# US 7,612,180 B2 · Humanized L243 antibodies

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- Number: US 7,612,180 B2
- Document ID: US7612180B2
- Status: Granted
- Kind: B2
- Application number: 11/368,296
- Filed: 2006-03-03
- Published: 2006-09-21
- Granted: 2009-11-03
- Earliest claimed: 2005-03-03
- Assignees: Immunomedics, Inc.
- Inventors: David M. Goldenberg; Hans J. Hansen; Zhengxing Qu; Chein-Hsing Chang
- Web page: https://patentgrep.com/patent/US7612180B2

## CPC

- C07K 16/2833: {against MHC-molecules, e.g. HLA-molecules}
- A61K 39/3955: {against proteinaceous materials, e.g. enzymes, hormones, lymphokines}
- A61K 39/39558: {against tumor tissues, cells, antigens}
- A61K 45/06: Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
- A61K 47/6849: {the antibody targeting a receptor, a cell surface antigen or a cell surface determinant}
- A61K 47/6867: {the tumour determinant being from a cell of a blood cancer}
- A61K 51/1027: {against receptors, cell-surface antigens or cell-surface determinants}
- A61K 51/1093: {conjugates with carriers being antibodies}
- A61P 3/00: Drugs for disorders of the metabolism (of the blood or the extracellular fluid A61P7/00)
- 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 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P 35/00: Antineoplastic agents
- A61P 35/02: specific for leukemia
- A61P 37/00: Drugs for immunological or allergic disorders
- A61P 37/02: Immunomodulators
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- C07K 16/28: against receptors, cell surface antigens or cell surface determinants
- C07K 16/2887: {against CD20}
- C07K 16/4241: {against anti-human or anti-animal Ig}
- C07K 19/00: Hybrid peptides {, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes}
- A61K 2039/505: {comprising antibodies}
- A61K 2039/507: {Comprising a combination of two or more separate antibodies}
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- C07K 2317/52: Constant or Fc region; Isotype
- C07K 2317/56: variable (Fv) region, i.e. VH and/or VL
- C07K 2317/565: Complementarity determining region [CDR]
- C07K 2317/71: Decreased effector function due to an Fc-modification
- C07K 2317/73: Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K 2317/732: Antibody-dependent cellular cytotoxicity [ADCC]
- C07K 2317/734: Complement-dependent cytotoxicity [CDC]
- C07K 2317/75: Agonist effect on antigen
- C07K 2317/92: Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
- 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)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

Humanized antibodies are provided that specifically bind HLA-DR. The antibodies recognize the epitope recognized by the murine monoclonal antibody L243. Processes for preparing such antibodies, pharmaceutical compositions containing such antibodies, and clinical therapuetic and diagnostic, as well as research-related uses for such antibodies, are provided.

## Claims

### Claim 1

A humanized L243 antibody or antigen-binding fragment thereof, comprising heavy chain variable domain complementarity determining region (CDR) sequences CDR1 (NYGMN, residues 31 to 35of SEQ ID NO: 4), CDR2 (WINTYTREPTYADDFKG, residues 50 to 66 of SEQ ID NO:4), and CDR3 (DITAVVPTGFDY, residues 99 to 110 of SEQ ID NO:4) and heavy chain framework residues F27, K38, K46, A68, and F91 and light chain variable domain CDR sequences CDR1 (RASENIYSNLA, residues 24 to 34 of SEQ ID NO:2), CDR2 (AASNLAD, residues 50 to 56 of SEQ ID NO:2), and CDR3 (OHFWTTPWA, residues 89 to 98 of SEQ ID NO:2) and light chain framework residues R37 , K39 , V48 and F49, wherein the remainder of the humanized L243 antibody framework region and constant region sequences are from one or more human antibodies, wherein the humanized L243 antibody has the ability to bind to HLA-DR on HLA-DR+ cells, and wherein said humanized L243 antibody induces apoptosis of said HLA-DR+ cells without inducing complement-mediated cytotoxicity (CDC) or antibody-dependent cell mediated cytotoxicity (ADCC).

### Claim 2 (depends on 1)

The humanized L243 antibody or antigen-binding fragment thereof of claim 1, wherein the humanized L243 antibody or fragment comprises human IgG4 constant region sequences.

### Claim 3 (depends on 2)

The humanized L243 antibody or antigen-binding fragment thereof of claim 2, further comprising a Ser241 Pro point mutation in the hinge region of the antibody or fragment thereof.

### Claim 4 (depends on 1)

The humanized L243 antibody or antigen-binding fragment thereof of claim 1, wherein humanized L243 antibody comprises the hL243VK amino acid sequence SEQ ID NO:6 and the hL243VH amino acid sequence SEQ ID NO:8.

### Claim 5 (depends on 1)

The humanized L243 antibody or antigen-binding fragment thereof of claim 1, wherein the humanized L243 antibody has a lower dissociation constant for HLA-DR+ cells than the murine L243 antibody.

### Claim 6 (depends on 1)

The humanized L243 antibody or antigen-binding fragment thereof of claim 1, wherein the humanized L243 antibody or fragment thereof is a naked antibody or fragment thereof.

### Claim 7 (depends on 1)

The humanized L243 antibody or antigen-binding fragment thereof of claim 1, wherein the humanized L243 antibody or fragment thereof is conjugated to at least one therapeutic or diagnostic agent.

### Claim 8 (depends on 7)

The humanized L243 antibody or antigen-binding fragment thereof of claim 7, wherein the therapeutic agent is selected from the group consisting of antibodies, antibody fragments, drugs, chemotherapeutic agents, toxins, enzymes, nucleases, hormones, hormone antagonists, immunomodulators, cytokines, oligonucleotides, interference RNA, chelators, boron compounds, photoactive agents, dyes and radioisotopes.

### Claim 9 (depends on 8)

The humanized L243 antibody or antigen-binding fragment thereof of claim 8, wherein the chemotherapeutic agent is a taxane, a nitrogen mustard, an ethylenimine, an alkyl sulfonate, a nitrosourea, a triazene, a folic acid analog, a pyrimidine analog, a purine analog, an antisense oligonucleotide, an interference RNA, an antibiotic, a platinum coordination complex, a COX-2 inhibitor, an apoptotic agent, a substituted urea, a methyl hydrazine, a steroid, a progestin, an estrogen, an antiestrogen, an androgen, actinomycin, azaribine, anastrozole, azacytidine, bleomycin, bryostatin- 1, busulfan, carmustine, Celebrex, chlorambucil, cisplatin, irinotecan (CPT- 11), carboplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine, docetaxel, dacarbazine, dactinomycin, daunorubicin, dexamethasone, diethyistilbestrol, doxorubicin, ethinyl estradiol, estramustine, etoposide, floxuridine, fludarabine, flutamide, 5-fluorouracil, fluoxymesterone, gemcitabine, hydroxyprogesterone caproate, hydroxyurea, idarubicin, ifosfamide, L-asparaginase, leucovorin, lomustine, mechlorethamine, medroprogesterone acetate, megestrol acetate, melphalan, mercaptopurine, methotrexate, mitoxantrone, mitomycin, mitotane, oxaliplatin, phenyl butyrate, prednisone, procarbazine, paclitaxel, pentostatin, semustine, streptozocin, SN-38, tamoxifen, taxanes, taxol, testosterone propionate, thalidomide, thioguanine, thiotepa, teniposide, topotecan, uracil mustard, vinblastine, vinorelbine or vincristine.

### Claim 10 (depends on 8)

The humanized L243 antibody or antigen-binding fragment thereof of claim 8, wherein the toxin is ricin, abrin, ribonuclease, DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, or Pseudomonas endotoxin.

### Claim 11 (depends on 8)

The humanized L243 antibody or antigen-binding fragment thereof of claim 8, wherein the immunomodulator is a cytokine, a stem cell growth factor, a lymphotoxin, a tumor necrosis factor (TNF), TNF-α, a hematopoietic factor, an interleukin, IL-1, IL-2, IL-3, IL-6, IL- 10, IL- 12, IL- 18, IL-21, a colony stimulating factor, G-CSF, GM-CSF, interferon-α, -β or -γ, the stem cell growth factor designated “S1 factor”, erythropoietin or thrombopoietin.

### Claim 12 (depends on 11)

The humanized L243 antibody or antigen-binding fragment thereof of claim 11, wherein the cytokine is interferon-α, interferon -β, interferon-γ or GM-CSF.

### Claim 13 (depends on 8)

The humanized L243 antibody or antigen-binding fragment thereof of claim 8, wherein the radioisotope is In-111, Lu-177, Bi-212, Bi-213, At-211, Cu-62, Cu-64, Cu-67, Y-90, I-125, I-131, P-32, P-33, Sc-47, Ag-111, Ga-67, Pr-142, Sm-153, Th-161, Dy-166, Ho-166, Re-186, Re-188, Re-189, Pb-212, Ra-223, Ac-225, Fe-59, Se-75, As-77, Sr-89, Mo-99, Rh-105, Pd-109, Pr-143, Pm-149, Er-169, Ir-194, Au-198, Au-199, Ac-225 or Pb-211.

### Claim 14 (depends on 1)

A bispecific antibody or antigen-binding fragment thereof comprising at least one first antibody or fragment thereof according to claim 1 and at least one second antibody or fragment thereof.

### Claim 15 (depends on 14)

The bispecific antibody or fragment thereof of claim 14, wherein the second antibody or second antibody fragment binds to a tumor-associated antigen.

### Claim 16 (depends on 15)

The bispecific antibody or fragment thereof of claim 15, wherein the tumor-associated antigen is selected from the group consisting of A3, antigen specific for A33 antibody, BrE3-antigen, CD1, CD1a, CD3, CD5, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD45, CD74, CD79a, CD80, HLA-DR, NCA95, NCA90, HCG, CEA (CEACAM-5), CEACAM-6, CSAp, EGFR, EGP-1, EGP-2, Ep-CAM, Ba 733, HER2/neu, hypoxia inducible factor (HIF), KC4-antigen, KS-1 antigen, KS 1-4, Le-Y, macrophage inhibition factor (MIF), MAGE, MUC1, MUC2, MUC3, MUC4, MUC16, PAM-4-antigen, PSA, PSMA, RS5, S100, TAG-72, p53, tenascin, IL-6, IL-8, insulin growth factor-1 (IGF-1), Tn antigen, Thomson-Friedenreich antigens, tumor necrosis antigens, VEGF, 17-1 A-antigen, an angiogenesis marker, ED-B fibronectin, an oncogene marker, an oncogene product, Ia, HM1.24, VEGF, ILGF, placental growth factor and carbonic anhydrase IX.

### Claim 17 (depends on 14)

The bispecific antibody or fragment thereof of claim 14, wherein the second antibody or second antibody fragment binds to a targetable conjugate that comprises at least one hapten moiety and at least one therapeutic agent.

### Claim 18 (depends on 1)

The humanized L243 antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or fragment induces apoptosis in cancer cell lines that are resistant to anti-CD20 antibodies.

### Claim 19 (depends on 18)

The humanized L243 antibody or antigen-binding fragment thereof of claim 18, wherein the antibody or fragment induces apoptosis in the Namalwa human lymphoma cell line.

### Claim 20 (depends on 1)

A pharmaceutical composition comprising a humanized L243 antibody or antigen-binding fragment thereof according to claim 1.

### Claim 21 (depends on 20)

The composition of claim 20, wherein the humanized L243 antibody or fragment thereof is conjugated to one or more peptides, lipids, polymeric carriers, micelles, nanoparticles, or a combination thereof.

### Claim 22 (depends on 20)

The composition of claim 20, further comprising at least one therapeutic agent.

### Claim 23 (depends on 22)

The composition of claim 22, wherein the therapeutic agent is a drug, a prodrug, a toxin, an enzyme, a radioisotope, an immunomodulator, a cytokine, a hormone, a second antibody or antigen binding fragment thereof, an oligonucleotide, an interference RNA, a photodynamic agent, or a combination thereof.

### Claim 24 (depends on 20)

The composition of claim 20, wherein the humanized L243 antibody or antigen-binding fragment thereof is conjugated to a chelator selected from the group consisting of NOTA, DOTA, DTPA, TETA, Tscg-Cys, Tsca-Cys, or a combination thereof.

### Claim 25 (depends on 22)

A kit comprising a composition according to claim 22.