCL) , Burkitt's lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia (ALL) , mycosis fungoides, anaplastic large cell lymphoma, T-cell lymphoma, and precursor T-lymphoblastic lymphoma, and any combinations of said cancers.
The method of any of claims 29-35, wherein the subject is human.
The method of any of claims 29-36, wherein the administration is via oral, nasal, intravenous, subcutaneous, sublingual, or intramuscular administration.
A method of modulating CTLA-4 activity in a CTLA-4-expressing cell, comprising exposing the CTLA-4-expressing cell to the bispecific antibody molecule of any of claims 1-22.
Use of the bispecific antibody molecule of any of claims 1-22 in the manufacture of a medicament for treating a disease or condition that would benefit from up-regulation of an immune response.
Use of the bispecific antibody molecule of any of claims 1-22 in the manufacture of a medicament for treating a disease or condition that is PD-1 and/or CTLA-4-related.