CA2774446A1 - Hiv-1 antibodies - Google Patents
Hiv-1 antibodies Download PDFInfo
- Publication number
- CA2774446A1 CA2774446A1 CA2774446A CA2774446A CA2774446A1 CA 2774446 A1 CA2774446 A1 CA 2774446A1 CA 2774446 A CA2774446 A CA 2774446A CA 2774446 A CA2774446 A CA 2774446A CA 2774446 A1 CA2774446 A1 CA 2774446A1
- Authority
- CA
- Canada
- Prior art keywords
- antibody
- cells
- antibodies
- hiv
- infection
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 210000004027 cell Anatomy 0.000 claims abstract description 75
- 238000000034 method Methods 0.000 claims abstract description 36
- 241000713772 Human immunodeficiency virus 1 Species 0.000 claims abstract description 27
- 208000010839 B-cell chronic lymphocytic leukemia Diseases 0.000 claims abstract description 23
- 208000032852 chronic lymphocytic leukemia Diseases 0.000 claims abstract description 9
- 101800001690 Transmembrane protein gp41 Proteins 0.000 claims abstract description 6
- 208000031422 Lymphocytic Chronic B-Cell Leukemia Diseases 0.000 claims abstract description 5
- 239000012634 fragment Substances 0.000 claims description 30
- 230000027455 binding Effects 0.000 claims description 25
- 239000000203 mixture Substances 0.000 claims description 18
- 208000015181 infectious disease Diseases 0.000 claims description 16
- 210000002540 macrophage Anatomy 0.000 claims description 9
- 206010015108 Epstein-Barr virus infection Diseases 0.000 claims description 3
- 210000000987 immune system Anatomy 0.000 claims description 2
- 230000002401 inhibitory effect Effects 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims 1
- 230000003472 neutralizing effect Effects 0.000 abstract description 16
- 210000003719 b-lymphocyte Anatomy 0.000 abstract description 10
- 230000000840 anti-viral effect Effects 0.000 abstract description 4
- 238000004113 cell culture Methods 0.000 abstract description 4
- 238000009877 rendering Methods 0.000 abstract description 3
- 108090000623 proteins and genes Proteins 0.000 description 29
- 108090000765 processed proteins & peptides Proteins 0.000 description 26
- 238000006386 neutralization reaction Methods 0.000 description 20
- 238000002965 ELISA Methods 0.000 description 14
- 238000004091 panning Methods 0.000 description 12
- 239000000523 sample Substances 0.000 description 12
- 241000700605 Viruses Species 0.000 description 11
- 210000004602 germ cell Anatomy 0.000 description 11
- 241000701044 Human gammaherpesvirus 4 Species 0.000 description 10
- 210000001744 T-lymphocyte Anatomy 0.000 description 10
- 235000018102 proteins Nutrition 0.000 description 10
- 102000004169 proteins and genes Human genes 0.000 description 10
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 9
- 102100036011 T-cell surface glycoprotein CD4 Human genes 0.000 description 8
- 210000004408 hybridoma Anatomy 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 208000031886 HIV Infections Diseases 0.000 description 7
- 101150117115 V gene Proteins 0.000 description 7
- 239000000427 antigen Substances 0.000 description 7
- 108091007433 antigens Proteins 0.000 description 7
- 102000036639 antigens Human genes 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- 238000012163 sequencing technique Methods 0.000 description 7
- 206010000807 Acute HIV infection Diseases 0.000 description 6
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 6
- 241001112090 Pseudovirus Species 0.000 description 6
- 238000003556 assay Methods 0.000 description 6
- 230000035772 mutation Effects 0.000 description 6
- 238000012216 screening Methods 0.000 description 6
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 5
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 5
- 239000002299 complementary DNA Substances 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 230000014509 gene expression Effects 0.000 description 5
- 238000002823 phage display Methods 0.000 description 5
- 238000000159 protein binding assay Methods 0.000 description 5
- 208000030507 AIDS Diseases 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 230000005540 biological transmission Effects 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 238000009472 formulation Methods 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 102100027723 Endogenous retrovirus group K member 6 Rec protein Human genes 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 208000005176 Hepatitis C Diseases 0.000 description 3
- 241000725303 Human immunodeficiency virus Species 0.000 description 3
- 230000009824 affinity maturation Effects 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 206010022000 influenza Diseases 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000011282 treatment Methods 0.000 description 3
- 230000003612 virological effect Effects 0.000 description 3
- 208000023275 Autoimmune disease Diseases 0.000 description 2
- 229930105110 Cyclosporin A Natural products 0.000 description 2
- PMATZTZNYRCHOR-CGLBZJNRSA-N Cyclosporin A Chemical compound CC[C@@H]1NC(=O)[C@H]([C@H](O)[C@H](C)C\C=C\C)N(C)C(=O)[C@H](C(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)N(C)C(=O)CN(C)C1=O PMATZTZNYRCHOR-CGLBZJNRSA-N 0.000 description 2
- 108010036949 Cyclosporine Proteins 0.000 description 2
- 101710091045 Envelope protein Proteins 0.000 description 2
- 108010002350 Interleukin-2 Proteins 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 101710188315 Protein X Proteins 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 238000012300 Sequence Analysis Methods 0.000 description 2
- 108010090804 Streptavidin Proteins 0.000 description 2
- 108010060818 Toll-Like Receptor 9 Proteins 0.000 description 2
- 102100033117 Toll-like receptor 9 Human genes 0.000 description 2
- 230000003466 anti-cipated effect Effects 0.000 description 2
- 230000036436 anti-hiv Effects 0.000 description 2
- 239000012736 aqueous medium Substances 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 235000014633 carbohydrates Nutrition 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 239000006071 cream Substances 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000003937 drug carrier Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 208000006454 hepatitis Diseases 0.000 description 2
- 231100000283 hepatitis Toxicity 0.000 description 2
- 230000000423 heterosexual effect Effects 0.000 description 2
- 230000006058 immune tolerance Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000004020 luminiscence type Methods 0.000 description 2
- 230000008774 maternal effect Effects 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000003641 microbiacidal effect Effects 0.000 description 2
- 210000004877 mucosa Anatomy 0.000 description 2
- 239000002674 ointment Substances 0.000 description 2
- 239000006072 paste Substances 0.000 description 2
- 239000008194 pharmaceutical composition Substances 0.000 description 2
- 230000002064 post-exposure prophylaxis Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000011321 prophylaxis Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000000392 somatic effect Effects 0.000 description 2
- 238000010561 standard procedure Methods 0.000 description 2
- 238000011200 topical administration Methods 0.000 description 2
- 206010069754 Acquired gene mutation Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 238000003737 Bright-Glo Luciferase Assay System Methods 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 102100038023 DNA fragmentation factor subunit beta Human genes 0.000 description 1
- 101710121417 Envelope glycoprotein Proteins 0.000 description 1
- 108010008177 Fd immunoglobulins Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 108700023372 Glycosyltransferases Proteins 0.000 description 1
- 101000950965 Homo sapiens DNA fragmentation factor subunit beta Proteins 0.000 description 1
- 101000989062 Homo sapiens Immunoglobulin heavy variable 5-51 Proteins 0.000 description 1
- 102100029414 Immunoglobulin heavy variable 5-51 Human genes 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 108010052090 Renilla Luciferases Proteins 0.000 description 1
- 108700008625 Reporter Genes Proteins 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 241000580858 Simian-Human immunodeficiency virus Species 0.000 description 1
- 230000005867 T cell response Effects 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 239000000556 agonist Substances 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 150000001413 amino acids Chemical group 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 230000005875 antibody response Effects 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000013357 binding ELISA Methods 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 230000011712 cell development Effects 0.000 description 1
- 238000003163 cell fusion method Methods 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 229960001265 ciclosporin Drugs 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 239000003636 conditioned culture medium Substances 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 229940059082 douche Drugs 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- ZINJLDJMHCUBIP-UHFFFAOYSA-N ethametsulfuron-methyl Chemical compound CCOC1=NC(NC)=NC(NC(=O)NS(=O)(=O)C=2C(=CC=CC=2)C(=O)OC)=N1 ZINJLDJMHCUBIP-UHFFFAOYSA-N 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- MUJOIMFVNIBMKC-UHFFFAOYSA-N fludioxonil Chemical compound C=12OC(F)(F)OC2=CC=CC=1C1=CNC=C1C#N MUJOIMFVNIBMKC-UHFFFAOYSA-N 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 102000045442 glycosyltransferase activity proteins Human genes 0.000 description 1
- 108700014210 glycosyltransferase activity proteins Proteins 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 208000010710 hepatitis C virus infection Diseases 0.000 description 1
- 238000012165 high-throughput sequencing Methods 0.000 description 1
- 230000028996 humoral immune response Effects 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000002794 lymphocyte assay Methods 0.000 description 1
- 125000000311 mannosyl group Chemical group C1([C@@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 230000000873 masking effect Effects 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000002864 mononuclear phagocyte Anatomy 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000005868 ontogenesis Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 210000001322 periplasm Anatomy 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- WQVJHHACXVLGBL-GOVYWFKWSA-N polymyxin B1 Polymers N1C(=O)[C@H](CCN)NC(=O)[C@@H](NC(=O)[C@H](CCN)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCN)NC(=O)CCCC[C@H](C)CC)CCNC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCN)NC(=O)[C@H](CCN)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]1CC1=CC=CC=C1 WQVJHHACXVLGBL-GOVYWFKWSA-N 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000002818 protein evolution Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000037439 somatic mutation Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 235000002374 tyrosine Nutrition 0.000 description 1
- 150000003668 tyrosines Chemical class 0.000 description 1
- 238000012056 up-stream process Methods 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 230000029812 viral genome replication Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1036—Retroviridae, e.g. leukemia viruses
- C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
- C07K16/1063—Lentiviridae, e.g. HIV, FIV, SIV env, e.g. gp41, gp110/120, gp160, V3, PND, CD4 binding site
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/18—Antivirals for RNA viruses for HIV
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/55—Fab or Fab'
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Virology (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- AIDS & HIV (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Oncology (AREA)
- Pharmacology & Pharmacy (AREA)
- Hematology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Animal Behavior & Ethology (AREA)
- Communicable Diseases (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Tropical Medicine & Parasitology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The present invention relates, in general, to HIV-1 specific antibodies and, in particular, to broadly neutralizing HIV-1 specific antibodies that target the gp41 membrane-proximal external region (MPER). The present invention also relates to a cell culture system, more specifically, to a method of rendering chronic lymphocytic leukemia B-cells immortal and to a method of isolating anti-viral antibodies from clones of such cells.
Description
This application claims priority from U.S. Provisional Application No. 61/272,349, filed September 16, 2009 and from U.S. Provisional Application No. 61/248,796, filed October 5, 2009, the entire contents of both of which are incorporated herein by reference.
This invention was made with government support under Grant No. Al 067854-05 and Grant No. UO1 Al 067854-02, awarded by the National Institutes of Health. The government has certain rights in the invention.
TECHNICAL FIELD
The present invention relates, in general, to HIV-1 specific antibodies and, in particular, to broadly neutralizing HIV-1 specific antibodies that target the gp41 membrane-proximal external region (MPER).
The present invention also relates to a cell culture system, more specifically, to a method of rendering chronic lymphocytic leukemia B-cells immortal and to a method of isolating anti-viral antibodies from clones of such cells.
BACKGROUND
The development of strategies to utilize human antibodies that potently inhibit HIV-1 infection of T cells and mononuclear phagocytes is a high priority for treatment and prevention of HIV-1 infection (Mascola et al, J. Virol.
79:10103-10107 (2005)). A few rare human monoclonal antibodies (mAbs) against gpl60 have been isolated that can broadly neutralize HIV-1 in vitro, and can protect non-human primates from SHIV infections in vivo (Mascola et al, Nat.
Med. 6:207-210 (2000), Baba et al, Nat. Med. 6:200-206 (2000)). These mAbs include antibodies 2F5 and 4E 10 against the membrane proximal external region (MPER) of gp4l (Muster et al, J. Virol. 67:6642-6647 (1993), Stiegler et al, AIDS
Res. & Hum. Retro. 17:1757-1765 (2001), Zwick et al, J. Virol. 75:10892-10905 (2001)), IgG l b 12 against the CD4 binding site of gp 120 (Roben et al, J.
Virol.
68:4821-4828 (1994)), and mAb 2G12 against gp120 high mannose residues (Sanders et al, J. Virol. 76:7293-7305 (2002)).
HIV-1 has evolved a number of effective strategies for evasion from neutralizing antibodies, including glycan shielding of neutralizing epitopes (Wei et al, Nature 422:307-312 (2003)), entropic barriers to neutralizing antibody binding (Kwong et al, Nature 420:678-682 (2002)), and masking or diversion of antibody responses by non-neutralizing antibodies (Alam et al, J. Virol.
82:115-125 (2008)). Despite intense investigation, it remains a conundrum why broadly neutralizing antibodies against either the gp120 CD4 binding site or the membrane proximal region of gp4l are not routinely induced in either animals or man.
One clue as to why broadly neutralizing antibodies are difficult to induce may be found in the fact that all of the above-referenced mAbs have unusual properties. The mAb 2G12 is against carbohydrates that are synthesized and modified by host glycosyltransferases and are, therefore, likely recognized as self carbohydrates (Calarese et al, Proc. Natl. Acad. Sci. USA 102:13372-13377 (2005)). 2G12 is also a unique antibody with Fabs that assemble into an interlocked VH domain-swapped dimers (Calarese et al, Science 300:2065-2071 (2003)). 2F5 and 4E10 both have long CDR3 loops, and react with multiple host antigens including host lipids (Zwick et al, J. Virol. 75:10892-10905 (2001), Alam et al, J. Immun. 178:4424-4435 (2007), Zwick et al, J. Virol. 78:3155-(2004), Sun et al, Immunity 28:52-63 (2008)). Similarly, IgGlbl2 also has a long CDR3 loop and reacts with dsDNA (Haynes et al, Science 308:1906-1908 (2005), Saphire et al, Science 293:1155-1159 (2001)). These findings, coupled with the perceived rarity of clinical HIV-1 infection in patients with autoimmune disease (Palacios and Santos, Inter. J. STD AIDS 15:277-278 (2004)), have prompted the hypothesis that some species of broadly reactive neutralizing antibodies are not made due to downregulation by immune tolerance mechanisms (Haynes et at, Science 308:1906-1908 (2005), Haynes et at, Hum. Antibodies 14:59-67 (2005)).
A corollary of this hypothesis is that some patients with autoimmune diseases may be "exposed and uninfected" subjects with some type of neutralizing antibody as a correlate of protection (Kay, Ann. Inter. Med. 111:158-167 (1989)).
A patient with broadly neutralizing antibodies that target the 2F5 epitope region of the MPER of gp41 has been defined (Shen et at, J. Virol. 83:3617-25 (2009)).
The present invention results, at least in part, from studies designed to identify 2F5-like mAbs from the patient of Shen et at (J. Virol. 83:3617-25 (2009)) that confer broad neutralizing activity. These studies involved preparing libraries from antibody fragments (scFv, scFab, Fab) displayed on yeast or phage from peripheral blood mononuclear cells (PBMCs) from an HIV-1 infected individual. The libraries were panned and screened with a peptide containing the 2F5 epitope, as well as with gpl40s. Antibodies were identified that bind specifically to the peptide, that binding being competed with 2F5.
SUMMARY OF THE INVENTION
In general, the present invention relates to HIV-1 specific antibodies.
More specifically, the invention relates to broadly neutralizing HIV-1 specific antibodies that target the gp4l MPER, and to methods of using same to both treat and prevent HIV-1 infection. The invention also relates to a method of rendering chronic lymphocytic leukemia B-cells immortal and to a method of isolating anti-viral antibodies from clones of such cells.
Objects and advantages of the present invention will be clear from the description that follows.
This invention was made with government support under Grant No. Al 067854-05 and Grant No. UO1 Al 067854-02, awarded by the National Institutes of Health. The government has certain rights in the invention.
TECHNICAL FIELD
The present invention relates, in general, to HIV-1 specific antibodies and, in particular, to broadly neutralizing HIV-1 specific antibodies that target the gp41 membrane-proximal external region (MPER).
The present invention also relates to a cell culture system, more specifically, to a method of rendering chronic lymphocytic leukemia B-cells immortal and to a method of isolating anti-viral antibodies from clones of such cells.
BACKGROUND
The development of strategies to utilize human antibodies that potently inhibit HIV-1 infection of T cells and mononuclear phagocytes is a high priority for treatment and prevention of HIV-1 infection (Mascola et al, J. Virol.
79:10103-10107 (2005)). A few rare human monoclonal antibodies (mAbs) against gpl60 have been isolated that can broadly neutralize HIV-1 in vitro, and can protect non-human primates from SHIV infections in vivo (Mascola et al, Nat.
Med. 6:207-210 (2000), Baba et al, Nat. Med. 6:200-206 (2000)). These mAbs include antibodies 2F5 and 4E 10 against the membrane proximal external region (MPER) of gp4l (Muster et al, J. Virol. 67:6642-6647 (1993), Stiegler et al, AIDS
Res. & Hum. Retro. 17:1757-1765 (2001), Zwick et al, J. Virol. 75:10892-10905 (2001)), IgG l b 12 against the CD4 binding site of gp 120 (Roben et al, J.
Virol.
68:4821-4828 (1994)), and mAb 2G12 against gp120 high mannose residues (Sanders et al, J. Virol. 76:7293-7305 (2002)).
HIV-1 has evolved a number of effective strategies for evasion from neutralizing antibodies, including glycan shielding of neutralizing epitopes (Wei et al, Nature 422:307-312 (2003)), entropic barriers to neutralizing antibody binding (Kwong et al, Nature 420:678-682 (2002)), and masking or diversion of antibody responses by non-neutralizing antibodies (Alam et al, J. Virol.
82:115-125 (2008)). Despite intense investigation, it remains a conundrum why broadly neutralizing antibodies against either the gp120 CD4 binding site or the membrane proximal region of gp4l are not routinely induced in either animals or man.
One clue as to why broadly neutralizing antibodies are difficult to induce may be found in the fact that all of the above-referenced mAbs have unusual properties. The mAb 2G12 is against carbohydrates that are synthesized and modified by host glycosyltransferases and are, therefore, likely recognized as self carbohydrates (Calarese et al, Proc. Natl. Acad. Sci. USA 102:13372-13377 (2005)). 2G12 is also a unique antibody with Fabs that assemble into an interlocked VH domain-swapped dimers (Calarese et al, Science 300:2065-2071 (2003)). 2F5 and 4E10 both have long CDR3 loops, and react with multiple host antigens including host lipids (Zwick et al, J. Virol. 75:10892-10905 (2001), Alam et al, J. Immun. 178:4424-4435 (2007), Zwick et al, J. Virol. 78:3155-(2004), Sun et al, Immunity 28:52-63 (2008)). Similarly, IgGlbl2 also has a long CDR3 loop and reacts with dsDNA (Haynes et al, Science 308:1906-1908 (2005), Saphire et al, Science 293:1155-1159 (2001)). These findings, coupled with the perceived rarity of clinical HIV-1 infection in patients with autoimmune disease (Palacios and Santos, Inter. J. STD AIDS 15:277-278 (2004)), have prompted the hypothesis that some species of broadly reactive neutralizing antibodies are not made due to downregulation by immune tolerance mechanisms (Haynes et at, Science 308:1906-1908 (2005), Haynes et at, Hum. Antibodies 14:59-67 (2005)).
A corollary of this hypothesis is that some patients with autoimmune diseases may be "exposed and uninfected" subjects with some type of neutralizing antibody as a correlate of protection (Kay, Ann. Inter. Med. 111:158-167 (1989)).
A patient with broadly neutralizing antibodies that target the 2F5 epitope region of the MPER of gp41 has been defined (Shen et at, J. Virol. 83:3617-25 (2009)).
The present invention results, at least in part, from studies designed to identify 2F5-like mAbs from the patient of Shen et at (J. Virol. 83:3617-25 (2009)) that confer broad neutralizing activity. These studies involved preparing libraries from antibody fragments (scFv, scFab, Fab) displayed on yeast or phage from peripheral blood mononuclear cells (PBMCs) from an HIV-1 infected individual. The libraries were panned and screened with a peptide containing the 2F5 epitope, as well as with gpl40s. Antibodies were identified that bind specifically to the peptide, that binding being competed with 2F5.
SUMMARY OF THE INVENTION
In general, the present invention relates to HIV-1 specific antibodies.
More specifically, the invention relates to broadly neutralizing HIV-1 specific antibodies that target the gp4l MPER, and to methods of using same to both treat and prevent HIV-1 infection. The invention also relates to a method of rendering chronic lymphocytic leukemia B-cells immortal and to a method of isolating anti-viral antibodies from clones of such cells.
Objects and advantages of the present invention will be clear from the description that follows.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Chronic HIV-1 infections in patients with high levels of bnAbS.
Antibodies in peripheral blood from a patient (SC44) with 2F5-like antibodies.
Figure 2. Ontogeny and isolation of broadly neutralizing HIV-1 antibodies.
Figure 3. M66 antibody binding data.
Figures 4A and 4B. IMGT V-Quest analysis of m66 VH and VL. The closet germline V gene segment is aligned with the VH as shown in Fig. 4A and VL as shown in Fig. 4B. The mutation sites differentiated from germline sequences were highlighted in bold font; the frameworks and CDRs also were defined and labeled according to IMGT database.
Figures 5A and 5B. Comparison of m66.6 and 2F5 on binding to both the MPER peptide and JRFL gp140 protein through ELISA. (Fig. 5A) Biotinylated MPER peptide was captured by coated strepatavidin on plate as target for the binding assay. (Fig. 5B) Purified soluble gp140 protein was coated directly on plate as target for binding assay.
Figure 6. SC44 patient derived IgG specific VH gene repertoire profiling through similarity match using 7 germline V genes from 7 heavy chain subfamilies as probes.
Figure 7. Phylogenetic tree of m66 variants.
Figure 1. Chronic HIV-1 infections in patients with high levels of bnAbS.
Antibodies in peripheral blood from a patient (SC44) with 2F5-like antibodies.
Figure 2. Ontogeny and isolation of broadly neutralizing HIV-1 antibodies.
Figure 3. M66 antibody binding data.
Figures 4A and 4B. IMGT V-Quest analysis of m66 VH and VL. The closet germline V gene segment is aligned with the VH as shown in Fig. 4A and VL as shown in Fig. 4B. The mutation sites differentiated from germline sequences were highlighted in bold font; the frameworks and CDRs also were defined and labeled according to IMGT database.
Figures 5A and 5B. Comparison of m66.6 and 2F5 on binding to both the MPER peptide and JRFL gp140 protein through ELISA. (Fig. 5A) Biotinylated MPER peptide was captured by coated strepatavidin on plate as target for the binding assay. (Fig. 5B) Purified soluble gp140 protein was coated directly on plate as target for binding assay.
Figure 6. SC44 patient derived IgG specific VH gene repertoire profiling through similarity match using 7 germline V genes from 7 heavy chain subfamilies as probes.
Figure 7. Phylogenetic tree of m66 variants.
Figure 8. Epstein-Barr virus (EBV) transformation of B cells (for B-CLL
cells).
Figure 9. Preparation of complete medium.
Figure 10. Electrofusion procedure.
Figure 11. EBV-transformation of B-CLL cells and production of monoclonal antibodies.
Figures 12A-12D. Comprehensive CLL screening results.
Figure 13. Luminex analysis of'gp41 reactive IgM from CLL 1075 hybridoma (CLL1075-2).
Figure 14. The J774A.1 feeder cells enhance growth and production of IgM from B-CLL cells (CLL246) after Epstein Barr virus (EBV) infection. After incubation with EBV, the B-CLL cells were plated at 5,000 cells/well (A) in the absence of J774A.1 cells or (B) in the presence of irradiated J774A.1 cells (50,000 cells/well). Three weeks after infection, levels of IgM were detected by ELISA and expressed in g/ml.
Figure 15. A B-CLL cell line, CLL246, produces IgM against both HIV-1 gp140 and hepatitis virus C E2 (HCV E2) envelope proteins. IgM against the test antigens was detected by ELISA and expressed in OD.
cells).
Figure 9. Preparation of complete medium.
Figure 10. Electrofusion procedure.
Figure 11. EBV-transformation of B-CLL cells and production of monoclonal antibodies.
Figures 12A-12D. Comprehensive CLL screening results.
Figure 13. Luminex analysis of'gp41 reactive IgM from CLL 1075 hybridoma (CLL1075-2).
Figure 14. The J774A.1 feeder cells enhance growth and production of IgM from B-CLL cells (CLL246) after Epstein Barr virus (EBV) infection. After incubation with EBV, the B-CLL cells were plated at 5,000 cells/well (A) in the absence of J774A.1 cells or (B) in the presence of irradiated J774A.1 cells (50,000 cells/well). Three weeks after infection, levels of IgM were detected by ELISA and expressed in g/ml.
Figure 15. A B-CLL cell line, CLL246, produces IgM against both HIV-1 gp140 and hepatitis virus C E2 (HCV E2) envelope proteins. IgM against the test antigens was detected by ELISA and expressed in OD.
Figure 16. Summary of B-CLL cultures. Purpose: i) to infect B-CLL
cells with EBV and generate the IgM-producing hybridomas, and ii) to profile reactivity and HIV-neutralizing activity of B-CLL IgM.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates, in one embodiment, to a method of inhibiting infection of cells (e.g., T-cells) of a subject by HIV-1. The invention also relates to a method of controlling the initial viral load and preserving the CD4+ T cell pool and preventing CD4+ T cell destruction. The method comprises administering to the subject (e.g., a human subject) an HIV-1 specific antibody (other than 2F5) that binds the 2F5 epitope, or fragment thereof, in an amount and under conditions such that the antibody, or fragment thereof, inhibits infection.
In accordance with the invention, the antibodies can be administered prior to contact of the subject or the subject's immune system/cells with HIV-1 or after infection of vulnerable cells. Administration prior to contact or shortly thereafter can maximize inhibition of infection of vulnerable cells of the subject (e.g., T-cells).
One preferred antibody for use in the invention is a mAb having the variable heavy and variable light sequences of the M66 antibody as set forth in Table 1. Libraries were prepared from antibody fragments (scFv, scFab, Fab) displayed on yeast or phage from peripheral blood mononuclear cells (PBMCs) from an HIV-1 infected individual. The libraries were panned and screened with a peptide containing the 2F5 epitope (see Fig. 1, "QQEKNEQELLELD-KWASLWN", as well as with gpl40s. Antibodies were identified that bound specifically to the peptide, that binding being competed with 2F5. The M66 antibody neutralized four out of four tested isolates from Glade B. The closest corresponding germline variable heavy (VH) gene of M66 is IGHV5-51 *01 and its heavy chain complementary determining region (CDR) is long (23 amino acid residues) comparable to that of 2F5 (24 residues). The degree of somatic hypermutation of the M66 VH gene is lower than that of 2F5. The variable heavy and variable light gene and amino acid sequences of the M66 antibody are set forth in Table 1. Table 2 includes details of identified CDR regions.
cells with EBV and generate the IgM-producing hybridomas, and ii) to profile reactivity and HIV-neutralizing activity of B-CLL IgM.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates, in one embodiment, to a method of inhibiting infection of cells (e.g., T-cells) of a subject by HIV-1. The invention also relates to a method of controlling the initial viral load and preserving the CD4+ T cell pool and preventing CD4+ T cell destruction. The method comprises administering to the subject (e.g., a human subject) an HIV-1 specific antibody (other than 2F5) that binds the 2F5 epitope, or fragment thereof, in an amount and under conditions such that the antibody, or fragment thereof, inhibits infection.
In accordance with the invention, the antibodies can be administered prior to contact of the subject or the subject's immune system/cells with HIV-1 or after infection of vulnerable cells. Administration prior to contact or shortly thereafter can maximize inhibition of infection of vulnerable cells of the subject (e.g., T-cells).
One preferred antibody for use in the invention is a mAb having the variable heavy and variable light sequences of the M66 antibody as set forth in Table 1. Libraries were prepared from antibody fragments (scFv, scFab, Fab) displayed on yeast or phage from peripheral blood mononuclear cells (PBMCs) from an HIV-1 infected individual. The libraries were panned and screened with a peptide containing the 2F5 epitope (see Fig. 1, "QQEKNEQELLELD-KWASLWN", as well as with gpl40s. Antibodies were identified that bound specifically to the peptide, that binding being competed with 2F5. The M66 antibody neutralized four out of four tested isolates from Glade B. The closest corresponding germline variable heavy (VH) gene of M66 is IGHV5-51 *01 and its heavy chain complementary determining region (CDR) is long (23 amino acid residues) comparable to that of 2F5 (24 residues). The degree of somatic hypermutation of the M66 VH gene is lower than that of 2F5. The variable heavy and variable light gene and amino acid sequences of the M66 antibody are set forth in Table 1. Table 2 includes details of identified CDR regions.
>M66VH.seq CAGATGCAGCTGGTGCAGTCTGGAGCAGAGGTGAA.A.AAGCCCGGGGAGTCTCTGAAGATCTCCTGTA
AGGTTTCTGGATACAACTTTGCCAGCGAATGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCT
GGAGTGGATGGGGATTATCTATCCTGGTGACTCTGATACCAAATACAGCCCGTCCTTCCAAGGCCAG
GTCATCATCTCAGCCGACAAGTCCATCAACACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGG
ACACCGCCATATATTACTGTGCGAGACAGAATCACTATGGTTCGGGGAGTTATTTCTACCGAACGGC
CTACTACTATGCTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA
>M66VH.pep QMQLVQSGAEVKKPGESLKISCKVSGYNFASEWIGWVRQMPGKGLEWMGIIYPGDSDTKYSPSFQGQ
VI ISADKSINTAYLQWSSLKASDTAIYYCARQNHYGSGSYFYRTAYYYAMDVWGQGTTVTVSS
>M66VL.seq GTGGCCCAGGCGGCCGACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACA
GAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACC
AGGGAAAGCCCCTAACCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCGTCAAGGTTC
AGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAA
CTTACTACTGTCAACAGAGTTACAATACCCCATTCACTCTTGGCCCGGGGACCAAGGTGGAGATCAA
A
>M66VL..pep VAQAADIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPNLLIYAASSLQSGVPSRF
SGSGSGTDFTLTISSLQPEDFATYYCQQSYNTPFTLGPGTKVEIKR
AGGTTTCTGGATACAACTTTGCCAGCGAATGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCT
GGAGTGGATGGGGATTATCTATCCTGGTGACTCTGATACCAAATACAGCCCGTCCTTCCAAGGCCAG
GTCATCATCTCAGCCGACAAGTCCATCAACACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGG
ACACCGCCATATATTACTGTGCGAGACAGAATCACTATGGTTCGGGGAGTTATTTCTACCGAACGGC
CTACTACTATGCTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA
>M66VH.pep QMQLVQSGAEVKKPGESLKISCKVSGYNFASEWIGWVRQMPGKGLEWMGIIYPGDSDTKYSPSFQGQ
VI ISADKSINTAYLQWSSLKASDTAIYYCARQNHYGSGSYFYRTAYYYAMDVWGQGTTVTVSS
>M66VL.seq GTGGCCCAGGCGGCCGACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACA
GAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACC
AGGGAAAGCCCCTAACCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCGTCAAGGTTC
AGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAA
CTTACTACTGTCAACAGAGTTACAATACCCCATTCACTCTTGGCCCGGGGACCAAGGTGGAGATCAA
A
>M66VL..pep VAQAADIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPNLLIYAASSLQSGVPSRF
SGSGSGTDFTLTISSLQPEDFATYYCQQSYNTPFTLGPGTKVEIKR
H
w E
z a v v a I N H
ri) U O
`^vl E
E
4 x -J x a U Q
A
y W
x - w ELI Ln U ~O
lI
41 %D
C A tO
Q H ~
w E
z a v v a I N H
ri) U O
`^vl E
E
4 x -J x a U Q
A
y W
x - w ELI Ln U ~O
lI
41 %D
C A tO
Q H ~
Another preferred antibody for use in the invention is a mAb having the variable heavy and variable light sequences as the M66.6 antibody set forth in Table 3. Details of the identification of the M66.6 antibody are provided in Example 3.
Table 3 M66.6H DNA sequence :
cagatgcagctggtgcagtctggagcagaggtgaaaaagcccggggagtctctgaagatctcctgt aaggtttctggatacaactttgccagcgaatggatcggctgggtgcgccagatgcccgggaaaggc ctggagtggatggggattatctatcctggtgactctgataccaaatacagcccgtccttccaaggc caggtcatcatctcagccgacaagtccatcaac accgcctacctgcagtggagcagcctgaaggcctcggacaccgccatatattactgtgcgagacag aatcactatggttcggggagttatttctaccgaacggcctactactatgctatggacgtctggggc caagggaccacggtcaccgtctcctca M66.6 VH protein sequence :
Q M Q L V Q S G A E V K K P G E S L K I S C K V S G Y N F A S E W I G W V R Q
M P G K
G L E W M G I I Y P G D S D T K Y S P S F Q G Q V I I S A D K S I N T A Y L Q
W S S L K A
S D T A I Y Y C A R Q N H Y G S G S Y F Y R T A Y Y Y A M D V W G Q G T T V T
V S S
M66.6 Light chain DNA sequences:
gacatccagttgacccagtctccatcttccctgtctgcatctttgggggacaaagtcacca.tcact tgccgggcaagtcagcacattaagaagtatttaaactggtatcagcagaaacctgggaaagcccct aaactcctgatctatggtgcactcaatttgcagagtggggtcccatcaaggttcagtggcagagga tctgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgt cagcagagttacagtaccccattcactttcggccctgggaccaaagtggatatcaaacga M66.6 VL Protein sequence DIQLTQSPSSLSASLGDKVTITCRASQHIKKYLNWYQQKPGK
APKLL IYGALNLQSGVPSRFSGRGSGT DFTLTISSLQPEDFAT
YYCQQSYSTPFTFGPGTKVDIKR
As indicated above, either the intact antibody or fragment (e.g., antigen binding fragment) thereof can be used in the method of the present invention.
Exemplary functional fragments (regions) include scFv, Fv, Fab', Fab and F(ab')2 fragments. Single chain antibodies can also be used. Techniques for preparing suitable fragments and single chain antibodies are well known in the art.
(See, for example, USPs 5,855,866; 5,877,289; 5,965,132; 6,093,399; 6,261,535;
6,004,555; 7,417,125 and 7,078,491 and WO 98/45331.) The invention also includes variants of the antibodies (and fragments) disclosed herein, including variants that retain the binding properties of the antibodies (and fragments) specifically disclosed, and methods of using same in the present method. For example, the invention includes an isolated human antibody or fragment thereof that binds selectively to gp4l MPER and that comprises one or more CDRs as set forth in Table 2 and Fig. 4. Modifications of M66 and M66.6 that can be used therapeutically in accordance with the invention include IgA, IgM and IgG1, 2, or 4 versions of the M66 M66.6 VH and VL chains.
The antibodies, and fragments thereof, described above can be formulated as a composition (e.g., a pharmaceutical composition). Suitable compositions can comprise the antibody (or antibody fragment) dissolved or dispersed in a pharmaceutically acceptable carrier (e.g., an aqueous medium). The compositions can be sterile and can in an injectable form. The antibodies (and fragments thereof) can also be formulated as a composition appropriate for topical administration to the skin or mucosa. Such compositions can take the form of liquids, ointments, creams, gels and pastes. Standard formulation techniques can be used in preparing suitable compositions. The antibodies can be formulated so as to be administered as a post-coital douche or with a condom.
The antibodies and antibody fragments of the invention show their utility for prophylaxis in, for example, the following settings:
i) in the setting of anticipated known exposure to HIV-1 infection, the antibodies described herein (or binding fragments thereof) can be administered prophylactically (e.g., IV or topically) as a microbiocide, ii) in the setting of known or suspected exposure, such as occurs in the setting of rape victims, or commercial sex workers, or in any heterosexual transmission with out condom protection, the antibodies described herein (or fragments thereof) can be administered as post-exposure prophylaxis, e.g., IV
or topically, iii) in the setting of Acute HIV infection (AHI), antibodies described herein (or binding fragments thereof) can be administered as a treatment for AHI
to control the initial viral load and preserve the CD4+ T cell pool and prevent CD4+ T cell destruction, and iv) in the setting of maternal to baby transmission whi8le the child is breastfeeding.
Suitable dose ranges can depend, for example, on the antibody and on the nature of the formulation and route of administration. Optimum doses can be determined by one skilled in the art without undue experimentation. Doses of antibodies in the range of I Ong to 20 .tg/ml can be suitable.
In another embodiment, the present invention provides a cell culture system that makes possible the production of monoclonal anti-viral antibodies from B-chronic lymphocytic leukemia (B-CLL) cell repertoires. In accordance with this embodiment of the invention, macrophage cells are used as feeder cells to grow B-CLL cells in culture following EBV-infection.
A specific example of a protocol suitable for use in generating immortalized clones of B-CLL cells is set forth in Figure 8. Generally, a macrophage cell line (e.g., a rodent macrophage cell line) is used as a feeder. A
preferred macrophage cell line is the mouse line J774A. 1. J774A. I macrophage cells have been shown to produce growth factors suitable for hybridoma growth and cloning (Rathjen and Geczy, Hybridoma 5:255-261 (1986)). Conditioned medium prepared from J774A.1 cells has been widely used for enhancing hybridoma viability. It has been also shown that co-culture of the J774A.1 cell line as a feeder with T cells, as well as EBV-infected B cells, helps clearance of apoptotic cells in vitro (Kagan et al, J. Immunol. 169:487-499 (2002)).
In accordance with the invention, the macrophage cell line is subjected to irradiation to prevent outgrowth of the feeder cells. The optimum y-irradiation dose can be determined experimentally, for example, by testing the cell line to determine the minimum dose required to completely inhibit cell proliferation (e.g., about 4,000 Rad). The irradiated cells can then be distributed into culture containers (for example, when 96-well plates are used, about 50,000 cells can distributed/well (about 100 l/well)). The optimum number of irradiated cells can be determined experimentally by testing the cells at different densities.
EBV infection of B-CLL cells can be effected using standard techniques.
For example, peripheral blood mononuclear cells (PBMCs) can be isolated from a blood sample from a CLL patient using standard techniques, the cells can then be washed in complete medium (see, for example, Fig. 9) and suspended in complete medium comprising, for example, PS 2006 and cyclosporine A. PS 2006, a Toll-like receptor 9 (TLR-9) agonist, can be added to stimulate B cells according to previously described methods (Lanzavecchia et al, Curr. Opin. Biotech. 18:523-528 (2007)). Cyclosporin A can be added to suppress any EBV-B cell-specific cytotoxic T cell response. Optimal concentrations can be determined experimentally by testing EBV-B cell stimulation potency.
In accordance with this embodiment of the invention, an EBV suspension is then added to the cell suspension. Following incubation at about 37 C
(e.g., for about I to about 24 hours, preferably, for about 4 hours) the EBV-infected cells can be resuspended in complete medium comprising, for example, PS2006, distributed into the culture container with the irradiated macrophage cells and incubated at about 37 C..The medium can be changed periodically and antibody production assessed using, for example, ELISA when the EBV-infected cells have grown sufficiently (e.g., about 3-about 4 weeks post-infection). IgM producing hybridoma cell lines derived from CLL cells can then be produced, for example, using a hybridoma cell fusion method, such as that described in Fig. 10, and cloned. (See, generally, Fig. 11) Three monoclonal B-CLL hybridoma cell lines have been derived from CLL246 (VH1-69, unmutated case), CLL1075 (VH1-69, mutated case), and CLL493 (VH1-69, unmutated case). (See Figs. 12 and 13.) Therapeutic antibodies (e.g., anti-HIV antibodies, anti-hepatitis C
antibodies or anti-influenza antibodies) produced from B-CLL cells immortalized in accordance with the method described above, or fragments thereof (e.g., antigen binding fragments - exemplary functional fragments (regions) include scFv, Fv, Fab', Fab and F(ab')2 fragments) can be formulated as a composition (e.g., a pharmaceutical composition). Suitable compositions can comprise the antibody (or antibody fragment) dissolved or dispersed in a pharmaceutically acceptable carrier (e.g., an aqueous medium). The compositions can be sterile and can be in an injectable form. The antibodies (and fragments thereof) can also be formulated as a composition appropriate for topical administration to the skin or mucosa. Such compositions can take the form of liquids, ointments, creams, gels, pastes or aerosols. Standard formulation techniques can be used in preparing suitable compositions.
The anti-HIV antibodies and fragments thereof show their utility for prophylaxis in, for example, the following settings:
i) in the setting of anticipated known exposure to HIV-1 infection, the antibodies described herein (or binding fragments thereof) can be administered prophylactically (e.g., IV or topically) as a microbiocide, ii) in the setting of known or suspected exposure, such as occurs in the setting of rape victims, or commercial sex workers, or in any heterosexual transmission with out condom protection, the antibodies described herein (or fragments thereof) can be administered as post-exposure prophylaxis, e.g., IV
or topically, iii) in the setting of Acute HIV infection (AHI), antibodies described herein (or binding fragments thereof) can be administered as a treatment for AHI
to control the initial viral load and preserve the CD4+ T cell pool and prevent CD4+ T cell destruction, and iv) in the setting of maternal to baby transmission while the child is breastfeeding.
Anti-hepatitis C and anti-influenza antibodies, or fragments thereof as described above, can be used to treat or prevent hepatitis C and influenza infection, respectively.
Suitable dose ranges can depend, for example, on the antibody, on the nature of the formulation, on the route of administration, and on the patient (e.g., a human patient). Optimum doses can be determined by one skilled in the art without undue experimentation. Doses of antibodies in the range of 1 Ong to g/ml can be suitable.
Certain aspects of the invention can be described in greater detail in the non-limiting Examples that follows. (See also U.S. Prov. Applns. 61/272,349 and 20 61/248,769, Shen et al, J. Virol. 83(8):3617-25 Epub 2009, Zhu and Dimitrov, Methods Mol. Biol. 525:129-142 (2009), Dimitrov and Marks, Methods Mol.
Biol. 525:1-27 (2009), Zhang et al, J. Virol. 82(14):6869-6879 (2008), Prabakaran et al, Advances in Pharmacology 55:33-97 (2007), Perez et al, J. Virol.
83(15):7397-7410 (2009)). (See also Chan et al, Blood 97:1023-1026 (2001);
Gorny et al, Mol. Immunol. 46:917-926 (2009)).
EXAMPLE I
IgM and IgG libraries from a patient with acute infection and a patient with 2F5-like antibodies were generated and analyzed by high throughput (454) sequencing and other methods. HIV-1-specific antibodies from IgG libraries derived from the blood of an acutely infected patient at two time points (40 days and 8 months) were identified. These antibodies bound envelope glycoproteins (Envs) with high affinity but did not neutralize a panel of 9 pseudoviruses.
Antibodies from samples at 40 days were not found at 8 months and antibodies from samples at 8 months did not bind a dominant Env at 40 days. Panning of bone marrow derived libraries from the same patient did not result in selection of any antibodies.
Novel antibodies were selected from IgM+IgG phage and yeast display libraries derived from a patient with 2F5-like antibodies. One of these antibodies, M66, has long (23 residues) heavy chain CDR3 and its VH gene has relatively low number of somatic mutations. It bound specifically to a gp41 MPER peptide containing the 2F5 epitope and cross-reactively neutralized HIV-1 isolates.
The other antibodies are being characterized. These results could have implications for understanding humoral immune responses and design of vaccine immunogens.
(See Figs. 1 and 2.) Supernatants from 293T cells stably transfected with M66 antibody genes were evaluated for binding to the 2F5 epitope by custom HIV-1 luminex. Serial dilutions of supernatant were assessed for binding to the epitope (readout of mean fluorescence intensity -MFI). Positive controls for the experiment included mAb, and HIVIG titrations and the negative control was the mock transfected supernatant. The binding data for the M66 antibody is presented in Fig. 3.
EXAMPLE -3) Experimental Details cDNA, antibodies, gp140s and peptides cDNA was prepared using the total RNA extracted from PBML taken from Patient SC44 at 12 month after enrollment (Shen et al, J. Virol. 83:3617-(2009)). HIV-1 gp4l MAbs 2F5 was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH (Hermann Katinger). Recombinant gp140s were kindly provided by C. Broder (Uniformed Services University of the Health Sciences, Bethesda, MD). Two biotinylated lo peptides containing the 2F5 epitope sequence (2F5 peptides) were used for panning and binding assay: SP62 QQEKNEQELLELDKWASLWN and SP62 scrambled peptide. These peptides were custom-made by Primm Biotech and CPC. Horseradish peroxidase (HRP)-conjugated anti-FLAG tag antibody and HRP-conjugated anti-human IgG (Fc-specific) antibody were purchased from Sigma-Aldrich (St. Louis).
Phage Display Fab library construction and library panning, screening Phage display Fab libraries were constructed primarily following a published protocol (Zhu et al, Methods Mol. Biol. 525:129-42, xv (2009)) using the cDNA prepared from PBMC of patient SC44 as template for the antibody gene repertoire cloning. Sequential pannings were performed using biotinylated Peptide SP62 and gp140 proteins with the first three rounds of panning on 1 g of biotinylated peptide on streptavidin conjugated magnetic bead and the fourth and fifth pannings on gp140 (JRFL) coated at 1 ug/well on 96 well ELISA plates.
Bound phage on the beads or plate wells were directly used to infect exponentially growing TGI cells and rescued by M13KO7 helper phage and amplified for the next round panning. 190 individual colonies after the fifth round panning were picked and inoculated into 2YT medium in 96-well plate for phage ELISA screening as described (Zhu et al, Methods Mol. Biol. 525:129-42, xv (2009)). Identified positive binders were sequenced and a unique clone was expressed and purified Fab was used for preliminary characterization.
Generation and selection of the light chain-shuffled phage display library The Fd fragment of m66 Fab construct was PCR amplified and fused with the light chain gene repertoire obtained during the original Fab library construction using overlapping PCR and cloned into a phagmid vector essentially as described (Zhu et al, Methods Mol. Biol. 525:129-42, xv (2009)). The amplified chain shuffling phage library was aliquoted and stored in 50%
glycerol in PBS at -80 C. One aliquot of phage library stock was precipitated following standard protocols and used for two rounds of panning against biotinylated MPER
peptide and followed by monoclonal phage ELISA screening using JRFL gp140 as target to isolate clones which could cross reactively bind to both MPER
peptide and JRFL gp140 protein.
Conversion from Fab to IgGI
M66 and m66.6 Fabs in pComb3X were cloned into pDR12, kindly provided by Dennis Burton (The Scripps Research Institute, La Jolla, CA), which allows simultaneous expression of the heavy chain and light chains. Briefly, the heavy chain variable region was first cloned into pDR12 via XbaI and SacI
sites.
The full light chain was then cloned into pDR12 via HindIII and EcoRI sites.
Expression of Fab and IgGI
HB2151 cells were transformed with plasmid containing m66 Fab sequences. Single fresh colonies were inoculated into 2YT medium + 100 Ag/mL
ampicillin + 0.2% glucose. The culture was shaken at 250 rpm at 37 C until = 0.5. Isopropyl-L-thio-h-D-galactopyranoside (1 mmol/L) was added to induce expression. After overnight growth at 30 C, the culture was harvested.
Bacteria were centrifuged at 5,000g for 15 minutes. The pellet was resuspended in PBS
with polymycin B (10,000units/mL). Soluble Fab was released from periplasm by incubating at room temperature for 45 minutes. The extract was clarified at 15,000g for 30 minutes. The clear supernatant was recovered for purification on a Nikel column from Qiagen.
M66 and m66.6 IgGI was expressed in 293 free style cells. 293Fectin was used to transfect 293 free style cells according to the instructions from manufacturer (Invitrogen). Four days after transfection, the culture supernatant was harvested. IgGl was purified on protein A column.
ELISA Binding Assay Antigens (streptavidin) for capturing the biotinylated peptide or gp 140) were coated on narrow-well, 96-well plate at 50 ng/well in PBS overnight at 4 C.
For phage ELISA, 1010 phage from each round of panning was incubated with antigen. Bound phage was detected with anti-M13- HRP polyclonal antibody (Pharmacia, Piscataway, NJ). For soluble Fab binding assay, anti-Flag HRP
conjugate was used to detect the binding. For IgGI binding ELISA, HRP
conjugated goat anti-human IgG antibodies was used for detecting.
Pseudovirus Neutralization Assay.
Viruses pseudotyped with HIV-1 Envs were prepared by cotransfection of 70-80% confluent 293T cells with pNL4-3.luc.E-R- and pSV7d constructs encoding HIV-1 Envs (a gift from G. Quinnan, USUHS, Bethesda, MD) by using the PolyFect transfection reagent (Qiagen) according to manufacturer's instruction. Pseudotyped viruses were obtained after 24 h by centrifugation and filtration of cell culture through 0.45- m filters. For neutralization, viruses were mixed with different concentrations of antibodies for 1 h at 37 C, and then the mixture was added to z1.5 x 104 HOS-CD4-CCR5 (used for all R5 and dual tropic viruses) or HOS-CD4-CXCR4 cells grown in each well of 96-well plates.
Luminesence was measured after 48 h by using the Bright-Glo Luciferase Assay System (Promega, Madison, WI) and a LumiCount microplate luminometer (Turner Designs). Mean relative light units (RLU) for duplicate wells were determined. Percentage inhibition was calculated by the following formula: (1 -average RLU of antibody-containing wells/average RLU of virus-only wells) x 100.
PBMC based neutralization assay.
Neutralization of HIV-1 in the PBMC assay was measured as a reduction in LucR reporter gene expression after multiple rounds of virus replication.
Virus was incubated with serial 3-fold dilutions of test sample (eight dilutions total) in duplicate in a total volume of 150 l of IL-2-containing growth medium for I h at 37 C in a 96-well U-bottom culture plate. One-day-old PHA-PBMCs (2 x 105 cells in 50 l of IL-2-containing growth medium) were added to each well. One set of control wells received cells plus virus (virus control) and another set received cells only (background control). After a 4-day incubation, 100.tl of cells was transferred to a 96-well white solid plates (Costar) for measurements of Renilla luciferase luminescence using the ViviRen Live Cell Substrate (Promega).
TZM/bl and 7ZM-bl/FcyRI based pseudovirus neutralization assay.
The TZM/bl cells and TZM-bl cells expressing FcyRI based pseudovirus neutralization assay was previously described in Perez et al (J. Virol.
83(15):7397-7410 (2009)).
454 sequencing of the whole IgG derived VHgene repertoire from patient SC44 The IgG-derived Fd fragments were PCR amplified by using the cDNA
isolated from the patient as a template. The sense primers used were described 1o previously (Zhu et al, Methods Mol. Biol. 525:129-42, xv (2009)); the antisense primer was IgGR (5'-ACTAGTTTTGTCACAAGATTTGGGCTCAACTBTCTTGTCCACCTTGGTG
TTGC-3'), which is shared by all IgG1-4. The PCR was performed in a volume of 50 l for 25 cycles. The products were gel-purified and then used as a template for additional 12 cycles of secondary PCR amplification with primers (HF12: 5'-CCATCTCATCCCTGCGTGTCTCCGACTCAGTACTGAGCTAGCTGCCCA
ACCAGCCATGGCC-3' and HR2:
5' CCTATCCCCTGTGTGCCTTGGCAGTCTCAGGTCACAAGATTTGGGCT
CAAC-3') containing 454 sequencing-specific adaptors. The resultant products were gel-purified and subjected to 454 sequencing as described (www.454.com).
Similarity search using m66VH and germline Vgene segments against VHgene repertoire obtained through 454 sequencing Similarity matches between the m66 VH, germline probes and the SC44 VH gene repertoire database were obtained by applying the standard Perl String:: Similarity module (CPAN String-Similarity-1.04 by Marc Lehmann) to each database entry. The single best score for each sequence comparison was obtained by successively applying the String:: Similarity algorithm to starting positions L n. A score that fell at or above the selected threshold (similarity percentage) was retained for later analysis.
Results Isolation and affinity maturation of m66 through phage display library After 5 rounds of panning against the MPER peptide and gp140 protein, 190 random clones were picked for monoclonal phage screening. Sequence analysis of those identified positive clones showed one unique clone designated as m66 was identified, the m66 VH gene was used as probe to search IMGT online database, As shown in Fig. 4, m66 VH is derived from VH51-1 V gene, there are 8 mutations in the heavy chain V gene segment; m66VL is derived from VK-1-39 there are 3 mutation in the light chain V gene segment. In contrast to 2F5 and 4E10, m66 VH and VL bear a significantly lower number of mutations from their germline predecessors, which might indicate m66 is still in the early stage of the antibody maturation process. It was observed that the heavy chain CDR3 has 23 residues with multiple tyrosines and one phenylalanine.
To further improve the binding affinity of m66, light chain shuffling library with size at 2x 108 was constructed as described above. A chain shuffling library was panned two rounds against MPER peptide followed by Phage ELISA
screening using gpl40 protein as target. Six unique clones were identified which share the same heavy chain as expected but paired with different light chains with several mutations from the same VL subfamily. ELISA data showed they all bind similarly well to both the peptide and gp140 (data not shown). Among the 6 clones, one which has 9 mutations in the light chain V gene segment was designated as m66.6 and converted to IgG 1 for further characterization.
Specific binding of m66 to both MPER peptide and gp140 As shown in Fig. 5, purified m66.6 IgGI from transiently expression in 293 free style cells was tested side by side with 2F5 IgG on the binding to both MPER peptide and JRFL gpl40. Data showed m66.6 and 2F5 IgGI bind similarly well to both MPER peptide and gp 140 protein in ELISA. In agreement with previous report, both m66.6 and 2F5 IgGI bound better to MP ER peptide than to gp 140 protein.
Neutralization activity of m66 Fab and 1gG1 It has been reported that binding to the MPER region does not necessarily mean the antibody will neutralize. The neutralization of m66 and m66.6 IgG
were further tested and the neutralization activity compared side by side with 2F5.
The results demonstrated that m66.6 neutralization breadth is very similar to 2F5.
As also shown in Table 4, m66.6 neutralization is dramatically increased compared to m66, although the binding activity of m66 and m66.6 IgGs to gp 140 protein is very similar (data not shown). Similarly, although m66.6 and 2F5 IgGIs bind equally well to both the MPER peptide and JRFL gp140 protein, overall, the neutralization potency of m66.6 is less than that of 2F5 in this set of neutralization assay.
Table 4 Neutralization activity of m66 and m66.6 against a serial of HIV
isolates.
Neutralization percentage at 10 g/ml Isolate Clade m66 IgG I m66.6 IgG I 2F5 IgG 15 Bal B 5 89 78 89.6 B 25 51 97 UG(A) A 79 62 95 15 Potentiated neutralization of m66.6IgG1 mediated by FcyRI receptor 20 It has been repeatedly shown that broadly neutralizing antibodies such as 2F5 and 4E 10, which target the MPER region, can neutralize pseudovirus infection with much higher potency when FcyRI is also expressed on the target cells in the neutralization assay (Perez et al, J. Virol. 83(15):7397-410 (2009), Epub 2009 May 20). To test whether m66.6 also shares the same feature, the 25 neutralization activity of m66.6 IgG 1 was also tested on TZM-bl/FcyRI
cells, m66Fab and an unrelated antibody, m 102.4, were used as control. The data shown in Table 5 demonstrated that the neutralization potency of m66.6 IgGI is increased than 1000 times, while no increase was observed for the control m66Fab, indicating the dramatic increase is attributed to the binding of the Fc portion of the m66.6 IgGI to the over expressed FcyRI on the target cell surface.
Table 5 Potent Neutralization of HIV Pseudoviruses infection of TZM-bl FcyRI
cells IC50 m1 Clade Tier m66 m66 m66.6 m66.6 m102.4 m102.4 TZM-bl TZM- TZM-bl TZM- TZM-bl TZM-bl/FcyRl b!FcyRI bl/FcyRI
cells cells cells SF162.LS B 1 >10 0.43 51.4 <0.0006 >50 >50 MW965.26 B I >10 >10 >100 >50 >50 >50 6535.3 B 2 >10 >10 75.2 <0.0006 >50 >50 QH0692.42 B 2 >10 >10 88.9 0.06 >50 >50 SC422661.8 B 2 >10 0.01 16.7 <0.0006 >50 >50 AC 10Ø29 B 2 >10 0.08 41.6 <0.0006 >50 >50 RHPA4259.7 B 2 >10 >10 >100 0.07 >50 >50 BB1006-11.03.1601 B 2 >10 79.4 <0.0006 >50 >50 Du156.12 C 2 >10 >100 >50 >50 >50 Du172.17 C 2 >10 >100 >50 >50 >50 Du422.1 C 2 >10 >100 >50 >50 >50 ZM197M.PB7 C - 2 >10 >100 0.02 >50 >50 CenvFs4 Pt2010 F5 C 2 >10 >100 2.9 >50 >50 CAP206.1.B5 C 2 >10 >100 >50 >50 >50 Q23.17 A 2 >10 26.0 <0.0006 >50 >50 Q842.d12 A 2 >10 >100 <0.0006 >50 >50 'Values are the concentration (pg/ml) at which relative luminescence units (RLUs) were reduced 50% compared to virus control wells (no test sample).
IgG specific VH gene repertoire analysis through 454 sequencing and sequence analysis It is expected that other m66 variants also exist in this repertoire but were not identified during the library construction and panning. To further confirm the existence of m66 like antibodies and also to explore the affinity maturation ' pathway of m66, 454 high throughput sequencing was performed using SC44 IgG
specific VH gene repertoire amplified from patient SC44 PBMC derived cDNA, total 392,198 functional unique VH sequences were obtained from the sequencing experiment. M66 VH was used as probe to search the VH gene repertoire and a series of m66 VH variants were also identified through similarity search as described above. A phylogenetic tree made from those sequences indicates that m66 VH is still at the early stage of the evolvement process (Fig. 7), which is consistent with a previous report (Shen et al, J. Virol. 83:3617-25 (2009)), where it was shown the MPER peptide binding activity of the serum antibodies at 12 months after enrollment is still relatively moderate comparing to the antibodies at later time points.
To profile the VH germline gene usage in the whole repertoire, 7 germline gene segments from each of 7 VH subfamilies namely VH 1-69, VI2-5, VH3-23, VH4-1, VH51-1, VH6-1 and VH7-l were used as probes to do similarity search against the whole VH gene repertoire. All the sequences from each search were ranked according to the similarity to the probes and were plotted against the similarity percentages and 7 probes as shown in Fig. 6. It was found that VH51-derived genes were disproportionally expanded in this repertoire at the specific time point when the blood sample was taken in this patient. Also large number of v genes in this family showed very high homology to the germline sequence, some of which even show identical V gene sequence to the germline sequence.
The fact that the HCDR3s of those clones also showed very diversified sequences (data not shown) indicated this unique IgG repertoire is derived from a pool of closely related B cells rather than single B cell. However, no such phenomenon was observed in other similarity searches using other V gene probes from the other subfamilies. This set of data might indicate this specific pool of B
cells were activated and went through class switch from IgM to IgG at very early stage of the B cell development process without going through somatic hypermuation and affinity maturation at the IgM stage. This might also explain how these polyspecific autoreactive antibodies overcome immune tolerance and evolve into broadly neutralizing antibodies against HIV in vivo.
As shown in Fig. 14, J774A.1 feeder cells enhance growth and production of IgM from B-CLL cells (CLL246) after Epstein Barr virus (EBV) infection.
After incubation with EBV, the B-CLL cells were plated at 5,000 cells/well (A) in the absence of J774A.1 cells or (B) in the presence of irradiated J774A.1 cells (50,000 cells/well). Three weeks after infection, levels of IgM were detected by ELISA and expressed in pg/ml.
As shown in Fig. 15, the CLL246 cells produce IgM against both HIV-1 gp140 and hepatitis virus C E2 (HCV E2) envelope proteins. IgM against the test antigens was detected by ELISA and expressed in OD. (See also Fig. 16.) * * *
All documents and other information sources cited above are hereby incorporated in their entirety by reference.
Table 3 M66.6H DNA sequence :
cagatgcagctggtgcagtctggagcagaggtgaaaaagcccggggagtctctgaagatctcctgt aaggtttctggatacaactttgccagcgaatggatcggctgggtgcgccagatgcccgggaaaggc ctggagtggatggggattatctatcctggtgactctgataccaaatacagcccgtccttccaaggc caggtcatcatctcagccgacaagtccatcaac accgcctacctgcagtggagcagcctgaaggcctcggacaccgccatatattactgtgcgagacag aatcactatggttcggggagttatttctaccgaacggcctactactatgctatggacgtctggggc caagggaccacggtcaccgtctcctca M66.6 VH protein sequence :
Q M Q L V Q S G A E V K K P G E S L K I S C K V S G Y N F A S E W I G W V R Q
M P G K
G L E W M G I I Y P G D S D T K Y S P S F Q G Q V I I S A D K S I N T A Y L Q
W S S L K A
S D T A I Y Y C A R Q N H Y G S G S Y F Y R T A Y Y Y A M D V W G Q G T T V T
V S S
M66.6 Light chain DNA sequences:
gacatccagttgacccagtctccatcttccctgtctgcatctttgggggacaaagtcacca.tcact tgccgggcaagtcagcacattaagaagtatttaaactggtatcagcagaaacctgggaaagcccct aaactcctgatctatggtgcactcaatttgcagagtggggtcccatcaaggttcagtggcagagga tctgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgt cagcagagttacagtaccccattcactttcggccctgggaccaaagtggatatcaaacga M66.6 VL Protein sequence DIQLTQSPSSLSASLGDKVTITCRASQHIKKYLNWYQQKPGK
APKLL IYGALNLQSGVPSRFSGRGSGT DFTLTISSLQPEDFAT
YYCQQSYSTPFTFGPGTKVDIKR
As indicated above, either the intact antibody or fragment (e.g., antigen binding fragment) thereof can be used in the method of the present invention.
Exemplary functional fragments (regions) include scFv, Fv, Fab', Fab and F(ab')2 fragments. Single chain antibodies can also be used. Techniques for preparing suitable fragments and single chain antibodies are well known in the art.
(See, for example, USPs 5,855,866; 5,877,289; 5,965,132; 6,093,399; 6,261,535;
6,004,555; 7,417,125 and 7,078,491 and WO 98/45331.) The invention also includes variants of the antibodies (and fragments) disclosed herein, including variants that retain the binding properties of the antibodies (and fragments) specifically disclosed, and methods of using same in the present method. For example, the invention includes an isolated human antibody or fragment thereof that binds selectively to gp4l MPER and that comprises one or more CDRs as set forth in Table 2 and Fig. 4. Modifications of M66 and M66.6 that can be used therapeutically in accordance with the invention include IgA, IgM and IgG1, 2, or 4 versions of the M66 M66.6 VH and VL chains.
The antibodies, and fragments thereof, described above can be formulated as a composition (e.g., a pharmaceutical composition). Suitable compositions can comprise the antibody (or antibody fragment) dissolved or dispersed in a pharmaceutically acceptable carrier (e.g., an aqueous medium). The compositions can be sterile and can in an injectable form. The antibodies (and fragments thereof) can also be formulated as a composition appropriate for topical administration to the skin or mucosa. Such compositions can take the form of liquids, ointments, creams, gels and pastes. Standard formulation techniques can be used in preparing suitable compositions. The antibodies can be formulated so as to be administered as a post-coital douche or with a condom.
The antibodies and antibody fragments of the invention show their utility for prophylaxis in, for example, the following settings:
i) in the setting of anticipated known exposure to HIV-1 infection, the antibodies described herein (or binding fragments thereof) can be administered prophylactically (e.g., IV or topically) as a microbiocide, ii) in the setting of known or suspected exposure, such as occurs in the setting of rape victims, or commercial sex workers, or in any heterosexual transmission with out condom protection, the antibodies described herein (or fragments thereof) can be administered as post-exposure prophylaxis, e.g., IV
or topically, iii) in the setting of Acute HIV infection (AHI), antibodies described herein (or binding fragments thereof) can be administered as a treatment for AHI
to control the initial viral load and preserve the CD4+ T cell pool and prevent CD4+ T cell destruction, and iv) in the setting of maternal to baby transmission whi8le the child is breastfeeding.
Suitable dose ranges can depend, for example, on the antibody and on the nature of the formulation and route of administration. Optimum doses can be determined by one skilled in the art without undue experimentation. Doses of antibodies in the range of I Ong to 20 .tg/ml can be suitable.
In another embodiment, the present invention provides a cell culture system that makes possible the production of monoclonal anti-viral antibodies from B-chronic lymphocytic leukemia (B-CLL) cell repertoires. In accordance with this embodiment of the invention, macrophage cells are used as feeder cells to grow B-CLL cells in culture following EBV-infection.
A specific example of a protocol suitable for use in generating immortalized clones of B-CLL cells is set forth in Figure 8. Generally, a macrophage cell line (e.g., a rodent macrophage cell line) is used as a feeder. A
preferred macrophage cell line is the mouse line J774A. 1. J774A. I macrophage cells have been shown to produce growth factors suitable for hybridoma growth and cloning (Rathjen and Geczy, Hybridoma 5:255-261 (1986)). Conditioned medium prepared from J774A.1 cells has been widely used for enhancing hybridoma viability. It has been also shown that co-culture of the J774A.1 cell line as a feeder with T cells, as well as EBV-infected B cells, helps clearance of apoptotic cells in vitro (Kagan et al, J. Immunol. 169:487-499 (2002)).
In accordance with the invention, the macrophage cell line is subjected to irradiation to prevent outgrowth of the feeder cells. The optimum y-irradiation dose can be determined experimentally, for example, by testing the cell line to determine the minimum dose required to completely inhibit cell proliferation (e.g., about 4,000 Rad). The irradiated cells can then be distributed into culture containers (for example, when 96-well plates are used, about 50,000 cells can distributed/well (about 100 l/well)). The optimum number of irradiated cells can be determined experimentally by testing the cells at different densities.
EBV infection of B-CLL cells can be effected using standard techniques.
For example, peripheral blood mononuclear cells (PBMCs) can be isolated from a blood sample from a CLL patient using standard techniques, the cells can then be washed in complete medium (see, for example, Fig. 9) and suspended in complete medium comprising, for example, PS 2006 and cyclosporine A. PS 2006, a Toll-like receptor 9 (TLR-9) agonist, can be added to stimulate B cells according to previously described methods (Lanzavecchia et al, Curr. Opin. Biotech. 18:523-528 (2007)). Cyclosporin A can be added to suppress any EBV-B cell-specific cytotoxic T cell response. Optimal concentrations can be determined experimentally by testing EBV-B cell stimulation potency.
In accordance with this embodiment of the invention, an EBV suspension is then added to the cell suspension. Following incubation at about 37 C
(e.g., for about I to about 24 hours, preferably, for about 4 hours) the EBV-infected cells can be resuspended in complete medium comprising, for example, PS2006, distributed into the culture container with the irradiated macrophage cells and incubated at about 37 C..The medium can be changed periodically and antibody production assessed using, for example, ELISA when the EBV-infected cells have grown sufficiently (e.g., about 3-about 4 weeks post-infection). IgM producing hybridoma cell lines derived from CLL cells can then be produced, for example, using a hybridoma cell fusion method, such as that described in Fig. 10, and cloned. (See, generally, Fig. 11) Three monoclonal B-CLL hybridoma cell lines have been derived from CLL246 (VH1-69, unmutated case), CLL1075 (VH1-69, mutated case), and CLL493 (VH1-69, unmutated case). (See Figs. 12 and 13.) Therapeutic antibodies (e.g., anti-HIV antibodies, anti-hepatitis C
antibodies or anti-influenza antibodies) produced from B-CLL cells immortalized in accordance with the method described above, or fragments thereof (e.g., antigen binding fragments - exemplary functional fragments (regions) include scFv, Fv, Fab', Fab and F(ab')2 fragments) can be formulated as a composition (e.g., a pharmaceutical composition). Suitable compositions can comprise the antibody (or antibody fragment) dissolved or dispersed in a pharmaceutically acceptable carrier (e.g., an aqueous medium). The compositions can be sterile and can be in an injectable form. The antibodies (and fragments thereof) can also be formulated as a composition appropriate for topical administration to the skin or mucosa. Such compositions can take the form of liquids, ointments, creams, gels, pastes or aerosols. Standard formulation techniques can be used in preparing suitable compositions.
The anti-HIV antibodies and fragments thereof show their utility for prophylaxis in, for example, the following settings:
i) in the setting of anticipated known exposure to HIV-1 infection, the antibodies described herein (or binding fragments thereof) can be administered prophylactically (e.g., IV or topically) as a microbiocide, ii) in the setting of known or suspected exposure, such as occurs in the setting of rape victims, or commercial sex workers, or in any heterosexual transmission with out condom protection, the antibodies described herein (or fragments thereof) can be administered as post-exposure prophylaxis, e.g., IV
or topically, iii) in the setting of Acute HIV infection (AHI), antibodies described herein (or binding fragments thereof) can be administered as a treatment for AHI
to control the initial viral load and preserve the CD4+ T cell pool and prevent CD4+ T cell destruction, and iv) in the setting of maternal to baby transmission while the child is breastfeeding.
Anti-hepatitis C and anti-influenza antibodies, or fragments thereof as described above, can be used to treat or prevent hepatitis C and influenza infection, respectively.
Suitable dose ranges can depend, for example, on the antibody, on the nature of the formulation, on the route of administration, and on the patient (e.g., a human patient). Optimum doses can be determined by one skilled in the art without undue experimentation. Doses of antibodies in the range of 1 Ong to g/ml can be suitable.
Certain aspects of the invention can be described in greater detail in the non-limiting Examples that follows. (See also U.S. Prov. Applns. 61/272,349 and 20 61/248,769, Shen et al, J. Virol. 83(8):3617-25 Epub 2009, Zhu and Dimitrov, Methods Mol. Biol. 525:129-142 (2009), Dimitrov and Marks, Methods Mol.
Biol. 525:1-27 (2009), Zhang et al, J. Virol. 82(14):6869-6879 (2008), Prabakaran et al, Advances in Pharmacology 55:33-97 (2007), Perez et al, J. Virol.
83(15):7397-7410 (2009)). (See also Chan et al, Blood 97:1023-1026 (2001);
Gorny et al, Mol. Immunol. 46:917-926 (2009)).
EXAMPLE I
IgM and IgG libraries from a patient with acute infection and a patient with 2F5-like antibodies were generated and analyzed by high throughput (454) sequencing and other methods. HIV-1-specific antibodies from IgG libraries derived from the blood of an acutely infected patient at two time points (40 days and 8 months) were identified. These antibodies bound envelope glycoproteins (Envs) with high affinity but did not neutralize a panel of 9 pseudoviruses.
Antibodies from samples at 40 days were not found at 8 months and antibodies from samples at 8 months did not bind a dominant Env at 40 days. Panning of bone marrow derived libraries from the same patient did not result in selection of any antibodies.
Novel antibodies were selected from IgM+IgG phage and yeast display libraries derived from a patient with 2F5-like antibodies. One of these antibodies, M66, has long (23 residues) heavy chain CDR3 and its VH gene has relatively low number of somatic mutations. It bound specifically to a gp41 MPER peptide containing the 2F5 epitope and cross-reactively neutralized HIV-1 isolates.
The other antibodies are being characterized. These results could have implications for understanding humoral immune responses and design of vaccine immunogens.
(See Figs. 1 and 2.) Supernatants from 293T cells stably transfected with M66 antibody genes were evaluated for binding to the 2F5 epitope by custom HIV-1 luminex. Serial dilutions of supernatant were assessed for binding to the epitope (readout of mean fluorescence intensity -MFI). Positive controls for the experiment included mAb, and HIVIG titrations and the negative control was the mock transfected supernatant. The binding data for the M66 antibody is presented in Fig. 3.
EXAMPLE -3) Experimental Details cDNA, antibodies, gp140s and peptides cDNA was prepared using the total RNA extracted from PBML taken from Patient SC44 at 12 month after enrollment (Shen et al, J. Virol. 83:3617-(2009)). HIV-1 gp4l MAbs 2F5 was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH (Hermann Katinger). Recombinant gp140s were kindly provided by C. Broder (Uniformed Services University of the Health Sciences, Bethesda, MD). Two biotinylated lo peptides containing the 2F5 epitope sequence (2F5 peptides) were used for panning and binding assay: SP62 QQEKNEQELLELDKWASLWN and SP62 scrambled peptide. These peptides were custom-made by Primm Biotech and CPC. Horseradish peroxidase (HRP)-conjugated anti-FLAG tag antibody and HRP-conjugated anti-human IgG (Fc-specific) antibody were purchased from Sigma-Aldrich (St. Louis).
Phage Display Fab library construction and library panning, screening Phage display Fab libraries were constructed primarily following a published protocol (Zhu et al, Methods Mol. Biol. 525:129-42, xv (2009)) using the cDNA prepared from PBMC of patient SC44 as template for the antibody gene repertoire cloning. Sequential pannings were performed using biotinylated Peptide SP62 and gp140 proteins with the first three rounds of panning on 1 g of biotinylated peptide on streptavidin conjugated magnetic bead and the fourth and fifth pannings on gp140 (JRFL) coated at 1 ug/well on 96 well ELISA plates.
Bound phage on the beads or plate wells were directly used to infect exponentially growing TGI cells and rescued by M13KO7 helper phage and amplified for the next round panning. 190 individual colonies after the fifth round panning were picked and inoculated into 2YT medium in 96-well plate for phage ELISA screening as described (Zhu et al, Methods Mol. Biol. 525:129-42, xv (2009)). Identified positive binders were sequenced and a unique clone was expressed and purified Fab was used for preliminary characterization.
Generation and selection of the light chain-shuffled phage display library The Fd fragment of m66 Fab construct was PCR amplified and fused with the light chain gene repertoire obtained during the original Fab library construction using overlapping PCR and cloned into a phagmid vector essentially as described (Zhu et al, Methods Mol. Biol. 525:129-42, xv (2009)). The amplified chain shuffling phage library was aliquoted and stored in 50%
glycerol in PBS at -80 C. One aliquot of phage library stock was precipitated following standard protocols and used for two rounds of panning against biotinylated MPER
peptide and followed by monoclonal phage ELISA screening using JRFL gp140 as target to isolate clones which could cross reactively bind to both MPER
peptide and JRFL gp140 protein.
Conversion from Fab to IgGI
M66 and m66.6 Fabs in pComb3X were cloned into pDR12, kindly provided by Dennis Burton (The Scripps Research Institute, La Jolla, CA), which allows simultaneous expression of the heavy chain and light chains. Briefly, the heavy chain variable region was first cloned into pDR12 via XbaI and SacI
sites.
The full light chain was then cloned into pDR12 via HindIII and EcoRI sites.
Expression of Fab and IgGI
HB2151 cells were transformed with plasmid containing m66 Fab sequences. Single fresh colonies were inoculated into 2YT medium + 100 Ag/mL
ampicillin + 0.2% glucose. The culture was shaken at 250 rpm at 37 C until = 0.5. Isopropyl-L-thio-h-D-galactopyranoside (1 mmol/L) was added to induce expression. After overnight growth at 30 C, the culture was harvested.
Bacteria were centrifuged at 5,000g for 15 minutes. The pellet was resuspended in PBS
with polymycin B (10,000units/mL). Soluble Fab was released from periplasm by incubating at room temperature for 45 minutes. The extract was clarified at 15,000g for 30 minutes. The clear supernatant was recovered for purification on a Nikel column from Qiagen.
M66 and m66.6 IgGI was expressed in 293 free style cells. 293Fectin was used to transfect 293 free style cells according to the instructions from manufacturer (Invitrogen). Four days after transfection, the culture supernatant was harvested. IgGl was purified on protein A column.
ELISA Binding Assay Antigens (streptavidin) for capturing the biotinylated peptide or gp 140) were coated on narrow-well, 96-well plate at 50 ng/well in PBS overnight at 4 C.
For phage ELISA, 1010 phage from each round of panning was incubated with antigen. Bound phage was detected with anti-M13- HRP polyclonal antibody (Pharmacia, Piscataway, NJ). For soluble Fab binding assay, anti-Flag HRP
conjugate was used to detect the binding. For IgGI binding ELISA, HRP
conjugated goat anti-human IgG antibodies was used for detecting.
Pseudovirus Neutralization Assay.
Viruses pseudotyped with HIV-1 Envs were prepared by cotransfection of 70-80% confluent 293T cells with pNL4-3.luc.E-R- and pSV7d constructs encoding HIV-1 Envs (a gift from G. Quinnan, USUHS, Bethesda, MD) by using the PolyFect transfection reagent (Qiagen) according to manufacturer's instruction. Pseudotyped viruses were obtained after 24 h by centrifugation and filtration of cell culture through 0.45- m filters. For neutralization, viruses were mixed with different concentrations of antibodies for 1 h at 37 C, and then the mixture was added to z1.5 x 104 HOS-CD4-CCR5 (used for all R5 and dual tropic viruses) or HOS-CD4-CXCR4 cells grown in each well of 96-well plates.
Luminesence was measured after 48 h by using the Bright-Glo Luciferase Assay System (Promega, Madison, WI) and a LumiCount microplate luminometer (Turner Designs). Mean relative light units (RLU) for duplicate wells were determined. Percentage inhibition was calculated by the following formula: (1 -average RLU of antibody-containing wells/average RLU of virus-only wells) x 100.
PBMC based neutralization assay.
Neutralization of HIV-1 in the PBMC assay was measured as a reduction in LucR reporter gene expression after multiple rounds of virus replication.
Virus was incubated with serial 3-fold dilutions of test sample (eight dilutions total) in duplicate in a total volume of 150 l of IL-2-containing growth medium for I h at 37 C in a 96-well U-bottom culture plate. One-day-old PHA-PBMCs (2 x 105 cells in 50 l of IL-2-containing growth medium) were added to each well. One set of control wells received cells plus virus (virus control) and another set received cells only (background control). After a 4-day incubation, 100.tl of cells was transferred to a 96-well white solid plates (Costar) for measurements of Renilla luciferase luminescence using the ViviRen Live Cell Substrate (Promega).
TZM/bl and 7ZM-bl/FcyRI based pseudovirus neutralization assay.
The TZM/bl cells and TZM-bl cells expressing FcyRI based pseudovirus neutralization assay was previously described in Perez et al (J. Virol.
83(15):7397-7410 (2009)).
454 sequencing of the whole IgG derived VHgene repertoire from patient SC44 The IgG-derived Fd fragments were PCR amplified by using the cDNA
isolated from the patient as a template. The sense primers used were described 1o previously (Zhu et al, Methods Mol. Biol. 525:129-42, xv (2009)); the antisense primer was IgGR (5'-ACTAGTTTTGTCACAAGATTTGGGCTCAACTBTCTTGTCCACCTTGGTG
TTGC-3'), which is shared by all IgG1-4. The PCR was performed in a volume of 50 l for 25 cycles. The products were gel-purified and then used as a template for additional 12 cycles of secondary PCR amplification with primers (HF12: 5'-CCATCTCATCCCTGCGTGTCTCCGACTCAGTACTGAGCTAGCTGCCCA
ACCAGCCATGGCC-3' and HR2:
5' CCTATCCCCTGTGTGCCTTGGCAGTCTCAGGTCACAAGATTTGGGCT
CAAC-3') containing 454 sequencing-specific adaptors. The resultant products were gel-purified and subjected to 454 sequencing as described (www.454.com).
Similarity search using m66VH and germline Vgene segments against VHgene repertoire obtained through 454 sequencing Similarity matches between the m66 VH, germline probes and the SC44 VH gene repertoire database were obtained by applying the standard Perl String:: Similarity module (CPAN String-Similarity-1.04 by Marc Lehmann) to each database entry. The single best score for each sequence comparison was obtained by successively applying the String:: Similarity algorithm to starting positions L n. A score that fell at or above the selected threshold (similarity percentage) was retained for later analysis.
Results Isolation and affinity maturation of m66 through phage display library After 5 rounds of panning against the MPER peptide and gp140 protein, 190 random clones were picked for monoclonal phage screening. Sequence analysis of those identified positive clones showed one unique clone designated as m66 was identified, the m66 VH gene was used as probe to search IMGT online database, As shown in Fig. 4, m66 VH is derived from VH51-1 V gene, there are 8 mutations in the heavy chain V gene segment; m66VL is derived from VK-1-39 there are 3 mutation in the light chain V gene segment. In contrast to 2F5 and 4E10, m66 VH and VL bear a significantly lower number of mutations from their germline predecessors, which might indicate m66 is still in the early stage of the antibody maturation process. It was observed that the heavy chain CDR3 has 23 residues with multiple tyrosines and one phenylalanine.
To further improve the binding affinity of m66, light chain shuffling library with size at 2x 108 was constructed as described above. A chain shuffling library was panned two rounds against MPER peptide followed by Phage ELISA
screening using gpl40 protein as target. Six unique clones were identified which share the same heavy chain as expected but paired with different light chains with several mutations from the same VL subfamily. ELISA data showed they all bind similarly well to both the peptide and gp140 (data not shown). Among the 6 clones, one which has 9 mutations in the light chain V gene segment was designated as m66.6 and converted to IgG 1 for further characterization.
Specific binding of m66 to both MPER peptide and gp140 As shown in Fig. 5, purified m66.6 IgGI from transiently expression in 293 free style cells was tested side by side with 2F5 IgG on the binding to both MPER peptide and JRFL gpl40. Data showed m66.6 and 2F5 IgGI bind similarly well to both MPER peptide and gp 140 protein in ELISA. In agreement with previous report, both m66.6 and 2F5 IgGI bound better to MP ER peptide than to gp 140 protein.
Neutralization activity of m66 Fab and 1gG1 It has been reported that binding to the MPER region does not necessarily mean the antibody will neutralize. The neutralization of m66 and m66.6 IgG
were further tested and the neutralization activity compared side by side with 2F5.
The results demonstrated that m66.6 neutralization breadth is very similar to 2F5.
As also shown in Table 4, m66.6 neutralization is dramatically increased compared to m66, although the binding activity of m66 and m66.6 IgGs to gp 140 protein is very similar (data not shown). Similarly, although m66.6 and 2F5 IgGIs bind equally well to both the MPER peptide and JRFL gp140 protein, overall, the neutralization potency of m66.6 is less than that of 2F5 in this set of neutralization assay.
Table 4 Neutralization activity of m66 and m66.6 against a serial of HIV
isolates.
Neutralization percentage at 10 g/ml Isolate Clade m66 IgG I m66.6 IgG I 2F5 IgG 15 Bal B 5 89 78 89.6 B 25 51 97 UG(A) A 79 62 95 15 Potentiated neutralization of m66.6IgG1 mediated by FcyRI receptor 20 It has been repeatedly shown that broadly neutralizing antibodies such as 2F5 and 4E 10, which target the MPER region, can neutralize pseudovirus infection with much higher potency when FcyRI is also expressed on the target cells in the neutralization assay (Perez et al, J. Virol. 83(15):7397-410 (2009), Epub 2009 May 20). To test whether m66.6 also shares the same feature, the 25 neutralization activity of m66.6 IgG 1 was also tested on TZM-bl/FcyRI
cells, m66Fab and an unrelated antibody, m 102.4, were used as control. The data shown in Table 5 demonstrated that the neutralization potency of m66.6 IgGI is increased than 1000 times, while no increase was observed for the control m66Fab, indicating the dramatic increase is attributed to the binding of the Fc portion of the m66.6 IgGI to the over expressed FcyRI on the target cell surface.
Table 5 Potent Neutralization of HIV Pseudoviruses infection of TZM-bl FcyRI
cells IC50 m1 Clade Tier m66 m66 m66.6 m66.6 m102.4 m102.4 TZM-bl TZM- TZM-bl TZM- TZM-bl TZM-bl/FcyRl b!FcyRI bl/FcyRI
cells cells cells SF162.LS B 1 >10 0.43 51.4 <0.0006 >50 >50 MW965.26 B I >10 >10 >100 >50 >50 >50 6535.3 B 2 >10 >10 75.2 <0.0006 >50 >50 QH0692.42 B 2 >10 >10 88.9 0.06 >50 >50 SC422661.8 B 2 >10 0.01 16.7 <0.0006 >50 >50 AC 10Ø29 B 2 >10 0.08 41.6 <0.0006 >50 >50 RHPA4259.7 B 2 >10 >10 >100 0.07 >50 >50 BB1006-11.03.1601 B 2 >10 79.4 <0.0006 >50 >50 Du156.12 C 2 >10 >100 >50 >50 >50 Du172.17 C 2 >10 >100 >50 >50 >50 Du422.1 C 2 >10 >100 >50 >50 >50 ZM197M.PB7 C - 2 >10 >100 0.02 >50 >50 CenvFs4 Pt2010 F5 C 2 >10 >100 2.9 >50 >50 CAP206.1.B5 C 2 >10 >100 >50 >50 >50 Q23.17 A 2 >10 26.0 <0.0006 >50 >50 Q842.d12 A 2 >10 >100 <0.0006 >50 >50 'Values are the concentration (pg/ml) at which relative luminescence units (RLUs) were reduced 50% compared to virus control wells (no test sample).
IgG specific VH gene repertoire analysis through 454 sequencing and sequence analysis It is expected that other m66 variants also exist in this repertoire but were not identified during the library construction and panning. To further confirm the existence of m66 like antibodies and also to explore the affinity maturation ' pathway of m66, 454 high throughput sequencing was performed using SC44 IgG
specific VH gene repertoire amplified from patient SC44 PBMC derived cDNA, total 392,198 functional unique VH sequences were obtained from the sequencing experiment. M66 VH was used as probe to search the VH gene repertoire and a series of m66 VH variants were also identified through similarity search as described above. A phylogenetic tree made from those sequences indicates that m66 VH is still at the early stage of the evolvement process (Fig. 7), which is consistent with a previous report (Shen et al, J. Virol. 83:3617-25 (2009)), where it was shown the MPER peptide binding activity of the serum antibodies at 12 months after enrollment is still relatively moderate comparing to the antibodies at later time points.
To profile the VH germline gene usage in the whole repertoire, 7 germline gene segments from each of 7 VH subfamilies namely VH 1-69, VI2-5, VH3-23, VH4-1, VH51-1, VH6-1 and VH7-l were used as probes to do similarity search against the whole VH gene repertoire. All the sequences from each search were ranked according to the similarity to the probes and were plotted against the similarity percentages and 7 probes as shown in Fig. 6. It was found that VH51-derived genes were disproportionally expanded in this repertoire at the specific time point when the blood sample was taken in this patient. Also large number of v genes in this family showed very high homology to the germline sequence, some of which even show identical V gene sequence to the germline sequence.
The fact that the HCDR3s of those clones also showed very diversified sequences (data not shown) indicated this unique IgG repertoire is derived from a pool of closely related B cells rather than single B cell. However, no such phenomenon was observed in other similarity searches using other V gene probes from the other subfamilies. This set of data might indicate this specific pool of B
cells were activated and went through class switch from IgM to IgG at very early stage of the B cell development process without going through somatic hypermuation and affinity maturation at the IgM stage. This might also explain how these polyspecific autoreactive antibodies overcome immune tolerance and evolve into broadly neutralizing antibodies against HIV in vivo.
As shown in Fig. 14, J774A.1 feeder cells enhance growth and production of IgM from B-CLL cells (CLL246) after Epstein Barr virus (EBV) infection.
After incubation with EBV, the B-CLL cells were plated at 5,000 cells/well (A) in the absence of J774A.1 cells or (B) in the presence of irradiated J774A.1 cells (50,000 cells/well). Three weeks after infection, levels of IgM were detected by ELISA and expressed in pg/ml.
As shown in Fig. 15, the CLL246 cells produce IgM against both HIV-1 gp140 and hepatitis virus C E2 (HCV E2) envelope proteins. IgM against the test antigens was detected by ELISA and expressed in OD. (See also Fig. 16.) * * *
All documents and other information sources cited above are hereby incorporated in their entirety by reference.
Claims (9)
1. A method of inhibiting infection of cells of a subject by HIV-1 comprising administering to said subject an HIV-1 specific antibody other than 2F5 that binds the 2F5 epitope, or binding fragment thereof, in an amount and under conditions such that said antibody, or fragment thereof, inhibits said infection.
2. The method according to claim 1 wherein said antibody is administered prior to contact of said subject or said subject's immune system with HIV-1 or after infection of vulnerable cells of said subject with HIV-1.
3. The method according to claim 1 wherein said antibody is a monoclonal antibody comprising variable heavy (VH) and variable light (VL) sequences of the M66 antibody.
4. The method according to claim 1 wherein said antibody is a monoclonal antibody comprising variable heavy and variable light sequences of the M66.6 antibody.
5. The method according to claim 1 wherein said antibody comprises IgA, IgM or IgG 1, 2, 3 or 4 versions of monoclonal antibody M66 or M66.6 VH
and VL chains.
and VL chains.
6. An isolated antibody, or fragment thereof, that binds selectively to gp41 MPER and that comprises one or more CDRs as set forth in Table 2 or Figure 4.
7. A composition comprising the antibody, or fragment thereof, according to claim 6 and a carrier.
8. A method of generating immortalized clones of B-chronic lymphocytic leukemia (B-CLL) cells comprising culturing said B-CLL cells in culture following EBV-infection with a macrophage feeder cell line.
9. The method according to claim 8 wherein said macrophage cell line is mouse line J774A.1.
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US27234909P | 2009-09-16 | 2009-09-16 | |
US61/272,349 | 2009-09-16 | ||
US24879609P | 2009-10-05 | 2009-10-05 | |
US61/248,796 | 2009-10-05 | ||
PCT/US2010/002515 WO2011034582A2 (en) | 2009-09-16 | 2010-09-16 | Hiv-1 antibodies |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2774446A1 true CA2774446A1 (en) | 2011-03-24 |
Family
ID=43759219
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA2774446A Pending CA2774446A1 (en) | 2009-09-16 | 2010-09-16 | Hiv-1 antibodies |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP2477658A2 (en) |
JP (1) | JP2013505236A (en) |
AU (1) | AU2010296058A1 (en) |
CA (1) | CA2774446A1 (en) |
WO (1) | WO2011034582A2 (en) |
Families Citing this family (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP3271022A4 (en) | 2015-03-19 | 2018-10-31 | Duke University | Hiv-1 neutralizing antibodies and uses thereof |
US11071783B2 (en) | 2015-03-19 | 2021-07-27 | Duke University | HIV-1 neutralizing antibodies and uses thereof |
JP7126573B2 (en) | 2018-07-03 | 2022-08-26 | ギリアード サイエンシーズ, インコーポレイテッド | Antibodies targeting HIV gp120 and methods of use |
CA3145791A1 (en) | 2019-07-16 | 2021-01-21 | Gilead Sciences, Inc. | Hiv vaccines and methods of making and using |
CR20230101A (en) | 2020-08-25 | 2023-04-28 | Gilead Sciences Inc | Multi-specific antigen binding molecules targeting hiv and methods of use |
TWI815194B (en) | 2020-10-22 | 2023-09-11 | 美商基利科學股份有限公司 | INTERLEUKIN-2-Fc FUSION PROTEINS AND METHODS OF USE |
WO2024015741A1 (en) | 2022-07-12 | 2024-01-18 | Gilead Sciences, Inc. | Hiv immunogenic polypeptides and vaccines and uses thereof |
WO2024097957A1 (en) * | 2022-11-03 | 2024-05-10 | Duke University | Novel antibodies for hiv and methods of making and using the same |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5811524A (en) * | 1995-06-07 | 1998-09-22 | Idec Pharmaceuticals Corporation | Neutralizing high affinity human monoclonal antibodies specific to RSV F-protein and methods for their manufacture and therapeutic use thereof |
US20040198661A1 (en) * | 2000-12-08 | 2004-10-07 | Bowdish Katherine S. | Polypeptides and antibodies derived from chronic lymphocytic leukemia cells and uses thereof |
WO2006110831A2 (en) * | 2005-04-12 | 2006-10-19 | Duke University | Method of inducing neutralizing antibodies to human immunodeficiency virus |
-
2010
- 2010-09-16 CA CA2774446A patent/CA2774446A1/en active Pending
- 2010-09-16 JP JP2012529742A patent/JP2013505236A/en not_active Ceased
- 2010-09-16 AU AU2010296058A patent/AU2010296058A1/en not_active Abandoned
- 2010-09-16 EP EP10817555A patent/EP2477658A2/en not_active Withdrawn
- 2010-09-16 WO PCT/US2010/002515 patent/WO2011034582A2/en active Application Filing
Also Published As
Publication number | Publication date |
---|---|
WO2011034582A3 (en) | 2011-09-15 |
WO2011034582A2 (en) | 2011-03-24 |
AU2010296058A1 (en) | 2012-05-03 |
JP2013505236A (en) | 2013-02-14 |
EP2477658A2 (en) | 2012-07-25 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20120269821A1 (en) | Hiv-1 antibodies | |
Xiao et al. | Germline-like predecessors of broadly neutralizing antibodies lack measurable binding to HIV-1 envelope glycoproteins: implications for evasion of immune responses and design of vaccine immunogens | |
McCoy et al. | Potent and broad neutralization of HIV-1 by a llama antibody elicited by immunization | |
Tudor et al. | HIV-1 gp41-specific monoclonal mucosal IgAs derived from highly exposed but IgG-seronegative individuals block HIV-1 epithelial transcytosis and neutralize CD4+ cell infection: an IgA gene and functional analysis | |
CA2774446A1 (en) | Hiv-1 antibodies | |
Zwick et al. | Broadly neutralizing antibodies targeted to the membrane-proximal external region of human immunodeficiency virus type 1 glycoprotein gp41 | |
CN102272158B (en) | Rapid expression cloning of human monoclonal antibodies from memory B cells | |
Kunert et al. | Characterization of molecular features, antigen-binding, and in vitro properties of IgG and IgM variants of 4E10, an anti-HIV type 1 neutralizing monoclonal antibody | |
Zhang et al. | Identification and characterization of a new cross-reactive human immunodeficiency virus type 1-neutralizing human monoclonal antibody | |
Zhang et al. | Broadly cross-reactive HIV neutralizing human monoclonal antibody Fab selected by sequential antigen panning of a phage display library | |
CA2809837A1 (en) | Human immunodeficiency virus (hiv)-neutralizing antibodies | |
Choudhry et al. | Cross-reactive HIV-1 neutralizing monoclonal antibodies selected by screening of an immune human phage library against an envelope glycoprotein (gp140) isolated from a patient (R2) with broadly HIV-1 neutralizing antibodies | |
US20110212106A1 (en) | Hiv-1 neutralizing antibodies and uses thereof | |
WO2008113833A1 (en) | Novel human anti-r7v antibodies and uses thereof | |
US20070292390A1 (en) | Broadly Cross-Reactive Hiv-1 Neutralizing Human Monoclonal Antibodies | |
Sun et al. | Brief introduction of current technologies in isolation of broadly neutralizing HIV-1 antibodies | |
WO2002059154A9 (en) | Neutralizing human monoclonal antibodies against hiv-1, their production and uses | |
Zhang et al. | Novel approaches for identification of broadly cross-reactive HIV-1 neutralizing human monoclonal antibodies and improvement of their potency | |
Dorgham et al. | Immunogenicity of HIV type 1 gp120 CD4 binding site phage mimotopes | |
Yuan et al. | Putative rhesus macaque germline predecessors of human broadly HIV-neutralizing antibodies: Differences from the human counterparts and implications for HIV-1 vaccine development | |
CN107383190B (en) | Humanized anti-HIV gp41 specific antibody and application thereof | |
Houimel et al. | Isolation and characterization of human neutralizing antibodies to rabies virus derived from a recombinant immune antibody library | |
CA2886448A1 (en) | Adcc-mediating antibodies, combinations and uses thereof | |
Wrotniak et al. | Antibody dependent cell cytotoxicity is maintained by the unmutated common ancestor of 6F5, a Gp41 conformational epitope targeting antibody that utilizes heavy chain VH1-2 | |
Li et al. | N-terminal residues of an HIV-1 gp41 membrane-proximal external region antigen influence broadly neutralizing 2F5-like antibodies |