WO2011159938A2

WO2011159938A2 - High affinity human antibodies to human cytomegalovirus (cmv) gb protein

Info

Publication number: WO2011159938A2
Application number: PCT/US2011/040761
Authority: WO
Inventors: Lawrence M. Kauvar; Stote Ellsworth; William Usinger; Krista Maureen Mccutcheon; Ying-ping JIANG; Fen Zhang; Bo Chen; Gizette Sperinde; Minha Park; Orit Foord
Original assignee: Trellis Bioscience, Inc.
Priority date: 2010-06-16
Filing date: 2011-06-16
Publication date: 2011-12-22
Also published as: JP2017038612A; RU2613421C2; AU2011268277A2; US9017668B2; CN103221063B; CA2839420A1; AU2011268277A1; AU2011268277B2; KR20130086036A; US10030069B2; RU2013101769A; BR112012032182A2; US20170204165A1; JP2013531991A; CA2839420C; KR101900435B1; EP2582389B1; IL223665B; US20150252099A1; JP6055763B2

Abstract

Antibodies to human Cytomegalovirus (CMV) gB protein have been isolated from human B cells. The affinities of these antibodies are higher than the best previously reported antibodies. Since high affinity is critical to prevention of virus transfer across the placenta, the invention antibodies are useful as therapeutic and prophylactic agents to prevent or ameliorate effects on the fetus of CMV infection during pregnancy.

Description

HIGH AFFINITY HUMAN ANTIBODIES TO HUMAN

CYTOMEGALOVIRUS (CMV) GB PROTEIN

Related Applications

[0001] This application claims priority to United States Provisional Patent Application Serial No. 61/355,499 filed on 16 June, 2010, the contents of which is incorporated in its entirety by reference herein.

Reference to Sequence Listing Submitted Via EFS-WEB

[0002] The entire content of the following electronic submission of the sequence listing the USPTO EFS-WEB server, as authorized and set forth in MPEP §1730 II.B.2(a)(C), is incorporated herein by reference in its entirety for all purposes. The sequence listing is identified on the electronically filed text file as follows:

Technical Field

[0003] The invention relates to human monoclonal antibodies (mAbs) against the gB protein of CMV, for therapeutic and prophylactic use to prevent or ameliorate the effects on the fetus of CMV infection during pregnancy, and to treat CMV infection in immunocompromised patients, including transplant patients.

Background Art

[0004] CMV is a major disease-causing agent in transplant patients, other

immunocompromised patients, and newborns. About 40,000 infants are born shedding CMV every year in the US. Of these, 8,000 are born with symptoms and/or severe handicaps and up to 8,000 more will later develop progressive hearing loss. About half of pregnant mothers have adequate immunity naturally. Thus it is known that effective mAbs exist in human blood. This is also shown by successful passive transfer of immunity by intravenously administered gamma globulin (IVIG), which has shown very high efficacy for protecting the fetus. This is in contrast to the limited efficacy observed for IVIG in the transplant setting, for which cellular immunity is apparently more important than humoral immunity. U 2011/040761

[0005] A substantial portion of the natural response to CMV is directed towards the gB protein (Park, J. W., et al, J. Korean Med. Sci. (2000) 15: 133-138). The Towne vaccine is an attenuated live virus vaccine passaged extensively in vitro, which induces antibodies that neutralize fibroblast infection, but not endothelial cell infection. This vaccine is known to be safe and has been studied for 20 years (Adler, S. P., et al, Pediatr. Infect. Dis. J. (1998) 17:200-206). Blood donors useful for isolating antibodies to gB as described below include seropositive individuals with previous exposure to CMV and seronegative subjects before and after vaccination with the Towne vaccine.

[0006] Antibodies to gB protein of CMV have been prepared (Nozawa N., et al, J. Clin. Virol. (2009) [Epub ahead of print], Nakajima, N., et al. (US2009/0004198 Al),

Lanzavecchia, A, et al. (US2009/0004198 Al), Ohlin, M., et al. (J Virol (1993) 67:703-710). A neutralizing antibody to the AD-2 domain of gB, ITC88, has been reported (Lantto, Virology (2003) 305:201-209). However, prior efforts to clone human antibodies against CMV, while successful, are limited in scope and no high affinity (sub-nanomolar) antibodies have been described. High affinity is a key parameter as weak affinity antibodies to CMV actually promote transmission across the human placenta (Nozawa, supra), an aspect of the pathology not seen in rodents. Human CMV has a double stranded DNA genome of approximately 236 kb and is a prototypical member of the β-herpesvirus family. The high complexity of the genome means that there are many potential antigens of interest. Efforts to characterize neutralizing antibodies and their associated epitopes resulted in a subunit vaccine based on

glycoprotein B (gB) that elicits an effective neutralizing response, but, when tested in a cohort of seronegative women has only 50% efficacy. This appears to be the highest efficacy of any CMV vaccine. Since vaccines typically induce antibodies with a range of affinities, the disappointing efficacy of the tested vaccines to date may be attributable to the requirement for high affinity antibodies, which argues in favor of supplying a high affinity mAb directly as a prophylactic strategy.

[0007] Failure to focus the immune response on the specific neutralizing epitopes has also been postulated as the cause of the poor efficacy (Marshall, B. C, et al, Viral Immunol (2003) 16:491-500. Another suspected technical problem in developing anti-CMV vaccines is that they have only been assessed for their ability to generate antibodies that neutralize fibroblast infection although infection of other cell types has increasingly become a focus for

understanding the viral pathology. This bias reflects technical obstacles with regard to growth of the virus in vitro. Repeated virus passage on fibroblast cells is believed to have caused many lab strains to lose tropism for endothelial and epithelial cells. During the last few years, this deficit has been associated with the loss of one or more components of the gH/gL/UL131- UL128 glycoprotein complex on the virus surface.

[0008] Clearly a need exists for a more effective anti-CMV prophylaxis strategy.

Disclosure of the Invention

[0009] Human antibodies that are specifically immunoreactive with the CMV gB protein, with improved affinity compared to prior antibodies (human or murine) and with neutralizing ability have been prepared. The humoral immune system is capable of producing millions of antibody structures with tens of thousands of well differentiated binding capabilities, yet the protective antibodies are only a very small subset of these. The present inventors have employed CellSpot™ technology (Harriman, W. D., et al, J. Immunol. Methods (2009) 34: 135-145, Collarini, E. J., et al, J. Immunol. (2009) 183:6338-6345), and U.S. patent 7,413,868), all incorporated herein by reference, to generate a panel of mAbs from blood of donors verified as having high titer to CMV.

[0010] Thus, in one aspect, the invention is directed to human monoclonal antibodies or immunoreactive fragments thereof that bind an epitope on the gB protein, with a preferred embodiment being binding to a conserved sequence therein. These antibodies display neutralizing capabilities in standard plaque forming assays for neutralization of CMV and demonstrate EC₅₀ in such assays of <500 ng/ml, preferably <200 ng/ml, more preferably <100 ng/ml. The antibodies of the invention also have affinities for the gB protein of CMV strain AD169 of <10 nM or <5 nM or <1 nM.

[0011] For use in the methods of the invention to treat CMV infection or to enhance resistance to CMV, the monoclonal antibodies or fragments of the invention may be

immunoreactive with a multiplicity of CMV strains and a single monoclonal antibody may suffice to have the desired effect. Alternatively, the subject to be treated or to be made resistant may be administered more than a single monoclonal antibody, which bind to the same or different CMV proteins.

[0012] The invention also includes pharmaceutical compositions useful for prophylaxis or treatment which contain as an active agent a single antibody or immunoreactive fragment of the invention, or no more than two antibodies or fragments of the invention.

[0013] Other aspects of the invention include methods of using the antibodies to treat CMV in human subjects or to induce resistance to infection in human subjects. [0014] The monoclonal antibodies of the invention may be produced recombinantly and therefore the invention also includes recombinant materials for such production as well as cell lines or immortalized cells and non-human multicellular organisms or cells thereof, or microbial cells, for the production of these antibodies. In one embodiment, cells obtained from human subjects are produced in "immortalized" form wherein they have been modified to permit secretion of the antibodies for a sufficient time period that they may be characterized and the relevant encoding sequence cloned.

Brief Description of the Drawings

[0015] Figures 1A and IB show the binding of 4A2 and 19B10 to gB protein and to the conserved region thereof.

[0016] Figures 2A and B show the neutralization of VR1814 by mAbs 4A2, 310, 313, 338, and 345 of HUVEC and HFF cells.

[0017] Figure 3 shows the neutralization of VR1814 by mAbs 4A2, 310, 313, 338, and 345 in HUVECs.

[0018] Figure 4 shows the neutralization of VR1814 by mAbs 4A2, 310, 313, 338, and 345 in HFF cells.

Modes of Carrying Out the Invention

[0019] As used herein, the term "treat" refers to reducing the viral burden in a subject that is already infected with CMV or to ameliorating the symptoms of the disease in such a subject. Such symptoms include retinitis and hepatitis.

[0020] The term "confers resistance to" refers to a prophylactic effect wherein viral infection by RS V upon challenge is at least reduced in severity.

[0021] "Immortalized cells" refers to cells that can survive significantly more passages than unmodified primary isolated cells. As used in the context of the present invention,

"immortalized" does not necessarily mean that the cells continue to secrete antibodies over very long periods of time, only that they can survive longer than primary cell cultures. The time over which secretion of antibody occurs need only be sufficient for its identification and recovery of the encoding nucleotide sequence.

[0022] Human antibodies, such as those herein isolated from human cells do not elicit a strong immune response. It is known that human antibodies do elicit a response in 5-10% of humans treated, even for antibodies that are isolated from humans, since there is a certain level of background "noise" in an immune response elicited. The immune response may be humoral or cellular or both. In particular, elevated levels of cytokines may be found in this percentage of individuals.

[0023] The gB Protein of CMV is synthesized as a precursor protein of 130 kDa, which is cleaved into fragments of 1 16 kDa (N-terminal) and 58 kDa (C -terminal) that remain covalently linked; the observed molecular weights may vary depending on glycosylation status. The AD-2 antigenic determinant refers to residues 67-82 of gpl 16. A considerable portion of natural immunity to CMV is accounted for by binding to AD-2 (i.e., can be blocked by a peptide covering this region), Ohlin (supra).

[0024] The antibodies of the invention have been recovered from CMV exposed human donors using the proprietary CellSpot™ method which is described in U.S. Patent

No. 7,413,868, PCT publications WO 2005/045396 and WO 2008/008858, all incorporated by reference, as set forth in Example 1.

[0025] Production of the human or humanized antibody of the invention is accomplished by conventional recombinant techniques, such as production in Chinese hamster ovary cells or other eukaryotic cell lines, such as insect cells. Alternatively, techniques are also known for producing recombinant materials, including antibodies, in plants and in transgenic animals, for example in the milk of bovines, or in microbial or plant or insect derived single cell systems or in cell free extracts of such cells.

[0026] In addition, since the nucleotide sequences encoding the antibodies are available, the relevant fragments which bind the same epitope, e.g. , Fab, F(ab')₂ or F_v fragments, may be produced recombinantly (or by proteolytic treatment of the protein itself) and the antibody may be produced in single-chain form. A variety of techniques for manipulation of recombinant antibody production is known in the art.

[0027] Chimeric, humanized and human antibodies are all within the scope of the present invention as are antibody mimics based on other protein scaffolds such as fibronectin, transferrin, or lipocalin. Likewise, multiple technologies now exist for making a single antibody-like molecule that incorporates antigen specificity domains from two separate antibodies (bi-specific antibody). Suitable technologies have been described by Macrogenics (Rockville, MD), Micromet (Bethesda, MD) and Merrimac (Cambridge, MA). (See, e.g., Orcutt KD, Ackerman ME, Cieslewicz M, Quiroz E, Slusarczyk AL, Frangioni JV, Wittrup KD. A modular IgG-scFv bispecific antibody topology. Protein Eng Des Sel. (2010) 23:221-228; Fitzgerald J, Lugovskoy A. Rational engineering of antibody therapeutics targeting multiple oncogene pathways. MAbs. (2011) 1 ;3(3); Baeuerle PA, Reinhardt C. Bispecific T-cell engaging antibodies for cancer therapy. Cancer Res. (2009) 69:4941-4944.) [0028] Thus, a single antibody with very broad strain reactivity can be constructed using the Fab domains of individual antibodies with reactivity to different CMV epitopes, such that for example, the bi-specific antibody has activity against both gB and the gH complex, or alternatively, may be reactive with gB proteins from the same or different strains. High affinity gH antibodies have been described, for example, by Macagno A, Bernasconi NL, Vanzetta F, Dander E, Sarasini A, Revello MG, Gerna G, Sallusto F, Lanzavecchia A. Isolation of human monoclonal antibodies that potently neutralize human cytomegalovirus infection by targeting different epitopes on the gH/gL/ULl 28-131 A complex. J Virol. (2010) 84:1005-1013. High affinity antibodies to gH proteins from other strains may also be generated and used.

[0029] For use in therapy, the recombinantly produced antibodies or fragments are formulated into pharmaceutical compositions using suitable excipients and administered according to standard protocols. The pharmaceutical compositions may have as their sole active ingredient a monoclonal antibody or fragment of the invention, especially a monoclonal antibody or fragment that is crossreactive with gB protein of all CMV strains. Alternatively, two monoclonal antibodies may be the sole active ingredients wherein one more strongly reacts with the one strain gB protein and the other more strongly with another strain gB protein. In all of these cases, additional therapeutic agents may be present including those binding to other CMV proteins. Also, the compounds may include nutritional substances such as vitamins, or any other beneficial compound other than an antibody.

[0030] In one embodiment, when the formulations for administration are used in order to increase resistance to infection, complete antibodies, including the complement-containing Fc region are employed. Typically, the antibodies are administered as dosage levels of

0.01-20 mg/kg of human subjects or in amounts in the range of 0.01-5 mg/kg or intermediate amounts within these ranges. In one embodiment, amounts in the range of 0.1-1.0 mg/kg are employed. Repeated administration separated by several days or several weeks or several months may be beneficial.

[0031] In another embodiment, for a therapeutic effect in order to reduce viral load, complete antibodies, containing the complement-containing Fc region are also employed. The amounts administered in such protocols are of the order of .001-50 mg/kg or intermediate values in this range such as 0.01, 1 or 10 mg/kg are employed. Repeated administration may also be used. The therapeutic treatment is administered as soon as possible after diagnosis of infection, although administration within a few days is also within the scope of the invention. Repeated administration may also be employed. In order to reduce the inflammatory response in the lungs, only the immunospecific fragments of the antibodies need be employed. Dosage levels are similar to those for whole antibodies. Administration of mixtures of immunospecific fragments and entire antibodies is also included within the scope of the invention.

[0032] Administration of the antibody compositions of the invention is typically by injection, generally intravenous injection. Thus, parenteral administration is preferred.

However, any workable mode of administration is included, including gene therapy (production of recombinant antibody in vivo).

[0033] The formulations are prepared in ways generally known in the art for administering antibody compositions. Suitable formulations may be found in standard formularies, such as Remington's Pharmaceutical Sciences, latest edition, Mack Publishing Co., Easton, PA, incorporated herein by reference. The formulations are typically those suitable for parenteral administration including isotonic solutions, which include buffers, antioxidants and the like, as well as emulsions that include delivery vehicles such as liposomes, micelles and nanoparticles.

[0034] The desired protocols and formulations are dependent on the judgment of the attending practitioner as well as the specific condition of the subject. Dosage levels will depend on the age, general health and severity of infection, if appropriate, of the subject.

[0035] The following examples are offered to illustrate but not to limit the invention.

Example 1

Isolation of Human B cells Secreting Antibody to CMV gB

[0036] Peripheral blood mononuclear cells from 50 adults with confirmed titer against CMV were surveyed for human B cells producing anti-viral antibodies. Subjects with the desired antibodies against CMV gB protein were used for cloning of specific mAbs. The result of the survey was that -10% of the subjects had a frequency of the desired cells greater than 1 in 50,000.

[0037] To accomplish the survey and recovery of rare favorable cells, we used the previously described CellSpot™ technology (U.S. 7,413,868, incorporated herein by reference). The CellSpot™ assay method effectively shrinks an ELISA equivalent assay down to a virtual well of nearly single cell dimensions by capturing secreted IgG from a single cell as a footprint in the vicinity of the cell, so that millions of cells can be readily analyzed. Further, by use of microscopic multiplexing reagents (combinatorially colored fluorescent latex microspheres, cf US 6,642,062, incorporated herein by reference), each clone's secreted antibody footprint can be characterized in detail for specificity and/or affinity using multiple biochemical probes. The fidelity of the quantitative assay is sufficient to enable rescue of extremely rare favorable cells from the survey population, with the cloned expression cell showing a phenotype consistent with the original identifying assay.

[0038] The screening criteria were binding to purified gB protein as well as to viral lysate. gB protein was purified from 293 cells infected with AD 169 strain of CMV. Affinity rank ordering of clones is accomplished by diluting the antigen on the bead with serum albumin. This reduces the chances for multi-dentate binding to the secreted IgG footprint (an "avidity" effect), thus selecting for higher intrinsic affinity.

[0039] Non-B cells were depleted from PBMCs in plasma of human donors using standard magnetic separation methods. Cells were resuspended in IMDM/20% HI-FCS at le6/ml; and immortalized with EBV (direct pelleted from the supernatant of infected B95-8 cells). EBV was added at 1 : 100 dilution, and the cells incubated 2 hr at 37°C. Excess EBV was washed away, and cells either:

(1) cultured at 2e6/ml in IMDM, 20% HI-FCS, 20% Giant cell tumor conditioned medium, 2 μg/ml CpG (ODN2006), and 10 ng/ml IL-10 for surveying only, or

(2) further selected for surface IgG using magnetic positive selection.

[0040] Cells were cultured at 200-300 cells/well on irradiated human lung cells (MRC-5, 5,000 cells/well) in IMDM, 20% HI-FCS, 20% Giant cell tumor conditioned medium, 2 μg ml CpG (ODN2006), and 10 ng/ml IL-10. Medium was supplemented every 2-3 days. One half of the contents of the wells were assayed in CellSpot™ at day 6. The remaining cells in the small number of wells positive by the survey assay were then diluted to 10, 5, 1, and 0.5 cells/well with the same feeder cells and culture conditions. After 4-5 days these limiting dilution plates were again assayed by ELISA or CellSpot™.

[0041] CellSpot™ nano-particles were conjugated with viral lysate or purified gB protein to screen for the desired antibodies. Lysate created from cells infected with the CMV AD 169 virus was purchased from Virusys (cat # CV046). (The lysate is produced in Normal Human Dermal Fibroblast (NHDF) cell line.) Recombinant CMV gB antigen was produced as His- tagged fusion protein in 293 cells and purified using a nickel chelation column. Purified gB protein was used for ELISA and CellSpot. The preparations of AD 169 lysate and gB purified protein were conjugated to nano-particles, respectively, as previously described, cf Harriman et al {supra) and Collarini et al (supra).

[0042] Contents of positive wells at limiting dilution were then processed using Reverse Transcriptase-PCR to recover the encoding mRNA for the antibody heavy and light chains. Total time from thawing PBMCs to recovery of the encoding mRNA sequence via RT-PCR was 10-12 days. Example 2

Cloning of Human Antibodies to CMV gB

[0043] Amplification of rearranged Ig Heavy and Ig Light genes from positive ELISA wells was accomplished using semi-nested polymerase chain reaction (PCR). For amplification of a previously unknown V-gene rearrangements, a collection of family-specific V-gene primers was constructed, which recognize nearly all V-gene segments in the human Ig Locus. The 5' primers were used together with primer mixes specific for the Ογ, CK and CX gene segments. The clonality of the limiting dilution CMV-gB specific B cells was unequivocally determined by sequence comparison of V-gene amplificates from distinct progeny cells, and the amplified full length V-gene rearrangements were cloned into IgG expression vectors.

[0044] In detail, total mRNA from the isolated human B cells was extracted using a commercially available RNA purification kit (RNeasy™; Qiagen (Germany)). Reverse transcription-PCR was done by using total RNA preparations and oligonucleotides as primers. Three PCR reactions were run for each sample: one for light chain kappa (κ) one for light chain lambda (λ), and one for gamma heavy chain (γ). The QIAGEN^® OneStep RT-PCR kit was used for amplification, (Qiagen Catalog No. 210212). In the coupled RT-PCR reactions, cDNA is synthesized with unique blend of RT enzymes (Omniscript™ and Sensiscript™) using antisense sequence specific primer corresponded to C-K, C-λ or to a consensus of the CHI regions of Cy genes, RT is preformed at 50°C for 1 hour followed by PCR amplification of the cDNA by HotStarTaq DNA Polymerase for high specificity and sensitivity. Each PCR reaction used a mixture of 5' sense primers. Primer sequences were based on leader sequences of VH, VK and VL. PCR reactions were run at 95°C for 15 minutes, initial hot start followed by 20 cycles of 95°C for 30 seconds (denaturation), 60°C for 45 seconds (annealing) and 72°C for 1 minute (elongation).

[0045] Nested PCR for detection and cloning of the variable Ig fragments into expression vectors. In the second round, an aliquot of 5 μΐ of the first amplification reaction was applied. The primers used carry the 5'BglII and 3' Xbal restriction sites. Thirty PCR cycles were performed. Identical conditions were used for the first and second rounds of amplification. Five microliters of each reaction were loaded and separated on a 1% agarose gel and then stained with ethidium bromide. The V-C PCR product is predicted to amplify rearranged fragments of VH and VL, 500 and 450 bp respectively. PCR bands with a molecular size of approximately 500 bp indicated a positive result. PCR products were purified (Qiagen gel purification kit catalog number 28704) and the extracted PCR products were directly sequenced using specific constant region primers. The sequences of the cloned fragments were confirmed by sequencing plasmids prepared for recombinant production.

[0046] The PCR fragments described above were digested and cloned into individual expression vectors carrying the constant region of human gamma 1, or of human kappa or lambda, for in vitro antibody production in mammalian cells. The expression vectors coding for heavy and light chains were co-transfected into the 293 (human kidney) cell line (Invitrogen). The expression plasmids were introduced with the use of a cationic lipid-based transfection reagent (293fectin™; Invitrogen). For each transfection reaction, 20 μg of purified plasmids and 40 xL of the 293fectin™ were mixed with 1 mL of Opti-MEM^® (Invitrogen) and incubated for 5 min at room temperature before being combined and allowed to form complexes for 20 min at room temperature. The DNA-293fectin complexes were added to 3x10⁶ cells seeded in 90 mm petri plates and incubated at 37°C, 8% C02. In the final procedure, the supernatant was harvested 72 hrs post-transfection by centrifugation (3,000 g, 15 min at 4°C), to recover the secreted antibodies.

[0047] From ~2 million lymphocytes, 45 clones were isolated which bound the AD 169 lysate. Of these, the majority also bound the recombinant gB protein.

[0048] Two of the niAbs that bound both AD 169 and gB (4A2 and 19B10) had neutralizing capability. One of these (4A2) binds the AD-2 peptide, which is a conserved site on the gB protein. An additional mAb (5C5) binds AD-2 but does not neutralize the virus.

[0049] The amino acid sequences of the heavy and light chains of 4 A2 and 19B 10, including variable region, the D and J joining regions, the framework (FR) and complementarity determining (CDR) regions, are shown below. The secretion signal sequence on the heavy chain is italicized, and CDRs 1-3 are underlined.

4A2 HC. VH3-30 nucleic acid (SEP ID NO:l and amino acid (SEP ID NO:2)

atggaattggggctgagctgggttttcgtcgttgctcttttaagaggtgtccagtgtcaa

M E L G L S N V F V V A L L R G V Q C Q

gtgttgttggaggagtctgggggaggcgtggtccagcctgggaggtctctgagactctcc

V L L E E S G G G V V Q P G R S L R L S

tgtgcaggctctggattcaccttcaataggcatggaattcactgggtccgccaggctcca

C A G S G F T F N R H G I H W V R Q A P

ggcaaggggctggagtgggtgactgttatatcatctgatggagcaaatcaacagtatgca

G K G L E W V T V I S S D G A N Q Q Y A

gagtccgtgaagggccgattcatcatctccagagacaattccaagaacacggtatatcta

E S V K G R F I I S R D N S K N T V Y L

gaaatgaatagcctgaggaatgacgacacgggtgtgtatttctgcgcgagagacggtcgt

E M N S L R N D D T G V Y F C A R D G R

tgtgaaggcgagaggtgctactccggtgtcacggacttctggggccagggaacactggtc

C E G E R C Y S G V T D F G Q G T L V 4A2 LC L6. IeKV3-l 1 nucleic acid (SEP ID NO:3) and amino acid (SEP ID N0:4) atggaagccccagcgcagcttctcttcctcctgctactctggctcccagataccaccgga

M E A P A Q L L F L L L L W L P D T T G

gaaattgtattgacacagtctccagccaccctgtctttgtctccaggggagagagccacc

E I V L T Q S P A T L S L S P G E R A T

ctctcctgcagggccagtcagaatattggcggctacttggcctggttccaacaaaaagct

L S C R A S Q N I G G Y L A W F Q Q K A

ggccaggctcccaggctcctcatctatgatgcatccatcagggccactggcatcccagcc

G Q A P R L L I Y D A S I R A T G I P A

aggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcct

R F S G S G S G T D F T L T I S S L E P

gaagattttgcagtttattactgtcagcagcgtaacagttggcctccactcactttcggc

E D F A V Y Y C Q Q R N S W P P L T F G

19B10 HC VH4-31, D2, J6 nucleic acid (SEP ID NP:5) and amino acid (SEP ID NP:6 atgaaacatctgtggttcttcctcctgctggtggcagctcccagatgggtcctgtcccag

M K H L W F F L L L V A A P R W V L S Q

gtgcagctgcagcagtcgggcccaggactggtgaagccttcacagaccctgtccctcacc

V Q L Q Q S G P G L V K P S Q T L S L T

tgcactgtctctggtggctccatcagtagcggtgatttttgctggaattggatccgccag

C T V S G G S I S S G D F C W N W I R Q

cccccagggaagggcctggagtggattgggtacatctgttacaccggggacacctactac

P P G K G L E I G Y I C Y T G D T Y Y

aacccgccccttaacagtcgagttaccatatcagtcgacaggtccaggaaccaaatctcc

N P P L N S R V T I S V D R S R N Q I S

ctgaggctgagttctgtgactgccgcagacacggccgtgtattattgtgccagagaggat

L R L S S V T A A D T A V Y Y C A R E D

aggagacaactacactctcgcccctacttctactacggtttggacgtctggggccgaggg

R R Q L H S R P Y F Y Y G L D V W G R G

accaaggtcaccgtc tec t cage ttccaccaagggcccatcggtcttccccctggtaccc

T K V T V S S A S T K G P S V F P L V P

tctagc

S S

19B10 LC, A3. IgKV2 nucleic acid (SEP ID NP:7 and amino acid (SEP ID NP:8) atgaggctccctgctcagcttctggggctgctaatgctctgggtctctggatccagtggg

M R L P A Q L L G L L M L W V S G S S G

gagattgtgatgactcagtctccgctctccctgcccgtcacccctggagagacggcctcc

E I V M T Q S P L S L P V T P G E T A S

atctcctgcaggtctagtcagagcctcctgcatagtaatggacacaactatttggattgg

I S C R S S Q S L L H S N G H N Y L D W

tatctgcagaagccagggcagtctccacacctcctgatctatttgggttctattcgggcc

Y L Q K P G Q S P H L L I Y L G S I R A

tccggggtccctgacaggttcagtggcagtggaacaggcacagattttacactgaaaatc

S G V P D R F S G S G T G T D F T L K I

agcagagtggaggctgaggatgttggggtttattactgcatgcaagctctacaaactcct

S R V E A E D V G V Y Y C M Q A L Q T P

aacacttttggccaggggaccaagctggagatcagacgaactgtggctgcaccatctgtc

N T F G Q G T K L E I R R T V A A P S V [0050] High affinity antibodies were generated by preparing CellSpot probes coated with full length gB protein or with the Ad-2 peptide at either high or low density on the fluorescent bead. Without being constrained by theory, since low density reduces the multi-dentate avidity effect, the search is biased in favor oa antibodies with high intrinsic affinity. Multiple high affinity antibodies were isolated and sequenced. The sequences of other monoclonal antibodies that are reactive to CMV were determined, and are shown as SEQ ID NOs:9-36, and 38-66. The nucleotide sequence of the human IgGl heavy chain constant region is shown in SEQ ID NO:37.

[0051] In antibodies of the invention, the heavy chain can have a CDR1 of GFTFNRHG (SEQ ID NO:67) or GSISSEDFC (SEQ ID NO:68); and/or a CDR2 region of SSDGANQ (SEQ ID NO:69) or ICYTGD (SEQ ID NO:70); and/or a CDR3 region of

ARDGRCEGERCYSGVTDF (SEQ ID NO:71) or AREDRRQLHSRPYFYYGLDV (SEQ ID NO:72). In other embodiments, the light chain has a CDR1 region of QNIGGY (SEQ ID NO:73) or QSLLHSNGHNY (SEQ ID NO:74); and/or a CDR2 region of DAS (SEQ ID NO:75) or LG (SEQ ID NO:76); and/or a CDR3 region of QQRNSWPPLT (SEQ ID NO:77) or QALQTPNT (SEQ ID NO:78).

Example 3

Affinity Determination

[0052] The affinity of the invention antibodies was determined by ForteBio^® (Menlo Park, CA) biosensor analysis. In this method, the carboxylic acid groups on Amine Reactive

Biosensors were activated with EDC/NHS. Antibody, diluted in MES buffer at pH 5, was attached to the activated surface of the probe and the remaining active carboxylated groups were blocked with ethanolamine. The gB protein was incubated with the Ab-coated probe and rates of association to and dissociation from the Ab-coated probe were determined by the ForteBio^® instrument.

[0053] In one experiment, 4A2 was found to have an affinity of 168 pM and 19B10 an affinity of 697 pM, as shown as corresponding IC₅₀ in μg/ml in Table 1. These affinity constants are substantially better than for published monoclonal antibodies to gB. P T/US2011/040761

Table 1

Comparison of neutralizing potency of mAbs

IC50

mAb (target) citation source

( g mL)

4A2 (gB) 0.02 human

19B10 (gB) 0.04 human

CHI 77 (gB) 0.23 Nozawa, N, et al. (2009) supra murine

G3D (gB) 0.50 Nakajima, N., et al. (2009) supra human

10C6 (gB) 0.30 Lanzavecchia, A., et al. (20Q9)supra human

[0054] The binding affinity of mAbs 310, 313, 345, and 4A2 to the AD-2 epitope of gB was determined. The gB binding kinetics are shown in Table 2. The mAbs 310, 313, and 338 have about lOx higher potency than mAb 4A2. The mAbs 323, 316, and 338 also have higher potency than mAb 4A2 (data not shown). The binding affinities of the remaining mAbs are also tested and also better than for published monoclonal antibodies to gB.

Table 2

gB binding kinetics

Example 4

Elisa Binding Assay and Epitope Mapping

[0055] The mAbs 4A2 and 19B10 were assessed for binding to purified gB protein and to a conserved peptide designated AD-2: NETIYNTTLKYGDV (SEQ ID NO:79). As shown in Figure 1, 4A2 binds well to both full length protein and peptide AD-2, whereas 19B10 only binds the protein. Example 5

Virus Neutralization Assay

[0056] The mAbs 4A2 and 19B 10 neutralized the AD 169 strain of CMV in MRC5 primary fibroblasts. Serial dilutions of the antibodies were mixed with an equal volume of AD 169

(10 /ml stock diluted to give 2000 infected cells per well) and incubated for 1 h at room temperature before addition to target cell monolayers in 96-well microplates. After 24 h, cells were fixed, permeabilized, and stained with monoclonal antibody against IE1 (Intermediate Early protein 1, also known as UL123, a marker of replicating virus) conjugated to HRP. The infected cells were detected following deposition of HRP substrate. The number of infected cells was plotted against the concentration of the antibody.

[0057] Virus neutralization was also assessed using the VR1814 strain (Revello, et al. , J Gen. Virol. (2001) 82:1429-1438). The mAbs 4A2, 310, 313, 338, and 345 neutralized the VR1814 strain in both human umbilical vein endothelial cells (HUVEC) and human foreskin fibroblast (HFF) cells. Figures 2A and 2B show the IC50 and IC90 values for each of mAbs 4A2, 310, 313, 338, and 345. Figures 3 and 4 show the neutralization of HUVEC and HFF cells, respectively, by each of the mAbs 4A2, 310, 313, 338, and 345. The results were tested in duplicate.

[0058] Other mAbs are also tested, which neutralize the AD 169 and VR1814 strains. SEQUENCE LISTING

4A2 HC, VH3-30 nucleic acid iSEQ ID NO:l) and amino acid (SEP ID NO:2)

atggaattggggctgagctgggttttcgtcgttgctcttttaagaggtgtccagtgtcaa

M E L G L S W V F V V A L L R G V Q C Q

gtgttgttggaggagtctgggggaggcgtggtccagcctgggaggtctctgagactctcc

V L L E E S G G G V V Q P G R S L R L S

tgtgcaggctctggattcaccttcaataggcatggaattcactgggtccgccaggctcca

C A G S G F T F N R H G I H W V R Q A P

ggcaaggggctggagtgggtgactgttatatcatctgatggagcaaatcaacagtatgca

G K G L E W V T V I S S D G A N Q Q Y A

gagtccgtgaagggccgattcatcatctccagagacaattccaagaacacggtatatcta

E S V K G R F I I S R D N S K N T V Y L

gaaatgaatagcctgaggaatgacgacacgggtgtgtatttctgcgcgagagacggtcgt

E M N S L R N D D T G V Y F C A R D G R

tgtgaaggcgagaggtgctactccggtgtcacggacttctggggccagggaacactggtc

C E G E R C Y S G V T D F W G Q G T L V 4A2 LC L6, IgKV3-l 1 nucleic acid iSEQ ID NO:3) and amino acid (SEP ID NO:4^) atggaagccccagcgcagcttctcttcctcctgctactctggctcccagataccaccgga . M E A P A Q L L F L L L L L P D T T G gaaattgtattgacacagtctccagccaccctgtctttgtctccaggggagagagccacc

E I V L T Q S P A T L S L S P G E R A T

ctctcctgcagggccagtcagaatattggcggctacttggcctggttccaacaaaaagct

L S C R A S Q N I G G Y L A W F Q Q K A

ggccaggctcccaggctcctcatctatgatgcatccatcagggccactggcatcccagcc

G Q A P R L L I Y D A S I R A T G I P A

aggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcct

R F S G S G S G T D F T L T I S S L E P

gaagattttgcagtttattactgtcagcagcgtaacagttggcctccactcactttcggc

E D F A V Y Y C Q Q R N S W P P L T F G

19B10 HC VH4-31, D2, J6 nucleic acid (SEQ ID NO:5 and amino acid (SEQ ID NO:6) atgaaacatctgtggttcttcctcctgctggtggcagctcccagatgggtcctgtcccag

M K H L W F F L L L V A A P R W V L S Q

gtgcagctgcagcagtcgggcccaggactggtgaagccttcacagaccctgtccctcacc

V Q L Q Q S G P G L V K P S Q T L S L T

tgcactgtctctggtggctccatcagtagcggtgatttttgctggaattggatccgccag

C T V S G G S I S S G D F C W N W I R Q

cccccagggaagggcctggagtggattgggtacatctgttacaccggggacacctactac

P P G K G L E W I G Y I C Y T G D T Y Y

aacccgccccttaacagtcgagttaccatatcagtcgacaggtccaggaaccaaatctcc

N P P L N S R V T I S V D R S R N Q I S

ctgaggctgagttctgtgactgccgcagacacggccgtgtattattgtgccagagaggat

L R L S S V T A A D T A V Y Y C A R E D

aggagacaactacactctcgcccctacttctactacggtttggacgtctggggccgaggg

R R Q L H S R P Y F Y Y G L D V W G R G

accaaggtcaccgtctcctcagcttccaccaagggcccatcggtcttccccctggtaccc

T K V T V S S A S T K G P S V F P L V P

tctagc

S S

19B10 LC, A3, IgKV2 nucleic acid rSEO ID NO:7> and amino acid (SEP ID NO:8 atgaggctccctgctcagcttctggggctgctaatgctctgggtctctggatccagtggg

M R L P A Q L L G L L M L W V S G S S G

gagattgtgatgactcagtctccgctctccctgcccgtcacccctggagagacggcctcc

E I V M T Q S P L S L P V T P G E T A S

atctcctgcaggtctagtcagagcctcctgcatagtaatggacacaactatttggattgg

I S C R S S Q S L L H S N G H N Y L D W

tatctgcagaagccagggcagtctccacacctcctgatctatttgggttctattcgggcc

Y L Q K P G Q S P H L L I Y L G S I R A

tccggggtccctgacaggttcagtggcagtggaacaggcacagattttacactgaaaatc

S G V P D R F S G S G T G T D F T L K I

agcagagtggaggctgaggatgttggggtttattactgcatgcaagctctacaaactcct

S R V E A E D V G V Y Y C M Q A L Q T P

aacacttttggccaggggaccaagctggagatcagacgaactgtggctgcaccatctgtc

N T F G Q G T K L E I R R T V A A P S V Human IgGl HC amino acid sequence of constant region (SEQ ID NO: 9)

ASTKGPSVFPLVPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCAAA DVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEY CKVSNKALPAPIEKTI SKAKGQPRE PQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

MAB297 HC variable domain amino acid sequence (SEQ ID NO: 10

QVQLVQSGGGWQPGRSLRLSCSASGFTFSNYNMHWVRQAPGKGPEWVAVISKDGNE KHYAESAKGRFTISRDNSK TLYMEMHSLTPEDTAMYYCTRDGRTDGTGYSGILDIW GQGTKVIVS

MAB309 and 318 HC variable domain amino acid sequence (SEQ ID NO:l 1)

QVQLVQSGGGWQPGTSLRLSCAASGFMFNTYNMHWVRQAPGKGLEWVAVISNDGTY KHFADSLKGRFSISRDDSKNTLYLHMNSLRPDDTAIYYCARDGRSVGGFSGILDPWG QGTLVTVSS

MAB31Q HC variable domain amino acid sequence (SEQ ID NO: 12)

QVQLVQSGGGWQPGTSLRLSCAASGFMFNTYNMHWVRQAPGKGLEWVAVISNDGTY KYSADSLKGRFS ISRDNSKNTLYLHMNSLRPDDTAVYYCARDGRSVGGFSGILDPWG QGTLVTVSS

MAB313 HC variable domain amino acid sequence (SEQ ID NO: 13)

QVQ LVQSGGGVIQPGRSLTLSCAASGFTFSAYSLHWVRQAPG GLQWVAVISFDGNF HFADSLRGRFTISRDNSKNRFYLQMNGLRGEDTAVYYCARDGRAVDGFSGILDFWG QGTLVSVSS

MAB314 HC variable domain amino acid sequence (SEQ ID NO: 14)

QVQLQESGGGLVQPGGSLKLSCAVSGFSFGGSAMHWVRQASGKGLEWIGHIRSGANN FETAYAPSLDGRFTISRDDSK TAYLHMNSLKTDDTAMYFCTTGLIASGDANFDYWG QGTQVTVSS

MAB316 HC variable domain amino acid sequence (SEQ ID NO: 15)

QVQLVQSGGGWQPGRSLTLSCAASGFTFSGFSLHWVRQAPGKGLQWVAVISFDGNH KHFADSLKGRFTISRDNSKNTLYLQINDLRGEDTAVYYCARDGRAVDGFSGILDFWG QGTLVSVSS

MAB319 HC variable domain amino acid sequence (SEQ ID NO: 16)

QVQLVESGGGWQPGRSLRLSCSASGFTFSDYNLHWVRQAPGKGLEWVAVIS IDGSD KHHADSVKGRFTVSRDNSK TVSLQMDSLRPEDTAVYYCARDGRSVGGYSGILDPWG QGTLVTVSS MAB321 HC variable domain amino acid sequence (SEP ID NO: 17

EVQLVESGAEVK PGESLKISCQGSGYRFTNYWIAWVRQMPGKGLEWMGI IYPGDSD TRYHPSFQGQVTI SSDKSLNTAYLQWSSLKPSDTAVYYCARHHCLSTNCQTAVAGYN DYWGQGNPGRRLLS

MAB322 HC variable domain amino acid sequence (SEQ ID NO: 18)

QVQLVQSGGGWQPGRSLRLSCSASGFTFTNYNMHWVRQAPGKGLEWVAVTSKDGNE KHFADSVKGRFTISRDNSKNTLYLEMNTLTAEDTAIYYCTRDGRTDGTGYSGILDIW GQGTKVTVSS

MAB323 HC variable domain amino acid sequence (SEQ ID NO: 19)

QVQLVQSGGGWQPGRSLRLSCAASGFTFSNFAMH VRQAPGKGLEWVAVISNAGRE THYADSVKGRFTVSRDNSKNMLSLQMNSLRGEDTAVYYCARDGRTDGSGYSGVLDIW AQGTLVTVSS

MAB338 HC variable domain amino acid sequence (SEQ ID NO:20)

QVQLVESGGGWQPGRSLRLSCSGSGFTFSDYNLHWVRQAPGKGLE VAVIS IDGTN KHHADSV GRFTISRDNSKNTVNLEMSRLKAEDTAVYYCVRDGRS IGGYSGIFDPWG QGTLVTVSS

MAB343 HC variable domain amino acid sequence (SEQ ID NO:21)

QVQLQESGGGWQPGRSLRLSCAASGFTFNTYNMHWVRQAPGKGLEWVAVISNDGTY KYS ADS VKGRFS I SRGNS KNTLYLQMNS LRPDDTAVY YC ARDGRS VGGF S G I LD PWG QGTLATVSS

MAB345 HC variable domain amino acid sequence (SEQ ID NO:22)

QVQLVESGGGWQPGRSLRLSCAASGFTFSDYNMHWVRQAPGKGLEWVAVIS IDGTY KYSADSVAGRFSLSRDNSKNTLYLQMNSLRPDDTAIYYCARDGRSVGGFSGILDPWG QGTLVTVSS

Human LC amino acid sequence of constant kappa region (SEQ ID NO:23)

TVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDS KDS TYS LS S TLTLS KAD YEKHKVYACE VTHQGLS S PVTKS FNRGE C

MAB297 and MAB322 LC variable domain amino acid sequence (SEP ID NO:24)

EIVMTQSPATLSLSPGERATLSCRASQSVGGYLAWYQQ PDQAPRLLIYDVSNRAAG IPARFSGSGSGTDFTLTI SSLEPEDFAVYYCQQRNTWPPLTFGGGTKVEIKR

MAB309 LC variable domain amino acid sequence (SEQ ID NO:25)

EIVLTQSPATLSLSPGDRATLSCRASQTVGRYLAWYQQKPGQAPRLLIYDASDRATG ISARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSSWPPLTFGGGTKVEIKR MAB310 LC variable domain amino acid sequence (SEQ ID NO:26)

E I VLTQS PATLS LS PGDRATLS CRAS QTVGRYLAWYQQKPGQAPRLL I YDASDRATG ISARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPLTFGGGTKVEIKR

MAB313 LC variable domain amino acid sequence (SEQ ID NO:27)

EIVMTQSPATLSLSPGERATLSCRASQSVGRYLTWFQQKPGQAPRLLIYDASERATG I PARFS GSGSGTDFTLT I S S LE PEDFAVYYCQQRAN PPLTFGGGTKVE I K

MAB314 LC variable domain amino acid sequence (SEQ ID NO:28

EIVMTQSPGTLSLFPGERATLSCRASQTVRNGYLAWYQQKPGQAPRLLIYGASIRAT GIPDRFSGSGSETDFTLS ITRVEPEDFAVYYCQQYGRLSSTFGQGTKLDLK

MAB316 LC variable domain amino acid sequence (SEQ ID NO:29

EIVMTQSPATLSLSPGERATLSCRASQSVGRYLTWFQQKPGQAPRLLIYDASERATG VPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPLTFGGGTKVEIK

MAB318 LC variable domain amino acid sequence (SEQ ID NO: 30)

EWLTQSPATLSLSPGDRATLSCRASQTVGRYLAWYQQKPGQAPRLLIYDASDRATG ISARFSGSGSGTDFTLTIGSLEPEDFAVYYCQQRSSWPPLTFGGGTKVEIK

MAB319 LC variable domain amino acid sequence (SEQ ID NO:31)

EI VLTQS PATLSLSPGERATLSCRASQSVGSYLAWYQQKPGQAPRLLIYDASERATG IPARFSGSGSGTDFTLTISSLEPEDVAVYYCQQRNNWPPLTFGGGTKVEIK

MAB321 LC variable domain amino acid sequence (SEQ ID NO:32

EIVMTQSPDSLAVSLGERATINCKSSQSILFSSKNQNHLAWYQQKPGQPPKLLIYWA STRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYNIPHTFGGGTKVEIK

MAB323 LC variable domain amino acid sequence (SEQ ID NO:33)

EIVLTQSPATLSLSPGERATLSCRASQSVNRYLAWFQHRPGQPPRLLIYDASKRATG IPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSN PLTFGGGTKVEIK

MAB338 LC variable domain amino acid sequence (SEQ ID NO:34)

E I VLTQS PATLS LS PGERATLS CRAS QS VDRYLAWYQQKPGQAPRLL I YDAS QRATG IPARFSGSGSGTDFTLAISSLEPEDVAVYYCQQRSNWPPLTFGGGTKIEIK

MAB343 LC variable domain amino acid sequence (SEQ ID NO:35)

EIVMTQSPATLSLSPGDRATLSCRASQSVGSYLAWYQQKPGQAPRLLI YDASDRATG IPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPLTFGGGTKVEIK MAB345 LC variable domain amino acid sequence (SEQ ID NO:36

EIVMTQSPATLSLSPGDRATLSCRASQSVGSYLAWYQQKPGQAPRLLMYDSSVRATG IPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRNNWPPLTFGGGTKVEIK

Human IgGl HC nucleotide sequence of constant region (introns are underlined) (SEQ ID NO.-37)

GCCTCCACCAAGGGCCCATCAGTCTTCCCCCTGGCACCCTCTACCAAGAGCACCTCT GGGGGCACAACGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACG GTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTA CAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTG GGC AC C C AGAC CTACATC TGC AACGTGAAT CAC AAGC C C AGCAAC AC C AAGGTGGAC AAGAGAGTTGGTGAGAGGCCAGCACAGGGAGGGAGGGTGTCTGCTGGAAGCCAGGCT CAGCGCTCCTGCCTGGACGCATCCCGGCTATGCAGTCCCAGTCCAGGGCAGCAAGGC AGGCCCCGTCTGCCTCTTCACCCGGAGGCCTCTGCCCGCCCCACTCATGCTCAGGGA GAGGGTCTTCTGGCTTTTTCCCCAGGCTCTGGGCAGGCACAGGCTAGGTGCCCCTAA CCCAGGCCCTGCACACAAAGGGGCAGGTGCTGGGCTCAGACCTGCCAAGAGCCATAT CCGGGAGGACCCTGCCCCTGACCTAAGCCCACCCCAAAGGCCAAACTCTCCACTCCC TCAGCTCGGACACCTTCTCTCCTCCCAGATTCCAGTAACTCCCAATCTTCTCTCTGC AGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGGTAAGCCAGC CCAGGCCTCGCCCTCCAGCTCAAGGCGGGACAGGTGCCCTAGAGTAGCCTGCATCCA GGGACAGGCCCCAGCCGGGTGCTGACACGTCCACCTCCATCTCTTCCTCAGCACCTG AACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCA TGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACC CTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAA AGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCC TGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCC TCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGTGGGACCCGTGGGGTGC GAGGGCCACATGGACAGAGGCCGGCTCGGCCCACCCTCTGCCCTGAGAGTGACCGCT GTACCAACCTCTGTCCCTACAGGGCAGCC C CGAGAAC CACAGGTGTAC AC C C TGC C C C CAT C C C GGGAGGAGATGAC C AAGAAC C AGGT C AG C C TGAC CTGCCTGGT C AAAGG C TTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAAC TACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAG CTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATG CATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAA TGA

MAB297 HC variable domain nucleotide sequence (SEQ ID NO:38)

CAGGTGCAACTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA CTCTCCTGTTCAGCCTCTGGATTCACCTTCAGCAACTATAATATGCACTGGGTCCGC CAGGCTCCAGGCAAGGGGCCGGAGTGGGTGGCAGTTATATCAAAAGATGGAAACGAA AAACACTATGCAGAGTCTGCGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAG AACACGCTGTATATGGAAATGCACAGCCTGACACCTGAGGACACGGCTATGTATTAC TGTACGAGAGATGGGCGAACCGATGGTACTGGGTACTCCGGTATTCTTGATATCTGG GGCCAAGGGACAAAGGTCATCGTCTCT MAB3Q9 and 318 HC variable domain nucleotide sequence (SEP ID NO:39

CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGACGTCCCTGAGA CTCTCCTGTGCAGCCTCTGGATTCATGTTCAATACCTATAATATGCACTGGGTCCGC CAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCAAATGATGGAACCTAT AAGCATTTCGCTGACTCCCTGAAGGGCCGATTCAGCATCTCCAGAGACGATTCCAAG AACACGCTGTATCTGCACATGAACAGCCTGAGACCTGACGACACGGCTATATATTAC TGTGCGAGAGATGGCCGTAGTGTTGGCGGGTTTAGTGGGATCCTCGACCCCTGGGGC CAGGGAACCCTGGTCACCGTCTCCTCAG

MAB310 HC variable domain nucleotide sequence (SEP ID NO:40)

CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGACGTCCCTGAGA CTCTCCTGTGCAGCCTCTGGATTCATGTTCAATACCTACAATATGCACTGGGTCCGC CAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCAAATGATGGAACCTAT AAGTACTCCGCTGACTCCCTGAAGGGCCGATTCAGCATCTCCAGAGACAATTCCAAG AACACGTTGTATCTGCACATGAACAGCCTGAGACCTGACGACACGGCTGTATATTAC TGTGCGAGAGATGGCCGTAGTGTTGGCGGGTTTAGTGGGATCCTCGACCCCTGGGGC CAGGGAACCCTGGTCACCGTCTCCTCAG

MAB313 HC variable domain nucleotide sequence (SEP ID NO:41 )

CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCGTGATCCAGCCTGGGAGGTCCCTGACA CTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGCCTATTCTCTACACTGGGTCCGC CAGGCTCCAGGCAAAGGGCTACAGTGGGTGGCGGTTATCTCATTTGATGGGAATTTT AAACACTTCGCAGACTCCCTGAGGGGCCGATTCACCATCTCCAGAGACAATTCCAAG AACAGATTCTATTTGCAAATGAATGGCCTGAGAGGTGAGGACACGGCTGTATATTAC TGTGCGAGAGATGGACGTGCTGTTGACGGGTTTAGTGGGATCCTCGACTTCTGGGGC CAGGGAACCCTAGTCAGCGTCTCCTCAG

MAB314 HC variable domain nucleotide sequence (SEP ID NO:42

CAGGTGCAGCTGCAGGAGTCGGGGGGAGGCTTGGTCCAGCCGGGGGGGTCCCTGAAA CTCTCCTGTGCAGTCTCTGGATTCTCCTTCGGTGGCTCTGCAATGCACTGGGTCCGC CAGGCTTCCGGGAAAGGGCTGGAGTGGATTGGCCATATTAGAAGCGGAGCTAATAAT TTCGAGACAGCATATGCTCCGTCGCTGGATGGCAGGTTCACCATCTCCAGAGACGAT TCAAAGAACACGGCGTATCTGCACATGAACAGCCTGAAAACCGATGACACGGCCATG TATTTCTGCACTACCGGACTTATAGCGTCAGGTGATGCAAATTTTGACTACTGGGGC CAGGGAACCCAGGTCACCGTCTCCTCGG

MAB316 HC variable domain nucleotide sequence (SEP ID NP:43

CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGACA CTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGGCTTTTCTCTACACTGGGTCCGC CAGGCTCCAGGCAAGGGGCTACAGTGGGTGGCGGTTATCTCATTTGATGGGAACCAT AAACACTTCGCAGACTCCCTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAG AACACATTGTATTTGCAAATTAATGACCTGAGAGGTGAGGACACGGCTGTATATTAC TGTGCGAGAGATGGACGTGCTGTTGACGGGTTTAGTGGGATTCTCGACTTCTGGGGC CAGGGAACCCTGGTCAGCGTCTCCTCAG

MAB319 HC variable domain nucleotide sequence (SEP ID ΝΡ:44Ί

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA CTCTCCTGTTCAGCCTCAGGATTCACCTTCAGTGACTATAATCTACACTGGGTCCGC CAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTCATCTCAATTGATGGAAGCGAT AAACACCACGCAGACTCCGTGAAGGGCCGATTCACCGTCTCCAGAGACAATTCCAAG AACACAGTGAGTCTACAAATGGACAGCCTGAGACCTGAAGACACGGCTGTATATTAC TGTGCGAGAGATGGCCGTAGTGTGGGCGGCTACAGTGGGATCCTCGACCCCTGGGGC CAGGGAACCCTGGTCACCGTCTCCTCAG

MAB321 HC variable domain nucleotide sequence (SEP ID NO:45

GAGGTGCAGCTGGTGGAGTCCGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAG ATCTCCTGTCAGGGTTCTGGATACAGGTTTACCAATTACTGGATCGCCTGGGTGCGC CAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGAT ACCAGATATCACCCGTCCTTCCAAGGCCAGGTCACCATCTCATCCGACAAATCCCTC AACACCGCCTACCTGCAGTGGAGCAGCCTGAAGCCCTCGGACACCGCCGTGTATTAC TGTGCGAGACACCACTGCCTTAGTACCAACTGCCAAACCGCAGTGGCTGGATATAAT GACTACTGGGGCCAGGGAAACCCTGGTCGCCGTCTCCTCAG

MAB322 HC variable domain nucleotide sequence (SEP ID NO:46)

CAGGTGCAGCTGGTGGAGTCCGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA CTTTCCTGTTCAGCCTCTGGATTCACCTTCACCAACTATAACATGCACTGGGTCCGC CAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTACGTCAAAAGATGGAAACGAA AAACACTTTGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAG AACACGCTGTATCTGGAAATGAACACCCTGACAGCTGAGGACACGGCGATATATTAC TGTACGAGAGATGGGCGAACCGATGGTACTGGGTACTCCGGTATTCTTGATATCTGG GGCCAAGGGACAAAGGTCACCGTCTCCTCA

MAB323 HC variable domain nucleotide sequence fSEP ID NO:47)

CAGGTGCAGCTGGTGCAGTCTGGGGGAGGGGTGGTCCAGCCTGGGAGGTCCCTGAGA CTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACTTTGCTATGCACTGGGTCCGC CAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCAAATGCTGGAAGGGAA ACACACTACGCAGACTCCGTGAAGGGCCGATTCACCGTCTCCAGAGACAATTCCAAG AATATGTTGTCTCTGCAAATGAACAGCCTGAGAGGTGAGGACACGGCTGTGTATTAC TGTGCGAGAGATGGGCGAACCGATGGTAGTGGCTATTCCGGTGTTCTTGATATCTGG GCCCAAGGGACACTGGTCACTGTCTCCTCA MAB338 HC variable domain nucleotide sequence (SEQ ID NO:48

CAGGTGCAGCTGGTGGAGTCCGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGA

GACTCTCCTGTTCAGGCTCTGGATTCACCTTCAGTGACTATAATCTACACTGGGTCC

GCCAGGCTCCAGGCAAGGGGCTGGAATGGGTGGCAGTCATTTCAATTGATGGAACT

AATAAACACCACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACTC

CAAGAATACAGTGAATCTGGAAATGAGTCGGCTGAAAGCAGAAGACACGGCTGTA

TATTACTGTGTGAGAGATGGGCGAAGTATTGGCGGCTACAGTGGAATCTTCGACCC

CTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

MAB343 HC variable domain nucleotide sequence (SEQ ID NO:49)

CAGGTGCAGCTGCAGGAGTCAGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA CTCTCCTGTGCAGCCTCTGGATTCACCTTCAATACCTACAATATGCACTGGGTCCGC CAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCAAATGATGGAACCTAT AAATACTCCGCTGACTCCGTGAAGGGCCGATTCAGCATCTCCAGAGGCAATTCCAAG AACACGTTGTATCTGCAGATGAACAGCCTGAGACCTGACGACACGGCTGTATATTAC TGTGCGAGAGATGGGCGTAGTGTTGGCGGGTTTAGTGGGATCCTCGACCCCTGGGGC CAGGGAACCCTGGCCACCGTCTCCTCA

MAB345 HC variable domain nucleotide sequence (SEQ ID NO:50)

CAGGTGCAGCTGGTGGAGTCCGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA CTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACAATATGCACTGGGTCCGC CAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATTTCAATTGATGGAACGTAT AAATACTCCGCTGACTCCGTGGCGGGCCGATTCAGTCTCTCCAGAGACAATTCCAAG AACACGTTGTATTTGCAGATGAATAGTCTGAGACCTGACGACACGGCTATATATTAT TGCGCGAGAGATGGGCGTAGTGTTGGCGGGTTTAGTGGGATCCTCGACCCCTGGGGC CAGGGAACCCTGGTCACCGTCTCCTCAG

Human LC nucleotide sequence of constant kappa region (SEQ ID NO:51)

ACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCT GGAACTGCTAGCGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTA CAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAG CAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCA GACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCG CCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

MAB297 and MAB322 LC variable domain nucleotide sequence (SEP ID NO:52)

GAAATTGTAATGACGCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCC ACCCTCTCCTGCAGGGCCAGTCAGAGTGTTGGCGGCTACTTAGCCTGGTACCAACAG AAACCTGACCAGGCTCCCAGGCTCCTCATCTATGATGTTTCCAATAGGGCCGCTGGC ATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGC AGCCTGGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGGAACACCTGGCCT CCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGA MAB309 LC variable domain nucleotide sequence (SEP ID NO:53)

GAAATTGTGTTGACGCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGATAGAGCC ACCCTCTCCTGCAGGGCCAGTCAGACTGTTGGCAGGTACTTAGCCTGGTACCAACAA AAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCTTCCGACAGGGCCACTGGC ATCTCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGATTTCACTCTCACCATCAGC AGCCTGGAGCCTGAAGATTTTGCAGTCTATTACTGTCAGCAGCGGAGCAGCTGGCCG CCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGA

MAB310 LC variable domain nucleotide sequence (SEQ ID NO:54)

GAAATTGTGTTGACTCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGATAGAGCC ACCCTCTCCTGCAGGGCCAGTCAGACTGTTGGCAGGTACTTAGCCTGGTACCAACAG AAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCTTCCGACAGGGCCACTGGC ATCTCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGATTTCACTCTCACCATCAGC AGCCTAGAGCCTGAAGATTTTGCAGTCTATTACTGTCAGCAGCGGAGCAACTGGCCT CCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGA

MAB313 LC variable domain nucleotide sequence (SEQ ID NO:55)

GAAATTGTGATGACTCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCC ACCCTCTCCTGCAGGGCCAGTCAGAGTGTTGGCAGATACTTAACTTGGTTCCAGCAG AAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCTTCCGAGAGGGCCACTGGC ATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGC AGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAACAGCGTGCTAACTGGCCT CCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGA

MAB314 LC variable domain nucleotide sequence (SEQ ID NO:56)

GAAATTGTGATGACCCAGTCTCCAGGCACCCTGTCCTTGTTTCCAGGGGAAAGAGCC ACCCTCTCCTGCAGGGCCAGTCAGACTGTTAGGAACGGCTACTTAGCCTGGTACCAG CAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCTTCCATCAGGGCCACT GGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGAGACAGACTTCACCCTCAGCATC ACCAGAGTGGAGCCTGAAGATTTTGCAGTTTATTACTGTCAACAGTATGGAAGGTTA TCGTCCACTTTTGGCCAGGGGACCAAGCTGGACCTCAAACGA

MAB316 LC variable domain nucleotide sequence (SEQ ID NO:57)

GAAATTGTGATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCC ACCCTCTCCTGCAGGGCCAGTCAGAGTGTTGGCAGATACTTAACTTGGTTCCAGCAG AAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCTTCCGAGAGGGCCACTGGC GTCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGC AGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAACAGCGTAGTAACTGGCCT CCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAC MAB318 LC variable domain nucleotide sequence (SEP ID NO:58^{^})

GAAGTTGTGCTGACGCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGATAGAGCC ACCCTCTCCTGCAGGGCCAGTCAGACTGTTGGCAGGTACTTAGCCTGGTACCAACAA AAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCTTCCGACAGGGCCACTGGC ATCTCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGATTTCACTCTCACCATCGGC AGCCTGGAGCCTGAAGATTTTGCAGTCTATTACTGTCAGCAGCGGAGCAGCTGGCCG CCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAC

MAB319 LC variable domain nucleotide sequence (SEP ID NO:59

GAAATTGTGTTGACGCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGGGCC ACCCTCTCCTGCAGGGCCAGTCAGAGTGTTGGCAGCTACTTAGCCTGGTATCAACAG AAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCGAGAGGGCCACTGGC ATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGC AGCCTAGAGCCTGAAGATGTTGCAGTTTATTACTGTCAGCAGCGTAACAACTGGCCT CCGCTCACCTTCGGCGGAGGGACCAAGGTGGAGATCAAAC

MAB321 LC variable domain nucleotide sequence (SEQ ID NO:60)

GAAATTGTGATGACCCAGTCTCCAGACTCCCTTGCTGTGTCTCTGGGCGAGAGGGCC ACCATCAACTGCAAGTCCAGTCAGAGTATTTTATTCAGCTCCAAGAATCAGAACCAC TTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTGATTTACTGGGCA TCTACCCGGGAATCCGGGGTCCCCGACCGATTCAGTGGCAGCGGGTCTGGGACAGAT TTCACTCTCACCATCAGCAGCCTCCAGGCTGAAGATGTGGCAGTTTATTACTGTCAG C AATATTAT AATATT C CTC ACACTTTCGGC GGAGGGAC C AAGGTGGAGAT C AAA

MAB323 LC variable domain nucleotide sequence (SEP ID NO:61

GAAATTGTGTTGACTCAGTCTCCAGCCACCTTGTCTTTGTCTCCAGGGGAAAGAGCC ACCCTCTCCTGCCGGGCCAGTCAGAGTGTTAACCGCTACTTAGCCTGGTTCCAACAC AGACCTGGCCAGCCTCCCAGGCTCCTCATCTATGATGCGTCCAAGAGGGCCACTGGC ATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGC AGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCG CTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAG

MAB338 LC variable domain nucleotide sequence (SEP ID NP:62)

GAAATTGTGTTGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCC ACCCTCTCCTGCAGGGCCAGTCAGAGTGTTGACAGGTACTTAGCCTGGTACCAACAG AAACCTGGCCAGGCTCCCAGACTCCTCATCTATGATGCATCCCAGAGGGCCACTGGC ATCCCAGCCAGGTTCAGTGGCAGTGGGTCCGGGACAGACTTCACTCTCGCCATCAGC AGCCTGGAGCCTGAAGATGTTGCAGTTTATTACTGTCAGCAGCGTAGTAACTGGCCT CCGCTCACCTTCGGCGGAGGGACCAAAATAGAGATCAAA MAB343 LC variable domain nucleotide sequence (SEP ID NO:63 )

GAAATCGTGATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGATAGAGCC ACCCTCTCCTGCAGGGCCAGTCAGAGTGTTGGCAGCTACTTAGCCTGGTACCAACAG AAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCTTCCGACAGGGCCACTGGC ATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGATTTCACTCTCACCATCAGC AGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCT CCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAC

MAB345 LC variable domain nucleotide sequence (SEP ID NO:64)

GAAATTGTGATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGATAGAGCC ACCCTCTCCTGCAGGGCCAGTCAGAGTGTTGGCAGCTACTTAGCCTGGTACCAACAG AAACCTGGCCAGGCTCCCAGGCTCCTCATGTATGATTCTTCCGTCAGGGCCACTGGC ATCCCAGCCAGGTTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGC AGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAACAACTGGCCT CCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA

Human LC nucleotide sequence of constant kappa region (SEP ID NP:65)

ACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCT GGAACTGCTAGCGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTA C AGTGGAAGGTGGATAAC GC C C TC C AATCGGGT AACT C C CAGGAGAGTGTC ACAGAG CAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCA GACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCG CCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

Human LC nucleotide sequence of constant lambda region (SEP ID NP:66)

GGTCAGCCCAAGGCTGCCCCCTCTGTCACTCTGTTCCCGCCCTCTAGCGAGGAGCTT CAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTG ACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACA CCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCT GAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACC GTGGAGAAGACAGTGGTCCCTGCAGAATGCTCT

Claims

1. An isolated monoclonal antibody (mAb) or immunoreactive

fragment thereof that:

(a) binds the gB protein of cytomegalovirus (CMV) strain AD 169 with an affinity of at least 10 nM;

(b) is a human antibody or fragment, and

(c) neutralizes CMV in fibroblast cell culture.

2. The mAb of claim 1 that is in the form of a complete antibody.

3. The mAb of claim 1 that is a bi-specific antibody.

4. The mAb or fragment of claim 1 that has an affinity for gB of the AD169 strain of CMV of at least 1 nM.

5. The antibody of claim 1 wherein the heavy chain has a CDR1 of GFTFNRHG (SEQ ID NO:67) or GSISSEDFC (SEQ ID NO:68); and/or

wherein the heavy chain has a CDR2 region of SSDGANQ (SEQ ID NO:69)or ICYTGD (SEQ ID NO:70); and/or

wherein the heavy chain has a CDR3 region of ARDGRCEGERCYSGVTDF (SEQ ID NO:71) or AREDRRQLHSRPYFYYGLDV (SEQ ID NO:72).

6. The antibody of claim 1 wherein the light chain has a CDR1 region of QNIGGY (SEQ ID NO:73) or QSLLHSNGHNY (SEQ ID NO:74); and/or

wherein the light chain has a CDR2 region of DAS (SEQ ID NO:75) or LG (SEQ ID NO:76); and/or

wherein the light chain has a CDR3 region of QQRNSWPPLT (SEQ ID NO:77) or QALQTPNT (SEQ ID NO:78).

7. The antibody or fragment of claim 6 which is 4A2, 19B 10, 313 , 338, or 345.

8. One or more nucleic acid molecules that comprise first nucleotide sequence(s) that encode(s) the mAb or fragment of any of claims 1-7 or one or more nucleic acid molecule(s) that comprise(s) second nucleotide sequence(s) complementary to said first nucleotide sequence(s) over its(their) entire length.

9. Recombinant host cells comprising expression systems that produce the mAb or fragment of any of claims 1-7, wherein the recombinant host cells are mammalian cells, microbial cells, insect cells or plant cells.

10. A method to produce an mAb or immunoreactive fragment thereof, which method comprises culturing the cells of claim 9 and recovering said mAb or fragment.

11. A pharmaceutical composition that comprises an isolated monoclonal antibody or fragment of any of claims 1 -7, along with a pharmaceutically acceptable excipient.

12. The pharmaceutical composition of claim 11 that further contains an additional pharmaceutical agent, along with a pharmaceutically acceptable excipient.

13. A method to treat CMV in a human subject infected with CMV, which comprises administering to a subject in need of such treatment an effective amount of the antibody or fragment of any of claims 1-7.

14. A method to enhance resistance to infection by CMV in a human subject, which comprises administering to a subject in need of such enhancement an effective amount of the antibody or fragment of any of claims 1-7.

15. The method of claim 13 or 14 wherein the subject is immunocompromised.

16. The method of claim 13 or 14 wherein the subject is a pregnant woman.