WO2022164791A1

WO2022164791A1 - Tumor necrosis factor alpha recombinant antibody

Info

Publication number: WO2022164791A1
Application number: PCT/US2022/013677
Authority: WO
Inventors: Hwei-Jiung WANG; Cheng-Chung Lee; Wen-Chih KUO
Original assignee: Pharmtekx Co., Ltd.
Priority date: 2021-01-29
Filing date: 2022-01-25
Publication date: 2022-08-04
Also published as: TWI821881B; TW202229341A

Abstract

The present invention discloses a recombinant antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises an amino acid sequence at least 50% but not 100% identical to a light chain variable region of adalimumab. The recombinant antibody is applicable for treatment of dog's osteoarthritis under administration of a low dosage without provoking side effects.

Description

TUMOR NECROSIS FACTOR ALPHA RECOMBINANT ANTIBODY FIELD OF THE INVENTION

The invention is related to an antibody and uses thereof, especially related to a recombinant antibody, and the antibody is for immunotherapy of tumor necrosis factor-a. BACKGROUND

Tumor necrosis factor-a (TNFa) is a potent pleiotropic cytokine and plays a central role to protect cells from microbial pathogens infection or endogenous stress. It is a homotrimeric type-II membrane protein of the TNF superfamily. After binding to the receptors, TNFa mediate apoptosis, differentiation or proliferation through the activation of pathways involving NK-kB, JUN N-terminal kinase (JNK), p42/p44 mitogen-activated kinase (MAPK) and p38 MAPK. Macrophages are the major producers of TNFa and are also highly responsive to TNFa. The normal function of TNFa such as immune responses, haematopoiesis and morphogenesis. It has also been implicated in tumorigenesis, transplant rejection, septic shock, viral replication, bone resorption, rheumatoid arthritis and diabetes.

Aberrant TNFa production and TNF receptor signaling have been associated with the pathogenesis of several diseases, which including rheumatoid arthritis, Crohn’s disease, atherosclerosis, psoriasis, sepsis, diabetes, and obesity. The increased presence of TNFa and its receptors in articular cartilage with mild osteoarthritic changes suggests a role in the development of early osteoarthritis (OA). Regulating TNFa may be an important component in the treatment of canine OA.

Arthritis is a common problem for many dogs, especially for older dogs. In fact, one in five dogs will experience arthritis in their lifetime. It is caused by the inflammation of the joints, which causes pain, discomfort and stiffness. Inside a dog’s joints, bone surfaces are normally covered with a thin layer of very smooth cartilage, lubricated with a small amount of joint fluid that allows the two surfaces to glide freely over one another with minimum friction. In dogs with arthritis, cartilage within the joint is damaged, causing discomfort to dog, and further deterioration to cartilage. Eventually it turns into a condition known as degenerative joint disease. It is difficult to remodel an arthritic joint without surgical intervention, but reduction of joint inflammation and pain can be attempted. Therefore, the main medications for arthritis dogs are the pain controlling. NASIDs (Nonsteroidal Anti-Inflammatory Drugs) like Etogesic, Rimadyl, Metacam and Deramaxx have been designed specifically for dogs. However, these NSAIDs can still cause gastrointestinal upset, and in rare cases, liver or kidney dysfunction. Other pain-relieving medications like tramadol, amantadine, prednisone, dexamethasone and other corticosteroids will markedly reduce swelling and inflammation in arthritic joints. However, there is a downside to the use of steroid for long-term palliation of arthritis, which is why veterinarians don’t prescribe corticosteroids for arthritis in dogs as often as they used to in the past. Therefore, it is actually necessary to develop exclusive canine medicines for dogs, which can not only have a good therapeutic response on the target diseases, but also reduce the adverse side effects that enter the dog’s body.

Among the monoclonal antibodies available for anti-inflammation therapy, there are several targeting TNFs in clinical use. Among them, Adalimumab (trade name Humira) is the best- selling drug with annual sales over USD 10 billion in 2019. It is a recombinant, fully human IgGl monoclonal antibody and subcutaneously administered biological disease modifier.

The second drug, Humira, was originally launched by Abbvie in the US and approved in 2002 by the FDA. This drug is available in a prefilled syringe form and convenient pen form for subcutaneous self-administered doses. However, due to the importance of TNF in host defense, one of the issues of major concern with all TNF-blockers, including adalimumab, is the increased risk of infections and malignancies.

SUMMARY OF THE INVENTION

Osteoarthritis is a progressive disease and there is no known cure for dogs. Preventing the development of osteoarthritis through diet, exercise and the use of protective joint supplements is the best way to keep dog’s joints healthy. However, supplementation usually has no significant effect on alleviating animal symptoms, and it is also a huge expense for the owner. In addition to the use of joint supplements, pain control is also a mainstay of osteoarthritis treatment in dogs. The most commonly used pain control medications for more severe osteoarthritis are Non-Steroidal Anti-Inflammatory Drugs (NSAIDs). NSAIDs can not only reduce pain, but also decrease inflammation in the joints. Nonetheless, in recent years, chronic use of NSAIDs has been linked to numerous side effects, including gastrointestinal (GI) bleeding, and renal and hepatic dysfunction. Antiinflammation drugs such as aspirin and ibuprofen are non-specific inhibitors of COX enzymes. They inhibit the production not only of inflammatory prostaglandins, but also of constitutive prostaglandins, resulting in side effects, such use GI bleeding. Therefore, safer therapy is needed for arthritic dogs.

As arthritis is a common disease in dogs, an anti-TNFa monoclonal would be useful. In the present invention, complementarity-determining regions (CDRs) of Humira are grafted on the canine antibody framework to make a new anti-TNFa monoclonal antibody for dogs.

In one aspect, the present invention provides a recombinant antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises an amino acid sequence at least 50% but not 100% identical to a light chain variable region of adalimumab.

In various embodiments, the heavy chain variable region comprises an amino acid sequence at least 50% but not 100% identical to a heavy chain variable region of adalimumab.

In various embodiments, the light chain comprises an amino acid comprising a first region at residue 1 to 12 from N-terminus, a second region at residue 18 to 54 from N- terminus, a third region at residue 56 to 84 from N-terminus and a fourth region at residue 86 to 108 from N-terminus, wherein the first region comprises an amino acid sequence identical to DIVMTQSPASLS; the second region comprises an amino acid sequence identical to TVTITCRASQGIRNYLAWYQQKPGQAPKLLIYAASTL; the third region comprises an amino acid sequence identical to TGVPSRFSGSGSGTDFSLTISSLEPEDVAVYYCQHYNRAPYTFGQGTKVELKR; the fourth region comprises an amino acid sequence identical to

YYCQHYNRAPYTFGQGTKVELKR. In some embodiments, the light chain further comprises a fifth region at residue 110 to 217 from N-terminus comprising an amino acid sequence identical to DAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGIQESV TEQDKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSECQRVD.

In preferred embodiments, the light chain comprises an amino acid sequence identical to a sequence selected from the group consisted of SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 18; more preferably, the light chain comprises an amino acid sequence identical to SEQ ID NO: 12.

In various embodiments, the heavy chain comprises an amino acid comprising a sixth region at residue 1 to 15 from N-terminus, a seventh region at residue 24 to 48 from N-terminus, an eighth region at residue 51 to 62 from N-terminus, a ninth region at residue 67 to 77 from N-terminus and a tenth region at residue 89 to 115, wherein the sixth region comprises an amino acid sequence identical to EVQLVESGGGLVQPG; the seventh region comprises an amino acid sequence identical to ASGFTFDDYAMHWVRQAPGKGLEWV; the eighth region comprises an amino acid sequence identical to ITWNSGHIDYAD; the ninth region comprises an amino acid sequence identical to RFTISRDNAKN; the tenth region comprises an amino acid sequence identical to EDTAVYYCAKVSYLSTASSLDYWGQGT.

In some embodiments, the heavy chain further comprises an eleventh region at residue 117 to 452 from N-terminus, wherein the eleventh region comprises an amino acid sequence identical to

VTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVH TFPSVLQSSGLHSLSSMVTVPSSRWPSETFTCNVVHPASNTKVDKPVFNECRCTD TPPCPVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFVDGK EVHTAKTQSREQQFNGTYRVVSVLPIEHQDWLTGKEFKCRVNHIDLPSPIERTISK ARGRAHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPERKH RMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVMHETLQNHYTDLSLSHSP GK. In preferred embodiments, the heavy chain comprises an amino acid sequence identical to a sequence selected from the group consisted of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO: 7 and SEQ ID NO: 9; more preferably, the heavy chain comprises an amino acid sequence identical to SEQ ID NO:6.

In further preferred embodiments, the heavy chain comprises an amino acid sequence identical to a sequence selected from the group consisted of SEQ ID NO: 3, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:9 and the light chain comprises an amino acid sequence identical to a sequence selected from the group consisted of SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 18; more preferably, the heavy chain comprises an amino acid sequence identical to SEQ ID NO: 6 and the light chain comprises an amino acid sequence identical to SEQ ID NO: 12.

In other aspect, the present invention provides a method for neutralizing TNFa, and the method comprises administrating an effective concentration of antibody to a subject in need, wherein the antibody comprises the aforementioned recombinant antibody.

Preferably, the effective concentration ranges from 0.04-1.0nM; more preferably, the effective ranges from 0.5-0.8nM.

In the present invention, the recombinant antibody can be obtained construction based on computer modeling for simulation of the binding orientation of desired antibody. The recombinant antibody demonstrated high specificity on canine TNFa and highly effective with low dosages.

The recombinant antibody disclosed in the present invention shows higher binding affinity stronger neutralization ability in comparison with commercialized monoclonal antibody in current market.

The recombinant antibody in the present invention could reduce probability of unexpected side effects and promote medication safety because a far less dosage is required for treatment of osteoarthritis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the sequence alignments of heavy chains among all designed recombinant antibody in the present invention. FIG. 2 shows the sequence alignments of light chains among all designed recombinant antibody in the present invention.

FIG. 3 shows the sequence alignments of heavy chains of Humivet series in examples. FIG. 4 shows the sequence alignments of light chains of Humivet series in examples.

FIG. 5 shows validation of the biological activity of recombinant canine TNFa in HEK- Dual™ TNFa cells.

FIG. 6 shows binding affinities of IgG variants, wherein ELISA assay was performed to compare the binding affinity in different version of IgGs; the IgGs were diluted from 117 to O.OnM and incubated on 96-well plates coated with TNFa for 60 mins.

FIG. 7 shows inhibitory effect of caninized antibody Version Humivet-3.0 series in HEK- Dual™ TNFa cells.

FIG. 8 shows inhibitory effect of caninized antibody Humivet-3.0 in HEK-Dual™ TNFa cells.

DETAILED DESCRIPTION

In one aspect, the present disclosure provides a recombinant antibody which specifically binds to canine TNFa; the recombinant antibody comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises an amino acid sequence at least 50% but not 100% identical to a light chain variable region of adalimumab. Preferably, the light chain variable region comprises an amino acid sequence 50, 60, 70, 80, 90 or 95% identical to a light chain variable region of adalimumab.

In various embodiments, the light chain comprises an amino acid comprising a first region at residue 1 to 12 from N-terminus, a second region at residue 18 to 54 from N- terminus, a third region at residue 56 to 84 from N-terminus and a fourth region at residue 86 to 108 from N-terminus, wherein the first region comprises an amino acid sequence identical to DIVMTQSPASLS; the second region comprises an amino acid sequence identical to TVTITCRASQGIRNYLAWYQQKPGQAPKLLIYAASTL; the third region comprises an amino acid sequence identical to TGVPSRFSGSGSGTDFSLTISSLEPEDVAVYYCQHYNRAPYTFGQGTKVELKR; the fourth region comprises an amino acid sequence identical to YYCQHYNRAPYTFGQGTKVELKR.

In some embodiments, the light chain further comprises a fifth region at residue 110 to 217 from N-terminus comprising an amino acid sequence identical to DAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGIQESV TEQDKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSECQRVD.

In various embodiments, the light chain comprises an amino acid sequence identical to a sequence selected from the group consisted of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO:20; please refer to FIG. 1 illustrating alignment of amino acid sequences of light chain among all designed recombinant antibody in the present invention.

In preferred embodiments, the light chain comprises an amino acid sequence identical to a sequence selected from the group consisted of SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 18.

In further preferred embodiments, the light chain comprises an amino acid sequence identical to SEQ ID NO: 12.

In another aspect, the present disclosure provides a recombinant antibody which specifically binds to canine TNFa; the recombinant antibody comprises a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence at least 50% but not 100% identical to a heavy chain variable region of adalimumab. Preferably, the heavy chain variable region comprises an amino acid sequence 50, 60, 70, 80, 90 or 95% identical to a heavy chain variable region of adalimumab.

VTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVH TFPSVLQSSGLHSLSSMVTVPSSRWPSETFTCNVVHPASNTKVDKPVFNECRCTD TPPCPVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFVDGK EVHTAKTQSREQQFNGTYRVVSVLPIEHQDWLTGKEFKCRVNHIDLPSPIERTISK ARGRAHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPERKH RMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVMHETLQNHYTDLSLSHSP GK.

In various embodiments, the heavy chain comprises an amino acid sequence identical to a sequence selected from the group consisted of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NOG, SEQ ID NO:4, SEQ ID NOG, SEQ ID NOG, SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO:9 and SEQ ID NO: 10; please refer to FIG. 2 illustrating alignment of amino acid sequences of heavy chain among all designed recombinant antibody in the present invention.

In preferred embodiments, the heavy chain comprises an amino acid sequence identical to a sequence selected from the group consisted of SEQ ID NOG, SEQ ID NOG, SEQ ID NOG and SEQ ID NO:9.

In further preferred embodiments, the heavy chain comprises an amino acid sequence identical to SEQ ID NOG.

In one another aspect, the present disclosure provides a recombinant antibody which specifically binds to canine TNFa; the recombinant antibody comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises an amino acid sequence at least 50% but not 100% identical to a light chain variable region of adalimumab while the heavy chain variable region comprises an amino acid sequence at least 50% but not 100% identical to a heavy chain variable region of adalimumab. Preferably, the light chain variable region comprises an amino acid sequence 50, 60, 70, 80, 90 or 95% identical to a light chain variable region of adalimumab; the heavy chain variable region comprises an amino acid sequence 50, 60, 70, 80, 90 or 95% identical to a heavy chain variable region of adalimumab.

In various embodiments, the light chain comprises an amino acid comprising a first region at residue 1 to 12 from N-terminus, a second region at residue 18 to 54 from N- terminus, a third region at residue 56 to 84 from N-terminus and a fourth region at residue 86 to 108 from N-terminus, wherein the first region comprises an amino acid sequence identical to DIVMTQSPASLS; the second region comprises an amino acid sequence identical to TVTITCRASQGIRNYLAWYQQKPGQAPKLLIYAASTL; the third region comprises an amino acid sequence identical to TGVPSRFSGSGSGTDFSLTISSLEPEDVAVYYCQHYNRAPYTFGQGTKVELKR; the fourth region comprises an amino acid sequence identical to YYCQHYNRAPYTFGQGTKVELKR; the heavy chain comprises an amino acid comprising a sixth region at residue 1 to 15 from N-terminus, a seventh region at residue 24 to 48 from N-terminus, an eighth region at residue 51 to 62 from N-terminus, a ninth region at residue 67 to 77 from N-terminus and a tenth region at residue 89 to 115, wherein the sixth region comprises an amino acid sequence identical to EVQLVESGGGLVQPG; the seventh region comprises an amino acid sequence identical to ASGFTFDDYAMHWVRQAPGKGLEWV; the eighth region comprises an amino acid sequence identical to ITWNSGHIDYAD; the ninth region comprises an amino acid sequence identical to RFTISRDNAKN; the tenth region comprises an amino acid sequence identical to EDTAVYYCAKVSYLSTASSLDYWGQGT.

In some embodiments, the light chain further comprises a fifth region at residue 110 to 217 from N-terminus comprising an amino acid sequence identical to DAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGIQESV TEQDKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSECQRVD; the heavy chain further comprises an eleventh region at residue 117 to 452 from N-terminus, wherein the eleventh region comprises an amino acid sequence identical to VTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVH TFPSVLQSSGLHSLSSMVTVPSSRWPSETFTCNVVHPASNTKVDKPVFNECRCTD TPPCPVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFVDGK EVHTAKTQSREQQFNGTYRVVSVLPIEHQDWLTGKEFKCRVNHIDLPSPIERTISK ARGRAHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPERKH RMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVMHETLQNHYTDLSLSHSP GK.

In preferred embodiments, the light chain variable region comprises an amino acid sequence identical to a sequence selected from the group consisted of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO:20 and the heavy chain variable region comprises an amino acid sequence identical to a sequence selected from the group consisted of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NOG, SEQ ID NO:4, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NO:9 or SEQ ID NO: 10.

In preferred embodiments, the heavy chain comprises an amino acid sequence identical to a sequence selected from the group consisted of SEQ ID NOG, SEQ ID NOG, SEQ ID NOG and SEQ ID NO: 9; the light chain comprises an amino acid sequence identical to a sequence selected from the group consisted of SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 18.

In further preferred embodiments, the light chain comprises an amino acid sequence identical to SEQ ID NO: 12; the heavy chain comprises an amino acid sequence identical to SEQ ID NOG.

In some embodiments, the light chain variable region further comprises canine k chain or its variant with light chain framework region (FR region); more preferably, the recombinant antibody further comprises canine IgG or its variant with light chain constant region. In some embodiments, the recombinant antibody further comprises a canine IgG or its variant with heavy chain FR region; more preferably, the recombinant antibody further comprises a canine IgG or its variant with heavy chain constant region.

In one more another aspect, the present invention provides a chimeric antibody comprising a light chain and a heavy chain, wherein the light chain variable region comprises an amino acid sequence at least 50% but not 100% identical to a light chain variable region of adalimumab while the heavy chain variable region comprises an amino acid sequence at least 50% but not 100% identical to a heavy chain variable region of adalimumab. Preferably, the light chain variable region comprises an amino acid sequence 50, 60, 70, 80, 90 or 95% identical to a light chain variable region of adalimumab; the heavy chain variable region comprises an amino acid sequence 50, 60, 70, 80, 90 or 95% identical to a heavy chain variable region of adalimumab.

In various embodiments, the light chain variable region comprises an amino acid sequence identical to a sequence selected from the group consisted of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO:20.

In various embodiments, the heavy chain further comprises an eleventh region at residue 117 to 452 from N-terminus, wherein the eleventh region comprises an amino acid sequence identical to

In some embodiments, the heavy chain variable region comprises an amino acid sequence identical to a sequence selected from the group consisted of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NOG, SEQ ID NO:4, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID N0:8, SEQ ID N0:9 or SEQ ID NO: 10.

In preferred embodiments, the heavy chain comprises an amino acid sequence identical to a sequence selected from the group consisted of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NOG and SEQ ID NO:9.

In further preferred embodiments, the heavy chain comprises an amino acid sequence identical to SEQ ID NO:6.

In preferred embodiments, the light chain variable region of the chimeric antibody further comprises canine k chain or its variant with light chain FR region; more preferably, the chimeric antibody further comprises canine IgG or its variant with light chain constant region.

In preferred embodiments, the chimeric antibody further comprises a canine IgG or its variant with heavy chain FR region; more preferably, the chimeric antibody further comprises a canine IgG or its variant with heavy chain constant region.

As an exemplary embodiment of the chimeric antibody, a grafting technique is required to create complementarity-determining regions (CDRs) on a canine antibody specific to tumor necrosis factor-a. To be specific, CDRs of Humira are grafted on a canine antibody framework to make a new anti-TNFa monoclonal antibody for dogs. For instance,

CDRs of Humira are grafted on a canine IgG IK framework. In some cases, CDRs are grafted to variable regions of a light chain or a heavy chain depending on the requirement of following medication.

Exemplarily, the IgG gene sequences are cloned into an expression vector, which is then transfected into host cells, such as E. coli cells, yeast cells or mammalian cells (e.g., human HEK293 cells or Chinese hamster ovary (CHO) cells) to produce the recombinant antibody of the present disclosure. According to preferred embodiments, the recombinant antibody is produced by mammalian cells. In one specific example, the recombinant antibody is produced by human HEK293 cells.

Another exemplary embodiment further illustrates construction of the chimeric antibody sequences by grafting CDRs of Humira on canine antibody framework, which can be performed with establishment of a yeast expression vector library. To be specific, the library of chimeric antibody sequences is constructed by grafting sequences encoding CDRs of Humira into the framework sequences of a canine antibody library. Through this grafting process, a library of chimeric antibody sequences is created, and the sequences encode a library of chimeric antibodies containing both canine and non-canine antibody sequences. Subsequently, the library of chimeric antibody sequences is cloned into a yeast expression vector to generate a yeast expression vector library containing caninized antibody sequences. The caninization strategy involves two selection steps for the sequential caninization of the light chain and the constant region of the heavy chain. Throughout these selections the only preserved sequences in the variable domains are CDRs of Humira on the light and/or the heavy chain variable regions.

The chimeric antibody can be manufactured by any other conventional method disclosed in prior arts, and the exemplary embodiment in the present invention aims to explain construction of the chimeric antibody rather than limiting the scope of the present invention.

In yet one another aspect, the present invention provides a caninized antibody which specifically binds to canine tumor necrosis factor-a, wherein the caninized antibody comprises a light chain and a heavy chain, wherein the light chain variable region comprises an amino acid sequence at least 50% but not 100% identical to a light chain variable region of adalimumab while the heavy chain variable region comprises an amino acid sequence at least 50% but not 100% identical to a heavy chain variable region of adalimumab.

In some embodiments, the heavy chain further comprises an eleventh region at residue 117 to 452 from N-terminus, wherein the eleventh region comprises an amino acid sequence identical to VTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVH TFPSVLQSSGLHSLSSMVTVPSSRWPSETFTCNVVHPASNTKVDKPVFNECRCTD TPPCPVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFVDGK EVHTAKTQSREQQFNGTYRVVSVLPIEHQDWLTGKEFKCRVNHIDLPSPIERTISK ARGRAHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPERKH RMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVMHETLQNHYTDLSLSHSP GK.

In various embodiments, the heavy chain variable region comprises an amino acid sequence identical to a sequence selected from the group consisted of SEQ ID NO:1, SEQ ID NOG, SEQ ID NOG, SEQ ID NO:4, SEQ ID NOG, SEQ ID NOG, SEQ ID NO:7, SEQ ID NOG, SEQ ID NO:9 or SEQ ID NO: 10.

In some embodiments, the caninized antibody further includes a canine IgG or its variant with light chain constant region.

In some embodiments, the caninized antibody further includes a canine IgG or its variant with heavy chain FR region.

In some other embodiments, the caninized antibody further includes a canine IgG or its variant with heavy chain constant region.

In some other embodiments, the caninized antibody includes a canine IgG with a canine k chain or its variant with light chain FR region.

In one or various embodiments, the caninized antibody further comprises canine IgGl, IgG2, IgG3, IgG4 and/or a heavy chain FR region variant.

In one or various embodiments, the caninized antibody comprises a light chain variable region further containing canine k, A, chain or a light chain FR region variant. In some embodiments, the caninized antibody consists of Fab, Fv, single chain variable fragment (scFv), F(ab’)2 and diabody.

In preferred embodiments, the caninized antibody further comprises canine IgGl, IgG2, IgG3, IgG4 and/or a heavy chain constant variant, preferred canine IgGl or IgG4.

One exemplary embodiment of the caninized antibody is to construct a library of fully canine antibody sequences. Firstly, a directed selection from two separate pools of fully canine antibody light chain and heavy chain sequences is performed. The selection is directed toward a chimeric antibody heavy chain comprising essential antigen-recognizing segments (e.g., VH or CDRs of the heavy chain) of a non-canine antibody (e.g., Humira) and a canine framework sequence (e.g., canine IgGl). For instance, a constant region of a canine antibody serves as a framework sequence to carry CDRs of Humira as described below.

Secondly, for assembly of the light chains from canine antibody and the chimeric heavy chains, individual gene pools are expressed in vivo to form a library of chimeric Fab. The library of double chain Fab containing the chimeric light chain is selected against the original antigen against which the non-canine antibody is elicited. The fully canine light chain(s) of the chimeric Fab(s) selected in this process is then matched with a pool of fully canine antibody heavy chain sequences to form a library of fully canine antibody sequences.

Thirdly, this library is screened against the original antigens again to select for those fully canine antibodies with high affinity toward original antigens. Consequently, fully canine antibodies with high affinity toward the antigens than original non-canine antibody should be obtained. Furthermore, such fully canine antibody should be less immunogenic than a chimeric antibody containing partially canine and partially non-canine sequences. Upon an administration to subjects in need, less side effects induced by such fully canine antibody could be anticipated.

In addition to antibody grafting by techniques described above, a protein expression system is further required for massive production. Preferably, sequences of the recombinant antibody, chimeric antibody or the caninized antibody are cloned into protein expression vectors and the vectors are subsequently transfected into host cells such as E. coli cells, yeast cells or mammalian cells (e.g., HEK293 cells or Chinese hamster ovary (CHO) cells). More preferably, mammalian cells are used as host cells for antibody production. In one preferred example, the antibody is produced by HEK293 cells.

The recombinant, chimeric or caninized antibody can be manufactured by any conventional method disclosed in the prior arts, and the exemplary embodiments in the present invention aims to explain construction of the antibodies within the scope of the present invention but rather than being limited by manufacturing methods as mentioned above.

In yet one more aspect, the present invention provides a method for neutralizing TNFa; the method comprises administrating an effective concentration of antibody to a subject in need, wherein the antibody comprises the aforementioned recombinant antibody, the chimeric antibody or the caninized antibody.

EXAMPLES

The following examples are provided to further explain and demonstrate some of the presently preferred embodiments and are not intended to limit the scope or content of the invention in any way.

MATERIAL AND METHODS

1. IgG Cloning, Expression and Purification

The heavy and light chain V-regions of Humira were cloned into canine IgG IK constant regions resulting in the ten individual caninized mAbs, named Humivet series. Construction of anti-canine TNFa antibody following the computer modeling designs according to the computer modeling of human TNFa. Ten canine IgGs (Humivet- 1, Humivet-2, Humivet-3.0, Humivet-3.1, Humivet- 3.1.1, Humivet-3.2, Humivet- 3.2.1, Humivet-3.2.2, Humivet- 3.2.3, Humivet-3.3) were designed with different heavy chain and light chain combinations. As for detailed information of Humivet sequences mentioned above, please refer to FIGS. 3 and 4 which list down amino acid sequences of heavy and light chains of Humivet series. 2. Synthesis and Biological Activity Test of Self-Expressed Recombinant Canine TNFa

DNA construction was performed for synthesis of the canine TNFa needed for subsequent experiments. Canine TNFa were synthesized separately in bacterial and mammalian system. HEK-Dual™ TNFa cells were used to test the biological activity of both synthesized resources compared with mouse TNFa as a positive control.

Please refer to FIG. 5, recombinant TNFa was diluted from 266.3nM to O.OnM to treat cells for 16 hours in 37°C; supernatant 100 pl was harvested and further analyzed following the recommend protocol. Mammalian expressed TNFa showed a higher activation potency than bacterial expressed. This result also indicated that glycosylation on TNFa had an important influence on physiological activity of itself. Meanwhile, mammalian expressed TNFa has the same physiological activity as mouse TNFa. Therefore, mammalian expressed TNFa would be used for the following verified experiments.

3. In Vitro TNFa Neutralization Assay

HEK-Dual™ TNFa cells (InvivoGen) allow detection of bioactive TNFa by monitoring the activation of the NK-KB pathways. Cells were maintained in DMEM supplemented with 10% FBS, lOOpg/mE Zeocin and 200pg/mE hygromycin B. When used 4 for TNFa neutralization assays, cells were seeded in 96- well plates at 3x10 cells/well in 200pE medium and incubated for 16 hours at 37°C in a 5% CO₂ humidified incubator. Cells then were treated with 294pM recombinant canine TNFa in the presence or absence of various concentrations of test antibodies for another 16 hrs. TNFa induced release of secreted embryonic alkaline phosphatase (SEAP) in the supernatant was then collected and assayed by adding QUANTI-Blue (InvivoGen) according to the manufacturer’s protocol.

4. ECso for Antibody- Antigen Interactions

The IgG EC50 was determined by the titration of IgG antibodies on immobilized 100 ng TNFa with EEISA. Briefly, the TNFa (lOOng/well) were coated with PBS buffer (pH 7.4) on NUNC 96-well Maxisorb immunoplates overnight at 4°C, and blocked with 5% skim milk in PBST [0.05% (v/v) Tween 20] for 1 hour at least. Simultaneously, IgGs in PBST with 5% milk were prepared at 11 concentrations by two-fold serial dilutions. After blocking, lOOpL diluted IgG samples were added to each well coated with TNFa, and incubated for another 1 hour under gentle shaking. The plates were washed 6 times with 300pL PBST and then added with lOOpL l:5000-diluted horseradish peroxidase/anti-Dog IgG (H+L) (Jackson ImmunoResearch, code:304-035-003) antibody conjugate in PBST with 5% milk for 30 mins incubation. The plates were washed six times with PBST buffer and twice with PBS, developed for 3 mins with 3,3’,5,5’-tereamethyl-benzidine peroxidase substrate (Kirkegaard & Perry Laboratories), quenched with 1.0M HC1 and read spectrophotometrically at 450nm. The ECso (ng/mL) was calculated according to Stewart and Watson method.

5. Statistical Analysis

Statistical analyses and graphical representation of data were carried out using GraphPad Prism version 6.0 (GraphPad Software). Data were presented as mean ± standard deviation (SD) of the mean of at least three independent experiments. Statistical significance was calculated using a multiple comparison t test. P- values less than 0.05 were considered to be statistically significant.

RESULTS

1

In this example, ELISA assay was performed to compare the binding affinity in different version of IgGs of Humivet-3.0 series. The IgGs were diluted from 117 to O.OnM and incubated on 96-well plates coated with TNFa for 60 mins. Please refer to FIG. 6, individual ECso was measured by ELISA assays and Humivet-3.0 showed the strongest binding affinity with canine TNFa compared with other IgG designs.

Example 2

In this example, in vitro TNFa neutralization assay was performed with HEK- Dual™ TNFa cells to determine which clone of antibody has the best neutralization ability for canine TNFa. Humivet-3.0 series clones were first incubated with different concentrations of TNFa for 30 mins. After incubation, the antibody unbound TNFa would bind on the HEK-Dual™ TNFa cells and transduce the activation signals. As shown in FIGS. 7 and 8, Humira served as a positive control. Serial dilutions of canine TNFa (294 to OpM) were used to induce NF-KB signaling in HEK-Dual™ TNFa cells. In FIGS 7 and 8, the y-axis of the plots shows the inhibitory percentage of corresponding IgGs, and the X-axis represents the log value of the concentration of IgGs. Humivet-3.0, Humivet-3.1, Humivet-3.2 and Humivet-3.3 all demonstrated higher inhibitory ability than that of Humira. Among all the clones, Humivet-3.0 demonstrated the highest inhibitory ability (IC50 = 0.04973nM) compared with other clones of Humivet series and Humira (IC50 = 0.21nM). Therefore, it is more determined that Humivet-3.0 should be a very effective drug for neutralizing excessive TNFa in dogs.

In the present invention, protein structure and computer modeling are used for simulating the binding orientation of designed antibody on canine TNFa. Ten IgGs were designed and tested in in vitro binding and neutralization assays. Among the tested antibodies, Humivet-3.0 demonstrated the highest binding affinity and the strongest neutralization ability among all designed IgGs. This is not the first time that caninized antibody has been used to treat dog diseases.

Lokivetmab is also a monoclonal antibody developed by Zoetis for the treatment of atopic dermatitis (AD) in dogs. Lokivetmab acts against a cytokine interleukin 31 (IL-31) which is involved in causing itchiness. Administration of Lokivetmab is via subcutaneous injection and each dose is effective for four to eight weeks. Notwithstanding, Lokivetmab has KD less than lOnM and IC50 is 15.2nM while the IC50 of Humivet-3.0 in the present invention is 0.04973nM, showing an effective concentration far lower than that of Lokivetmab and being much potential for osteoarthritis treatment. The recombinant antibody in the present invention has superior ability to neutralize TNFa, and it can treat dogs with a far less amount in comparison to current commercialized product. Furthermore, lower dosage of treatment should reduce the probability of unexpected side effects, and promote medication safety.

Claims

WHAT IS CLAIMED IS:

1. A recombinant antibody comprising: a light chain variable region and a heavy chain variable region; wherein the light chain variable region comprises an amino acid sequence at least 50% but not 100% identical to a light chain variable region of adalimumab.

2. The recombinant antibody as claimed in claim 1, wherein the heavy chain variable region comprises an amino acid sequence at least 50% but not 100% identical to a heavy chain variable region of adalimumab.

3. The recombinant antibody as claimed in claim 1, wherein the light chain comprises an amino acid comprising a first region at residue 1 to 12 from N-terminus, a second region at residue 18 to 54 from N-terminus, a third region at residue 56 to 84 from N-terminus, a fourth region at residue 86 to 108 from N-terminus and a fifth region at residue 110 to 217 from N-terminus, wherein the first region comprises an amino acid sequence identical to DIVMTQSPASLS; the second region comprises an amino acid sequence identical to TVTITCRASQGIRNYLAWYQQKPGQAPKLLIYAASTL; the third region comprises an amino acid sequence identical to TGVPSRFSGSGSGTDFSLTISSLEPEDVAVYYCQHYNRAPYTFGQGTKVELK R; the fourth region comprises an amino acid sequence identical to YYCQHYNRAPYTFGQGTKVELKR; the fifth region comprises an amino acid sequence identical to DAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGIQE SVTEQDKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSECQR VD.

4. The recombinant antibody as claimed in claim 3, wherein the light chain comprises an amino acid sequence identical to a sequence selected from the group consisted of SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 18.

22 The recombinant antibody as claimed in claim 4, wherein the light chain comprises an amino acid sequence identical to SEQ ID NO: 12. The recombinant antibody as claimed in claim 2, wherein the heavy chain comprises an amino acid comprising a sixth region at residue 1 to 15 from N-terminus, a seventh region at residue 24 to 48 from N-terminus, an eighth region at residue 51 to 62 from N-terminus, a ninth region at residue 67 to 77 from N-terminus, a tenth region at residue 89 to 115 and an eleventh region at residue 117 to 452 from N- terminus, wherein the sixth region comprises an amino acid sequence identical to EVQLVESGGGLVQPG; the seventh region comprises an amino acid sequence identical to ASGFTFDDYAMHWVRQAPGKGLEWV; the eighth region comprises an amino acid sequence identical to ITWNSGHIDYAD; the ninth region comprises an amino acid sequence identical to RFTISRDNAKN; the tenth region comprises an amino acid sequence identical to EDTAVYYCAKVSYLSTASSLDYWGQGT; the eleventh region comprises an amino acid sequence identical to VTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTS GVHTFPSVLQSSGLHSLSSMVTVPSSRWPSETFTCNVVHPASNTKVDKPVFN ECRCTDTPPCPVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEV QISWFVDGKEVHTAKTQSREQQFNGTYRVVSVLPIEHQDWLTGKEFKCRVN HIDLPSPIERTISKARGRAHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDV EWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAV MHETLQNHYTDLSLSHSPGK. The recombinant antibody as claimed in claim 6, wherein the heavy chain comprises an amino acid sequence identical to a sequence selected from the group consisted of SEQ ID NOG, SEQ ID NO:6, SEQ ID NOG and SEQ ID NO:9. The recombinant antibody as claimed in claim 7, wherein the heavy chain comprises an amino acid sequence identical to SEQ ID NO: 6. The recombinant antibody as claimed in claim 7, wherein the light chain comprises an amino acid sequence identical to a sequence selected from the group consisted of SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 18. The recombinant antibody as claimed in claim 9, wherein the heavy chain comprises an amino acid sequence identical to SEQ ID NO: 6 and the light chain comprises an amino acid sequence identical to SEQ ID NO: 12. A method for neutralizing TNFa comprising: administrating an effective concentration of recombinant antibody to a subject in need, wherein the recombinant antibody comprises a light chain variable region and a heavy chain variable region; wherein the light chain variable region comprises an amino acid sequence at least 50% but not 100% identical to a light chain variable region of adalimumab. The method as claimed in claim 11, wherein the heavy chain variable region comprises an amino acid sequence at least 50% but not 100% identical to a heavy chain variable region of adalimumab. The method as claimed in claim 11, wherein the light chain comprises an amino acid comprising a first region at residue 1 to 12 from N-terminus, a second region at residue 18 to 54 from N-terminus, a third region at residue 56 to 84 from N- terminus, a fourth region at residue 86 to 108 from N-terminus and a fifth region at residue 110 to 217 from N-terminus, wherein the first region comprises an amino acid sequence identical to DIVMTQSPASLS; the second region comprises an amino acid sequence identical to TVTITCRASQGIRNYLAWYQQKPGQAPKLLIYAASTL; the third region comprises an amino acid sequence identical to TGVPSRFSGSGSGTDFSLTISSLEPEDVAVYYCQHYNRAPYTFGQGTKVELK R; the fourth region comprises an amino acid sequence identical to YYCQHYNRAPYTFGQGTKVELKR; the fifth region comprising an amino acid sequence identical to DAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGIQE SVTEQDKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSECQR VD.

14. The method as claimed in claim 13, wherein the light chain comprises an amino acid sequence identical to a sequence selected from the group consisted of SEQ ID

NO: 12, SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 18.

15. The method as claimed in claim 14, wherein the light chain comprises an amino acid sequence identical to SEQ ID NO: 12.

16. The method as claimed in claim 12, wherein the heavy chain comprises an amino acid comprising a sixth region at residue 1 to 15 from N-terminus, a seventh region at residue 24 to 48 from N-terminus, an eighth region at residue 51 to 62 from N- terminus, a ninth region at residue 67 to 77 from N-terminus, a tenth region at residue 89 to 115 and an eleventh region at residue 117 to 452 from N-terminus, wherein the sixth region comprises an amino acid sequence identical to EVQLVESGGGLVQPG; the seventh region comprises an amino acid sequence identical to ASGFTFDDYAMHWVRQAPGKGLEWV; the eighth region comprises an amino acid sequence identical to ITWNSGHIDYAD; the ninth region comprises an amino acid sequence identical to RFTISRDNAKN; the tenth region comprises an amino acid sequence identical to EDTAVYYCAKVSYLSTASSLDYWGQGT; the heavy chain further comprises wherein the eleventh region comprises an amino acid sequence identical to VTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTS GVHTFPSVLQSSGLHSLSSMVTVPSSRWPSETFTCNVVHPASNTKVDKPVFN ECRCTDTPPCPVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEV QISWFVDGKEVHTAKTQSREQQFNGTYRVVSVLPIEHQDWLTGKEFKCRVN HIDLPSPIERTISKARGRAHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDV

25 EWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAV MHETLQNHYTDLSLSHSPGK.

17. The method as claimed in claim 16, wherein the heavy chain comprises an amino acid sequence identical to a sequence selected from the group consisted of SEQ ID NOG, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:9.

18. The method as claimed in claim 17, wherein the heavy chain comprises an amino acid sequence identical to SEQ ID NO:6.

19. The method as claimed in claim 18, wherein the light chain comprises an amino acid sequence identical to SEQ ID NO: 12. 20. The method as claimed in claim 11, wherein the effective concentration ranges from

0.04-1.0nM.

26