CA2774446A1 - Hiv-1 antibodies - Google Patents

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.

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 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.

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.

>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

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.

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.

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.