WO2019179390A1

WO2019179390A1 - Novel bispecific pd-1/egfr antibody molecules

Info

Publication number: WO2019179390A1
Application number: PCT/CN2019/078483
Authority: WO
Inventors: 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-EGFR/PD-1 bispecific antibody molecules, polynucleotides encoding the same, pharmaceutical compositions comprising the same, and the uses thereof.

Description

NOVEL BISPECIFIC PD-1/EGFR ANTIBODY MOLECULES

PRIORITY CLAIM

The present application claims the priority to PCT Application Number PCT/CN2018/079491, 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 EGFR.

BACKGROUND

EGFR belongs to ERBB receptor tyrosine kinase superfamily. Binding of EGF to EGFR leads to tyrosine phosphorylation and receptor dimerization with other family members resulting in enhanced uncontrolled proliferation. EGFR is overexpressed in several types of cancer, where EGFR and its family members are the major contributors of complex signaling cascades that modulate growth, signaling, differentiation, adhesion, migration and survival of cancer cells. Therefore EGFR and its family members have emerged as attractive targets for anti-cancer therapy.

Two EGFR-targeted antibodies, cetuximab (Erbitux) and panitumumab (Vectibix) , have been approved by the US Food and Drug Administration for the treatment of several solid cancer, including colorectal, head and neck cancer, NSCLC, pancreatic cancer. These antibodies block the binding of ligands to EGFR and downstream signals, therefore mediate antitumor immune responses. These antibodies also induce the internalization and degradation of EGFR, thereby leading to signal termination. Nevertheless, initial effectiveness of anti-EGFR therapy frequently dampened by resistance to such targeted therapy, mainly due to EGFR mutations. There is a clear unmet medical need for more effective therapy.

Despite of the development of therapeutics targeting the targets respectively, there is a significant need for novel bispecific therapeutics 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/EGFR 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 EGFR-binding domain and a PD-1-binding domain, wherein:

the EGFR-binding domain comprises:

1, 2, or 3 heavy chain complementarity determining region (CDR) sequences selected from the group consisting of: SEQ ID NOs: 1-3; 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: 8, 10, 12, 19, 21, 23; and/or

1, 2, or 3 light chain CDR sequences selected from: SEQ ID Nos: 9, 11, 13, 20, 22, 24, 25,

the EGFR-binding domain comprises a VHH domain; and

the PD-1-binding domain comprises a Fab.

In certain embodiments, the EGFR-binding domain comprises a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 1-3.

In certain embodiments, the EGFR-binding domain comprises a heavy chain variable region selected from SEQ ID NO: 4, SEQ ID NO: 6, and a homologous sequence thereof having at least 80%sequence identity yet retaining specific binding affinity to EGFR.

In certain embodiments, the EGFR-binding domain comprises a heavy chain variable region comprising SEQ ID NO: 6.

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

a) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected the group consisting of from SEQ ID NOs: 19, 21, 23; and

b) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected the group consisting of from SEQ ID NOs: 8, 10 and 12, and/or

a light chain variable region selected from:

c) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from the group consisting of SEQ ID NOs: 20, 22 and 24;

d) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from the group consisting of SEQ ID NOs: 20, 22 and 25; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from the group consisting of SEQ ID NOs: 9, 11 and 13.

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

e) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from the group consisting of SEQ ID NOs: 19, 21 and 23; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from the group consisting of SEQ ID NOs: 20, 22 and 24;

f) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from the group consisting of SEQ ID NOs: 19, 21 and 23; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from the group consisting of SEQ ID NOs: 20, 22 and 25; or

g) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from the group consisting of SEQ ID NOs: 8, 10 and 12; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from the group consisting of SEQ ID NOs: 9, 11 and 13.

In certain embodiments, the PD-1-binding domain comprises a heavy chain variable region selected from the group consisting of SEQ ID NOs: 14 and 26 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: 16, 18, 28 and 30, 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:

h) a heavy chain variable region comprising SEQ ID NO: 26 and a light chain variable region comprising SEQ ID NO: 28;

i) a heavy chain variable region comprising SEQ ID NO: 26 and a light chain variable region comprising SEQ ID NO: 30;

j) a heavy chain variable region comprising SEQ ID NO: 14 and a light chain variable region comprising SEQ ID NO: 16; or

k) a heavy chain variable region comprising SEQ ID NO: 14 and a light chain variable region comprising SEQ ID NO: 18.

In certain embodiments, the EGFR-binding domain further comprises one or more amino acid residue substitutions or modifications yet retains specific binding affinity to EGFR, 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 comprising an immunoglobulin constant region, optionally a constant region of human Ig, or optionally a constant region of human IgG.

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

In certain embodiments, the EGFR-binding domain comprises the sequence of SEQ ID NO: 6, and the PD-1-binding domain comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 14 and a light chain variable region comprising the sequence of SEQ ID NO: 18.

In certain embodiments, the EGFR-binding domain comprises the sequence of SEQ ID NO: 6, and the PD-1-binding domain comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 26 and a light chain variable region comprising the sequence of SEQ ID NO: 30.

In certain embodiments, the EGFR-binding domain is operably linked to the N terminus of the light 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, associated with a light chain in the format of VHH (anti-EGFR) -spacer-VL (anti-PD-1) -CL.

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: 35.

In certain embodiments, the bispecific antibody molecule comprising a heavy chain comprising the sequence of SEQ ID NO: 33 and a light chain comprising the sequence of SEQ ID NO: 32.

In certain embodiments, the EGFR-binding domain and/or the PD-1-binding domain is 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: 5, 7, 15, 17, 27, 29 and 31, 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 upregulation 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 upregulation 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 EGFR-related.

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

In certain embodiments, the EGFR-related disease or condition is cancer or inflammatory disease.

In certain embodiments, 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 EGFR-related disease or condition is cancer or inflammatory disease, and wherein the cancer is colorectal cancer, skin cancer, head and neck cancer, non-small cell lung cancer, gastrointestinal cancer, glioblastoma, melanoma, breast cancer, cervical cancer, ovarian cancer, endometrial cancer, prostate cancer, renal cancer, bladder cancer, esophageal cancer, brain cancer, liver cancer, pancreatic cancer, hepatocellular cancer, or squamous cell carcinoma; and wherein the inflammatory disease is Alzheimer's disease, asthma, atopic allergy, allergy, atherosclerosis, bronchial asthma, eczema, glomerulonephritis, graft vs. host disease, hemolytic anemias, osteoarthritis, psoriasis, sepsis, stroke, transplantation of tissue and organs, vasculitis, diabetic retinopathy or ventilator induced lung injury.

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 EGFR activity in a EGFR-expressing cell, comprising exposing the EGFR-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 EGFR-related.

BRIEF DESCRIPTION OF FIGURES

Figure 1 shows the result of W3366-T2U3-D5 and W3366-T2U8 transient production. W3366-T2U3-D5 and W3366-T2U8 are produced with high purity (98.35 and 98.05%, respectively) and good yield (35.5 and 264 mg/L) in transient production.

Figure 2A shows the result of Human PD-1 binding. W3366-T2U3-D5 and W3366-T2U8 bind to cell surface human PD-1 with EC ₅₀ of 0.50 and 0.39 nM, respectively, in comparison to WBP305BMK1 (EC ₅₀ of 0.28 nM) , as measured by FACS.

Figure 2B shows the result of Mouse PD-1 binding. W3366-T2U3-D5 binds to cell surface mouse PD-1with EC ₅₀ of 2.207 nM, as measured by FACS.

Figure 2C shows the result of Cyno PD-1 binding. W3366-T2U3-D5 and W3366-T2U8 bind to cell surface cyno PD-1 with EC ₅₀ of 1.26 and 0.92 nM, respectively, in comparison to WBP305BMK1 (EC ₅₀ of 1.03 nM) , as measured by FACS.

Figure 3A shows the result of Human EGFR Binding. W3366-T2U3-D5 and W3366-T2U8 bind to cell surface human EGFR with EC ₅₀ of 1.09 and 0.84 nM, respectively, in comparison to WBP336-hBMK1 (EC ₅₀ of 0.39 nM) , as measured by FACS.

Figure 3B shows the result of cyno EGFR Binding. W3366-T2U3-D5 and W3366-T2U8 bind to cell surface cyno EGFR with EC ₅₀ of 0.40 nM, in comparison to WBP336-hBMK1 (EC ₅₀ of 0.2044 nM) , as measured by FACS.

Figure 3C shows the result of mouse EGFR binding. W3366-T2U3-D5 and W3366-T2U8 bind to cell surface mouse EGFR with EC ₅₀ of 1.53 and 1.54 nM, respectively, as measured by FACS.

Figure 4A shows the result of Simultaneous dual target Binding. W3366-T2U3-D5 and W3366-T2U8 simultaneously bind to PD-1 and EGFR with EC ₅₀ of 0.030 and 0.018 nM respectively, when ELISA plate was coated with EGFR. W3366-T2U3-D5 and W3366-T2U8 simultaneously bind to PD-1 and EGFR with EC ₅₀ of 0.026 and 0.009 nM, respectively, when ELISA plate coated with PD-1.

Figure 4B shows the result of Simultaneous dual target binding as measured by FACS. W3366-T2U3-D5 and W3366-T2U8 can simultaneously bind to two targets (EGFR and PD-1) on cell surface.

Figure 5A shows the result of Human PD-L1/PD-1 blocking. W3366-T2U3-D5 and W3366-T2U8 block the binding of human PD-L1 to cell surface human PD-1 with IC ₅₀ of 0.31 and 0.34 nM, respectively, in comparison to WBP305BMK1 (IC ₅₀ of 0.25 nM) , as measured by FACS.

Figure 5B shows the result of Mouse PD-L1/PD-1 blocking. W3366-T2U3-D5 blocks the binding of mouse PD-L1 to cell surface mouse PD-1 with IC ₅₀ of 9.46 nM, as measured by FACS.

Figure 6A shows the result of Human EGF/EGFR blocking. W3366-T2U3-D5 and W3366-T2U8 block the binding of human EGF to cell surface human EGFR with IC ₅₀ of 2.709 and 3.27 nM, respectively, in comparison to WBP336-hBMK1 (IC ₅₀ of 1.599 nM) , as measured by FACS.

Figure 6B shows the result of mouse EGF/EGFR blocking. W3366-T2U3-D5 and W3366-T2U8 block the binding of mouse EGF to cell surface mouse EGFR with IC ₅₀ of 4.32 and 3.45 nM, respectively, in comparison to WBP336-hBMK1 (IC ₅₀ >200 nM) , as measured by FACS.

Figure 7A shows the result of full kinetic affinity to human PD-1. The affinities of W3366-T2U3-D5 and W3366-T2U8 to human PD-1 are 7.44E-09 and 5.58E-10 M, respectively, as measured by SPR.

Figure 7B shows the result of affinity to cell surface EGFR. The affinities of W3366-T2U3-D5 and W3366-T2U8 to cell surface human EGFR are 8.5E-10 and 7.0E-10 M, respectively, as measured by FACS.

Figure 8A shows the result of Human IL-2 production in allo-MLR assay. W3366-T2U3-D5 and W3366-T2U8 promote IL-2 production in a dose-dependent manner in human allo-MLR assay.

Figure 8B shows the result of Human IFN-γ production in allo-MLR assay. W3366-T2U3-D5 and W3366-T2U8 promote IFN-γ production in a dose-dependent manner in human allo-MLR assay.

Figure 9 shows the result of EGFR phosphorylation inhibition in A431 cells. W3366-T2U3-D5 and W3366-T2U8 inhibit EGFR phosphorylation in A431 cells with IC ₅₀ of 23.70 and 15.71 nM, respectively, in comparison to WBP336-hBMK1 (IC ₅₀ of 5.412 nM ) .

Figure 10 shows the result of A431 proliferation inhibition. W3366-T2U3-D5 and W3366-T2U8 inhibit A431 cell proliferation with IC ₅₀ of 3.418 and 4.201 nM, respectively, in comparison to WBP336-hBMK1 (IC ₅₀ of 4.494 nM) .

Figure 11A-F shows the result of ADCC and CDC assays on PD-1 expressing cells. W3366-T2U3-D5 and W3366-T2U8 do not induce ADCC (Figure 11A-B) and CDC (Figure 11D-E) on PD-1 expressing cells (activated CD4+ T and hPD-1 transfected cells) , while the positive control did induce ADCC (Figure 11C) or CDC (Figure 11F) .

Figure 11G-H shows the result of ADCC and CDC assays on A431 cells. W3366-T2U3-D5 and W3366-T2U8 do not induce ADCC and CDC on EGFR expressing cells. WBP336-hBMK1. IgG1 is a positive control antibody.

Figure 12A and 12B shows the result of serum Stability test as measured by FACS. W3366-T2U3-D5 and W3366-T2U8 are stable in human serum at 37℃ for at least 14 days.

Figure 13 shows the result of thermal stability by DSF. W3366-T2U3-D5 and W3366-T2U8 have normal DSF profile and Tm are 62.3 ℃ and 64.3 ℃, respectively.

Figure 14 shows the tumor growth curve of MBT-2 tumor bearing mice post administration of testing antibodies (W3052-2E5, W5626-2C10-z5, W3052-2E5 + W5626- 2C10-z5 and W3366-T2U3-D5, respectively) and the body weight changes of the tumor-bearing mice in the different groups. Data points represent group mean tumor volume and mean body weight, respectively. Error bars represent standard error of the mean (SEM) .

Figure 15 shows the mean plasma concentration of W3366-T2U3-D5 and W3366-T2U8 in mouse PK study.

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) ; N.R. 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. EGFR-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’ .

“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 V _H 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 “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.

“EGFR” as used herein, can be 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 EGFR includes human EGFR protein (Genbank accession No. : GI: 110002567 and GI: 63101670) . Exemplary sequence of EGFR includes Macaca fascicularis (monkey) EGFR protein (Genbank accession No. : GI: 544419950, GI: 544419948, GI: 544419952) ; Mus musculus (mouse) EGFR protein Genbank accession No. : GI: 10880776 and GI: 5524153) ; Rattus norvegicus (Rat) EGFR protein (Genbank accession No.: GI: 315227975, GI: 315227973, GI: 6478868) .

The term “EGFR” as used herein is intended to encompass any form of EGFR, for example, 1) native unprocessed EGFR molecule, “full-length” EGFR chain or naturally occurring variants of EGFR, including, for example, splice variants or allelic variants; 2) any form of EGFR 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 EGFR subunit generated through recombinant method.

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

“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 EGFR 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 EGFR and an anti-EGFR 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, “uIgG4” means an antibody with human constant region of a IgG4 isotype, “SP” refers to mutation of S228P in the constant region of human IgG4 (i.e. S228P) , “hAb” or “uAb” means human antibody, “z” means humanized antibody..

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 “EGFR-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 EGFR. In some embodiments, the EGFR related condition is cancer and inflammatory disease.

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 EGFR-binding domain and an PD-1-binding domain capable of specifically binding to PD-1, and the EGFR-binding domain comprises a VHH domain; and the PD-1-binding domain comprises a Fab.

i. EGFR-binding domain

In certain embodiments, the EGFR-binding domain comprises one or more (e.g. 1, 2, or 3) CDR sequences of an anti-EGFR single domain antibody W5626-2C10 and W5626-2C10-z5.

“W5626-2C10” as used herein refers to a VHH antibody having a heavy chain variable region of SEQ ID NO: 4.

“W5626-2C10-z5” as used herein refers to a humanized VHH antibody based on W5626-2C10 that comprises a heavy chain variable region of SEQ ID NO: 6. W5626 -2C10-z5 has comparable affinity to the antigen as compared with its parent antibody W5626-2C10.

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

Table 1 shows the CDR sequences of the anti-EGFR 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 EGFR-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 3 CDRs are indispensable or unchangeable. In other words, it is possible to replace or change or modify one or more CDRs in anti-EGFR single domain antibody W5626-2C10, yet substantially retain the specific binding affinity to EGFR.

In certain embodiments, the anti-EGFR antibody polypeptides provided herein comprise a heavy chain CDR3 sequence of SEQ ID NO: 3 (i.e. the heavy chain CDR3 sequence of anti-EGFR antibody W5626-2C10 or W5626-2C10-z5) .

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 EGFR-binding domains provided herein comprise any suitable framework region (FR) sequences, as long as the antigen-binding domains can specifically bind to EGFR. 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 EGFR-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 EGFR-binding domains provided herein comprise one or more FR sequences of W5626-2C10-z5.

The two exemplary humanized anti-EGFR single domain antibodies W5626-2C10-z5 retains the specific binding affinity to EGFR, 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 EGFR-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 EGFR-binding domain provided herein comprise a heavy chain variable domain sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, and a homologous sequence thereof having at least 80%sequence identity yet retaining specific binding affinity to EGFR. In certain embodiments, the EGFR-binding domain provided herein comprise a heavy chain variable domain sequence comprising SEQ ID NO: 6.

In some embodiments, the EGFR-binding domains provided herein comprise all or a portion of the heavy chain variable domain. In one embodiment, the EGFR-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) and comprises a Fab.

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, W3052-2E5 (F41Y) , W3055-1.103.11 hAb, and W3055-1.103.11-v2 hAb.

“W3052-2E5” as used herein refers to a humanized monoclonal antibody having a heavy chain variable region of SEQ ID NO: 14, and a kappa light chain variable region of SEQ ID NO: 16.

“W3052-2E5 (F41Y) ” as used herein refers to a humanized monoclonal antibody having a heavy chain variable region of SEQ ID NO: 14, and a kappa light chain variable region of SEQ ID NO: 18 (which contains a F41Y mutation as compared with SEQ ID NO: 16) .

“W3055-1.103.11 hAb” as used herein refers to a fully human monoclonal antibody having a heavy chain variable region of SEQ ID NO: 26, and a kappa light chain variable region of SEQ ID NO: 28.

“W3055-1.103.11-v2 hAb” as used herein refers to a fully human monoclonal antibody having a heavy chain variable region of SEQ ID NO: 26, and a kappa light chain variable region of SEQ ID NO: 30 (which contains an Asn93Serine mutation (Kabat Numbering) ) as compared with SEQ ID NO: 28) .

In certain embodiments, the PD-1-binding domain comprises a heavy chain CDR1 comprising the sequence of SEQ ID NO: 8, a heavy chain CDR2 comprising the sequence of SEQ ID NO: 10, and a heavy chain CDR3 comprising the sequence of SEQ ID NO: 12, and/or a light chain CDR1 comprising the sequence of SEQ ID NO: 9, a light chain CDR2 comprising the sequence of SEQ ID NO: 11, and a light chain CDR3 comprising the sequence of SEQ ID NO: 13.

In certain embodiments, the PD-1-binding domain comprises a heavy chain CDR1 comprising the sequence of SEQ ID NO: 19, a heavy chain CDR2 comprising the sequence of SEQ ID NO: 21, and a heavy chain CDR3 comprising the sequence of SEQ ID NO: 23, and/or a light chain CDR1 comprising the sequence of SEQ ID NO: 20, a light chain CDR2 comprising the sequence of SEQ ID NO: 22, and a light chain CDR3 comprising the sequence selected from SEQ ID NO: 24 and 25.

Table 4 shows the CDR sequences of these 3 anti-PD-1 antibodies. The heavy chain and light chain variable region sequences are also provided below in Table 5 and Table 6 (variant sequences are also included) .

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 SEQ ID NO: 12 or 23 (i.e. the HCDR3 of the anti-PD-1 antibodies W3052-2E5, W3052-2E5 (F41Y) , W3055-1.103.11 hAb and W3055-1.103.11-v2 hAb.

In certain embodiments, the PD-1-binding domains provided herein are fully human. For example, the PD-1-binding domains derived from W3055-1.103.11 hAb and W3055-1.103.11-v2 hAb are 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 and W3052-2E5 (F41Y) 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 and W3052-2E5 (F41Y) 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: 14, SEQ ID NO: 26, 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: 16, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 30, 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 EGFR-binding domain comprising one or more (e.g. 1, 2, or 3) CDR sequences of SEQ ID NOs: 1-3 (i.e. derived from W5626-2C10, W5626-2C10-z5) 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: 8-13 and 19-25 (i.e. derived from W3055-1.103.11 hAb, W3055-1.103.11-v2 hAb, and W3052-2E5, and W3052-2E5 (F41Y) ) , and the EGFR-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 EGFR-binding domain comprising:

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

a PD-1-binding domain comprising:

b) a heavy chain CDR1 comprising the sequence of SEQ ID NO: 8, a heavy chain CDR2 comprising the sequence of SEQ ID NO: 10, and a heavy chain CDR3 comprising the sequence of SEQ ID NO: 12, and/or a light chain CDR1 comprising the sequence of SEQ ID NO: 9, a light chain CDR2 comprising the sequence of SEQ ID NO: 11, and a light chain CDR3 comprising the sequence of SEQ ID NO: 13; or

c) heavy chain CDR1 comprising the sequence of SEQ ID NO: 19, heavy chain CDR2 comprising the sequence of SEQ ID NO: 21, and heavy chain CDR3 comprising the sequence of SEQ ID NO: 23, and/or light chain CDR1 comprising the sequence of SEQ ID NO: 20, light chain CDR2 comprising the sequence of SEQ ID NO: 22, and light chain CDR3 comprising the sequence selected from SEQ ID NO: 24 and 25, and the EGFR-binding domain comprises a VHH domain, and the PD-1-binding domain comprises a Fab.

In certain embodiments, the EGFR-binding domain comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 4, 6, or a homologous sequence thereof having at least 80%sequence identity yet retaining specific binding affinity to EGFR (e.g. human EGFR) .

In certain embodiments, the PD-1 binding domain comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 14, 26, 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: 16, 18, 28, 30 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 EGFR-binding domain comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 6 (i.e. derived from W5626-2C10-z5) , and the PD-1 binding domain comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 14 and a light chain variable region comprising the sequence of SEQ ID NO: 18 (i.e. derived from W3052-2E5 (F41Y) ) (such bispecific antibody molecules are also referred to as “W3366-T2U3-D5” herein) .

In certain embodiments, the EGFR-binding domain comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 6 (i.e. derived from W5626-2C10-z5) , and the PD-1 binding domain comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 26 and a light chain variable region comprising the sequence of SEQ ID NO: 30 (i.e. derived from W3055-1.103.11-V2) (such bispecific antibody molecules are also referred to as “W3366-T2U8” herein) .

The EGFR-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) , and screening from a phage display library as discussed above (e.g. for ScFv) . Other techniques for the production of antibody fragments will be apparent to a skilled practitioner.

In certain embodiments, the EGFR-binding domain is a VHH domain. In certain embodiments, the EGFR-binding VHH domain comprises the sequence of SEQ ID NO: 4, or 6. 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: 14 and a light chain variable region comprising the sequence of SEQ ID NO: 18 or 16; or a heavy chain variable region comprising the sequence of SEQ ID NO: 26 and a light chain variable region comprising the sequence of SEQ ID NO: 30 or 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 EGFR-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 EGFR-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, EGFR-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 EGFR-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 EGFR-binding domain may be bivalent (2 VHHs) , trivalent (3 VHHs) , tetravalent (4 VHHs) or have a higher valency molecules.

In certain embodiments, the EGFR-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 EGFR-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 and a Fab. In certain embodiment, the spacer comprises 1, 2, 3, 4 or more sequential or tandem repeats of SEQ ID NOs: 38-41. 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 EGFR-binding domain and the PD-1-binding domain, as long as both the EGFR-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 EGFR-binding domain comprises a VHH domain and the PD-1 binding domain comprises a Fab or IgG. In certain embodiments, the EGFR-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-spacer-VHH (anti-EGFR) or VHH(anti-EGFR) -spacer-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) refers respectively to the heavy and light chain variable domain of the anti-PD-1 antibody provided herein (e.g. W3052-2E5, W3052-2E5 (F41Y) , W3055-1.103.11-V2, and W3055-1.103.11) ; VHH (anti-EGFR) refers to a VHH derived from the anti-EGFR antibody provided herein (e.g. W5626-2c10-z5) , 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-EGFR) -spacer-VL (anti-PD-1) -CL or VL (anti-PD-1) -CL-spacer-VHH (anti-EGFR) , and a heavy chain VH (anti-PD-1) -CH1-Hinge-CH2-CH3, by the same token.

The EGFR-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 comprises a heavy chain in the format of: VH (anti-PD-1) -CH1-Hinge-CH2-CH3, and a light chain in the format of: VHH(anti-EGFR) -spacer-VL (anti-PD-1) -CL, wherein the VH (anti-PD-1) comprises the sequence of SEQ ID NO: 14 or 26; the VHH (anti-EGFR) comprises the sequence of SEQ ID NO: 4 or 6; the VL (anti-PD-1) comprises the sequence of SEQ ID NO: 16, 18, 28, or 30. In certain embodiments, the VH (anti-PD-1) comprises the sequence of SEQ ID NO: 14; the VHH(anti-EGFR) comprises the sequence of SEQ ID NO: 6; the VL (anti-PD-1) comprises the sequence of SEQ ID NO: 18. In certain embodiments, the VH (anti-PD-1) comprises the sequence of SEQ ID NO: 26; the VHH (anti-EGFR) comprises the sequence of SEQ ID NO: 6; the VL (anti-PD-1) comprises the sequence of SEQ ID NO: 30. In certain embodiments, the spacer comprises the sequence of SEQ ID NO: 38. In certain embodiments the heavy chain constant region (CH1-Hinge-CH2-CH3) is of human IgG4 isotype, and optionally contains mutations of S228P and/or L235E. In certain embodiments, the heavy chain constant region (CH1-Hinge-CH2-CH3) comprises the sequence of SEQ ID NO: 34. In certain embodiments, the light chain constant region (CL) comprises the sequence of SEQ ID NO: 37.

In certain embodiments, the bispecific antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 33 and a light chain comprising the amino acid sequence of SEQ ID NO: 32. This antibody is also called W3366-T2U3-D5 in the present disclosure.

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: 35. This antibody is also called W3366-T2U8 in the present disclosure.

Table 7 and 8 show the combination of heavy chain and light chain sequences of the bispecific antibody molecules of W3366-T2U3-D5, and W3366-T2U8.

“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 (F41Y) or W3055-1.103.11-v2.

“Anti-EGFR” refers to anti-EGFR antibody, in particular, the sequence provided in the table is the sequence derived from anti-EGFR antibody W5626-2C10-z5.

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, or 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, or a constant region of IgG4 or IgG2 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 or IgG2 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 EGFR. The bispecific antibody molecules provided herein retain the specific binding affinity to both PD-1 and EGFR, 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 EGFR which is sufficient to provide for diagnostic and/or therapeutic use.

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

In 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: 50x10 ^-9 M, 40x10 ^-9 M, 35x10 ^-9 M, 30x10 ^-9 M, 25x10 ^-9 M, 20x10 ^-9 M, 10x10 ^-9 M, 9x10 ^-9 M, 8x10 ^-9 M, 7x10 ^-9 M, 5x10 ^-9 M, 4x10 ^-9 M, 3x10 ^-9 M, 2x10 ^-9 M, 1x10 ^-9 M, 0.9x10 ^-9 M, 0.8x10 ^-9 M, 0.7x10 ^-9 M, or 0.6x10 ^-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 EGFR with a binding affinity (K _D) of no more than: 100x10 ^-10M, 90x10 ^-10M, 80x10 ^-10M, 70x10 ^-10M, 60x10 ^-10M, 50x10 ^-10M, 40x10 ^-10M, 30x10 ^- ¹⁰M, 20x10 ^-10M, 10x10 ^-10M, 9x10 ^-10M, 8x10 ^-10M, or 7x10 ^-10M as measured by surface plasmon resonance (SPR) .

Binding of the bispecific antibody molecules can also 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, flow cytometry assay, and other binding assay.

In certain embodiments, the bispecific antibody molecules provided herein specifically bind to human EGFR/EGFR-vIII at an EC ₅₀ of no more than: 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, or 0.8 nM as measured by flow cytometry assay.

In certain embodiments, the bispecific antibody molecules provided herein specifically bind to cynomolgus monkey EGFR at 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 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, or 0.4 nM as measured by flow cytometry assay.

In certain embodiments, the bispecific antibody molecules provided herein specifically bind to mouse EGFR with an EC ₅₀ of no more than: 20 nM, 19 nM, 18 nM, 17 nM, 16 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1.5 nM as measured by flow cytometry assay.

In certain embodiments, the bispecific antibody molecules provided herein cross-react with Cynomolgus monkey PD-1 and/or mouse PD-1. In certain embodiments, the bispecific antibody molecules to Cynomolgus monkey PD-1 or mouse PD-1 with a binding affinity similar to that of human PD-1.

In certain embodiments, the PD-1-binding domains provided herein specifically bind to human PD-1 at 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 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, or 0.4 nM by FACS.

In certain embodiments, the bispecific antibody molecules provided herein specifically bind to mouse PD-1 at an EC ₅₀ of no more than: 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 certain embodiments, the bispecific antibody molecules provided herein specifically bind to monkey PD-1 at an EC ₅₀ of no more than: 30 nM, 25 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, or 0.9 nM as measured by FACS.

In certain embodiments, the bispecific antibody molecules provided herein specifically bind to both human EGFR/EGFR-vIII and human PD-1, by binding to human EGFR/EGFR-vIII at 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, or 0.02 nM as measured by ELISA, and binding to human PD-1 at 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, 0.02 nM, or 0.01 nM as measured by ELISA.

In some embodiments, the bispecific antibody molecules provided herein are capable of blocking the binding between human PD-1 and human PD-L1. In certain embodiments, the bispecific antibody molecules provided herein specifically block the binding between human PD-1 and human PD-L1 at an IC ₅₀ (i.e. 50%blocking 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, or 0.3 nM by FACS.

In certain embodiments, the PD-1-binding domains provided herein specifically block the binding between mouse PD-1 and mouse PD-L1 at an IC ₅₀ of no more than: 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 15 nM, or 10 nM as measured by FACS.

In some embodiments, the bispecific antibody molecules provided herein are capable of specifically blocking the binding between human EGF and human EGFR. In certain embodiments, the bispecific antibody molecules provided herein specifically block the binding between human EGF and human EGFR 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, or 2 nM as measured by FACS.

In certain embodiments, the bispecific antibody molecules provided herein specifically block the binding between mouse EGF and mouse EGFR at an IC 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, 4 nM, or 3 nM as measured by FACS.

In some embodiments, the bispecific antibody molecules provided herein are capable of inhibiting EGFR phosphorylation. In certain embodiments, the bispecific antibody molecules provided herein inhibiting EGFR phosphorylation at an IC ₅₀ of no more than: 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, or 15 nM as measured by ELISA.

In some embodiments, the bispecific antibody molecules provided herein are capable of inhibiting proliferation of EGFR expressing cell 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, or 3 nM as measured by ELISA.

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, as measured by human allogeneic mixed lymphocyte reaction (MLR) .

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 EGFR 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 EGFR-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 EGFR-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, 2, or 3, and in the meantime retain the binding affinity to EGFR at a level similar to or even higher than its parent antibody.

In certain embodiments, the anti-EGFR-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 NOs: 4 or 6, and in the meantime retain the binding affinity to EGFR 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: 4 or 6. 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 any of SEQ ID NOs: 8-13 and 19-25, 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 NOs: 14, 16, 18, 26, 28 or 30, 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 NOs: 14, 16, 18, 26, 28 or 30,. 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 Fe 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 IN NOs: 5, 7, 15, 17, 27, 29, 31, 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 encode 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 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 EGFR-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., a PD-1 mono-specific antibody) and/or a PD-L1 inhibitor (e.g., a PD-L1 antibody) .

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 EGFR-related condition or a disorder. In some embodiment, the EGFR-related condition or a disorder is cancer, or inflammatory disease.

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.

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, psoriasis, sepsis, stroke, transplantation of tissue and organs, vasculitis, diabetic retinopathy and ventilator induced lung injury.

In certain embodiments, the EGFR-related condition or a disorder is also associated with EGFR variant III (EGFRvIII) , for example, glioblastoma.

In some embodiments, the subject has been identified as being likely to respond to an EGFR antibody. The presence, level, subtype and/or mutation of EGFR on an interested biological sample can be indicative of whether the subject from whom the biological sample is derived could likely respond to an EGFR antibody. In some embodiments, the test biological sample is derived from a cancer cell or tissue, or tumor infiltrating immune cells. In certain embodiments, presence or up-regulated level of the EGFR 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 EGFR in the test sample, as compared to the EGFR protein level in a reference sample as detected using the same method. 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 non-responsive to EGFR inhibitor mono-therapy (e.g. mono-specific anti-EGFR antibody) . For example, the subject can be one who progressed (e.g., experienced tumor growth) during therapy with an EGFR inhibitor mono-therapy (e.g., a mono-specific anti-EGFR antibody) . In some embodiments, the subject has been identified as having at least one of KRAS, NRAS, BRAF, and PIK3CA mutations.

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 EGFR 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 target both PD-1 and EGFR (including EGFRvIII) . The bispecific antibodies provided herein are superior to monospecific anti-PD1 antibodies, or monospecific anti-EGFR antibodies, or combinations thereof. The bispecific antibodies provided herein are also advantageous in that they are cross-reactive to mouse PD1 and EGFR, facilitating studies in mouse syngeneic tumor models. The bispecific antibodies may be used to treat the patients who are resistant to or relapse from anti-PD1 or anti-EGFR monotherapy. The bispecific antibodies may also increase the response rate comparing with anti-PD1 or anti-EGFR alone. The bispecific antibodies may also reduce the toxicity of anti-EGFR or anti-PD1 by using lower therapeutic dose.

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, plays a major role in limiting the activity of T cells that provides 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 regulates immune response and damp anti-tumor immunity. It is found that cancer cells are capable of evading immune destruction by upregulating PD-1/PDL1 pathway in the tumor microenvironment [Boussiotis 2016 N Engl J Med] . This mechanism is in particular found in tumors with activating mutations in the EGFR gene. As an evidence, high PDL1 expression is found in tumors of patients with EGFR mutations [Azuma 2014 Ann Oncol; Ramalingam 2016 J Thorac Oncol] . In addition, It has been reported that EGFR signaling affects PD-L1 expression in tumor cells [Akbay 2013 Cancer Discov, Azuma 2014 Ann Oncol, Chen 2015 J Thorac Oncol and Okita 2017 Cancer Immunol Immunother] . All these evidences suggest that targeting both EGFR pathway and PD-1/PD-L1 pathway may provide more effective therapy than targeting individual EGFR or PD-1 pathway for treatment of various tumors. The anti-PD-1 and anti-EGFR bispecific antibody not only block both EGFR and PD-1 pathways, but also the bispecific antibody can assist directing T cells to tumor cells, further improve the anti-tumor immunity. Here we show that our novel anti-PD-1 and anti-EGFR bispecific antibodies can effectively block PD-1 and EGFR pathways by several in vitro assays. In addition, we provide evidence by an in vivo mouse antitumor efficacy study, where our bispecific antibody displayed more potent antitumor activity than targeting individual EGFR or PD-1.

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.

EXAMPLES

EXAMPLE 1: Generation and characterization of PD-1 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: 14, a kappa light chain variable region of SEQ ID NO: 16, and a human IgG4 constant region. Its variant, W3052-2E5 (F41Y) , contains a F41Y mutation in the light chain variable region and had comparable effects as to W3052-2E5.

As described in PCT application No. : WO2018053709A1, W3052-2E5 bound to human PD-1 transfected CHO-Scells with EC ₅₀ of 2.20 nM, to mouse PD-1 transfected 293F cells with EC50 of 12.9 nM and to activated cynomolgus PBMC in a dose dependent way, as determined by flow cytometry. W3052-2E5 bound 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 bound 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-Scells 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 blocked human PD-L2 binding to PD-1 in a dose-dependent manner, as determined by ELISA. The affinity ability of W3052-2E5 to human PD-1 by SPR assay was 6.13 nM. The affinity ability of W3052-2E5 to mouse PD-1 by SPR assay was 3.99 nM. The affinity ability of W3052-2E5 to human PD-1 by FACS assay was 0.23 nM. The affinity ability of W3052-2E5 to mouse PD-1 by FACS assay was 29 nM.

Result of epitope binning test showed that the parent antibody of W3052-2E5 (i.e. W3052_r16.88.9) , was in the same or close epitope bin as benchmark antibodies nivolumab (clone of 5C4 from BMS 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 were functional in binding human PD-1 and blocking human PD-L1, they had 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-γsecretion and the T-cell proliferation.

W3052-2E5 did not mediate 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 W3055-1.103.11 PD-1 hAb

Fully human W3055-1.103.11 hAb was obtained as described in PCT application No. : PCT/CN2016/094624, having a heavy chain variable region of SEQ ID NO: 26, a kappa light chain variable region of SEQ ID NO: 28, and a human IgG4 constant region. Its variant W3055-1.103.11-v2 hAb had a heavy chain variable region of SEQ ID NO: 26, a kappa light chain variable region of SEQ ID NO: 30, and a human IgG4 constant region. W3055-1.103.11-v2 hAb had an Asn93Serine mutation (Kabat Numbering) in the light chain CDR3 of W3055-1.103.11 hAb, so as to reduce the risk of glycosylation on the CDR residue, and W3055-1.103.11-v2 hAb was expected to have comparable result to that of W3055-1.103.11 hAb, which was confirmed by cell-based functional assays that the mutation did not affect any binding capability. For example, the binding of 1.103.11-v2 hAbs in different solutions (either in formulation buffer or in 1xPBS (pH 7.4) ) to PD-1 expressing CHO cell was tested by FACS (EC ₅₀ at 2.52nM and 3.12 nM, respectively) or by ELISA (0.04889 nM and 0.05472 nM, respectively) . 1.103.11-v2 hAbs in both solutions bound to cell surface PD-1 on the CHO cell and there was no significant difference in binding affinity to human PD-1 between the two conditions.

As disclosed in PCT application No. : PCT/CN2016/094624, the affinities of W3055-1.103.11 hAb for recombinant human PD-1 was 0.175 nM by SPR. W3055-1.103.11 hAb bound to cynomolgus monkey PD-1 but did not bind to murine PD-1 as measured by FACS. W3055-1.103.11 hAb bound specifically to PD-1, but not to CD28 and CTLA4 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 W3055-1.103.11 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 W3055-1.103.11 hAb enhanced concentration dependent T cell proliferation.

Human CD4 ⁺ T Cells were stimulated with allogenenic dendritic cells (DCs) in the presence of W3055-1.103.11 hAb, which increased IL-2 secretion, IFNγ secretion in a dose manner by ELISA. W3055-1.103.11 hAb enhanced concentration dependent CMV ⁺-CD4 ⁺ T cell proliferation stimulated with CMV pp65 peptide-loaded autologous DC, as assessed by [ ³H] thymidine incorporation. W3055-1.103.11 hAb abrogated Treg’s suppressive function and restored responding T cell proliferation and IFNγ secretion, as assessed by [3H] thymidine incorporation.

W3055-1.103.11 hAb had no ADCC and CDC function.

In the epitope mapping, W3055-1.103.11 hAb and pembrolizumab had different epitopes in binding hPD-1 and blocking hPD-L1. The epitope of pembrolizumab did not intersect the PD-L1 binding site at all. In contrast, W3055-1.103.11 hAb had direct overlap with the hPD-L1 binding site.

EXAMPLE 3: Generation and characterization of anti-EGFR VHH antibody of W5626-2C10

Humanized W5626-2C10-z5 was obtained as described in PCT application No. : PCT/CN2018/079487, having a heavy chain variable region of SEQ ID NO: 9, and a human IgG1 constant region. As disclosed in PCT application No. : PCT/CN2018/079487, camelid animals were immunized with recombinant hFc tagged human EGFR ECD proteins. Peripheral blood mononuclear cells (PBMCs) from the 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 293F 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. The obtained lead VHH-hIgG1 Fc fusion antibody was named W5626-2C10-z5, and the obtained VHH antibody was named W5626-2C10. VHH.

W5626-2C10-z5 bound to cell surface human EGFR (EGFR transfected cells and A431 cell line) and EGFR-vIII in a dose dependent manner, as assessed by FACS. Specifically, W5626-2C10-z5 bound to cell surface human EGFR with an EC ₅₀ of 0.99 nM (vs. Erbitux with an EC ₅₀ of 0.55 nM) , to cell surface monkey EGFR with an EC ₅₀ of 0.58 nM (vs. WBP562-BMK3 (huML66 in Patent US20140023662 with human IgG1) has an EC ₅₀ of 1.47 nM) , to cell surface mouse EGFR with an EC ₅₀ of 1.06 nM (vs. WBP562-BMK3 has an EC ₅₀ of 2.42 nM) , and W5626-2C10. VHH bound to cell surface human EGFR-vIII with an EC ₅₀ of 1.08 nM (vs. Erbitux-scFv) (as assessed by FACS) . W5626-2C10-z5 could compete hEGF binding to cell surface hEGFR with IC ₅₀ at 2.58 nM, and that Erbitux has an IC ₅₀ of 1.01 nM, as measured by FACS.

W5626-2C10-z5 could inhibit EGFR phosphorylation in EGFR-expressing A431 cells with IC ₅₀ of 10.4 nM, as measured by ELISA. W5626-2C10-z5 induced EGFR internalization in a Fab-ZAP assay using luminescence measurement with EC ₅₀ at 0.21 nM, whereas Erbitux was tested to have an EC ₅₀ at 0.10 nM.

In the epitope binning test, W5626-2C10. VHH did not share the similar epitope bin with Erbitux or WBP562-BMK3 (based on the sequence of huML66 in US Patent US20140023662) .

The affinity of W5626-2C10-z5 to cell surface human EGFR was with a KD value of 1.1 nM, to cell surface monkey EGFR was with a KD value of 0.26 nM, to cell surface mouse EGFR was with a KD value of 0.51 nM, and to cell surface human hEGFRvIII was with a KD value of 0.24 nM, as measured by FACS. The affinity of W5626-2C10 to human EGFR extracellular domain were detected by SPR assay, and W5626-2C10 showed a KD value of 0.162 nM, whereas Erbitux showed a KD value of 0.308 nM.

W5626-2C10 was stable in thermal stability test measured by DSF assay and showed Tm value of 64.5 ℃. W5626-2C10 was also stable in serum stability test. No non-specific binding is observed to W5626-2C10 in a non-specific binding ELISA.

EXAMPLE 4: 1. Generation and characterization of BsAb antibodies

1.1 Generate soluble antigens

DNA sequences encoding the extracellular domain sequences of human EGFR (Uniport No. : P00533) , human PD-1 (Uniport No. : Q15116) , mouse PD-1 (Uniport No. : Q02242) , human PD-L1 (Uniport No. : Q9NZQ7) , mouse PD-L1 (Uniport No. : Q9EP73) were synthesized in Sangon Biothech (Shanghai, China) , and then subcloned into modified pcDNA3.3 expression vectors with different tag (such as 6xhis, human Fc, or mouse Fc) at C-terminus.

Expi293 cells (Invitrogen-A14635) were transfected with the purified expression vectors. Cells were cultured for 5 days and supernatant was collected for protein purification using Ni-NTA column (GE Healthcare, 175248) or Protein A column (GE Healthcare, 175438) or Protein G column (GE Healthcare, 170618) . The obtained human EGFR, human PD-1, mouse PD-1, human PD-L1, mouse PD-L1 were analyzed by SDS-PAGE and SEC, and then stored at -80 ℃.

1.2 Generation of parental antibody

Anti-EGFR VHH antibody W5626-2C10-z5 was obtained as described in Example 3, after immunizing camelid animals with human EGFR and mouse EGFR, and then was humanized and converted to human IgG1 format.

Anti-PD-1 antibody W3052-2E5 was generated as described in Example 1, after immunizing rats with human PD-1 and mouse PD-1, and then was humanized and converted to human IgG4 (S228P) format. Anti-PD-1 antibody W3055-1.103.11-v2 was generated as described in Example 2, after immunizing Omni rats with human PD-1, and then was converted to human IgG4 (S228P) format.

1.3 Generation of benchmark antibody

DNA sequence encoding the variable region of the benchmark anti-EGFR (WBP336-hBMK1, Erbitux, based on the sequence of clone “C225” in US Patent US7060808; WBP562-BMK3, based on the sequence of huML66 in US Patent US20140023662) , and anti-PD-1 antibodies ( “WBP305-BMK1” , i.e., Nivolumab (disclosed as clone of 5C4 in US9084776B2) were synthesized by Sangon Biotech (Shanghai, China) , then subcloned into modified pcDNA3.3 expression vectors with constant region of human IgG4 (S228P) . The IgG1 counterpart of WBP336-hBMK1 was also used, and referred to as WBP336-hBMK1 (IgG1) . The vectors were co-transfected into Expi293 cells. Cells were cultured for 5 days. Supernatant was collected for protein purification using the Protein A column (GE Healthcare, 175438) or the Protein G column (GE Healthcare, 170618) . The produced antibodies were tested by SDS-PAGE and SEC then stored at -80℃. An antibody that did not bind EGFR nor PD-1 and was of human IgG4 isotype was used as an isotype control.

1.4 Generation of Stable cell lines

Using Lipofectamine 2000, CHO-S or 293F cells were transfected 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 and mouse PD-1 high expression stable cell line were obtained by limited dilution.

The genes of human EGFR, human EGFRvIII, and macaca fascicularis EGFR were respectively inserted into modified expression vector pcDNA 3.3. The plasmids were then transfected to CHO-K1 cells by using Lipofectamine 2000, respectively. Stable single cell clones were isolated by limited dilution and screened by FACS using anti-EGFR antibodies.

1.5 Obtain and culture target-expressing tumor cell lines

A431 was purchased from ATCC (ATCC number: CRL-1555) and cultured in DMEM media with 10%fetal bovine serum (FBS) . The cells were incubated at 37 ℃, 5%CO ₂ incubator with routine subculturing. For long term storage, the cells were frozen in complete growth medium supplemented with 5% (v/v) DMSO and stored in liquid nitrogen vapor phase.

Bispecific antibody Generation

1.6 Construct expression vectors

Construction of bispecific antibodies: DNA sequence encoding the VHH of W5626-2C10-z5 at the N-terminus, followed by GGGGS linker (SEQ ID NO: 38) and the light chain of either W3052-2E5 (resulting in W3366-T2U3) or W3055-1.103.11-v2 (resulting in W3366-T2U8) at the C-terminus was cloned into modified pcDNA3.3 expression vector. DNA sequence encoding the heavy chain of W3052-2E5 or W3055-1.103.11-v2 was also cloned into modified pcDNA3.3 expression vector. Each of the bispecific antibodies was operably linked to human antibody constant region of an IgG4 isotype with S228P mutation.

1.7 Optimize bispecific antibodies

Based on the original construction (1.6) , the sequences of W3366-T2U3 was optimized to have a single F41Y mutation in the FR2 region of light chain of W3052-2E5, resulting in W3052-2E5 (F41Y) . The optimized BsAb was named as W3366-T2U3-D5.

1.8 Transfection, expression and purification

The sequences of W3366-T2U3-D5 and W3366-T2U8 were listed in Table 8, the DNA sequences of which were synthesized at Genewiz (Shanghai) and cloned into modified pcDNA3.3 expression vector. Heavy chain and light chain expression plasmids were co-transfected into Expi293 cells using Expi293 expression system kit (ThermoFisher-A14635) according to the manufacturer’s instructions. 5 days after transfection, the supernatant was collected and used for protein purification using Protein A column (GE Healthcare-17543802) . Antibody concentration was measured by Nano Drop. The purity of proteins was evaluated by SDS-PAGE and HPLC-SEC. Two bispecific antibodies, i.e. W3366-T2U3-D5 and W3366-T2U8 were obtained after expression and purification.

The result of W3366-T2U3-D5 and W3366-T2U8 transient production was shown in Figure 1. W3366-T2U3-D5 and W3366-T2U8 are produced with high purity and good yield in transient production.

2. In vitro characterization

2.1 Human PD-1-binding FACS

For FACS binding, human PD-1 transfected cells were seeded at 1×10 ⁵ cells/well in U-bottom 96-well plates. Serial dilutions of testing antibodies were added to the cells. Plates were incubated at 4 ℃ for 1 hour. After washing, PE-labeled goat anti-human IgG antibody was added to each well and the plates were incubated at 4℃ for 1 hour. The binding of the antibodies to the cell surface PD-1 was tested by flow cytometry and the mean fluorescence intensity (MFI) was analyzed by FlowJo. Four-parameter non-linear regression analysis was used to obtain EC ₅₀ values for cell binding using GraphPad Prism software.

The result of Human PD-1 binding (FACS) was shown in Figure 2A. W3366-T2U3-D5 and W3366-T2U8 bound to cell surface human PD-1 with EC ₅₀ of 0.50 and 0.39 nM, respectively, in comparison to WBP305BMK1 (EC ₅₀ of 0.28 nM) , as measured by FACS.

2.2 Mouse and cynomolgus PD-1-binding FACS

Mouse PD-1 and cyno PD-1 transfected cells were used for detecting the binding of W3366-T2U3-D5 and W3366-T2U8 to cell surface mouse and cyno PD-1, respectively, as described above.

The result of Mouse PD-1 binding (FACS) was shown in Figure 2B. W3366-T2U3-D5 bound to cell surface mouse PD-1. The result of Cyno PD-1 binding (FACS) was shown in Figure 2C. W3366-T2U3-D5 and W3366-T2U8 bound to cell surface cyno PD-1.

2.3 EGFR FACS binding

Binding of W3366-T2U3-D5 and W3366-T2U8 to EGFR expressing cells was determined by flow cytometry. Briefly, 1×10 ⁵ cells/well of A431 cells or cyno monkey EGFR transfected stable cell line or mouse EGFR transfected stable cell line were incubated for 60 minutes at 4 ℃ with serial diluted testing antibodies. After washing, cell surface bound antibodies were detected by incubating the cells with Fluorescence-labeled anti-human IgG antibody for 30 minutes at 4 ℃. The mean fluorescence (MFI) of stained cells was measured using a FACS Canto II cytometer (BD Biosciences) and analyzed by Flowjo. Four-parameter non-linear regression analysis was used to obtain EC ₅₀ values for cell binding using GraphPad Prism software.

The result of Human EGFR Binding (FACS) was shown in Figure 3A (W336-hBMK1 (IgG1) was used) . W3366-T2U3-D5 and W3366-T2U8 bound to cell surface human EGFR. The result of cyno EGFR Binding (FACS) was shown in Figure 3B. The result of mouse EGFR Binding (FACS) was shown in Figure 3C. W3366-T2U3-D5 and W3366-T2U8 bind to cell surface mouse EGFR.

2.4 PD-1 and EGFR dual binding (ELISA and FACS)

96-well ELISA plates (Nunc MaxiSorp, ThermoFisher) were coated overnight at 4 ℃ with antigen-1 (0.5 μg/mL of EGFR extracellular domain (ECD) ) or (0.5 μg/mL of biotinylated PD-1 ECD) in Carbonate-bicarbonate buffer. After blocking with 2%BSA-PBS for 1 hour, serial diluted testing antibodies were added into the plates and incubate for 1 hour at room temperature. After washing with PBS containing 0.5% (v/v) Tween 20 (PBST) , Antigen-2 (0.1μg/mL of biotinylated PD-1 ECD) or (0.25μg/mL of his tagged EGFR ECD) was added to plates respectively and incubated for 1 hour. After washing the plates for three times, Streptavidin-HRP (Invitrogen, #SNN1004) or anti-His-HRP (GenScript, #A00612) was added and incubated for 1 hour at room temperature. Finally, after washing, Tetramethylbenzidine (TMB) Substrate (Sigma-860336-5G) was added and the color reaction was stopped after approximate 10 minutes with 2 M HCl. The absorbance of the wells was measured at 450 nm with a multiwell plate reader (

M5 ^e) .

The ability of W3366-T2U3-D5 and W3366-T2U8 to bridge two target cells was tested by flow cytometry. 1×10 ⁶cells/mL of A431 cells or PD1 transfected stable cells were labeled with 50nM Calcein-AM (Invitrogen-C3099) or 20nM FarRed (Invitrogen-C34572) respectively for 30 minutes at 37 ℃. After washing, the two labeled cell lines were mixed to a final concentration of 1×10 ⁶ cells/ml at the ratio of 1: 1. Testing antibodies were added to the cells followed by gentle mixing and one hour incubation. Bridging %was calculated as the percentage of events that are simultaneously labeled Calcein-AM and FarRed.

The result of simultaneous dual target (EGFR and PD-1) binding by ELISA was shown in Figure 4A and by FACS is shown in Figure 4B. W3366-T2U3-D5 and W3366-T2U8 simultaneously bound to immobilized EGFR and PD-1 in solution with EC ₅₀ of 0.030 and 0.018 nM, respectively, and bound to immobilized PD-1 and EGFR in solution with EC ₅₀ of 0.026 and 0.009 nM, respectively, as measured by ELISA. W3366-T2U3-D5 and W3366-T2U8 can also simultaneously bound to two targets (EGFR and PD-1) on cell surface, as measured by FACS.

2.5 PD-1 /PD-L1 blocking (FACS)

Inhibition of human PD-L1 binding to human PD-1 expressing cells by W3366-T2U3-D5 and W3366-T2U8 was determined by flow cytometry. PD-1 transfected cells were seeded at 1×10 ⁵ cells/well in U-bottom 96-well plates. Serial diluted testing antibodies pre-mixed with 5μg/ml mouse Fc-tagged human PD-L1 protein (in house) were added to the cells. Plates were incubated at 4 ℃ for 1 hour. After washing, the binding of mouse Fc-tagged human PD-L1 to cell surface human PD-1 was detected by PE-labeled goat anti-mouse antibody. The competition binding of W3366-T2U3-D5 and W3366-T2U8 to the cells was tested by flow cytometry and the mean fluorescence intensity (MFI) was analyzed by FlowJo.

Inhibition of mouse PD-L1 binding to mouse PD-1 expressing cells by W3366-T2U3-D5 and W3366-T2U8 was performed as described above. Mouse PD-1 transfected cells and mouse Fc-tagged mouse PD-L1 protein (in house) were used.

As shown in Figure 5A, W3366-T2U3-D5 and W3366-T2U8 blocked the binding of human PD-L1 to cell surface human PD-1 with IC ₅₀ of about 0.3 nM. As shown in Figure 5B, W3366-T2U3-D5 did and W3366-T2U8 did not block the binding of mouse PD-L1 to cell surface mouse PD-1. This was consistent with its monoclonal counterpart antibodies.

2.6 EGFR /EGF blocking (FACS)

Inhibition of EGF binding to EGFR expressing cells by W3366-T2U3-D5 and W3366-T2U8 was determined by flow cytometry. Briefly, 1×10 ⁵ A431 (human EGFR+) cells or Mouse EGFR transfected cell line were incubated for 60 minutes at 4℃ with serial diluted testing antibodies and 0.1 μg/mL of biotin labeled EGF (Life Technology, #E3477, W562-hL1-Biotin) . After washing twice with 1%BSA-PBS, cell surface bound EGF was detected by incubating the cells with Streptavidin PE (Affymetrix, #12-4317-87) for 30 minutes at 4℃. MFI of stained cells was measured using a FACS Canto II cytometer (BD Biosciences) . Four-parameter non-linear regression analysis was used to obtain IC ₅₀ values for cell binding using GraphPad Prism software.

The result of Human EGF/EGFR blocking by FACS was shown in Figure 6A. The result of W3366-T2U3-D5 and W3366-T2U8 in Mouse EGF/EGFR blocking by FACS was shown in Figure 6B (W336-hBMK1 (IgG1) was used) . W3366-T2U3-D5 and W3366-T2U8 blocked the binding of EGF to cell surface human and mouse EGFR.

2.7 Affinity to PD-1 by SPR

Antibody binding affinity to human PD-1. ECD. his was detected by using surface plasmon resonance (SPR) assay using Biacore 8K. Each antibody was captured on anti-human IgG Fc antibody immobilized CM5 sensor chip (GE) . Different concentrations of human PD. 1. ECD. His were injected over the sensor chip at a flow rate of 30 uL/min for an association phase of 120 s, followed by 240 s or 600 s dissociation. The chip was regenerated by 10 mM glycine (pH 1.5) after each binding cycle.

The sensorgrams of blank surface and buffer channel were subtracted from the test sensorgrams. The experimental data were fitted by 1: 1 binding model. Molecular weight of 50 kDa was used to calculate the molar concentration of analyte human PD-1. ECD. his.

The result of affinity to PD-1 (SPR) was shown in Figure 7A. The affinities of W3366-T2U3-D5 and W3366-T2U8 to human PD-1 had KD values of 7.44x10 ^-09 M and 5.58 x10 ^-10 M.

2.8 Affinity to EGFR by FACS

A431 cells were transferred to 96-well U-bottom plates (BD) at a density of 5x10 ⁴ cells/well. Testing antibodies were 1: 3.16-fold serially diluted in 1%BSA-PBS and incubated with cells at 4 ℃ for 1 hr. The secondary antibody, Goat Anti-Human IgG Fc-FITC (Jackson Immunoresearch Lab 109-095-098) , was added to resuspended cells and incubated at 4 ℃ in the dark for 1 hr. The cells were then washed once and resuspended in 1%BSA-PBS for analysis by flow cytometry (BD) . Fluorescence intensity was converted to bound molecules/cell based on the quantitative beads (QuantumTM MESF Kits, Bangs Laboratories) . KD was calculated using Graphpad Prism5.

The affinities of W3366-T2U3-D5 and W3366-T2U8 to cell surface human EGFR had KD values of 8.5 x10 ^-10 M and 7.0 x10 ^-10 M (Figure 7B) .

2.9 Mixed lymphocyte reaction (MLR)

Human PBMCs were freshly isolated from healthy donors using Ficoll-Paque 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 according to the manufacturer’s instructions. Cells in complete RPMI-1640 medium supplemented with 800 U/mL recombinant human GM-CSF and 50 ng/mL rhIL-4. was seeded at a concentration of 2×10 ⁶ cells/mL, 2.5 mL/well in 6-well plates. The cells were cultured for 5 to 7 days to differentiate into DCs. Cytokines were replenished every 2-3 days by replacing half of the media with fresh media supplemented with cytokines. Eighteen to twenty-four hours before MLR, 1 μg/mL LPS was added to the culture to induce DCs maturation.

Human CD4 ⁺ T cells were isolated using Human CD4 ⁺ T cell Enrichment kit according to the manufacturer’s protocol.

For human allogeneic MLR, purified CD4 ⁺ T cells were co-cultured with immature or mature allogeneic DCs (iDCs or mDCs) . MLR was set up in 96-well round bottom plates using complete RPMI-1640 medium. CD4 ⁺ T cells, various concentrations of antibodies, and iDC or mDC were mixed and added to the plates. The plates were incubated at 37℃, 5%CO ₂. IL-2 and IFN-γ production was determined at day 3 and day 5, respectively.

Human IL-2 and IFN-γ releases in the culture system were measured by ELISA using matched antibody pairs. Recombinant human IFN-γ and IL-2 were used as standards, respectively. The plates were pre-coated with human IL-2 or IFN-γ capture antibody overnight at 4℃. After blocking, 100 μL of 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 for corresponding cytokine were added to the wells and incubated for 1 hour. HRP-streptavidin was then added to the wells for 30 minutes incubation at ambient temperature. The color was developed by dispensing 100 μL of TMB substrate, and then stopped by 100 μL of 2M HCl. The absorbance was read at 450 nM using a Microplate Spectrophotometer.

The result of Human IL-2 production in Allo-MLR assay was shown in Figure 8A. W3366-T2U3-D5 and W3366-T2U8 promoted IL-2 production in a dose-dependent manner in human MLR assay. The result of Human IFN-γ production in Allo-MLR assay was shown in Figure 8B. W3366-T2U3-D5 and W3366-T2U8 promoted IFN-γ production in a dose-dependent manner in human MLR assay.

2.10 EGFR phosphorylation inhibition in A431 by pELISA assay

A431 cells in culture were trypsinized and diluted to 5×10 ⁵ cells/mL. 100μL of cell suspension was then added to each well of a 96-well clear flat bottom microplate (Corning-3599) to give a final density of 5×10 ⁴ cells/well. A431 cells were allowed to attach for approximately 18 hours before the media was exchanged for starvation media without fetal bovine serum. All plates were incubated overnight at 37 ℃ prior to treatment with testing antibodies in the presence of 200 ng/ml EGF (Sino Biological-10605-HNAE) for 2 hours at 37 ℃. All media was gently aspirated and the cells were washed with ice-cold DPBS (GE-Healthcare-SH30028) . The cells were then lysed by ice-cold lysis buffer (R&D System-DYC002) supplemented with 10 μg/ml Aprotinin (Thermo-Prod78432) and Leupeptin hemisulfate (Santa Cruz Biotechnology-SC-295358) on ice for 15 minutes. The cell lysates were stored at -80 ℃ until analysis with pELISA assay.

For pELISA assay, 96-well ELISA plates (Nunc MaxiSorp, ThermoFisher) were coated overnight at room temperature with 8 μg/mL of human EGFR capture antibody (R&D Systems-DYC1095B) . After washing and blocking with 1%BSA-PBS, the lysate supernatants collected by centrifugation were added into the plates and incubated for 2 hours at room temperature. After washing the plates with PBST, phosphorylated EGFR was detected using anti-Phospho-tyrosine-HRP (R&D Systems-DYC1095B) by incubating at room temperature for 1 hour. The wells were washed with PBST for three times and substrate mixture (R&D Systems-DY999) was added. The reaction was stopped after approximate 10 minutes with 2 M HCl. The absorbance of each well was measured at 450 nm with a multiwall plate reader (

M5 ^e) . Four-parameter non-linear regression analysis was used to obtain IC ₅₀ values for EGFR phosphorylation inhibition using GraphPad Prism software.

The result of EGFR phosphorylation inhibition in A431 was shown in Figure 9. W3366-T2U3-D5 and W3366-T2U8 inhibited EGF induced EGFR phosphorylation in A431 cells with an IC ₅₀ of 23.70 nM and 15.71 nM, respectively, which was slightly higher than that of WBP336-hBMK1 (IC ₅₀ of 5.412 nM) .

2.11 Tumor cell proliferation inhibition assay

In vitro growth inhibition of tumor cells were assayed with Cell Titer Glo Kit (Promega-G7573) . In brief, A431cells were trypsinized and diluted to 5×10 ⁴ cells/mL with growth media and then plated into 96-well clear bottom black plates (Greinier-655090) at a density of 2500 cells/well. Serial dilutions of testing antibodies were added and incubated for 5 days. For assessing cell viability, 50μL of Cell Titer Glo solution was added to each well and incubated at room temperature for 10 minutes. The amount of luminescence was determined using Envision (PerkinElmer) . The extent of growth inhibition obtained with each antibody was calculated by comparing the luminescence values obtained from cell only control wells.

The result of A431 proliferation inhibition was shown in Figure 10. W3366-T2U3-D5 and W3366-T2U8 inhibited A431 cell proliferation with IC ₅₀ of 3.418 nM and 4.201 nM, respectively, in comparison to WBP336-hBMK1 (IC ₅₀ 4.494 nM) .

2.12 ADCC and CDC assays on PD-1 ⁺ cells

In order to test ADCC effect, the target cells (activated human CD4 ⁺ T cells or human PD-1-transfected cells) were pre-mixed with various concentrations of the testing antibodies and incubated in 96-well plate for 30 minutes. Then fresh isolated PBMCs as effector cells were added at the effector/target ratio of 20: 1. The plate was kept at 37℃ in a 5%CO ₂ incubator for 4 hours. Target cell lysis was determined by LDH-based cytotoxicity detection kit. The absorbance was read at 492 nM using a Microplate Spectrophotometer.

For CDC assay, human activated CD4 ⁺ T cells or human PD-1 transfected cells and various concentrations of testing antibodies were mixed in 96-well plate. The human complement was then added into the plates at the dilution ratio of 1: 50 and the plates were incubated at 37℃ in a 5%CO ₂ incubator for 2 hours. Target cell lysis was determined by CellTiter-Glo.

The result of ADCC and CDC assays on PD-1 expressing cells was shown in Figure 11A-F. W3366-T2U3-D5 and W3366-T2U8 did not induce ADCC (Figure 11A-B) and CDC (Figure 11D-E) on PD-1 expressing cells (activated CD4+ T and hPD-1 transfected cells) , while the positive control did induce ADCC (Figure 11C) or CDC (Figure 11F) .

2.13 ADCC and CDC assays on A431 cells

For ADCC assay, in brief, human NK cells, which was extracted from human PBMC by Stem cell CD56 positive selection kit, were incubated overnight in RPMI1640 medium containing 10%fetal bovine serum, 1%penicillin/streptomycin solution and 50 unit/mL hIL-2. The next day, NK cells were used as effector cells and A431 was used as target cells. NK cells and A431 cells at a E/T ratio of 10: 1 were mixed and incubated at 37℃for 4 hours. Cell death was then evaluated using LDH Cytotoxicity Detection Kit (Roche) according to manufacturer’s instructions.

For CDC assay, 4x10 ⁴ A431 cells in 50 μL RPMI1640 (no phenol) medium containing 1%FBS were added per well in a 96-well U-bottom plate. Then, serial-diluted antibodies were added in the plates and followed by adding of 50 fold dilution of complement. After incubation at 37℃ for 4 hours, cell death was evaluated using LDH Cytotoxicity Detection Kit (Roche) according to manufacturer’s instructions.

The results of ADCC and CDC assays on A431 cells were shown in Figure 11G-H. W3366-T2U3-D5 and W3366-T2U8 did not induce ADCC and CDC on EGFR expressing cells, but the positive controls WBP336-hBMK1. IgG1 induced ADCC (Figure 11G) .

2.14 Serum stability

Freshly collected human blood was statically incubated in polystyrene tubes without anticoagulant for 30 minutes at room temperature. Serum was collected after centrifugation the blood at 4000 rpm for 10 minutes. The centrifugation and collection steps were repeated until the serum was clarifying. The antibodies gently mixed with serum at 37 ℃ for 14 days, and aliquots were drawn at the indicated time points: 0 day, 1 day, 4 days, 7 days and 14 days, and the aliquots were quickly-frozen into liquid nitrogen and store them at -80℃ until use. The samples were used to assess their binding ability to PD-1 transfected cells and EGFR expressing A431 cells. PE-labeled goat anti-human IgG was used to detect the binding of antibodies to the cells. MFI was measured by a flow cytometer and analyzed by FlowJo.

The results of Serum Stability were shown in Figures 12A and 12B. W3366-T2U3-D5 and W3366-T2U8 were stable in human serum at 37℃ for at least 14 days.

2.15 Thermal stability by DSF assay

A DSF assay was performed using Real-Time Fluorescent Quantitative PCR (QuantStudio 7 Flex, Thermo Fisher Scientific) . Briefly, 19 μL of antibody solution was mixed with 1 μL of 62.5 X SYPRO Orange solution (Invitrogen) and added to a 96 well plate (Biosystems) . The plate was heated from 26 ℃ to 95 ℃ at a rate of 2 ℃/min, and the resulting fluorescence data were collected. The negative derivatives of the fluorescence changes with respect to different temperatures were calculated, and the maximal value was defined as melting temperature T _h. If a protein had multiple unfolding transitions, the first two T _h were reported, named as Tm ₁ and Tm ₂. Tm ₁ was always interpreted as the formal melting temperature Tm to facilitate comparisons between different proteins. Data collection and T _h calculation were conducted automatically by its operation software. (QuantStudio Real-Time PCR PCR Software v1.3) .

The result of thermal stability by DSF was shown in Figure 13. W3366-T2U3-D5 and W3366-T2U8 had normal DSF profile and Tm were 62.3 and 64.3 ℃, respectively.

2.16 Non-specific binding (ELISA/FACS)

Non-specific binding ELISA was performed in 96-well high binding plates (Nunc-Immuno Plate, Thermo Scientific) . The plate was coated with various antigens at 2 μg/mL overnight at 4℃. After blocking with 2%BSA-PBS, 10 μg/ml antibodies were added to the plate and incubated for 2 hours. The plates were subsequently incubated with the secondary antibody goat anti human IgG Fc-HRP (Bethyl) for additional 1 hour. The HRP signal was detected by adding TMB peroxidase substrate and the reaction was stopped after 12 minutes using 2M HCl. The absorbance at 450 nm was read using a microplate reader (Molecular Device) . All incubation steps were performed at room temperature. The plate was washed with PBST between steps.

For non-specific binding FACS, various cell lines were used. Briefly, the viable cells were centrifuged at 1500rpm for 4 minutes and then re-suspended in an appropriate volume of 1%BSA-PBS to the concentration of 1x 10 ⁶ cell/ml. 100 μl cell suspension was added into each well of 96-well U-plate. After centrifugation, the cells were re-suspended with 100 μl/well diluted antibodies at 10 μg/ml in 1%BSA-PBS. After incubation at 4℃ for 1 hour, the cells were washed twice with 1%BSA-PBS then incubated with 5 μg/ml goat anti-human IgG Fc-PE (Jackson, 109-115-098 &126973) at 4℃ for 30 minutes. After two times of washing, the cells were re-suspended in 100 μl/well 1%BSA-PBS and kept at 4℃ in the dark until FACS analysis (BD Canto II) . W3366-T2U3-D5 and W3366-T2U8 have no non-specific binding tested by ELISA and FACS as shown in Table 9.

Table 9: the summary of Non-specific binding (ELISA, FACS)

3. In vivo efficacy study in MBT-2 syngeneic model

In this study, the therapeutic efficacy of antibody W3366-T2U3-D5 was evaluated in mouse MBT-2 cancer model, since this antibody cross-reacts with mouse targets. Female C3H mice (Shanghai Slack Laboratory Animal Co., Ltd) of 6-8 week-old were used in the study. The MBT-2 tumor cells were maintained in vitro as a monolayer culture in RPMI-1640 medium supplemented with 10%fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin at 37℃ in an atmosphere of 5%CO ₂ in air. The tumor cells were routinely sub-cultured twice weekly by trypsin-EDTA treatment. The cells growing in an exponential growth phase were harvested and counted for tumor inoculation.

For the therapeutic efficacy study, each mouse was inoculated subcutaneously at the right axillary (lateral) with MBT-2 tumor cell (4×10 ⁵) in 0.1 ml of PBS for tumor development. The animals were randomly grouped as 6 mice per group when the average tumor volume reached 78 mm ³, then treatment started for the efficacy study. The treatment groups include vehicle PBS control, equal molar dose of anti-PD-1 antibody W3052-2E5, anti-EGFR antibody W5626-2C10-z5, W3366-T2U3-D5, as well the combo of fore-mentioned anti-PD-1 antibody W3052-2E5 and anti-EGFR antibody W5626-2C10-z5. The animals were treated with PBS or antibodies intra-peritoneally two time per week for total 6 injections. The day of the first injection was considered as day 0.

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

The major endpoint is to see if the tumor growth can be delayed or mice can be cured. Tumor sizes will be measured three times weekly in two dimensions using a caliper, and the volume will be expressed in mm ³ using the formula: V = 0.5 a x b ² where a and b are the long and short diameters of the tumor, respectively. Data was collected using the Stud Director ^TM (version 3.1.399.19, vendor Study log System, Inc., S. San Francisco, CA, USA) software. The results were represented by mean and the standard error (Mean ± SEM) . Data were analyzed using Two way ANOVA Bonferroni posttests with Prism and p<0.05 was considered to be statistically significant.

As shown in Figure 14, all mice were normal during the experiment and slowly gained weight, suggesting that antibodies (W3052-2E5, W5626-2C10-z5, W3052-2E5 +W5626-2C10-z5 and W3366-T2U3-D5, respectively) were not toxic. At the 15 days after the first dosing, the tumor volume of vehicle group reached ～3000mm ³, which indicated MBT2 model in C3H mice was well established. Compared with vehicle control, all antibody (W3052-2E5, W5626-2C10-z5, W3052-2E5 + W5626-2C10-z5 and W3366-T2U3-D5, respectively) treatment could significantly inhibit tumor growth from day 11 to day 15. The combination of anti-PD-1 and anti-EGFR (W3052-2E5 + W5626-2C10-z5) , as well as W3366-T2U3-D5 displayed synergic antitumor effects.

4. A Pharmacokinetics Study Following Single Intravenous Administration to mouse

10 C57BL/6 mice (5 animal/group) were divided into two groups and administered with W3366-T2U3-D5 and W3366-T2U8 at 10 mg/kg once by intravenous bolus administration, respectively. The formulations were formulated in 20mM Histidine, 8% Sucrose, 0.02%PS80, pH5.5. PK blood samples were collected Blank, 5 min, 0.5 h, 2 h, 6 h, 24h, Day 2, Day 3, Day 7, Day 14, Day 21. Serum concentrations of W3366-T2U3-D5 and W3366-T2U8 were determined by ELISA.

The concentration of W3366-T2U3-D5 and W3366-T2U8 in all formulations met the acceptance criteria. All animals tolerated W3366-T2U3-D5 and W3366-T2U8 well during the entire course of the study. No adverse effects were observed during the in-life phase of the study. The concentrations of W3366-T2U3-D5 and W3366-T2U8 in serum were determined by using a fully validated bioanalytical ELISA method. The serum concentration of W3366-T2U3-D5 and W3366-T2U8 in mouse was subjected to a non-compartmental pharmacokinetic analysis by using the Phoenix WinNonlin software (version 6.3, Pharsight, Mountain View, CA) . The linear/log trapezoidal rule was applied in obtaining the PK parameters.

All BLQ values were excluded from the PK parameters calculations. Individual BLQ was excluded from the calculation of the mean concentrations. The nominal dose levels and nominal sampling times were used in the calculation of all pharmacokinetic parameters. The summary for PK parameters was listed in the Table 10. The mean plasma concentration of W3366-T2U3-D5 and W3366-T2U8 were shown in Figure 15.

Table 10. The summary of mouse PK parameters (mean)

The results showed that the systemic exposure for T _1/2 was 26.7h and 109h for W3366-T2U3-D5 and W3366-T2U8 at the dosage of 10 mg/kg. The C _max was 208 μg/mL and 147 μg/mL. The AUC _0-last was 1582 h*μg/mL and 1492 h*μg/mL.

Abbreviations

AUC The area under the serum concentration-time curve

AUC _0-last The area under the serum concentration-time curve from time zero to the last

quantifiable concentration

AUC _0-inf The area under the serum concentration-time curve from time zero

extrapolated to infinity were calculated using the linear/log trapezoidal rule

C ₀ Maximum serum concentration

CL Total body clearance

MRT Mean residence time

MRT _0-last Mean residence time from time zero to the last quantifiable concentration

MRT _0-inf Mean residence time from time zero to infinity

T _1/2 Half-life

T _max Time to reach C _max

Vdss Volume of distribution at steady state

Claims

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

the EGFR-binding domain comprises:

1, 2, or 3 heavy chain complementarity determining region (CDR) sequences selected from the group consisting of: SEQ ID NOs: 1-3; 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: 8, 10, 12, 19, 21, 23; and/or

1, 2, or 3 light chain CDR sequences selected from: SEQ ID Nos: 9, 11, 13, 20, 22, 24, 25,

the EGFR-binding domain comprises a VHH domain; and

the PD-1-binding domain comprises a Fab.
The bispecific antibody molecule of claim 1, wherein the EGFR-binding domain comprises a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 1-3.
The bispecific antibody molecule of any of the preceding claims, wherein the EGFR-binding domain comprises a heavy chain variable region selected from SEQ ID NO: 4, SEQ ID NO: 6, and a homologous sequence thereof having at least 80%sequence identity yet retaining specific binding affinity to EGFR.
The bispecific antibody molecule of any of the preceding claims, wherein the EGFR-binding domain comprises a heavy chain variable region comprising SEQ ID NO: 6.
The bispecific antibody molecule of any of the preceding claims, wherein the PD-1-binding domain comprises a heavy chain variable region selected from:

a) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 19, 21, 23; and

b) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 8, 10 and 12, and/or

a light chain variable region selected from:

c) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 20, 22 and 24;

d) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 20, 22 and 25; and

a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 9, 11 and 13.
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: 19, 21 and 23; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 20, 22 and 24;

b) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 19, 21 and 23; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 20, 22 and 25; or

c) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 8, 10 and 12; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 9, 11 and 13.
The bispecific antibody molecule of any of the preceding claims, wherein the PD-1-binding domain comprises a heavy chain variable region selected from SEQ ID NOs: 14 and 26 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 SEQ ID NOs: 16, 18, 28 and 30, 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: 26 and a light chain variable region comprising SEQ ID NO: 28;

b) a heavy chain variable region comprising SEQ ID NO: 26 and a light chain variable region comprising SEQ ID NO: 30;

c) a heavy chain variable region comprising SEQ ID NO: 14 and a light chain variable region comprising SEQ ID NO: 16; or

d) a heavy chain variable region comprising SEQ ID NO: 14 and a light chain variable region comprising SEQ ID NO: 18.
The bispecific antibody molecule of any of the preceding claims, wherein the EGFR-binding domain further comprises one or more amino acid residue substitutions or modifications yet retains specific binding affinity to EGFR, 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 claim 10, 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 VL sequences but not in any of the CDR sequences.
The bispecific antibody molecule of any of the preceding claims, further comprising 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 EGFR-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 EGFR-binding domain comprises the sequence of SEQ ID NO: 6, and the PD-1-binding domain comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 14 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 EGFR-binding domain comprises the sequence of SEQ ID NO: 6, and the PD-1-binding domain comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 26 and a light chain variable region comprising the sequence of SEQ ID NO: 30.
The bispecific antibody molecule of any of the preceding claims, wherein the EGFR-binding domain is operably linked to the N terminus of the light 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, associated with a light chain in the format of VHH (anti-EGFR) -spacer-VL (anti-PD-1) -CL.
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: 35.
The bispecific antibody molecule of claim 17, comprising a heavy chain comprising the sequence of SEQ ID NO: 33 and a light chain comprising the sequence of SEQ ID NO: 32.
The bispecific antibody molecule of any of the preceding claims, wherein the EGFR-binding domain and/or the PD-1-binding domain is humanized.
The bispecific antibody molecule of any of the preceding claims, which is 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: 5, 7, 15, 17, 27, 29 and 31, 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 30 under the condition at which the vector of claim 33 is expressed.
A method of treating a disease or condition in a subject that would benefit from upregulation 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 upregulation 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 any of claims 29 or 30, wherein the disease or condition is PD-1-related and/or EGFR-related.
The method of any of claims 29-31, wherein the disease or condition is PD-1 related disease or condition is cancer, autoimmune disease, inflammatory disease, or infectious disease.
The method of any of claims 29-32, wherein the EGFR-related disease or condition is cancer or inflammatory disease.
The method of claim 32 or 33, wherein 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 claim 34, wherein the EGFR-related disease or condition is cancer or inflammatory disease, and wherein the cancer is colorectal cancer, skin cancer, head and neck cancer, non-small cell lung cancer, gastrointestinal cancer, glioblastoma, melanoma, breast cancer, cervical cancer, ovarian cancer, endometrial cancer, prostate cancer, renal cancer, bladder cancer, esophageal cancer, brain cancer, liver cancer, pancreatic cancer, hepatocellular cancer, or squamous cell carcinoma; and wherein the inflammatory disease is Alzheimer's disease, asthma, atopic allergy, allergy, atherosclerosis, bronchial asthma, eczema, glomerulonephritis, graft vs. host disease, hemolytic anemias, osteoarthritis, psoriasis, sepsis, stroke, transplantation of tissue and organs, vasculitis, diabetic retinopathy or ventilator induced lung injury.
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 EGFR activity in an EGFR-expressing cell, comprising exposing the EGFR-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 EGFR-related.