# US 2014/0193400 A1 · Stable and soluble antibodies inhibiting tnf alpha

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- Number: US 2014/0193400 A1
- Document ID: US20140193400A1
- Status: Published application
- Kind: A1
- Application number: 14/134,779
- Filed: 2013-12-19
- Published: 2014-07-10
- Earliest claimed: 2008-06-25
- Assignees: ESBA Tech, an Alcon Biomedical Research Unit LLC
- Applicants: ESBA Tech, an Alcon Biomedical Research Unit LLC
- Inventors: Leonardo Borras; Tea Gunde; David Urech
- Web page: https://patentgrep.com/patent/US20140193400A1

## CPC

- C07K 16/241: {Tumor Necrosis Factors}
- A61P 1/00: Drugs for disorders of the alimentary tract or the digestive system
- A61P 1/02: Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
- A61P 1/04: for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
- A61P 1/16: for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
- A61P 1/18: for pancreatic disorders, e.g. pancreatic enzymes
- A61P 3/00: Drugs for disorders of the metabolism (of the blood or the extracellular fluid A61P7/00)
- A61P 3/06: Antihyperlipidemics
- A61P 3/10: for hyperglycaemia, e.g. antidiabetics
- A61P 9/00: Drugs for disorders of the cardiovascular system
- A61P 9/04: Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
- A61P 9/10: for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
- A61P 9/12: Antihypertensives
- A61P 9/14: Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
- A61P 11/00: Drugs for disorders of the respiratory system
- A61P 11/02: Nasal agents, e.g. decongestants
- A61P 11/06: Antiasthmatics
- A61P 13/02: of urine or of the urinary tract, e.g. urine acidifiers
- A61P 13/10: of the bladder
- A61P 13/12: of the kidneys
- A61P 15/00: Drugs for genital or sexual disorders (for disorders of sex hormones A61P5/24); Contraceptives
- A61P 15/08: for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
- A61P 17/00: Drugs for dermatological disorders
- A61P 17/02: for treating wounds, ulcers, burns, scars, keloids, or the like
- A61P 17/06: Antipsoriatics
- A61P 17/08: Antiseborrheics
- A61P 17/10: Anti-acne agents
- A61P 19/02: for joint disorders, e.g. arthritis, arthrosis
- A61P 19/06: Antigout agents, e.g. antihyperuricemic or uricosuric agents
- A61P 19/08: for bone diseases, e.g. rachitism, Paget's disease
- A61P 21/00: Drugs for disorders of the muscular or neuromuscular system
- A61P 21/04: for myasthenia gravis
- A61P 25/00: Drugs for disorders of the nervous system
- A61P 25/02: for peripheral neuropathies
- A61P 25/04: Centrally acting analgesics, e.g. opioids
- A61P 25/14: for treating abnormal movements, e.g. chorea, dyskinesia
- A61P 25/16: Anti-Parkinson drugs
- A61P 25/28: for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
- A61P 27/00: Drugs for disorders of the senses
- A61P 27/02: Ophthalmic agents
- A61P 27/06: Antiglaucoma agents or miotics
- A61P 27/12: for cataracts
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P 31/04: Antibacterial agents
- A61P 31/06: for tuberculosis
- A61P 31/10: Antimycotics
- A61P 31/12: Antivirals
- A61P 31/18: for HIV
- A61P 35/00: Antineoplastic agents
- A61P 35/04: specific for metastasis
- A61P 37/00: Drugs for immunological or allergic disorders
- A61P 37/02: Immunomodulators
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- A61P 37/08: Antiallergic agents (antiasthmatic agents A61P11/06; ophthalmic antiallergics A61P27/14)
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C07K 2317/21: from primates, e.g. man
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- C07K 2317/55: Fab or Fab'
- C07K 2317/565: Complementarity determining region [CDR]
- C07K 2317/567: Framework region [FR]
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2317/92: Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
- C07K 2317/94: Stability, e.g. half-life, pH, temperature or enzyme-resistance
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

The present invention relates to particularly stable and soluble scFv antibodies and Fab fragments specific for TNF, which comprise specific light chain and heavy chain sequences that are optimized for stability, solubility, in vitro and in vivo binding of TNF, and low immunogenicity. Said antibodies are designed for the diagnosis and/or treatment of TNF-mediated disorders. The nucleic acids, vectors and host cells for expression of the recombinant antibodies of the invention, methods for isolating them and the use of said antibodies in medicine are also disclosed.

## Claims

### Claim 1

An immunobinder which specifically binds human TNFα, the immunobinder comprising:
(a) a human heavy chain variable framework sequence and CDR H1, CDR H2 and CDR H3 sequences stemming from a rabbit immunobinder, wherein the human heavy chain variable region framework has at least 90% identity to SEQ ID NO:1; and
(b) a human light chain variable framework sequence and CDR L1, CDR L2 and CDR L3 sequences stemming from a rabbit immunobinder, wherein the human light chain variable region framework sequence has at least 85% identity to SEQ ID NO:2.

### Claim 2 (depends on 1)

The immunobinder of claim 1, wherein the human heavy chain variable region framework is or comprises SEQ ID NO:1, SEQ ID NO: 89, or SEQ ID NO: 90, and the human light chain variable region framework sequence is or comprises SEQ ID NO: 2 or SEQ ID NO: 91.

### Claim 3 (depends on 2)

The immunobinder of claim 2, comprising one or more substitutions in the heavy chain framework (VH) at a position from the group consisting of positions H24, H25, H56, H82, H84, H89 and H108; and/or a substitution in the light chain framework (VL) at position L87 according to the AHo numbering system.

### Claim 4 (depends on 3)

The immunobinder of claim 3, wherein the substitution is selected from the group consisting of threonine (T) at position H24, valine (V) at position H25, glycine (G) or alanine (A) at position H56, lysine (K) at position H82, threonine (T) at position H84, valine (V) at position H89 and arginine (R) at position H108 and threonine (T) at position L87 according to the AHo numbering system.

### Claim 5 (depends on 2)

The immunobinder of claim 2, wherein immunobinder comprises a solubility enhancing substitution in at least one of heavy chain amino positions 12, 103 and 144 (AHo numbering).

### Claim 6 (depends on 5)

The immunobinder of claim 5, wherein the solubility enhancing substitution is selected from the group consisting of: (a) Serine (S) at position 12; (b) Threonine (T) at position 103; and (c) Threonine (T) at position 144.

### Claim 7 (depends on 1)

The immunobinder of claim 1, further comprising:
(a) one or more CDR sequences being at least 80% identical to a sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8;
(b) one or more CDR sequences being at least 80% identical to a sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14;
(c) one or more CDR sequences being at least 80% identical to a sequence selected from the group consisting of 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;
(d) one or more CDR sequences being at least 80% identical to a sequence selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26;
(e) one or more CDR sequences being at least 80% identical to a sequence selected from the group consisting of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32;
(f) one or more CDR sequences being at least 80% identical to a sequence selected from the group consisting of SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 38;
(g) one or more CDR sequences being at least 80% identical to a sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44; or
(h) one or more CDR sequences being at least 80% identical to a sequence selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49 and SEQ ID NO: 50.

### Claim 8 (depends on 7)

The immunobinder of claim 7, wherein the one or more CDR sequences being at least 80% identical to a sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, and has a substitution in at least one of positions 22, 74, 95, 97 and 99 of the light chain variable region (VL) according to the AHo numbering system.

### Claim 9 (depends on 8)

The immunobinder of claim 8, wherein the substitution is threonine (T) at position L22, phenylalanine (F) or tyrosine (Y) at position L74, glutamate (E) L95 or alanine (A) at position L99, or a combination thereof.

### Claim 10 (depends on 7)

The immunobinder of claim 7, comprising:
(a) a heavy chain variable region (VH) having at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NO:51, SEQ ID NO:53 and SEQ ID NO: 55, and a light chain variable region (VL) having at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO:58 and SEQ ID NO: 59;
(b) a heavy chain variable region (VH) having at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NO:60 and SEQ ID NO:62, and/or a light chain variable region (VL) having at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NO:61 and SEQ ID NO:63;
(c) comprising a heavy chain variable region (VH) having at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NO:64 and SEQ ID NO:66, and/or a light chain variable region (VL) having at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NO:65 and SEQ ID NO:67;
(d) a heavy chain variable region (VH) having at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NO:64 and SEQ ID NO:66, and/or a light chain variable region (VL) having at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NO:65 and SEQ ID NO:67;
(e) a heavy chain variable region (VH) having at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NO:68 and SEQ ID NO:70, and/or a light chain variable region (VL) having at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NO:69 and SEQ ID NO:71;
(f) a heavy chain variable region (VH) having at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NO:73 and SEQ ID NO:75, and/or a light chain variable region (VL) having at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NO:74 and SEQ ID NO:76;
(g) a heavy chain variable region (VH) having at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NO:77 and SEQ ID NO:79, and/or a light chain variable region (VL) having at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NO:78 and SEQ ID NO:80;
(h) a heavy chain variable region (VH) having at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NO:81 and SEQ ID NO:83, and/or a light chain variable region (VL) having at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NO:82 and SEQ ID NO:84; or
(i) a heavy chain variable region (VH) having at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NO:85 and SEQ ID NO:87, and/or a light chain variable region (VL) having at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NO:86 and SEQ ID NO:88.

### Claim 11 (depends on 7)

The immunobinder of claim 7, having at least 90% sequence identity to, preferably 100% to any of SEQ ID NO: 94 to SEQ ID NO: 121.

### Claim 12 (depends on 11)

The immunobinder of claim 11, having at least 90% sequence identity to, preferably 100% to SEQ ID NO: 103, SEQ ID NO: 104 or SEQ ID NO:105, and also having a substitution in at least one of positions 87, 89 and 92 of the light chain variable region (VL) according to the AHo numbering system.

### Claim 13 (depends on 12)

The immunobinder of claim 12, having at least 90% sequence identity to, preferably 100% to any of SEQ ID NO: 21 to SEQ ID NO: 50, wherein the heavy chain framework has deletions at framework positions 85 and 88 according to the AHo numbering system.

### Claim 14 (depends on 13)

The immunobinder of claim 13, having at least 90% sequence identity to, preferably 100% to any of SEQ ID NO: 27 to SEQ ID NO: 32, further having a substitution in at least one of positions 86 and 87 of the light chain variable region (VL) according to the AHo numbering system, preferably threonine (T) at position L87 and glutamine (Q) at position L88.

### Claim 15 (depends on 13)

The immunobinder of claim 13, having at least 90% sequence identity to, preferably 100% to any of SEQ ID NO: 33 to SEQ ID NO: 38, further, having a substitution in at least one of positions 15, 48, 90 of the light chain variable region (VL) according to the AHo numbering system.

### Claim 16 (depends on 13)

The immunobinder of claim 13, comprising one or more CDR sequences being at least 80% identical to a sequence selected from the group consisting of SEQ ID NO: 39 to SEQ ID NO: 44, having a substitution in at least one of positions 57 and 87 of the light chain variable region (VL) according to the AHo numbering system, preferably to valine (V) at position L57 and threonine (T) at position L87.

### Claim 17 (depends on 16)

The immunobinder of claim 16, having a stability enhancing substitution in at least one of positions 1, 3, 4, 10, 47, 57, 91 and 103 of the light chain variable region according to the AHo numbering system, preferably, a stability enhancing substitution selected from the group consisting of (a) glutamic acid (E) at position 1, (b) valine (V) at position 3, (c) leucine (L) at position 4; (d) Serine (S) at position 10; (e) Arginine (R) at position 47; (e) Serine (S) at position 57; (f) phenylalanine (F) at position 91; and (g) Valine (V) at position 103.

### Claim 18 (depends on 7)

The immunobinder of claim 7, comprising one or more CDR sequences being at least 80% identical to a sequence selected from the group consisting of SEQ ID NO: 45 to SEQ ID NO: 50, having deletions at framework positions 85 and 88 according to the AHo numbering system and having a stability enhancing substitution in at least one of positions 1, 3, 4, 10, 47, 57, 91 and 103 of the light chain variable region according to the AHo numbering system, preferably a stability enhancing substitution selected from the group consisting of (a) glutamic acid (E) at position 1, (b) valine (V) at position 3, (c) leucine (L) at position 4; (d) Serine (S) at position 10; (e) Arginine (R) at position 47; (e) Serine (S) at position 57; (f) phenylalanine (F) at position 91; and (g) Valine (V) at position 103.

### Claim 19 (depends on 7)

The immunobinder of claim 7, which is an antibody, scFv, Fab or Dab.

### Claim 20 (depends on 7)

An immunobinder having CDRs that are different from any CDR of SEQ ID NOs: 3-50, that competes for binding to human TNFα with the immunobinder of claim 7.

### Claim 21 (depends on 7)

An immunobinder having CDRs that are different from any CDR of SEQ ID NOs: 3-50, that binds the same epitope on human TNFα as the immunobinder of claim 7.

### Claim 22 (depends on 7)

A composition comprising the immunobinder of claim 7, and a pharmaceutically acceptable carrier.

### Claim 23 (depends on 7)

An isolated nucleic acid molecule encoding a variable heavy (VH) chain region and/or variable light (VL) chain region of claim 7.

### Claim 24 (depends on 23)

An expression vector comprising the nucleic acid molecule of claim 23.

### Claim 25 (depends on 24)

A host cell comprising the expression vector of claim 24.

### Claim 26 (depends on 7)

A method of treating or preventing a human TNFα-mediated disease in a subject, comprising administering to a subject the antibody of claim 7 such that the subject is treated for the TNFα-mediated disease.

### Claim 27 (depends on 26)

The method of claim 26, wherein the TNFα-mediated disease is selected from the group consisting of chronic and/or autoimmune states of inflammation in general, immune mediated inflammatory disorders in general, inflammatory CNS disease, inflammatory diseases affecting the eye, joint, skin, mucuous membranes, central nervous system, gastrointestinal tract, urinary tract or lung, states of uveitis in general, retinitis, HLA-B27+ uveitis, Behcet's disease, dry eye syndrome, glaucoma, Sjögren syndrome, diabetes mellitus (incl. diabetic neuropathy), insulin resistance, states of arthritis in general, rheumatoid arthritis, osteoarthritis, reactive arthritis and Reiter's syndrome, juvenile arthritis, ankylosing spondylitis, multiple sclerosis, Guillain-Barre syndrome, myasthenia gravis, amyotrophic lateral sclerosis, sarcoidosis, glomerulonephritis, chronic kidney disease, cystitis, psoriasis (incl. psoriatic arthritis), hidradenitis suppurativa, panniculitis, pyoderma gangrenosum, SAPHO syndrome (synovitis, acne, pustulosis, hyperostosis and osteitis), acne, Sweet's sydrome, pemphigus, Crohn's disease (incl. extraintestinal manifestastations), ulcerative colitis, asthma bronchiale, hypersensitivity pneumonitis, general allergies, allergic rhinitis, allergic sinusitis, chronic obstructive pulmonary disease (COPD), lung fibrosis, Wegener's granulomatosis, Kawasaki syndrome, Giant cell arteritis, Churg-Strauss vasculitis, polyarteritis nodosa, burns, graft versus host disease, host versus graft reactions, rejection episodes following organ or bone marrow transplantation, sytemic and local states of vasculitis in general, systemic and discoid lupus erythematodes, polymyositis and dermatomyositis, sclerodermia, pre-eclampsia, acute and chronic pancreatitis, viral hepatitis, alcoholic hepatitis, postsurgical inflammation such as after eye surgery (e.g. cataract (eye lens replacement) or glaucoma surgery), joint surgery (incl. arthroscopic surgery), surgery at joint-related structures (e.g. ligaments), oral and/or dental surgery, minimally invasive cardiovascular procedures (e.g. PTCA, atherectomy, stent placement), laparoscopic and/or endoscopic intra-abdominal and gynecological procedures, endoscopic urological procedures (e.g. prostate surgery, ureteroscopy, cystoscopy, interstitial cystitis), or perioperative inflammation (prevention) in general, Alzheimer disease, Parkinson's disease, Huntington's disease, Bell' palsy, Creutzfeld-Jakob disease. Cancer-related osteolysis, cancer-related inflammation, cancer-related pain, cancer-related cachexia, bone metastases, acute and chronic forms of pain, irrespective whether these are caused by central or peripheral effects of TNFα and whether they are classified as inflammatory, nociceptive or neuropathic forms of pain, sciatica, low back pain, carpal tunnel syndrome, complex regional pain syndrome (CRPS), gout, postherpetic neuralgia, fibromyalgia, local pain states, chronic pain syndroms due to metastatic tumor, dismenorrhea. Bacterial, viral or fungal sepsis, tuberculosis, AIDS, atherosclerosis, coronary artery disease, hypertension, dyslipidemia, heart insufficiency and chronic heart failure.

### Claim 28 (depends on 22)

The composition of claim 22, formulated for topical, oral, nasal, rectal or parental administration.

### Claim 29 (depends on 22)

A method of treating a TNF α-mediated disease in a mammal, in particular in a human, comprising administering to a subject the composition of claim 22 by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical, or inhalation routes.